Symposium SB12—Biomaterials for Regenerative Engineering

The treatment of bone injuries that necessitate bone regeneration continues to be a major challenge for the orthopaedic surgeon. This burden is compounded by supply constraints and associated morbidity with autograft tissues, the gold standard of repair. The use of allografts, xenografts, or metal and ceramic implants overcomes many of these limitations, but fail to provide a viable solution. Over the past 30 years, we have worked to engineer bone with a focus on biomaterial selection, material matrix development, cell/material interaction studies, and the development of inducible materials.

We have developed poly(ester), poly(anhydride), and poly(phosphazene) biomaterials for bone engineering applications through biocompatibility, degradation, and mechanical analyses. We developed a novel three-dimensional sintered microsphere matrix possessing a pore structure and mechanical strength within the range of human cancellous bone, a version of which is now clinically available. We also reported on attachment, growth, proliferation, and differentiation of osteoblasts, mesenchymal-, and marrow-derived stem cells on several of the material matrices we have developed. We have demonstrated regenerative bone formation in vivo using these biomaterial technologies. This entire body of work over more than thirty years has largely been responsible for the awarding of the highest honor for technological achievement in the United States, The National Medal of Technology and Innovation, and the American Association for the Advancement of Science’s highest award, the Philip Hauge Abelson Prize for ‘signal contributions to the advancement of science in the United States’.

Human ventricles are made up of helically arranged myofibers, that transition across the ventricle wall from a left- to right-handed helix. For more than fifty years, it has been argued that this helical structure is critical for achieving the large ejection fractions (60-80%) observed during healthy cardiac contraction while maintaining minimal ventricular strain. However, testing this fundamental structure-function relationship has been challenging, as in vivo studies are often characterized by concomitant changes in protein expression and metabolism, while in vitro studies have difficulty in reproducing the complex three-dimensional (3D) structures of the heart. Addressing this challenge, here we show how additive textile manufacturing approaches, such as Focused Rotary Jet Spinning (FRJS), can be used for the rapid manufacture of micro/nanofibers scaffolds with controlled alignments and helical architectures. As aligned micron-scale fibers can be used to direct tissue orientation and subsequent morphogenesis, we reasoned this approach would allow for the biofabrication in vitro models that are reminiscent of early simulations of cardiac contraction. Producing both helically and circumferentially aligned 3D biohybrid models of the left ventricle, we show that helically aligned ventricles displayed increased strain uniformity, axial shortening, cardiac output, and ejection fractions as compared to circumferential models. Additionally, we demonstrate how helically aligned scaffolds preserve some features of ventricle twist, a phenomenon observed in clinical readouts, but not previously reported in engineered biohybrid systems. Overall, this work shows that myofibril alignment plays a critical role in regulating cardiac performance, with important implications towards cardiac disease. Additionally, this approach suggests that additive textile manufacturing may serve as a valuable approach for future biofabrication, which can be used in conjunction with, or as a potential alternative to, more traditional methods such as 3D bioprinting.

The selection of a proper material to be used as a scaffold, as a proper matrix, or as a bioink in 3D bioprinting approaches to support or encapsulate cells is both a critical and a difficult choice that will determine the success of failure of any tissue engineering and regenerative medicine (TERM) strategy.

In our research group we have been mainly using natural origin polymers, including a wide range of marine origin materials, for many different approaches that allow for the regeneration of different tissues. Several innovative bioinks with quite specific properties were developed and proposed for several specific uses. We have also been optimizing the respective formulations for using these novel materials in distinct biomanufacturing strategies. This will be presented and discussed during the present keynote talk.

Furthermore, an adequate cell source should be selected. In many cases efficient cell isolation, expansion and differentiation methodologies should be developed and optimized. We have been using different human cell sources namely: mesenchymal stem cells from bone marrow, mesenchymal stem cells from human adipose tissue, human cells from amniotic fluids and membranes and cells obtained from human umbilical cords.

The potential of each materials/cells combination, as related to different manufacturing technologies, with details when appropriated focusing on bioprinting, to be used to develop novel useful regeneration therapies will be discussed. Several examples of TERM strategies to regenerate different types of tissues will be presented. The use of different cells and new ways to assess their interactions with different natural origin degradable scaffolds and bioinks will be described. A unique high-throughput platform to better understand material/cells interactions and optimise their performance and biological performance will be discussed. This rather innovative platform is based on the use of unique microfluidics-based approaches.

INTRODUCTION: Despite serving as the clinical “gold standard” treatment for critical size bone defects, decellularized allografts suffer from long-term failure rates of ~60% due to the absence of the periosteum. To promote allograft healing, our lab has pioneered the tissue engineered periosteum (TEP) based on poly(ethylene glycol) (PEG) hydrogels. The hydrogels entrap mouse mesenchymal stem cells (mMSCs) and mMSC derived osteoprogenitors (mMSC-OPs) and surround allografts to emulate native periosteum cell populations and subsequent paracrine factor production to coordinate periosteum-mediated healing. Despite robust angiogenic factor production, the TEP showed only modest increases in vascular volume versus unmodified allograft controls. Additionally, even though the maximum torque of TEP modified allografts was 2-fold higher versus unmodified allografts after 9 weeks, it was only ~40% of autografts, indicating delayed healing. These results are attributed to limited vascular recruitment within the hydrolytically degradable TEP (hydro-TEP), as its mesh size (~15 nm) is smaller than that of leading filipodia of migrating cells (0.2-0.4 µm). Therefore, to investigate the hypothesis that the degradation mechanism of the TEP is critical for coordinating host tissue infiltration necessary for successful bone, this study compared healing of Hydro-TEP, which bulk degrades, with that of the matrix metalloproteinase (MMP) degradable TEP (MMP-TEP), which enables host cell-dictated degradation.

MMP-degradable hydrogels supported endothelial cell migration in vitro whereas no migration was observed in hydrolytically degradable hydrogels. At week 3 post-implantation, ~2.5 and 1.8-fold, and ~3.5 and 2.6-fold greater vessel and nerve densities with high levels of vessel/nerve co-localization was observed using MMP-TEP-modified allografts versus unmodified and Hydro-TEP-modified allografts, respectively. At 3, 6, and 9 weeks post-implantation, the graft-localized vascular volumes of MMP-TEP-modified allografts (0.22 ± 0.8, 0.20 ± 0.8, and 0.17 ± 0.07 mm³) were significantly greater than those of unmodified allografts (0.002 ± 0.0004, 0.05 ± 0.04, and 0.12 ± 0.03 mm³) and Hydro-TEP-modified allografts (0.06 ± 0.004, 0.02 ± 0.001, and 0.05 ± 0.02 mm³). Furthermore, MMP-TEP-modified allografts showed 4.5, 4.6, and 2.8-fold increase in bone volume versus unmodified allografts after 3, 6, and 9 weeks respectively, and enhanced mineralization density of bone callus after 9 weeks versus unmodified and Hydro-TEP-modified allografts. MMP-TEP-modified allografts exhibited approximately 8 and 2-fold enhancements in maximum torque strength versus Hydro-TEP-modified allografts after 6 and 9 weeks respectively, reaching a comparable maximum torque to that of autografts at 9-week post-surgery.

Bone regeneration is synergistically mediated by angiogenesis and innervation. Consistent with this synergy, endothelial sprouting within MMP-degradable hydrogels in vitro paralleled significantly increased and co-localized vessel and nerve densities observed in vivo, and longitudinal graft-localized vascularization. Furthermore, the robust early stage neurovascularization of MMP-modified allografts significantly increase bridging endochondral callus formation with greater mineralization versus unmodified and Hydro-TEP modified allografts, leading to enhanced maximum torque, which was comparable to that of autografts at 9 weeks post-surgery. In summary, these data indicate that the MMP-TEP effectively coordinates allograft healing via early stage recruitment and support of host neurovasculature. The MMP-TEP improved allograft healing, while overall outcomes still lagged behind those of autografts. In future studies, additional matrix cues, such as alternative extracellular matrix-derived peptides and MMP-degradable crosslinkers can be explored to further orchestrate improved healing.

According to the World Health organization, there were 2.26 million women diagnosed with breast cancer and 1.41 million men with prostate cancer in 2020. Further, over a million deaths were reported due to breast cancer and prostate cancer globally in 2020. Both breast cancer and prostate cancer have the propensity to metastasize to bone, leading to a terminal prognosis for the patient. As of 2020, breast or prostate cancer that has metastasized to the bone is essentially incurable, and only palliative treatments exist. In the event of bone metastasis, skeletal failures are often the cause of death in patients. The inefficacy of anticancer drugs and failure of animal models necessitates the need for in vitro models. We have developed a novel bone mimetic scaffold that uses tissue engineering approaches to generate bone mimetic scaffolds for generation of bone that is in a remodelling stage; a preferred niche to which breast and prostate cancer migrate towards. Thus besides providing valuable opportunities for bone regenerative treatment the tissue engineered bone has additional uses as a testbed to evaluate bone metastasis of cancer. The bone mimetic scaffold utilizes unnatural amino acid modified nanoclays to mineralize hydroxyapatite mimicking the process of biomineralization. We report the generation of bone metastasized tumors at the stage of mesenchymal to epithelial transition on seeding the bone scaffold with commercial cell lines of breast cancer and also prostate cancer. We have developed cell lines from patient derived tissues that are also seeded onto the bone mimetic scaffolds to develop patient derived in vitro tumors of breast cancer bone metastasis. Further, we evaluate the mechanisms of influence of cancer cells on osteogenesis pathways (the Wnt bcatenin pathway) and demonstrate the specific role of cancer cell factors DKK and ET-1 on osteogenesis at the bone site. We have also developed unique mechanics-based biomarkers that use direct nanoindentation to evaluate the change in elastic and viscoelastic properties of cancer cells as a measure of progression of bone metastasis with the use of the testbeds. We also report the use of the testbed to screen new drugs for treatment of bone metastasis as well as report their influence on bone. Overall, the use of tissue engineered constructs present a useful testbed for evaluation of cancer.

Introduction: Across various areas of regenerative engineering, there have been limitations with controlling cell adhesion, stem cell differentiation, viability, growth, and blood-material compatibility. Conversion of simple and abundant items to advanced cell culture substrates addresses some of the current challenges in regenerative engineering. The departure from complex fabrication processes to the applications of unusual and ubiquitous materials provides another dimension to the engineering of viable multiscale tissue constructs and regenerative technologies.^1-4 In addition, the use of sustainable constructs and novel fabrication strategies has the potential to increase access to regenerative engineering technologies on a global scale. In this work, we used unconventional biomaterials such as eggshells and paper for tissue repair.
Materials and Methods: We fabricated eggshell micro/nanoparticle (ESP) reinforced gelatin-based hydrogels to obtain mechanically stable and biologically active three-dimensional (3D) constructs that can differentiate stem cells into osteoblasts. The ESP-reinforced gels were then subcutaneously implanted in a rat model to determine their biocompatibility and degradation behaviors. In addition, these composite scaffolds were used to regenerate critical sized cranial defects in a rat model. We also used mineralized paper scaffolds through an origami-inspired approach to test their osteoinductivity and potential for tissue repair in in vitro and in vivo studies.
Results and Discussion: The ESP-reinforced scaffolds enabled the differentiation of stem cells without the use of specialized osteogenic growth medium. The ESP-reinforced gels exhibited significant enhancement in mineralization by the cells. Our findings indicated that the ESP composites exhibited superior mechanical properties and showed a favorable in vivo response by subcutaneous implantation in a rat model. These scaffolds were highly responsive to cells, and did not elicit inflammatory responses in vivo. The implants were easily accepted by the host, allowed for cellular infiltration in 3D, and highly vascularized. Implantation of ESP-reinforced scaffolds into critical sized calvarial defects in a rat model resulted in significant bone regeneration in 12 weeks. The resulting bone volume and bone density were as high as the native bone using these composite scaffolds as determined by micro-computed tomography analyses.
We also fabricated origami-inspired paper-based scaffolds for biomineralization. Material properties of the paper-based mineralized scaffolds were determined to be highly tunable. The tensile modulus of the scaffolds increased significantly after the mineralization process. Gene
expression results for the osteogenic differentiation markers revealed the osteoinductivity of the mineralized paper scaffolds. Subcutaneous implantation of the samples in rats demonstrated biocompatibility, vascularization, and integration in vivo.
Conclusions: Unconventional scaffolds that are readily available and adapted from nature exhibited biomimetic characteristics including porosity, structure, and bioactivity resulting in physiologically relevant constructs. The use of existing naturally derived materials for regenerative engineering provides an inexpensive and sustainable approach that benefits the economy and environment while providing unique solutions to unmet clinical needs. Many of the unconventional biomaterials are overlooked and under-studied for biomedical applications, partially for their simplicity as mundane items.
References: ¹Nguyen, M.A., Camci-Unal, G., Trends in Biotechnology, 38(2): 178-190, 2020. ²Suvarnapathaki, S., Wu, X., Lantigua, D., Nguyen, M.A., Camci-Unal, G., Nature Asia Materials, 11(1): 1-18, 2019. ³Wu, X., Stroll, S.I., Lantigua, D., Suvarnapathaki, S., Camci-Unal, G., Biomaterials Science, 7, 2675-2685, 2019. ⁴Ahmed, A.R., Gauntlett, O., Camci-Unal, G., ACS Omega, 6(1): 46-54, 2021.

Introduction 3D bioprinting has the potential for becoming a breakthrough method in the implementation of skeletal muscle (SM) tissue engineering. Bioink chemical cues and mechanical properties have been widely investigated in the last decade¹. Less explored is the incorporation of exogenous stimulations (e.g. electrical, magnetic, mechanical) to boost cell differentiation. Piezoelectric nanoparticles (NPs) have been used as nanoscale transducers able to convert mechanically-induced deformation into an electrical cue when invested by an ultrasound wave (acting as a wireless source of mechanical energy). This paradigm has shown beneficial effects on different cell types, accelerating the differentiation of neural and muscle precursors^2,3. No research groups have yet explored the inclusion of piezoelectric NPs in a bioink for SM cells 3D bioprinting. This work shows preliminary results on the properties of a piezoelectric bioink based on Pluronic/alginate doped with barium titanate NPs (BTNPs) and the biological response of C2C12 cells encapsulated in it.
Methods BTNPs (diameter=~60 nm) were added to propylene glycol alginate (PGA, ratio 1:1). A hydrogel of 20% w/w Pluronic F127 and 2% w/w alginate was prepared as described in⁴. Four bioinks (one bare bioink and three ones with BNTPs at 100, 250, 500 μg/mL respectively) were loaded with 2x10⁶ C2C12 cells/mL, printed with a 3D Bioplotter (Envisiontec GmbH) in one layer of parallel fibers (7x7 mm², nozzle diameter=250 μm, 37°C, pressure=0.4 bar, speed=10 mm/s), and then crosslinked (25 mM CaCl₂, 10 min). Hydrogels were characterized through SEM, EDX and rheometry. Viability tests at 72 h were carried out using LIVE/DEAD and PrestoBlue assays. For differentiation experiments, two constructs (non-doped and doped with 250 μg/mL BNTPs) were cultured in growth medium (5 days) and for 4 days in differentiation medium (DM=DMEM, 1% ITS, 1% FBS, 1% P/S). Half of the constructs were treated with low-intensity pulsed ultrasound (LIPUS; frequency=1 MHz; spatial average pulse average intensity=250 mW/cm²; pulse repetition frequency=1 kHz; duty cycle=20%; exposure time=5 min) in DM, through a dedicated setup allowing precise control of the LIPUS dose at the cells⁵, while the other half was not stimulated. After 4 days, the expression levels of MYOG, CSRP3, MYH2 and GAPDH (reference gene) were assessed through real-time qRT-PCR. Cells stained with TRITC-phalloidin and DAPI were imaged using a confocal microscope. All experiments were performed in triplicate for each condition.
Results and Discussion SEM images and EDX results showed that BNTPs were uniformly dispersed in the printed hydrogels, even at high concentrations of BTNPS. Rheometric results highlighted no significant differences between the different bioinks in terms of storage, loss moduli and viscosity. Viscosity decreased under shear strain, showing a shear-thinning behavior. High cell viability was observed up to a BNTP concentration of 250 μg/mL. Constructs with 500 μg/mL BNTPs showed lower cell viability, therefore were excluded for further studies. Differentiation tests showed that the LIPUS-treated doped bioink (250 μg/mL) promoted a significantly higher expression of MYOG, CSRP3 and MYH2 (key genes in early myogenesis), with respect to the control (bare bioink).
Conclusion The preliminary results here reported, demonstrate the potential of piezoelectric bioinks for the development of 3D printed SM constructs and the myogenic effect of the interaction between LIPUS stimulation and piezoelectric nanomaterials, in this context.
Acknowledgments This work received financial and technical support by INAIL, in the framework of the project MIO-PRO.
References [1] Zhuang P., et al. (2020). Mater. Des., 108794
[2] Ricotti L., et al. (2013). PloS one, 8(8), e71707
[3] Marino A., et al. (2015). ACS Nano, 9(7), 7678-7689
[4] Mozetic P., et al (2017). J Biomed Mater Res, Part A, 105(9), 2582-2588
[5] Fontana F., et al. (2021). Ultrasonics, 106495

Dental demineralization, tooth resorption and endodontic treatments have a high impact on dentistry. Bioglass and calcium phosphate 3D printed scaffolds with suitable degradability and compatibility characteristics are a suitable choice for your solution. However, due to oral lesions, it is necessary to consider the behavior of these materials against pathogens in the dental area. These effects can be reduced by doping with elements associated with the biological processes of bone repair and / or regeneration such as boron (B) and magnesium (Mg), which also affects the reabsorption process of the material due to the release of ions that they generate. an antimicrobial effect. The objective of this work is to compare the antimicrobial activity of calcium phosphate powders obtained by sol-gel and self-combustion routes and scaffolds formed with these powders by 3D printing against C. albicans. and S. mutans. Calcium phosphates in sol-gel processes present a mixture of various types of crystalline phases, whereas self-combustion leads to a single β-TCP phase. The in vitro antimicrobial activity study showed that calcium phosphate obtained via self-combustion and magnesium doped (β-TCP / Mg) had a better effect than calcium phosphate obtained via sol-gel (CP-S) with and without doping against C. albicans. For S. mutans no inhibitory effect was found. In addition to the co-cultivation of these two species, it showed an inhibitory effect on the evaluated materials. These results made it possible to formulate a ceramic paste based on the β-TCP / Mg / bioglass system by Design of Experiment (DOE) to obtain 3D printing scaffolds. These ceramic materials show promise as devices in the regeneration of dental tissues.

INTRODUCTION A stable adhesion to the cartilage tissue is a crucial requisite for hydrogels used in the fields of cartilage substitution and regeneration. Indeed, the presence of a weak interface between the cartilage and the implanted material may lead to an early detachment, thus causing the failure of the cartilage repair or regeneration process [1]. Using an adhesive primer, namely a coating applied to the cartilage surface to promote the adhesion of materials, may overcome such an issue. Different primers (fibrin glue (FG) [2], cellulose nanofibers (CNF) [3] and catecholamines (CAT) [4]) have been proposed in the state-of-the-art. However, no studies have systematically compared their performance. This study aims at evaluating the adhesion strength between the cartilage tissue and gellan gum (GG)-based hydrogels, crosslinked using ions or, after methacrylation, through a light source.
METHODS GG was dissolved in d-H₂O at 2 % w/v by stirring for 1 h at 37°C, while gellan gum methacrylate (GGMA) was dissolved in PBS at 2 % w/v for 1 h at 37 °C. Then, ruthenium (Ru) and sodium persulfate (SPS) were added to GGMA at a concentration of 0.2 and 2 mM, respectively.
FG was deposited evaluating different waiting times before hydrogel deposition, namely 30 s, 5 min, 15 min and, 30 min. CNFs, dispersed in d-H₂O, were added to GG and GGMA at two concentrations (0.1 and 0.5% w/v) to form a CNF-based primer to be added before GG and GGMA. CAT-modified gelatin was dissolved in a Tris-buffered saline buffer at two concentrations (100 and 125 mg/mL). Then, the solutions were mixed with 1 mM Ru and 20 mM SPS and then crosslinked through a white LED for 30 s.
Before performing adhesion tests, GG or GGMA were cast on each primer and crosslinked for 10 min with MgCl₂ (1 % w/v in d-H₂O) (GG) or with the LED light (GGMA).
To evaluate the adhesion strength, two cartilage-hosting parts were used to block bovine cartilage samples and a top hydrogel-hosting part allowed pouring the primer and the hydrogel solution onto the cartilage. A traction test was performed through an Instron machine.
RESULTS Results showed that the photo-crosslinked GGMA showed higher adhesion strength than GG in absence of primers. However, the adhesion strength of both GG and GGMA improved in the presence of FG with respect to the control. In particular, the waiting times of 5 min for GGMA (9.80 ± 3.03 kPa for GGMA) and 15 min for both the hydrogels (12.64 ± 0.87 kPa for GG and 10.99 ± 3.14 kPa for GGMA) guaranteed to match the clinical goal (10 kPa) [5]. The adhesion strength using CNFs and GG hydrogels reached a value of 12.13 ± 4.69 kPa (clinically acceptable) in the case of 0.1 % w/v CNFs, while the strength was smaller for the higher concentration of CNFs.
The adhesion strengths obtained with the CAT-based gelatin for GG increased from 6.16 ± 1.87 kPa (100 mg/mL) to 8.85 ± 2.40 kPa (125 mg/mL) and for GGMA from 2.93 ± 1.52 kPa (100 mg/mL) to 8.06 ± 1.38 kPa (125 mg/mL), without reaching the clinically accettable value.
CONCLUSIONS We found that the use of primers can guarantee an increased adhesion of GG-based hydrogels onto the cartilage. In the case of FG primer the best condition was represented by a waiting time of 15 min between primer pouring and subsequent hydrogel deposition on the cartilage, in all cases. In the case of embedded CNFs, the fiber concentration of 0.1 % w/v considerably increased the adhesion strength of GG and GGMA hydrogels. The CAT-modified gelatin layer was less effective in promoting the adhesion between the hydrogels and the cartilage.
ACKNOWLEDGMENTS This work received funding from the European Union's Horizon 2020 research and innovation program, grant agreement No 814413, project ADMAIORA.
REFERENCES [1] L. Zhang et al., Crit. Rev. Biomed. Eng., 2009
[2] F. Scognamiglio et al., 2016
[3] P. Karami et al., 2018
[4] Y. Liu et al., Colloids Surfaces B Biointerfaces, 2018
[5] D. A. Wang et al., Nat. Mater., 2007

Cryopreservation of patient immune cells is an essential step in the scaled-up production of cell therapies such as stem cell and T cell therapies. Despite the rapidly expanding importance of cryopreservation for cell therapy, clinical best practices remain inadequate, unreliable, and harmful to patients. Cell recovery remains a critical problem: insufficient T cell yield results in manufacturing failure rates of chimeric antigen receptor T cell therapy (for 13%-20% of patients in recent trials). Also, the co-injection of approx. 10 mL of dimethyl sulfoxide (Me2SO) along with thawed cell therapy product is widely acknowledged to be responsible for the nausea and vomiting experienced by up to 60% of patients and is implicated in sometimes life-threatening neurological complications. Regrettably, this exposure is medically unnecessary and only permitted due to the unacceptably low and unreliable recovery rate of exchanging the cryo media with centrifugal cell washing at the point of care (for hematopoietic stem cells, 60% on average). A central challenge is that cryoprotective agents must be diluted gradually (i.e. over the course of several minutes) to prevent hypotonic shock and cell lysis.

We have recently developed a technology to gradually exchange cryoprotective agents (CPAs) from cell suspensions in a distributed manner based on hydrogel beads. We show that these beads exchange common cell-penetrating cryoprotective agents (CPAs)—glycerol and dimethyl sulfoxide (DMSO)—with the surrounding solution at a rate prescribed by the agent’s coefficient of diffusion within the gel and bead size.

Calcium alginate hydrogel beads of uniform controlled size (CV 2%) were synthesized by dripping alginic acid into a calcium alginate bath with an adjustable sheath flow of compressed air. Rates of CPA release with the solution were measured by pre-loading the beads with CPA, then gently mixing them with an agent-free solution and measuring the osmolarity of the solution over time. Rates of CPA uptake from solution were measured similarly. Hydrogel beads with volume 25 μL had an equilibration rate constant of about 1 minute for both uptake and release of glycerol, and about 45 seconds for DMSO. The rates of CPA exchange were well described by numerical models of diffusion.

We used these uncoated hydrogel beads to mediate glycerol unloading from Jurkat cells (a CD4 T cell line), in a model system of cryoprotective agent-induced hypo-osmotic damage. In this model system, Jurkat cells were loaded with cryo-media containing 15% glycerol, and then unloaded by either immediate dilution in hypotonic phosphate buffer, or by mixing with hypotonic (i.e., glycerol-free) hydrogel beads. The beads gradually diluted the CPA, preventing hypotonic shock and cell lysis. Altogether cell recovery and viability after 4 hours was increased from about 55% (single-step dilution) to about 90% (hydrogel bead mediated dilution).

We further explored the use of polyelectrolyte multilayer films on the beads to allow controllable permeability independent of bead size. Alternating layers of poly(allylamine) and dextran sulfate were adsorbed onto the negatively charged alginate beads, resulting in films which were less permeable to glycerol and DMSO than to water. These semipermeable membranes resulted in the beads acquiring a characteristic shrink-swell behavior when immersed in hypertonic cryoprotective agent solutions, allowing indirect measurement of membrane permeability to both water and the cryoprotective agent by measurement of the bead size over time and fitting with a two-compartment Kedem-Katchalsky formula.

Altogether, the simplicity and control afforded by this system may provide an attractive alternative to centrifugal cell washing for controlled-rate addition and removal of CPAs for large volumes of fragile and precious cell samples.

Cell function is influenced by the biochemical and biophysical cues provided by the extracellular matrix (ECM) and neighboring cells. [1,2] To study the role of geometric and mechanical signals from the ECM on cell fate, we developed a single cell culture platform that offers robust control over properties of the individual cell environments. [3] This microwell platform allowed for a decoupled investigation of geometric and mechanical cues. We used our microwell platform to investigate how niche volume and stiffness affect human mesenchymal stem cell (hMSC) function, with an emphasis on the role of 3D confinement. We found that viability and proliferation of hMSCs were affected by confinement of 3D niches compared with unconfined 2D culture. Observed differences in hMSC fate correlated to YAP localization. These results demonstrate that 3D geometric confinement is an important regulator of cell fate.
Single cell niches with controlled geometry and stiffness were patterned on PEG hydrogels through soft lithography. The patterned hydrogels were formed via thiol–ene photopolymerization between norbornene-functionalized star PEG and thiolated linear monomers. The network was functionalized with the RGD adhesion motifs to enable cell attachment. hMSCs were seeded and cultured in the niches, and their fate was assessed using cell viability and proliferation assays. Mechanical activation in hMSCs was assessed using the primary antibody against YAP.
We investigated cell function in three niche volumes: small, intermediate and large (35, 60, and 125 x 10³ µm³); and three stiffnesses: low, medium and high (5, 16, and 30 kPa, respectively). The cells were cultured in the niches for 24 and 72 h and the viability and proliferation rates were quantified. The cells in small niches expressed high death rates that increased with decreasing elastic modulus of the hydrogel; cells expressed maximum viability at high stiffness. The proliferation rate was markedly low in 3D confinement than for 2D controls. Observed differences in hMSC viability and proliferation correlated to YAP localization. hMSCs displayed primarily cytoplasmic YAP localization [Nuclear/Cytoplasmic ratio (N/C) < 1.7], indicating reduced mechanical activation upon confinement.
3D confinement regulated cell function and the effect was influenced by other biophysical parameters of the niche, such as stiffness. We anticipate that our platform, with defined 3D single cell niche arrays, will provide new insights on the role of microenvironmental control of cell fate.

References:
[1] V. Vogel et al., Nat. Rev. Mol. Cell Biol., 7, 265, (2006).
[2] M.W. Tibbitt et al., Biotechnol. Bioeng., 103, 655, (2009).
[3] O.Y. Dudaryeva et al., bioRxiv, (2021). DOI:10.1101/2021.05.02.442094

A bioengineered 3D neural tissue construct mimicking the structural and functional characteristics of native neural tissue offers the possibility of recapitulating biological events in-vitro, and acts as a potential tool to understand basic biology and human disease. A major bottleneck in modelling 3D neural tissues in-vitro is to control the interaction between neuronal cells and ECM within these complex 3D arrangements, both across space and time. As a result, several 3D platforms focus on modelling short range circuitry, which makes them less suited to modelling; for example, spinal cord circuitry, where motor axons span vast lengths and interacts with different environments. Recent advancements in 3D bioprinting offer precise control over direct positioning and timing of the cell-ECM interaction, allowing to create better models of these long-range connections.

In the present work, we introduce a biofabrication technique that combines 3D bioprinting, microfabrication, and human induced pluripotent stem cell (hiPSC) populations. This allows us to engineer a functional in-vitro spinal cord like construct capable of modelling physiological motor axons guidance and development. These spinal cord constructs were fabricated via extrusion-based multi-material 3D bioprinting, wherein aggregates of hiPSC-derived motor neuron progenitors (MNPs) were precisely positioned within 3D printed hydrogels. The location for placing these MNPs within a bio-compatible hydrogel construct is controlled using a dot-dispensing printing method, both across space (positional arrangement) and time (different timepoints) within one experiment. Mechanical stability of 3D printed neural construct is ensured by printing MNP-laden hydrogels comprising of biodegradable polymers. Moreover, the biofabricated neural tissue construct facilitated diffusion of essential nutrients and oxygen required for cell survival and differentiation. The optimized 3D printable bio-inks facilitated successful neuronal differentiation and axonal elongation throughout hydrogel-based spinal cord construct, and the activity of these 3D neuronal networks is confirmed by physiological spontaneous calcium flux studies.

Future development of these 3D spinal cord constructs is directed towards modelling complex PNS tissue architectures to address clinically relevant approaches for addressing motor neuron diseases and spinal cord defects. Techniques developed in this research work can also be extended for bioprinting other neuronal and glial cells from CNS and PNS with smart hydrogel materials to determine mutualistic interaction between cells in co-culture and ECM matrix. Taken together, our experimental efforts lead towards better control over complex cell/tissue culture environment for understanding the possibility of recapitulating biological events to answer fundamental questions in neural circuit engineering.

In this study, we developed a novel scaffold for oral mucosa tissue regeneration, which is safer and more biomimetic. Fish scale collagen was used as a scaffold to avoid the risk of transmissible infectious diseases for human clinical use. In order to mimic the unique and complex structure that biomedical tissue possesses, a high-precision 3D printing technology was employed to deliver maximized design flexibility. The results showed that 3D printer was capable of manufacturing micro-sized structure with the error rate less than 10% from the original design. The structure from 3D printed mold was transferred to PDMS with soft lithography, and collagen scaffold was fabricated by molding fish scale collagen solution onto PDMS mold. The histological evaluation of the tissue-engineered oral mucosa confirmed the successful formation of the epithelial layer mimicking oral mucosa in vivo. This study not only established a method for fabricating a new type of safe scaffold material, but also showed high potential of 3D printing technology as a tool in tissue engineering. By adjusting the structure’s geometry with 3D printer, we believe our scaffold derived from tilapia can be applicable in the area of extraoral grafting such as skin as well.

Collagen derived from human or animal tissues have proved to be a success in healing damaged oral mucosa. However, the use of animal-derived collagen is restricted in many countries due to ethical reasons and the risk of transmitting infectious diseases cannot be ignored. Instead, considerable attention has been drawn to fish-derived collagen as a potential material for biomedical applications because the risk of transmitting zoonotic is considerably low compared with animal-derived collagen. The interface area between epithelial layer and connective tissue has a unique three-dimensional undulated structure, also known as the dermal-epidermal interface (DEJ). A lot of studies have shown that mimicking DEJ is crucial in tissue engineering. In our previous study, micro electro mechanical systems technology was used to mimic this structure. However, the result showed a lack of mechanical stability between epithelial layer and connective tissue due to rectangular configuration. We established a technique for fabricating micropatterned fish scale collagen scaffold using 3D printing technology for achieving safety concerns and structural optimization.

The undulated micro-sized structures were manufactured using 3D printing technology. The 3D printed mold was then exposed to UV-ozone irradiation to improve wettability, and mold release agent was coated on its surface. After applying release agent, liquid polydimethylsiloxane (PDMS) was poured directly onto the mold for soft lithography replication. PDMS was specifically chosen for this process because of its biocompatible and gas permeable properties. Soft lithography was again used to fabricate the structures by molding fish scale collagen solution on the PDMS mold. Finally, to create tissue engineered oral mucosa, human oral keratinocytes were seeded on the micropatterned fish scale collagen and cultured for 11 days.

Scanning electron microscopy (SEM) was used to observe 3D printed molds, PDMS molds, and fish scale collagen scaffolds, and optical coordinate measuring machine (μCMM, Bruker Alicona) was used to determine geometrical measurements of 3D printed molds and PDMS molds. The measurement of the molds’ geometries demonstrated high accuracy with the error rate less than 10% from the original design. In addition, SEM images of PDMS molds and fish scale collagen scaffolds showed the structures of the 3D printed molds were successfully replicated. The histological images of the tissue engineered oral mucosa revealed the successful formation of epithelial cell layer along the undulated structures.

Impaired regeneration of damaged skin tissue is a common complication of diabetes, resulting in decreased quality of life and increased mortality. Despite decades of research, few advances have been made in treating these burdensome wounds. Most current treatments do not address the underlying molecular pathophysiological processes, such as impaired angiogenesis. Adequate vascularization is necessary to bring oxygen and nutrients to the healing wound. Non-coding microRNAs (miRNAs or miRs) play a role in regulating the angiogenic state through post-transcriptional modulation of gene expression, and 4 miRNAs - miRNA-26a, -92a, -135a-3p, and -615-5p - have all been implicated in the anti-angiogenic state. One promising approach to promote the regeneration of non-healing wounds is through delivery of miRNA inhibitors (anti-miRs), which block the effects of these anti-angiogenic miRNAs; however, current application of anti-miRs in pre-clinical studies requires numerous intradermal injections of excessive amounts of anti-miR. A packaging and controlled release strategy could enhance the translational potential of this approach. Specifically, we have investigated the coating of wound dressings with electrostatically assembled thin films containing anti-miRs and cationic transfection polymer to enable anti-miR transfection of endothelial cells (ECs). We have demonstrated the emergence of pro-angiogenic phenotypes in a 3D EC spheroid sprouting assay and an EC scratch assay after treatment with each of the 4 anti-miRs delivered using our cationic transfection polymer. Additionally, we have shown the ability to coat woven Tegaderm wound dressings with anti-miRs and to retain the bioactivity of these anti-miRs coated onto wound dressings. Finally, we have begun to explore potential synergies between these pro-angiogenic anti-miRs when dosed in combinations, which could further enhance wound regeneration. Ultimately, this work will enable translational pro-angiogenic anti-miR delivery to promote the regeneration of non-healing diabetic wounds.

SynOss Putty is a biocompatible synthetic calcium phosphate mineral matrix with type I collagen derived from bovine Achilles tendon. SynOss has been associated with the clinic use of regeneration of cell tissue in teeth and bones in periodontal, oral and maxillofacial surgery. Previous studies have determined that a more predictable tissue regeneration was found in the majority of samples using SynOss and blood as a scaffold. However, the mechanism of its properties in reconstruction of teeth and bones remains unknown. Analyzing the impact of SynOss on the proliferation of dental pulp stem cells helps to bring insight into the clinical applications of SynOss such as the recovery of dental pulp after dental surgery. The proliferation assay shows that the dental pulp stem cells treated with SynOss reduce the cell number initially but have a higher growth rate compare to the control group. This leads to the belief that the SynOss material promotes the proliferation of the cells.
SynOss was then measured using the XRF to decide the elemental content. The spectra from the XRF shows that there is a large quantity of calcium and phosphate which provides a ratio of 2.5 +/- 0.011, higher to that of bones which has a ratio of 2.2. This would help with the mineralization since it has a similar ratio and therefore would provide similar properties to bones. And traces of cadmium and tungsten were also found in the material which may help to explain why the cells with SynOss treatment have lower cell counts than the control group in the day 0 of proliferation. Cadmium and tungsten have properties that are carcinogenic which proves to be detrimental to the survival of the cells. To analyze genetic expression of the dental pulp stem cells with SynOss Putty, a 28-day reverse transcription polymerase chain reaction (RT-PCR) test was performed for the 3 groups: control, SynOss treated and SynOss with fibrin. The cells were lysed and RNA were collected on day-0, day-14, day-21 and day-28.
An acellular test is undergoing with different conditions are made to check the mineralization of SynOss. The conditions are: deionized(DI) water, phosphate buffered saline(PBS), PBS with fibrinogen, osteogenic differentiation medium, osteogenic differentiation medium with fibrinogen, osteogenic differentiation medium with fibrinogen and thrombin to simulate the environment of blood.
Future works will include the Raman spectroscopy test to analyze the chemical structure and morphology of SynOss Putty and the impact of SynOss with different amount on metabolic status of dental pulp stem cells .

Titanium dioxide(TiO₂) is commonly used in consumer applications such as cosmetics, electronics, and sunscreen. Previous studies have determined that in combination with UV light, titanium dioxide has the capability of killing bacteria and microorganisms Given that endothelial cells aid in angiogenesis which provides nutrients and oxygen to newly formed tissue, evaluating the effect of titanium dioxide on endothelial cells could help to realize the impact on the healing factor in humans. In this study, we aim to investigate the effect of TiO₂ nanoparticles on network formation of endothelial cells. Two types of TiO₂ were used to test the effect of TiO₂ on cellular network formation and cell migration of endothelial cells: regular rutile TiO₂ and lecithin coated rutile TiO₂ to reduce the toxicity. Human Umbilical Vein Endothelial Cells (HUVECs) were treated with rutile TiO₂ and lecithin-coated rutile respectively in a concentration of 0.1mg/mL for 24 hours. The angiogenesis tube formation assay on Matrigel was performed for 16 hours. Timelapses spanning the whole process with time interval of 10 minutes were taken for the both treatment group, along with the control group. Cell migration speed and network quantification by total length of branches and number of nodes were analyzed.
Our assumption was that, TiO₂ nanoparticles may cause tangling of actin fibers, making it difficult for cells to stretch out, make connections and create tubes. However, the results from the network quantification indicate that rutile had significantly more network formation than the lecithin-coated rutile and control at all hours. Next was the lecithin-coated rutile with control having the least amount of networks. This data suggests that TiO₂ might be a pro-angiogenic factor as treating the cells with rutile and lecithin-coated rutile, both containing TiO₂ nanoparticles, resulted in more network formation than the control group. It is very likely that TiO₂ increase the cholesterol level of HUVECs, will in turn soften HUVECs and make the cells easier to migrate and reach out. Future tests will re-run the experiment to make sure that TiO₂ is a pro-angiogenic factor, and have cholesterol test and further investigation of HUVECs’ metabolic status under the impact of TiO₂.

In this study, we aimed to investigate the effect of varying concentrations of titanium dioxide(TiO₂) on the viability of Human Umbilical Vein Endothelial Cells (HUVECs). HUVECs were plated in a 24-well plate. Triplicates were made respectively with Rutile TiO₂ treatment, Lecithin-coated Rutile TiO₂ treatment, and a control group with no treatment was used for comparison. The cells were treated with 4 different concentrations: 0.05 mg/ml, 0.1 mg/ml, 0.2 mg/ml, and 0.4 mg/ml. Afterwards, the cells were left in the incubator at a temperature of 37 ^οC with 5% CO₂ and 80% humidity for 24 hours. From the test results, 0.05mg/mL has no impact compare to the control group since the concentration is too small. For the other concentrations, the impact of TiO₂ start to become effective at 0.1mg/mL, a decrease in the cell count can be seen as the concentration increases for both treatments. This demonstrates that titanium dioxide nanoparticles inhibit the growth and proliferation of the HUVEC cells to a certain extend.
To test TiO₂’s antibacterial properties, after 24 hours treatment of 0.1 mg/mL rutile TiO₂ and 0.1 mg/mL lecithin-coated rutile TiO₂, along with control group, HUVECs were then infected with Staphylococcus aureus bacteria for another 24 hours in order to determine TiO₂’s effect on the infection. The alive and dead cells and bacteria for the bacteria infection test were analyzed, the results shows that live bacteria per cell for the control is greater than that of the rutile TiO₂ test which shows that TiO₂ may have antibacterial properties.
Future tests will look at the impact of TiO₂ nanoparticles on the doubling time of HUVEC cells, as well as study the cell modulus by atomic force microscope(AFM) to gain further insight on the impact that TiO₂ has on cell survival and proliferation.

The delivery of therapeutic short interfering RNA (siRNA) directly into a localized area holds significant potential for the treatment of non-healing, cancerous, and other difficult-to-treat lesions. siRNA has the ability to downregulate genes that may be overexpressed in these disease states; however, its delivery is challenged by the need to enter the cell for bioactivity and its degradation by nucleases. Previously, we have shown that siRNA can be coated onto a non-degradable wound dressing using Layer-by-Layer (LbL) assembly, which creates a thin polyelectrolyte film that directly incorporates siRNA and cationic polymer to aid transfection into cells. We have noted that removal of these dressings upon termination of lesion treatment can lead to destruction of the underlying tissue. Biodegradable scaffolds circumvent the need to remove the dressing and thus the associated local tissue damage. Films containing poly(β-aminoester) (Poly2), siRNA, and other polymer excipients were assembled on Endoform^TM, a biodegradable dressing comprised of reconstituted ECM and collagen. We show that biodegradable scaffolds can be coated with a thin polyelectrolyte film using LbL technology at an average loading 3.7 times greater than the loading on non-biodegradable scaffolds per layer, depending on assembly and substrate conditions. We have also characterized the coating of Endoform^TM with siRNA using surface measurement techniques. Finally, we have demonstrated that fluorescently labeled siRNA released from coated Endoform^TM dressings can be taken up by mammalian cells and have begun investigating the bioactivity of delivered siRNA from Endoform^TM films. Ultimately, this work will enhance the localized delivery of therapeutic siRNAs to localized lesions while preventing damage to the surrounding tissue caused by dressing removal.

Graphene oxide (GO), an oxidized form of graphene, a carbon allotrope, is a very attractive nanomaterial with potential biomedical applications in tissue engineering owing to its electrochemical activity. However, it lacks the ability to form freestanding films that can actively function as an electrochemical substrate. To work around its inability to do so, different methods have been reported such as annealing, ultrafiltration, electrophoresis, and mid-infrared polarizers. Two of the popular techniques to synthesize free standing films are electrospinning and electropolymerization through cyclic voltammetry (CV). In recent years nanofibrous substrates have found unique applications in the field of biomedical engineering, especially in wound healing. Nanofibrous films are ideal for preventing bacterial contamination in open wounds and in conjunction with viable materials can serve as biocompatible skins for wound dressings. These films can also be coated or mixed with different antibacterial topical medications that can aid in further protection from infections. Similarly, CV is a popular electrochemical technique that is utilized to investigate the reduction and oxidation processes of a species and to synthesize novel substrates for biomedical applications. This work deals with utilizing these two techniques of electrospinning and CV to synthesize nanocomposite GO films with polyvinyl alcohol (PVA) and polypyrrole (Ppy) polymers acting as scaffolds. PVA, which is a biocompatible, water-soluble, synthetic polymer, is simultaneously electrospun with GO to create a scaffold capable of supporting the GO film. Polypyrrole, which is both chemically and thermally stable and has good biocompatibility both in-vivo and in-vitro, can significantly impact cell adhesion, proliferation and differentiation, as well as tissue regeneration and repair. It is theoretically and experimentally supported that Ppy functionalized GO greatly improves the biocompatibility of GO. First, PVA/GO nanocomposite is electrospun using a 5 wt. % solution of PVA and 0.5mg/ml concentration of GO at 25kV and 0.5 ml/hr flow rate. The obtained GO/PVA nanofibrous film is then used as a substrate for the electropolymerization of pyrrole monomers, thereby forming a PVA/GO/Ppy nanocomposite film. After electropolymerization of pyrrole, the thin films are peeled off to test for impedance using electrochemical impedance spectroscopy (EIS). SEM analysis, together with EIS and CV data agrees well with the molecular understanding of the interactions at the interface of PVA, GO and Ppy from the simulated trajectories. The developed composite improves the mechanical properties of tissue engineering scaffolds and is anticipated to be utilized as a wound dressing material.

By and large the research on human machine interface is constantly attracted by the idea to act in a contactless and wireless way. In particular biological and medical areas can benefit from optical method for introducing innovative ways to probe and stimulate the biological matter. Conjugated molecules and polymers offers a promising platform to interface with cells and living organisms thanks to their high optical absorption/emission cross section, chemical synthesis’ easiness and relatively low toxicity.
In this work, we developed a simple and fast method to fabricate and a micro-machining technique to pattern an interface with a double functionality made possible by the blend of photoactive poly(3-hexylthiophene) and polyethylene. The pattern on the surface of the film is designed to mimic the physiological condition and to help living cells to organize in a tissue. At the same time, the active component is able to transduce light into a stimulus for excitable cells. The mechanical properties and easiness of process are due to the presence of polyethylene while the optoelectronic properties are given by the poly(3-hexylthiophene).
We demonstrate that the obtained scaffold combines he capability to align with that to photostimulate living cells. The ultimate goal of this project is to produce an artificial tissue for future applications in the robotic, pharmacological and regenerative medicine fields.

The market share of zirconia as metal-free dental restoratives continues to grow globally, especially 3 mol% Y₂O₃ stabilized tetragonal zirconia (3YZ). However, premature failure of zirconia femoral heads and subsequent recall of hip implants by the manufacturers in 2001, triggered concern on whether zirconia dental prosthesis could incur similar failure. Here, we investigate the mechanical properties (hardness and biaxial flexural strength), optical properties (translucency and gloss), and low-temperature degradation (LTD) of 3–6 mol% yttria-stabilized zirconia. The specimens were prepared by conventional solid-state sintering at 1400 °C. The mechanical and optical properties were evaluated for sintered and polished specimens. X-ray diffractometer (XRD) and scanning electron microscope (SEM) were used, respectively, for structural and microstructural investigation. LTD was determined by XRD analysis of the samples aged in a hydrothermal autoclave at 134 °C for 5–108 h. We found the mechanical and optical properties are found to be microstructure sensitive, and the LTD due to tetragonal to monoclinic phase transformation decreased with increasing Y₂O₃ content. The mechanical strength, translucency, and LTD were lowest for 6 mol% Y₂O₃ stabilized zirconia.

Bone is a dynamic, innervated and vascularized tissue with the inherent ability to regenerate after injury. Nevertheless, non-union fractures remain a major issue in orthopedic surgery. Autografts and allografts are the gold standard for repair but can be associated to donor site morbidity, donor material availability, and increased operative times. Alternative biomaterial-based solutions are promising, including synthetic bone fillers. However, these fillers do not allow refined control of graft geometry, and seldom offer adequate porosity for rapid cell colonization, efficient nutrient exchange, or implant degradation. Further, these approaches usually do not actively promote bone growth, nor account for the complexity of bone, and in particular the neurovascular network.

We developed a silk-ceramic bioink to 3D print porous constructs for bone regeneration. The resulting material was mechanically close to bone, promoting osseointegration. These structures were biocompatible, their shape well-controlled even for complex geometries, and their porosity could be precisely tuned for efficient osteogenesis.

Our constructs supported osteoblastic differentiation in vitro, and a rapid cell response and efficient neoosteogenesis when combined with osteoinductive factors (BMP-2). We further loaded this bioink with angiogenic and neurotrophic morphogens (VEGF and NGF), and precisely patterned these biomolecules, promoting the migration, organization and differentiation of human umbilical vein endothelial cells and human induced neural stem cells, respectively. Coculture conditions showed that the multiple morphogens and cell types promoted the osteogenic differentiation of human mesenchymal stem cells.

Finally, our bioink could be loaded with antibiotics, providing an encouraging solution for the prophylactic treatment of osteomyelitis in non-union fractures.

Embedded bioprinting can recreate the complexity of the in vivo microenvironment by spatially patterning multiple cell types and perfusable vasculature within biological matrices. However, most biological inks and matrices that permit cell growth, spreading, and migration exhibit a narrow “printing window”. Here, we report a new class of porous, printable biological matrices for creating 3D vascularized human tissues and tumor models. These matrices contain sacrificial microparticles that both broaden the printing window and generate microporosity upon their removal after printing. We have demonstrated that this porous collagen-based matrix enables the embedded printing of a murine melanoma cell ink in the form of a 3D tumor model. Confocal microscopy reveals that the resulting collagen network is fibrillar, an important in vivo feature of collagen type I. Importantly, we find that melanoma cells readily spread and proliferate, and that effector T cells migrate within this collagen-based matrix. Our biofabrication platform enables the formation of a 3D microenvironment that is conducive to cell growth, spreading, and migration – key cellular processes involved in anti-tumor immunity.

This paper explores the actin cytoskeletal structures and the statistical variations in the actin fluorescence intensities and viscoelastic properties of non-tumorigenic breast cells and triple-negative breast cancer cells at different stages of metastases. The actin contents of the cell cytoskeletal structures are shown to decrease significantly with cell progression from non-tumorigenic to more metastatic states. The corresponding viscoelastic properties of the nuclei and the cytoplasm (Young’s moduli, viscosities, and relaxation times) of the cells are also measured using Digital Image Correlation (DIC) and shear assay techniques. These are shown to exhibit statistical variations that are well characterized by normal distributions. The changes in the mean properties of individual cancer cells are tested using Fisher pairwise comparisons and the analysis of variance (ANOVA). The probabilistic implications of the results are then discussed for the development of shear assay techniques and mechanical biomarkers for the detection of triple-negative breast cancer at different stages of progression.

This paper presents results using immunohistochemical techniques for prostate cancer tumor characterization in males of African ancestry using Nigeria as a case study.100 paraffin-embedded prostate tissues blocks were collected from the tissue repository of National Hospital Abuja, following ethical approval from the Institutional Review Board. Hematoxylin and Eosin staining were carried out to determine tissues with suitable characteristics for inclusion in the study. Benign prostate Hyperplasia tissues were used as control while prostatic adenocarcinomas were studied to identify underexplored biomarkers of interest as drug targets. The tissues were stained with tissue-specific proteins to determine over-expression of selected biomarkers; an investigation as to its use as targeted drug therapy. Comparative analysis of tumour biomarkers were carried out using statistical tools to determine the optimal choice of tissue specific proteins. The results reveal significant differences in the individual intensities that predispose different proteins as tumor specific biomarkers for targeted drug delivery.

This paper presents the synthesis of inorganic mesoporous silica nanobiomaterial functionalized with a boronic group and capped with a polymer. The functionalized duo were then characterized and thereafter loaded with cancer drug (doxorubicin). The kinetics study was carried out using the Zero, First, second order, Higuchi and Korsmeyer and Peppas model in different buffers of pH ranging from 5.00 to 7.40. The drug release profile reveals anomalous transport from nanoparticle and supper case II transport from the polymer capped nanoparticle. The release exponent, n, of the silica nanoparticles were found and the diffusion coefficients were also obtained. The suitable phases along the drug release profile was explored in administering the drug loaded silica nanoparticles to act as a drug delivery cargo. Computational models using Comsol multiphysics was carried out to reveal the flux of the doxorubicin from the mesoporous silica nanoparticles. The nanoparticle was characterised using SEM, FTIR, TGA and TEM analysis and the diffusion coefficient. Further test would be carried out to reveal the effect of the nanobiomaterial to inhibit the growth of a prostate cancer cell.

While reducing foreign body capsule formation and increasing regenerative infiltration are central goals in the development of implants, there is surprisingly little clarity as to the effects of biomaterial properties on spatially localized protein expression, which drives implant success. Desired is that wound healing and tissue regeneration are optimally supported by the implant, adsorbed proteins, innate and adaptive immune cells, and fibroblasts. Cells play a central role in repair and functional recovery through the production and regulation of proteins. It is not fully understood, however, how an implant differentially drives the spatial quantity of individual proteins both in the interior of the implant and in the exterior tissue surrounding it. Here we apply GeoMx® digital spatial profiling to site-specifically investigate protein production in porous implants. Data is collected on the location and quantity of 40+ proteins from formalin-fixed, paraffin-embedded tissue slides of anisotropic tissue scaffolds (n=18) with differing pore sizes (35 µm, 53 µm) and implantation durations (2, 14, 28 days). In parallel, matched bulk high-plex gene expression data (700+ genes) was collected for the identical implant samples. Notably, we discover fundamental spatial relationships in protein localization in the interior and exterior of implants that are uniquely independent or dependent of implant microstructure. In particular, dendritic cell marker CD11c and fibronectin significantly dominate the scaffold interior of the scaffolds, while cell-to-cell adhesion marker CD34 and anti-inflammatory M2 polarization marker CD163 localize in the scaffold exterior. Lastly, collating spatial and bulk information, we report unique spatiotemporal expression patterns for markers such as fibronectin that were otherwise hidden in differential bulk expression analyses and only uncoverable through spatial profiling. Together, these discoveries motivate the critical importance of quantifying and discovering spatial expression patterns for implants, facilitating a paradigm shift in the iterative design, mechanistic understanding, and rapid assessment of biomaterials.

Hydrogel particles (microgels) generated using microfluidic methods have superb properties such as high size uniformity and precise control over degradation and release profiles, making them useful for applications in wound healing and injectable drug delivery. However, the throughput of microfluidics is constrained by the physics governing the flow of immiscible fluids confined within microchannels. This throughput tends to be several orders of magnitude lower than what would be necessary for commercial and clinical applications. Here, we demonstrate the scaling up of on-chip synthesis of microgels by parallelizing the microfluidic channels. Taking advantage of the established fabrication technologies developed by the semiconductor industry and a high flow control system, a 4-inch silicon microfluidic chip integrating more than 4,000 microfluidic devices is developed. By incorporating a high energy flood UV source, this chip allows the synthesis of poly (ethylene glycol) diacrylate microgel particles with diameter down to 30 µm at a throughput above 1kg/hr. By using photomasks that enable milli-second scale control of the UV exposure, the stiffness of microgels can be varied between 10³ to 10⁴ Pa. Large-scale production of microgels will enable construction of large-scale tissue scaffold with well-defined physiochemical properties, and will provide scalability for translation to clinical settings.

Introduction Recently, the number of patients with heart failure has been increasing. When pharmacological treatment does not bring the expected effects, there may be a need for mechanical circulatory support (MCS). One possible solution is to use blood pumps such as Ventricular Assist Devices (VAD). This devices are nowadays widely used as an effective treatment for patients with advanced heart failure and are the only alternative to heart transplantation. Over the years many new devices were introduced to the clinic. However, based on clinical experience, despite undeniable evidences of VAD effectiveness, many components could still be improved. The ongoing optimization performed by many producers does not concern only improvements in terms of their function, but also in terms of patient comfort and reduced mortality. According to the information provided by INTERMACS, patient survival drops to 50% after four years of support. However, the VADs, are designed to be wear-resistant and in some cases are used as a destination therapy. It is therefore important to further improve the devices biocompatibility in order to reduce the occurrence of complications. One of possible complications may be the pump thrombosis and inflow obstruction, caused by the ingrowth of tissue into the lumen of inflow cannula. According to reports from clinics and literature it seems that the solution to this problem may be the use of surface modification. Appropriate topography allows for controlled scar tissue formation, which results in reduction of inflammatory processes and appearance of thromboembolic events.
Materials and Methods The paper presents surface modification performed using vacuum sintering intended for use in an VAD inflow cannula. The study presents an analysis of the relationship between surface parameters on the susceptibility of cells to proliferate and the strength of their adhesion to the implant. Samples were prepared from titanium alloy Ti6Al7Nb in form of cylinders Ø14mm x H 3mm. During the initial research the base material was verified for compliance with the standard including the microstructure study, the chemical composition analysis and the study of mechanical properties. Then samples were subjected to tumbling before performing modification. Roughness was measured with the use of contact profilometry and was characterised by Ra=1,5µm and Rz=12,5µm. The sintering process included modifications with the use of Comercially Pure Titanium (Cp-Ti) powder with two different grain morphologies - spherical and irregular. The grain size was changed in rage of 50 to 250 µm. The obtained surfaces were then analysed by scanning electron microscope [SEM]. Additionally the porosity of obtained surfaces was determined. All samples revealed high roughness with the potential for cell anchoring and scar tissue formation. Fibroblasts were then applied to the samples for three periods of time. The number of cells was assessed on the basis of the stained cell nuclei and the presence of adhesive molecules. In addition, in vivo studies were performed in which samples were implanted into the dorsal muscle of New Zealand rabbits. After 4 and 8 weeks, the specimens were deplanted and the force of detachment of formed tissue from the implant surface was tested.
Results and Discussion The results have shown that surface after powder sintering is characterized by high porosity and complex 3D morphology. The obtained roughness was in rage Ra=21-36 µm. The porosity was in rage 27-49% depending on the size and shape of the powder grains. The detachment force differed by 0.5N depending on the shape of used powder grains.
Conclusions The presented modification has the potential to anchor cells and form controlled scar tissue on the surface of the implant, both in the context of cardiac and other medical implants.
Acknowledgments Project supported by:
NCBiR: RH-ROT/266798/STRATEGMED-II
National Science Centre, Poland: 2018/31/N/ST8/01085

Although advances in wireless signal acquisition systems have reduced the invasiveness of many implantable medical and research devices, wire-based systems continue to be necessary for applications directed at treating or analyzing certain areas of the body that are incompatible with wireless device use or conventional surgical approaches. Traditional metal wires provide the high conductivity that is needed for in vivo electrophysiological signal transmission. However, due to their rigidity, metal based wires are susceptible to mechanical damage and discomfort to the subject. The ideal bioelectronic wire is thus both highly conducting as well as mechanically and chemically similar to live tissue. In this work, we transform surgical sutures into conducting and biocompatible wires (e-sutures) that can be concomitantly used for wound suturing, electrophysiological signal acquisition, transmission, and power delivery. We use a solution of the conducting polymer PEDOT:PSS together with solvents and a cross-linking agent to coat surgical sutures and make them electrically conductive. Our coating method increases the e-suture conductivity such that we can record ECG and EMG data from rodents, as well as stimulate muscles and evoke movements. We evaluate additional methods to further increase the conductivity of the e-suture such as acid treatment and nanolayer metal deposition. We develop an insulating layer (wire jacket) for the e-suture using a combination of parylene-C, polyurethane and silicone elastomer and characterize its electrical properties over time in an in vivo environment using electrochemical impedance spectroscopy. The resulting e-suture is highly conductive, electrochemically stable and biocompatible. The high yield and ease of creating e-sutures combined with the widespread use of sutures in medicine suggests high translational potential to a broad range of medical procedures that require surgical intervention and monitoring or electrical modulation of tissue.

Stable, long-term interfaces between implanted devices and the brain remain a challenge. Properly interfaced neural implants provide the potential for recording brain activity, enabling the generation of neurally-driven prosthetics, treatment of neuroses, and a deeper understanding of brain function, among other applications. One of the major issues inhibiting long-term recordings of the brain is the lack of integration between an implant and surrounding tissue. This lack of integration often results in an inflammatory and subsequent scarring response driven by mechanical mismatches between the probe and adjacent tissue. Tissue engineered implants provide a solution to this problem through their superior integrative capacity in vivo but traditionally lack any interactive components. To generate a tissue engineered neural probe, we have combined the recording capabilities of bioelectronic devices with the regenerative capacity of tissue engineered implants to create a new type of device designed for long-term implantation. Here, we discuss the design, development, and testing of a tissue engineered implant with an integrated microscale conducting polymer-based recording system for monitoring in the brain.

We aimed to build a device that contains a series of discrete recording sites within a tissue engineered gel. The active portion of the probe was fabricated to consist of individually articulated electrodes on the scale of single neurons. Implants were microfabricated using photolithographic procedures to produce a flexible bioelectronic recording device. The device consists of gold tracks, insulated with parylene-C, which terminate in exposed pads with poly(ethylene dioxythiophene):poly(styrene sulfonate) (PEDOT:PSS)-based coatings to enhance recording efficacy. To embed these devices in gels, a mold was produced to encompass the entirety of the active portion of the device. Collagen was then injection molded around the device, resulting in a three-dimensional spread of the recording sites within the gel. To test the integrative capacity of these implants, we chose a subcutaneous intramuscular model to examine the ability of tissue to grow between the individual leads. Initially, implants without gels were positioned within the musculature of rats. Rats were sacrificed at days 3, 7, and 14 post-implantation to examine the healing response around the implant. By day 14, tissue was observed growing between the individual leads, and tissue around the probes appeared indistinguishable from tissue elsewhere at the insertion site. This result indicates excellent potential for widespread recording at the implantation site and for integration of these implants into the body.

In this study, we have developed a tissue engineered neural implant to promote integration with surrounding tissue. Our next step will be the implantation of gel-based probes to examine the ability of the tissue engineered probe body to promote tissue ingrowth. Ultimately, we will incorporate cells into these devices and move towards intracortical implantations in the brain. Overall, we have generated a new type of neural implant, utilizing tissue engineering and bioelectronic principles, to enhance regeneration at the implantation site and enable long-term recordings of the nervous system.

In the field of cell mimicry, bottom-up approaches are explored to resemble cells, organelles and enzymes using both natural and synthetic materials. Micro/Nanoreactors that can perform encapsulated catalysis are popular examples for the former cases. However, the first examples of their integration with mammalian cells only start to emerge. For instance, we recently demonstrated that microreactors loaded with catalase could support HepG2 cells in 3D cell aggregates when stressed with hydrogen peroxide. The artificial moieties did only their task for only up to 24 h because the encapsulated enzyme lost its activity.

Here, we use the superoxide dismutase/catalase mimetic Eukarion (EUK) as the active unit in alginate-based microreactors to alleviate the oxidative pressure in 3D cell aggregates. To this end, we first synthesized an EUK derivative that was water-soluble and could be conjugated to alginate resulting in EUK-Alg. Droplet microfluidics was used to fabricate microreactors with incorporated EUK-Alg. Then, the long-term activity of molecular EUK, EUK-Alg and the microreactors was assessed considering both, the catalase- and superoxide dismutase activity. The microreactors were coated with e.g., poly(L-lysine) or cell membrane vesicles, to facilitate integration in 3D cell aggregates with HepG2 cells. The viability of these bionic tissues was followed over time and the integration of the natural and synthetic units was visualized. Finally, bionic tissues could tolerate higher levels of hydrogen peroxide over longer times compared to pristine HepG2 cell spheroids.

Taken together, we advanced the concept of bionic tissue by illustrating the benefits the integration of the biological and synthetic world could have.

Preterm labour, representing nearly 20% of births in the United States, is one of the leading and rising causes of infant mortality and nearly 40% of these cases are linked with preterm premature rupture of membrane (pPROM). Despite this, there are no treatments currently available aside from antibiotics, which do not address the cause of pPROM. This is a life-threatening condition that can sometimes be reversed by spontaneous sealing of the membrane, suggested recently to be mediated by fetal macrophage recruitment and polarization. However, the role in the recruitment and polarization of macrophages of amnion-derived mesenchymal stem cells (MSCs), a major constituent of the most loadbearing part of the fetal membrane, is not well understood. We demonstrate that understanding the mechanics of fetal membranes with regard to rupture and repair can drive secretion of proteins from MSCs. This in turn affects recruitment and programming of macrophages, and as a result, pPROM healing. Collecting patient samples from both full-term and pre-term birth patients, we demonstrate we can make large-scale physical property maps of the amnion such as dynamic rheological maps of Storage and Loss Modulus, as well as millimeter-scale topographical maps to characterize defects in the fetal membrane. We characterized of both sides of the fetal membrane (chorion and amnion), as the intrinsic mechanical cues and resident mechanosensitive resident cells differed. We found that there were substantial topological differences in the amnion due to embrittlement of the fetal membrane, and the viscoelastic properties varied depending on the delivery mechanism. These viscoelastic properties had significant immunomodulatory effects on resident macrophages, responsible for inflammation and overall remodeling in the fetal membrane during birth.

Devices for micro-prosthetic applications must maintain a reduced footprint in small implant sites while still repairing or restoring a specific function lost following trauma or pathology. We focus on the prosthetic for the middle ear, for which piston-like struts replace the complex arrangement of bones and soft tissues to recover hearing. The selection of a suitable material for such applications is critical in order to generate devices that meet stringent mechanical and biological requirements. Silk-Hydroxyapatite (S-HA) composites have been extensively used in the biomedical field as materials to replace bone-like structures, due to their mechanical properties, biocompatibility, and fabrication versatility via additive manufacturing. Most current medical applications have employed compositions with high percentages of HA over S, leading to stable constructs that, however, have limited toughness. Our study aimed at unveiling correlations between S-HA compositions, rheology, and printability to improve the final part properties with this composite system, in view of micro-prosthetic applications for which geometrical precision and accuracy play a key role in the final device functions.
Two batches of composite materials with different compositions were prepared and studied. The first consisted of HA microspheres (diameter less than 1 µm) with different contents of water, between 1:1 and 1:2 (w/v). The second was composed of S-HA, using silk extracted for 60 min and concentrated to 15% (w/v). In this case, the ratio of solid/liquid was held constant and values of S and W varied from 1:0.8:0.4 up to a ratio of 1:1:1 (w/v/v). Rheology was performed with a parallel plate geometry to mimic forces exerted during printing. In particular, we evaluated the elastic (G') and viscous (G'') moduli across strain to find the main mechanical parameters (yield stress and strain) of the inks. We further monitored the evolution of moduli in a recovery step after the strain was removed. The same inks were printed with a displacement-driven 3D extrusion printer and printed parts were imaged and analyzed to quantify feature fidelity.
The results showed a reduction of elastic properties of the inks with increased water content. The HA showed values of G' above those having S in the composition, up to three orders of magnitude. Yield stresses were also higher, with associated strains two orders of magnitude lower than those of the S-HA composites. This difference highlights the higher toughness for the HA-S composite inks, an important factor for customizations of the constructs. All inks showed a recovery over time for the main viscoelastic parameters (G', G''), resulting in a stable solid-like behavior when the solid/liquid ratio was below 1:2 (w/v).
Linking rheology and printability, we observed a direct relationship between the main rheology outcomes and the geometric quality of the constructs. Specifically, high printability with high yield stress independently of the concentration of S was observed. Moreover, increasing the concentration of S reduced the elastic moduli of the inks, making them more extrudable through fine nozzles at achievable pressures. In addition, while large amounts of S reduced the stability of the final products, compositions with about 0.13 ml/ml (dry silk/total water) show geometric precision comparable to those of HA alone, but with significantly higher toughness. In addition, this analysis allowed us for the first time to assess printability for ceramic-polymeric inks with varied critical yield strains.
These results provide an insightful picture of the relationships between rheology and printability of S-HA composites, drawing clear relationships about the influence of S on the composition of the ink in view of micro-prosthetic applications.
This work has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement COLLHEAR No 794614.

Retinal detachment is a serious condition where the thin retina separates from the back of the eye causing immediate vision problems and permanent issues if left untreated. Treatment is aimed at relieving the retina traction and sealing the causative tears. Often this is achieved surgically by removing the existing vitreous from the eye and reattaching the retina into place by drainage of the subretinal fluid through a fluid air exchange and sealing the tears with laser induced retinopexy. However, the sealing of retinopexy is not instant and the retina needs to be supported during healing by an agent commonly referred to as “tamponade”. Tamponades have been used since 1911, and today both gas and liquid materials are used. Gas tamponades reabsorb over time in the eye, replaced with fluid spontaneously. For liquid tamponades, silicone oil is most commonly used but requires surgically removal. Neither material is density matched and patients are therefore often required to maintain a face-down prone position for weeks to maintain contact of the tamponade against the retina tear and prevent fluid migration in the subretinal space.
The fluid that comprises the majority of the volume of the eye is known as the vitreous humor. This material is a colorless, transparent crosslinked viscoelastic hydrogel. It is ~99% water, the rest composed largely of collagen, proteoglycans and proteins. If vitreous is removed in patients, the natural fluid turnover in the eye gradually refills the vitreal cavity. The ideal device for support of the healing retina would be one that mimics the properties of the healthy vitreous, at least over the time required to successfully heal. Although a number of attempts to developed permanent replacement vitreous materials have been tried, few have tried to develop a material that will only be present long enough to aid healing. Here we describe a degradable hydrogel for support of the healing retina during recovery from detachment surgery. The added benefit of a designed temporary residence is that regulatory approval is simpler if the device is not considered permanent.
We present work on the development of a thiolated PVA utilizing a polyethylene glycol diacrylate (PEG-DA) crosslinker. This system (termed a Thiogel) reacts through Michael addition, without heat release and formation of by-products, and does not require use of toxic initiators or a UV source. This system is therefore amenable to a mix-and-gel strategy in the operating room and has very low potential toxicity. We will discuss two Design of Experiments (DoE) structured to first screen for an optimal functionalization level, and then to determine the optimal composition for transparency, refractive index, gelation time and tuned degradation kinetics.
Finally, we will describe a pilot rabbit study (New Zealand White (NZW) Rabbits) to examine safety and degradation kinetics of the material in vivo. Animals received a vitrectomy using a conventional three port vitrectomy with either Thiogel or control air in the right eye while the left eye was left as the non-surgical control. Full ophthalmic exams, gross ocular exams, Intraocular pressure (IOP), and fundus images were performed to document the response at timepoints out to 8 weeks. Both test and controls exhibited expected IOP reductions immediately following surgery, with the IOP levels returning close to baseline by 3 weeks and thereafter statistically similar.
To attempt to determine the clearance rate of the gel we developed a liquid chromatography based technique to allow us to track the components over time in the excised tissue. This involves separation of the components from the physiological tissue through a purification step, followed by analysis on a liquid chromatography system. These data demonstrate that the gel is almost completely degraded at 14 days and is almost entirely cleared by 28 days.

Human adipose stem cells (hASCs) provide a research tool for regenerative medicine in various capacities. There are major research needs for new therapies for bone-related diseases such as osteoporosis, osteomyelitis, and severe trauma. Human ASCs can self-renew for extended durations outside of the body and provide an ideal platform to study therapeutics for the formation of bone-specific modalities or wound healing applications.
Combining hASCs with nanomaterials is a growing area of research that requires additional studies. Halloysites (HNTs) are hollow aluminosilicate nanotubes with an exterior negative charge and a positively charged lumen. These nanoparticles range between 100-900 nanometers and the lumen spanning roughly 20 nanometers across. HNTs have demonstrated biocompatibility in cell and animal-based studies, with additional capabilities for vacuum loading of drugs and coating metal ions such as silver, zinc, strontium, and copper (mHNTs). Metal ions provide synergistic antimicrobial effects on prokaryotes, allowing healthy cells to form in infection risk areas. These unique properties allow a varying of methods to be developed in cell-based therapeutics at the tissue level.
The primary focus of my research is to investigate the therapeutic potential of metalized HNTs with a linked tripeptide; GHK (glycyl-L-histidyl-L-lysine) is known to be essential in the wound healing process. Ultimately, studying these interactions will lead to better bandages to be hemostatic, alleviate pain and inflammation, and cost-effectively possess antimicrobial features. Enhanced wound healing can have drastic implications on military personnel and people without proper medical access. Bone and wound analysis studies were conducted on hASCs after the incorporation of such nanoparticles. Results introducing GHK with metalized HNTs showed increased proliferation of hASCs, collagen production, and an increase in the genes ki67 and runx2, a proliferation and bone marker, respectively. Strontium-coated HNTs increased osteochondral differentiation after analysis by phase-contrast imaging, gene expression, and unique morphological changes of hASCs exhibiting trabecular-like bone formations in culture.
Copper-coated HNTs coupled with GHK increased wound closure after artificial wound analysis, further validating gene expression and proliferation studies. Human ASCs were additionally encapsulated in alginate hydrogels with GHK and mHNTs that increased cell viability and collagen production, leading to differentiated fibroblast clusters within the hydrogel constructs. Given these results, expanding the use of mHNTs with GHK in animal wound healing studies can profoundly impact wound healing if incorporated into FDA-approved materials.

Many of the proposed alternative strategies to bone grafts for the repair of non-union bone defects rely on the development of synthetic scaffolds for bone regeneration. However, these scaffolds are limited by their structure and scale-up/cost implications. With its well-organized, hierarchical, and functionally graded structure, mechanical strength, and potential for large-scale cultivation for bone tissue engineering applications, decellularized bamboo offers a cost-effective, scalable alternative for scaffold development. In the first part of this work, we demonstrate that decellularized bamboo scaffolds can support the growth of human fetal osteoblasts and the regeneration of bone tissue in vitro. “Lucky” bamboo stems are decellularized into scaffolds using detergents (sodium dodecyl sulfate and Triton X-100), washed with deionized water and Tris buffer, and functionalized with dopamine-conjugated RGD peptides (RGDOPA). Human fetal osteoblast (hFOB) cells are cultured for 28 days on the scaffolds, and cell proliferation is evaluated through DNA content and alamar blue assays. Alkaline phosphatase (ALP) activity is used as an early marker of osteogenesis, while ECM production is quantified by estimating the de novo total protein contents using the BCA protein assay. Mineralization is detected through measurements of de novo calcium contents (calcium colorimetric assay) followed by alizarin red S staining. Scanning electron microscopy is used to visualize the morphologies of the cell-scaffold constructs. However, the major drawback of using decellularized bamboo scaffolds for bone tissue engineering is their limited biodegradability when implanted into a patient, since human enzymes outside the digestive system have limited ability to break down the cellulose, hemicellulose, and lignin macromolecules found in bamboo. Hence, in the second part of this work, we present injectable cellulase-encapsulated poly(lactic-co-glycolic acid) (PLGA) microspheres as a strategy to facilitate the degradability of the decellularized bamboo scaffolds. The cellulase-encapsulated PLGA microspheres are synthesized via the single emulsion solvent evaporation technique. These microspheres are then characterized using scanning electron microscopy, dynamic light scattering, Fourier-transform infrared spectroscopy, thermogravimetric analysis, and differential scanning calorimetry. The amounts of cellulase enzyme released from the PLGA microspheres over a period of 50 days are monitored using the BCA protein assay. Degradation of the decellularized bamboo scaffolds in phosphate-buffered saline (pH 7.4, 37°C) containing the cellulase eluting PLGA microspheres is assessed via changes in weight, compressive mechanical properties, and glucose content due to enzymatic breakdown of the cellulosic macromolecules by cellulase. Strain mapping is also used to study the local strain distributions in the scaffolds under compressive deformation. The implications of the results are then discussed for the production of these sophisticated, and yet, readily-available decellularized bamboo scaffolds to circumvent the challenges associated with conventional bone tissue engineering scaffolds in an environmentally and economically sustainable way.

The International Agency for Research on Cancer (IARC) of the World Health Organization reports 1,276,106 incidences and 358,989 fatalities resulting from prostate cancer worldwide in 2018. Prostate cancer is the second leading cause of cancer death among men in the United States. Prostate cancer has the propensity to migrate to or metastasize to bone. Although curative treatments are available for primary prostate cancer tumors, advanced-stage prostate cancer that has metastasized to bone often results in a poor prognosis. The primary cause of morbidity due to prostate cancer is the metastasis to bone. The effects of various biochemical factors in the bone microenvironment have been demonstrated in the disease progression to metastasis; however, the role of physical factors such as mechanical cues induced by the interstitial fluid flow around the bone is poorly explored. It has been established that interstitial flow has a pro-migratory and biomechanical stimulatory effect on cancer cell invasion. To address this issue, we designed a 3D in-vitro model to evaluate the progression of prostate cancer cells at metastasis condition, colonizing the bone site under dynamic conditions. In this study, we developed a 3D dynamic in-vitro model employing nanoclay-polycaprolactone-hydroxyapatite scaffolds and a specially designed perfusion bioreactor. Initially, we differentiated human mesenchymal stem cells (hMSCs) on scaffolds under dynamic culturing conditions to duplicate an accurate bone-like microenvironment. Next, we utilized these bone mimetic scaffolds to grow prostate cancer cells under identical dynamic conditions to recapitulate prostate cancer metastasis to bone. Finally, we performed various cellular assays and immunocytochemistry studies to establish the feasibility of dynamic culture. We observed a significant increase in bone growth under dynamic culture conditions evaluated by bone-related biomarkers (ALP, RUNX2, and OCN) compared to static culture. We also noticed upregulation in E-cadherin and downregulation of vimentin levels under dynamic conditions that confirms the occurrence of mesenchymal to epithelial (MET) transition of prostate cancer cells at the metastatic bone site. We further examined the influence of fluid-induced mechanical cues on cells by monitoring alteration in orientation and morphology of hMSCs and prostate cancer cells. Overall, we observed significant differences in cellular morphology and gene expressions of bone cells and prostate tumors grown under flow conditions. The established perfusion bioreactor based 3D dynamic in-vitro model can be further utilized to elucidate the mechanisms of prostate cancer metastasis and screen novel drug therapies geared towards bone metastatic prostate cancer.

Bioactive glass nanoparticles have been used for a wide range of biomedical applications including bone regeneration due to its high specific surface area and reactivity. Inspired by the composite nature of bone, bioactive glass nanoparticles (nBG) derived from a SiO₂-CaO-P₂O₅ ternary system may be incorporated into polymer blend (PLGA:PCL) fibers, and this study explore nBG synthesis and evaluates the bioactivity and osteogenic potential of this composite fiber platform. The development of a Ca-P phase on the polymer-nBG fibers were monitored longitudinally under physiological conditions using FTIR-ATR, XRD and SEM. It is hypothesized that the nanoparticle infused polymeric fibers will be bioactive, and provide structural template for mineralization in situ. Here, A coprecipitation sol-gel method was used to fabricate the nBG (55%SiO₂-40%CaO-5%P₂O₅, wt%). For the fabrication of polymer-nBG fibers, 5 wt% of nBG were added to a polymeric blend of PLGA:PCL (5:1) and electrospun at 10 kV. To test the bioactivity and mineralization potential of the composite fibers, the samples with corresponding controls were immersed in DMEM (1mg/mL) and collected at Day 0, 1, 3, 7 and 14. One-way ANOVA and Tukey-Kramer HSD post-hoc tests were used to study significant differences (p<0.05) of pH levels between different experimental groups.
The nBG appeared globular and are 10 – 20 nm in diameter under SEM. The fibers with or without nBG were 2.21±0.64 μm and 2.24±0.58 μm in diameter, respectively. FTIR analysis reveals a peak at 1100 cm^-1 representing the Si-O bond of the silica network. After immersion in DMEM for 14 days, pH level remained similar across all experimental groups except for day 14 where particle-free control measured significantly lower pH compared to all other groups, indicative of polymer degradation. Coupling the polymer fiber with nBG served to neutralize the negative impact on pH and allow maintainence of physiological pH. XRD patterns showed crystallization peaks at 31.8^o and 45^o suggesting crystallization of phosphate andthe early formation of carbonated apatite. This was confirmed by FTIR spectra showing growing peaks at 603 and 568 cm^-1 and 960 cm^-1 suggesting dissociation of Si-O bonds and mineral crystal formation by day 14. These bulk and surface characterization analysis confirm the bioactivity and mineralized in this novel composite system. Future studies will assess both osteoconductive and osteoinductive potential of these composites for bone regeneration.

Brain aneurysms are weak-walled pathological dilations occurring at cerebral vasculatures. If left untreated, they can easily rupture, resulting in stroke or even death. Current prominent treatment methods involve the permanent implantation of metallic devices, flow diversion, and liquid embolic agents (LEA). However, unfavorable primary outcomes occur in 20% to 30% of the treated cases, and the success rate can even be significantly lower depending on the type and location of the aneurysms. This project aims to improve patient outcomes through the development of a novel LEA that is injectable, visible under angiography, has proper mechanical strength, promotes cell adhesion, and is stable within an aneurysm sac. To achieve these properties, the LEA was comprised of the hydrogel F127-DMA, a reverse thermo-responsive, easily injectable, cross-linkable, and shape-conforming polymer system with proper mechanical strength; the catalyst FeCl2, which gives the polymer long term stability by allowing chemical crosslinking; and the biocompatible contrast agent iohexol.

To determine the mechanical strength of the LEA, rheology tests (single-frequency time sweeps and amplitude sweeps) were performed using a Bohlin Gemini 150 HR Nano rheometer. The tests showed that 29% F127-DMA hydrogel exhibited an elastic modulus of 202.1 kPa after chemical crosslinking, and the average wall shear stress of the carotid artery— 0.85 ± 0.2 Pa —was within the gel’s linear viscoelastic region[1]. These results prove that the LEA will be able to withstand pressure from blood flow.

Differential Scanning Calorimetry (DSC) is traditionally used to measure phase transitions, where energy is absorbed or released by a substance. Here, it is shown that DSC can also accurately measure the ordering temperature of the micelles in the gel. This corresponds to the gelation temperature of the F127-DMA polymer, in which the micelles were found to form an FCC lattice. This lattice formation found through DSC corresponds to the drastic rise in modulus found through rheology. In this manner, the LEA composition could be tailored to achieve the desired modulus and viscosity during the injection. Additionally, both DSC and rheology confirmed that the contrast agent iohexol did not interfere with the gel’s phase transition ability or lattice formation.

Experiments were also conducted to determine the extent to which cells adhered to the LEA. While F127 alone has been shown by multiple authors not to be cell adhesive, it was found that once cross-linked, the F127-DMA was able to support cell adhesion[2]. This indicates that the LEA can support the migration of endothelial cells, which is required for the healing of aneurysms. Ongoing experiments are being conducted to pinpoint how the hydrogel system acquires its cell adhesive properties once crosslinked. This includes testing samples of F127-DMA with F127, F127-DMA with FeCl2 and F127, F127-DMA alone, F127 alone, and F127-DMA with FeCl2.

The radiopacity of F127-DMA was also tested in animal injection experiments (with IACUC approval). The iohexol contrast agent was visible through angiography, and current testing is focused on determining gel stability in vivo.

In addition to the aforementioned ongoing experiments, future development of the LEA will involve monitoring the long-term performance of the agent within animals, enabling the LEA to promote endothelialization, and implementing biodegradability. The results obtained thus far indicate that Pluronic polymers are successful candidates for designing LEAs by meeting the requirements of injectability, radiopacity, and promotion of neointimal layer regrowth.

[1] Sui, B., et al. “Assessment of Wall Shear Stress in the Common Carotid Artery of Healthy Subjects Using 3.0-Tesla Magnetic Resonance.” Acta Radiologica.
[2] Rodriguez, Natalia M., et al. “Micropatterned Multicolor Dynamically ADHESIVE Substrates to Control Cell Adhesion and Multicellular Organization.” Langmuir.

Clinical islet transplantation, the intrahepatic infusion of allogeneic islets, has the potential to provide physiological blood glucose control for insulin-dependent diabetics. The success of clinical islet transplantation, however, is hindered by the location of the implant site, which is prone to mechanical stresses, inflammatory responses, and exposure to high drug and toxin loads, as well as the strong inflammatory and immunological responses to the transplant in spite of systemic immunosuppression. To address these challenges, our research has focused on three primary strategies: the development of scaffolds to house islets at alternative transplant sites; the fabrication of encapsulation protocols for the immuno-camouflage of the transplant; and the production of bioactive biomaterials for the local delivery of oxygen and immunomodulatory drugs and/or cells. Three-dimensional scaffolds can serve to create a more favorable islet engraftment site, by ensuring optimal distribution of the transplanted cells, creating a desirable niche for the islets, and promoting vascularization. Encapsulation can substantially decrease the need for systemic immunosuppression of the recipient, by preventing host recognition of surface antigens. Finally, localization of supportive agents to the site of the transplant can serve to enhance efficacy, while minimizing the side effects commonly observed with systemic delivery. Success in these strategies should increase the efficacy of islet transplantation for the treatment of Type 1 Diabetes, whereby the long-term survival and engraftment of the transplanted islets are significantly improved.

Pathological matrix remodelling plays a central role in many human diseases, but is challenging to study as in vitro models often cannot replicate the complex 3D cell-matrix interactions that drive pathologies. Here, we describe a 3D model of the human gut that allowed us to uncover an unexpected role for a rare immune cell type called Type-1 innate lymphoid cells (ILC1) in driving gut fibrosis in patients with inflammatory bowel diseases. ILC1 are enriched in patient mucosa with active inflammatory bowel disease (IBD), but the impact of this accumulation remains elusive, and therapeutics against ILC1’s signature cytokine IFNγ lack clinical efficacy.

We used molecular dynamics simulations to design tetra-PEG hydrogels that cross-link quickly, but can still mimic the stiffness of normal intestinal tissue. We then co-cultured encapsulated human intestinal organoids with ILC1, and using a combination of atomic force microscopy force spectroscopy and multiple particle tracking microrheology, found that ILC1 drive intestinal matrix remodelling through a balance of MMP9-mediated matrix degradation and TGFβ1-driven fibronectin deposition. Our findings demonstrate the potential of using hydrogels in disease modelling, and open the possibility of unravelling how pathological matrix remodelling contributes to disease.


Aims and Hypothesis: We hypothesized that ILC1 accumulation in IBD is not simply a driver of inflammation, but rather that ILC1 play a role in driving disease pathology itself. Therefore, the aim of this work was to use reductionist models based on intestinal organoid-ILC1 co-cultures to uncover how ILC1 impact the intestinal epithelium and matrix.

Methods: We established co-cultures of murine small intestinal organoids (SIO) with ILC1, and human induced pluripotent stem cell (iPSC)-derived intestinal organoids (HIO) with patient-derived ILC1. We also developed a functionalized, PEG-based synthetic hydrogel system designed to form efficient networks at low polymer concentrations.

Results: Pico-SMARTSeq2 transcriptomics on SIO co-cultures revealed that IFNγ sensitizes epithelial cells to Fas-mediated apoptosis. However, ILC1 also drive expansion of the epithelial stem cell crypt through p38γ phosphorylation and aberrant Cd44v6 expression, which is unexpectedly regulated by ILC1-derived TGFβ1, not IFNγ. We next established that human ILC1 also secrete TGFβ1, and drive CD44v6 expression in both HIO epithelium and the surrounding mesenchyme, though notably this phenotype is only recapitulated by ILC1 from patient biopsies with active inflammation. As TGFβ1 is a master regulator of fibrosis, the leading indicator for surgery in IBD, we next characterized the ability of ILC1 to regulate matrix remodelling. We created modifiable PEG hydrogels that cross-link quickly but at low stiffnesses and harnessed this platform to perform multiple particle tracking microrheology and atomic force microscopy force spectroscopy on encapsulated HIO. We show that ILC1 drive matrix stiffening and degradation, which we posit occurs through a balance of MMP9 degradation and TGFβ1-induced fibronectin deposition.

Conclusion: Our synthetic organoid co-culture system enabled us to tease apart an important role for intestinal ILC1 in epithelial and matrix remodelling, which may drive either wound healing, tumor formation or fibrotic pathologies in IBD. Moreover, this controlled 3D microenvironment provides a broader platform for dissecting interactions between complex human iPSC-derived tissues and rare cell subtypes in development and disease.

Current clinical translation of molecule delivery strategies requires distinctive design of materials that can offer innovative solutions to address the limitations associated with biomedical problems. Glycan-based hydrogels are of great promise in terms of creating new vehicles with responsive behavior and tunable properties for various clinical applications. For example, we developed anthracene-functionalized alginate to create both chemically and physically crosslinked polymer networks. Due to reversible dimerization of anthracene in response to light with alternate wavelengths, where we were able to tune the mechanical properties of the hydrogels precisely, while controlling the gelation behaviour in a light-responsive manner. The same approach can be applied to chitosan, another promising glycan-based polymer, where chitosan was used for dual-crosslinking with the anthracene and photopolymerization was carried out in the absence of a toxic photoinitiator. The nature of dual-crosslinking was modulated to adjust the visco-elastic modulus of both hydrogels. Furthermore, it was shown that alterations on moduli significantly influenced the morphology of seeded cells in a 3D environment. Finally, chitosan can be crosslinked through ionic gelation with negatively charged DNA to produce nanoparticles with gene delivery potential. In summary, glycan-based materials hold significant potential for tunable, reproducible, and responsive biomaterials, which can then be utilized as robust systems to be used in drug delivery applications.

The specific properties of the zeolites such as their nanoporous structure, stability in biological environments, textural properties and high adsorption capacity made them useful as drug carrier. The performance as carrier mainly depends on zeolite framework, affinity zeolite - drug molecule and the pH of the media. For these reasons in the present work it was evaluated the behavior of LTL nanozeolite as doxorubicin carrier. The LTL nanozeolite was synthesized by hydrothermal method. The performance as nanocarrier was evaluated at different concentrations of doxorubicin in order to evaluate the doxorubicin adsorption capacity on the nanozeolite. In a second step, the doxorubicin –loaded nanozeolite was submitted to a releasing process at two different pH conditions. The nanozeolite was characterized by XRD, SEM and FT-IR techniques. The concentration of doxorubicin was monitored by UV-Vis spectroscopy at wavelength of 480 nm. The LTL nanozeolite was obtained as unique crystalline phase and presented a rod-like morphology. By FT-IR was evidence the loading of doxorubicin on the LTL nanozeolite. The LTL nanozeolite exhibited a high doxorrubicin adsorption capacity at all the concentrations evaluated. The doxorubicin releasing rate was influenced by the pH of the media, a faster releasing was observed at pH of 7.4 while a slower and controlled doxorubicin releasing was reached at pH of 5.5. These findings allow to infer that LTL nanozeolite has the potential to be used as doxorubicin nanocarrier.

Functional nerve recovery following substantial injury is crucial in the field of neural regeneration. Even though various microfluidics and printing techniques have been established, effective axonal guidance over long distances has still remained challenging. Among numerous biomaterials, conjugated polymers such as poly(3,4-ethylenedioxythiophene) (PEDOT) have demonstrated promising results in engineered scaffolds due to their soft mechanical properties, mixed electronic / ionic conductivity, and excellent chemical stability in physiological environment. Laminin, a substrate-bound protein in the extracellular matrix, is a well-known chemoattractant which fosters neurite outgrowth. In this work, we introduce a novel technique to fabricate various profiles of laminin gradients on thin PEDOT films.

To fabricate the device, first a layer of gold (thickness: 150 nm) was deposited on silicon wafers via electron beam evaporation and laser-cut masks. Using AUTOLAB, a thin layer of PEDOT was then electropolymerized gold-coated silicon substrates at charge deposition density of 0.18 C cm^-2. Surface of PEDOT films was treated with Poly (L, Lysine) to improve laminin adhesion. Laminin gradients were first printed on the surface of a 2 wt% agarose hydrogel slab (thickness: 10 mm), using micro-scale motorized X-Y-Z stages and a nano-syringe pump. The droplets of laminin solutions with different laminin concentrations were deposited at injection volume of 17.5 nl next to each other (vertical and horizontal distances were 400 µm and 800 µm, respectively). Finally, the printed laminin pattern was transferred onto the PEDOT film using molecular stamping. Laminin was fluorescently tagged via immunohistochemistry; and fluorescent imaging was utilized to visualize and quantify various laminin gradients.

To showcase the potential of the developed methodology, linear and hill gradients were created and quantified. The linear gradient consisted of 10 lines (8 mm-long) with a solution concentration range of 10-55 µg ml^-1. In the hill gradient (7.2 mm-long, 9 deposited lines), initially the solution concentration was 10 µg ml^-1 which linearly increased until it reached a maximum value of 90 µg ml^-1, then symmetrically decreased. Measured fluorescent intensity followed a linear trend (R-square= 0.99) with respect to laminin concentrations in the linear gradient. Similarly, both symmetrical sides of the hill gradient showed a good linear fit (R-sqaure= 0.98 and 0.99, respectively) with almost equal absolute slope values.

We aim to assess the neurite response to various gradients and find the optimum gradient type and concentration range for effective axonal regeneration. The outcome of this study will pave the way towards development of more effective engineered conduits for nerve regeneration and will help us unravel fundamental questions of axonal regeneration in the nervous system.

While organoid culture has the potential to revolutionize our understanding of human biology, current protocols rely on the use of Matrigel, a complex, heterogeneous material with large batch-to-batch variations that hinder reproducibility. In response, several groups have begun designing synthetic hydrogel systems to enable the reproducible culture of organoids. Recently, the matrix stress relaxation rate (i.e. the ability of a hydrogel to remodel its network connectivity in response to an applied stress) has been demonstrated to have profound effects on encapsulated cells. To date, the role of matrix stress relaxation on organoid cultures has not been explored. Here we present the design of a family of double-network hydrogels that undergo two stages of crosslinking: the first stage uses reversibly dynamic covalent chemistry bonds, while the second stage reinforces the hydrogel through thermal-induced protein-polymer aggregation. This double-network of physical interactions results in a gel with a broad dynamic range of tunable mechanical properties, where the gel stiffness is set by the number of crosslinks and the gel stress relaxation rate is independently set by the kinetics of the crosslink binding and unbinding. These novel, double-network hydrogels have been used to study the role of mechanotransduction in the culture of patient-derived, human intestinal organoids. In this system, we find that the organoid cultures display strong phenotypic responses to matrix stress relaxation, which are dependent on cell-matrix interactions with both CD44 and integrin cell-surface receptors.

Defects in craniofacial bones of the skull occur congenitally, after high-energy impacts, and during the course of treatment for stroke and cancer. They affect a broad segment of the US population, are large and irregularly shaped, and heal poorly. Autologous bone or alloplastic implants are the clinical gold-standards for repair. However, limited quantities and the need for time-intensive intraoperative fitting of autologous bone, the non-regenerative nature of alloplastic implants, and surgical challenges that stem from irregular defect margins and the quality of the surrounding bone all contribute to poor healing and high complication rates. Advances in the fields of tissue engineering and regenerative medicine require biomaterials that instruct, rather than simply permit, a desired cellular response. Contemporary tissue engineering efforts combining exogenous mesenchymal stem cells (MSCs), morphogens, and biomaterials have not attained wide clinical use due to the cost and time for in vitro MSC expansion and MSC-biomaterial culture as well as the need for supraphysiological morphogen doses. A unique opportunity exists to develop degradable biomaterials that actively instruct, rather than passively permit, osteogenic differentiation and subsequent bone regeneration. Our laboratory has developed a class of mineralized collagen scaffold to promote MSC osteogenic differentiation and subsequent CMF bone regeneration in the absence of exogenous growth factor supplements. I will describe recent advances to our collagen scaffold system to address barriers preventing regeneration of craniomaxillofacial bones and orthopedic insertions. Ongoing efforts are developing platform technologies to address challenges such as poor biomaterial-defect contact, inflammatory response and reduced cell bioactivity in large defects, and poor matrix remodeling. We are using selective alteration to scaffold structural alignment and proteoglycan content as well as the incorporation of bioinspired structural motifs to create composite biomaterials able to improve cell bioactivity and mechanical competence in order to address mechanistic and translational challenges.

Chronic respiratory disease is the third leading cause of death worldwide. Prevalence of respiratory conditions is rising and expected to increase further as a direct result of lung damage sustained by COVID-19. Unfortunately, there are currently no effective treatments for the majority of advanced lung diseases. Biomaterials engineered to replicate the intricate geometry and dynamic mechanical properties of the lung could accelerate discovery of new treatments based on pulmonary regenerative medicine.

Toward this goal of engineering tissue-informed models for lung disease and regeneration research we have employed a photopolymerizable poly(ethylene glycol)-norbornene (PEG-NB) hydrogel and new microfabrication techniques to create geometrically accurate 3D lung models that closely mimic the mechanical properties of native lung tissue. Briefly, eight-arm PEG-NB macromers were crosslinked with an MMP3-degradable dithiol crosslinker via emulsion polymerization to create hydrogel microspheres. A design of experiments approach determined the optimal emulsion conditions to achieve a median microsphere diameter (d=180.8 µm) that approximates the size of a human lung alveolus (d~200 µm). Bioactive peptide sequences mimicking fibronectin and laminin were incorporated into the microspheres to promote cell attachment and magnetic nanoparticles were included to enable magnetic aggregation into structures replicating distal lung architecture. The microspheres were seeded with magnetically labeled primary murine primary alveolar epithelial type II (ATII) cells and aggregated with a magnetic levitating drive to resemble an alveolar air sac. These aggregates were coated with primary murine fibroblasts to replicate interactions between epithelial cells and fibroblasts that drive many chronic pulmonary diseases. These acinar structures were next embedded in a PEG-NB hydrogel that mimics the mechanics of healthy or diseased lung parenchyma to create a robust, physiologically relevant acinar model.

We envision that ongoing work to incorporate patient-derived induced-pluripotent stem cells (iPSCs) into tissue-informed lung models could lead to pulmonary regenerative medicine advances that bridge the gap between outputs measured in traditional in vitro models and outcomes in human clinical trials.

Injectable hydrogels can be delivered via needle-syringe injection into the human body with low invasiveness. They have found significant use in many branches of medicine. Despite extensive efforts in the field, there remain long-lasting challenges concerning the mass transport and mechanical properties of injectable hydrogels. First, most existing injectable hydrogels are not perfusable. This issue limits the rapid transport of oxygen and nutrients (diffusion depth ~ 600 µm), as well as the trafficking of native or transplanted cells. Immediate vascularization is therefore imperative but difficult to realize through injection. Second, injectable hydrogels show limited mechanical toughness and robustness. They tend to crack and fail under large or repeated force excitation. An extreme case is the vocal folds, where implants in the vocal fold lamina propria are exposed to up to 50% strains at a fundamental frequency that is on the order of 10² Hz. Currently, patients with vocal fold injuries suffer from repeated hydrogel injections, due in part to the fracture-induced short lifetime of the hydrogel implants under the dynamic loadings. By contrast, many biological tissues are perfusable and yet tough to tolerate extreme biomechanical stimulations as part of their normal functions, as exemplified by the vocal fold and heart. To close the gap between injectable hydrogels and biological tissues, strategies to achieve a combination of high permeability and mechanical toughness are highly desired.

Here, we describe a design and methodology to fabricate new injectable hydrogels featuring in-situ pore formation, strengthened mechanical performance, and excellent cytocompatibility. We achieved this by orchestrating stepwise gelation and phase separation processes. The resulting hydrogels contain porous double-networks, thereby termed PDNs, which differ from previously reported injectable hydrogels that consist of either nanoporous or preformed porous networks. PDNs can form interconnected cell-sized pores in-situ upon injection. The highly porous matrices enable rapid media perfusion and support cell spreading and trafficking. The permeability of PDNs was at least 2 to 4 order of magnitudes greater than that of most existing hydrogels and comparable to many biological tissues. Such perfusable hydrogels were demonstrated to support cell survival in organ-sized scaffolds with a dimension beyond 60 mm. To our knowledge, this is the first material reported to support cell viability in centimeter-scale avascular constructs. Meanwhile, unlike many porous hydrogels that are weakened by their pores, PDNs are tough and resist the propagation of moderately-sized cracks below 500 µm. Their fracture toughness was up to 40-fold greater than those of commonly used nanoporous or porous single-network hydrogels. They can be stretched more than 200% of strain without failure. The stiffness of PDNs is tunable between ~ 3 to 10 kPa, which spans the range of various soft biological tissues such as the vocal folds, lungs, heart, and gastrointestinal tract. They also exhibited a fast stress relaxation response within 10 to 100 seconds similar to organs and extracellular matrix.

Thanks to the facile injectability, PDNs can be easily delivered through fine needles and incorporated into 3D cell culture perfusion systems with complex mechanical loadings, such as microfluidic chips and bioreactors. We demonstrated that PDNs can withstand over 6,000,000 cycles of high-frequency (~ 120 Hz) biomechanical stimulations without rupture. Our method is also generalizable to commonly used material systems, such as chitosan and gelatin. With the unparalleled combination of interconnected pores, toughness, cytocompatibility, and injectability, the described material systems and method may open new opportunities for regenerative medicine and serve as biomimetic in vitro 3D cell culture platforms for a broader range of applications.

Introduction: Wound healing is a highly regulated complex biological process that includes homeostasis, inflammation, proliferation, and maturation stages to regenerate damaged tissue. The coverage of the open wound by a dressing is the general treatment to prevent bacterial invasion and possible further injuries. Recent wound dressings have been improved by providing multifunctional features to better control the wound and the healing process. There are various key features of wound dressings such as retention of wound exudate, creation of a moist environment around the wound, and prevention of inflammation by anti-inflammatory components. Hydrogels have been widely used in wound dressings due to their ability to retain high amounts of water. For practical and rapid wound treatments, conventional dressings demonstrate limitations with inadequate amounts of oxygen, poor fitting onto the wound, and additional need for antimicrobial treatment. To address these issues, we have developed a sprayable hydrogel dressing with antimicrobial properties using naturally-derived materials and inexpensive components.

Methods: In this work, sprayable, antimicrobial, and cost-effective hydrogel wound dressings were developed for rapid treatment of burn wounds. These hydrogels are composed of photocurable gelatin, which is hydrolyzed collagen (GelMA) as the bioactive component and solid peroxide microparticles as the oxygen forming antimicrobial component. GelMA was synthesized and composite hydrogels were fabricated using 5-10% (w/v) GelMA and different concentrations of peroxide microparticles (0-15 mg/mL). The physical characteristics of the composite hydrogels were investigated by mechanical testing, swelling, degradation, and porosity analyses. The oxygen formation kinetics were measured using an oxygen sensing probe. The antimicrobial capabilities of composite hydrogels were tested against the growth of Escherichia coli bacteria. The cellular responses were evaluated by performing viability, adhesion, proliferation, metabolic activity, and cytotoxicity assays upon culturing human dermal fibroblasts with the composite hydrogels.

Results: We developed practical, sprayable, and antimicrobial composite wound dressings. 10% (w/v) of GelMA concentration was chosen as an optimum concentration. The composite prepolymer solutions with 10% (w/v) GelMA have shown less hydrophilic characteristics compared to the 5% (w/v) concentration when they were sprayed on an ex vivo pig skin model. The gelatin and peroxide containing composite hydrogels have shown tunability in physical and biological properties. The composite hydrogels have generated oxygen up to two weeks which was beneficial to the in vitro cell culture. The metabolic stress of human dermal fibroblast cells was relieved by the sustained formation of oxygen from the peroxide microparticles. The generation and supply of additional oxygen reduced cell death as well as providing antimicrobial properties in this novel sprayable wound dressing.

Conclusion: The sprayable, antimicrobial, and composite hydrogels that are composed of GelMA and solid peroxide microparticles were successfully fabricated. Their physical, chemical, and biological characteristics including swelling, degradation, porosity, mechanical strength, oxygen formation kinetics, antimicrobial activities against bacteria, and cytocompatibility with human dermal fibroblasts were evaluated. Our sprayable hydrogel wound dressings can be applied on wounds without contact, eliminating the risk of infection via handling, and are easily conformable to the morphology of the wound allowing for generation of personalized dressings. The fabricated composite hydrogel dressing will not require a frequent change due to its stability on the wound. In addition to pre-treatment of burn wounds, the developed sprayable and antimicrobial hydrogel dressing can be useful in plastic surgery and dermatology, applications.

Our long-term goal is to tissue engineer and build pediatric vascular patches that will grow with a child to address unmet needs of surgical solutions for children with congenital heart defects. A major cell type of blood vessels are vascular smooth muscle cells (VSMCs) which largely comprise the medial layer of blood vessels. Vascular smooth muscle cell behavior is modulated by various physicochemical factors and their phenotype changes during normal developmental processes and during pathophysiology. Examples of vascular smooth muscle cell phenotype involve differences in extracellular matrix molecule production, cell migration, cell proliferation, contractility.
Our laboratory has investigated the effects of a variety of physicochemical stimuli on vascular smooth muscle cell behavior with the goal of understanding key relationships between VSMC phenotype and the underlying substrate. Using these model systems, we have elucidated the effects of substrate mechanical, chemical, and topographical properties on VSMC phenotype. Furthermore, to better represent physiological and pathophysiological conditions, we have investigated the interplay of multiple stimuli on VSMC phenotype.
From this information, we have developed several technologies such as micropatterned cell sheet engineering to form stacked VSMC patches and have assessed their mechanical strength. We have validated computational modeling of these stacked cell sheets in which we investigate the role of structural orientation on their mechanical properties. Computational modeling enables us to interrogate a large experimental variable space in silico.
Finally, we are developing several strategies for using different stem cell sources from patients with clinical translation in mind.

Type 1 diabetes results from the auto-immune destruction of inulin secreting cells of the pancreas – the beta cells within Islets of Langerhans. Exogenous supply of insulin is a commonplace procedure in regulating blood glucose levels in diabetic patients. Alternately, cell replacement therapies such as pancreas and islet transplants offer a more permanent solution to maintain blood glycemic control. However cell therapy is restricted by the availability of donor tissue, which can be overcome by deriving insulin producing cells from a regenerative cell source, like pluripotent stem cells (PSCs). With the current advancement of PSC-derived cell therapy from the laboratory to Phase 1 clinical trials, there is an enhanced emphasis on deriving mature and functional islets from hPSCs in a robust and reproducible manner. In parallel to regenerative therapy, there is also a strong emphasis to reproduce disease phenotypes in vitro, using microphysiology systems (MPS) models in tissue chip platforms. Once developed and validated, these models will be invaluable platforms for interrogating disease mechanisms as well as supplementing drug discovery and drug testing activities. Appropriate functioning of the MPS models, however, will largely rely upon successful derivation of mature and functional cells/ tissues/ organs from hPSCs.

Our research focuses on a range of biomaterial strategies for deriving pancreatic islet like cells from hPSCs. We have developed cell encapsulation strategies for scalable culture of hPSCs and its subsequent differentiation to islet like clusters. We have introduced systems engineering techniques to identify robust biophysical conditions for hPSC propagation. In a collaborative team we have developed biomaterial substrate to synthesize controlled, multicellular organoids from hPSCs resembling pancreatic islets. We are currently developing strategies to induce in-vitro microvascular network formation within the stem cell derived islet organoids. This talk will highlight ways in which our laboratory has integrated natural and synthetic materials to engineer the cellular environment to closely mimic the natural islet environment. Our current efforts on integrating islet organoid with microfluidic device towards developing Diabetes MPS models for Diabetes will also be discussed.

Cells can sense the physical stimuli that may arise from the composition of the material, from the extracellular matrix (ECM) that they surrounded, or the topography and the stiffness of the surface that they interact with. Cells respond to these stimuli by triggering certain metabolic pathways and altering their metabolic activities, which may lead to a change in gene expression and protein synthesis as well as promoting growth and differentiation.
Generally, the transformation of these physical stimuli into biochemical signals is called “mechanotransduction”. There are several methods and mediators to create physical stimuli and induct cells. Magnetic particles and magnetic scaffolds are one of the most convenient methods to construct a mechano-sensitive system as these scaffolds can respond to external magnetic fields. Our research on magnetic cryogels reveal that these scaffolds can induce bone and cartilage differentiation of mesenchymal stem cells both in low and high magnetic strength.
Conductive scaffolds are one of the other alternatives to use as a mechano-sensitive system. Our research on electrically conductive decellularized muscle/polyaniline/ polycaprolactone blend scaffolds possess favorable properties to serve as a matrix for the regeneration of skeletal muscle tissue.
Surface modification is also a very convenient method to obtain desirable surfaces and topography. Polydimethylsiloxane (PDMS) is a widely used polymer due to its biocompatibility, high oxygen permeability and ease of fabrication. Our research on protein-modified PDMS and bone surface mimicked PDMS shows enhanced bone cell-related metabolism.
Cells are surrounded by their own ECM. Therefore, the composition of this ECM may contain several important cues to regulate or affect cell behavior. Engineered decellularized matrices are one of the latest approaches, as decellularization aims to preserve the structural and functional biomacromolecules of ECM while removing the cellular components. We reported both animal and plant derived modified decellularized matrices can provide a suitable biomechanical microenvironment for better cell attachments and proliferation.
Whether using a mechano-sensitive system, modified bio-mimicked surface, or even a decellularized matrices, cells will sense and alter their metabolism as they always do. It should be also noted that tracking and exploring the mechanistic pathways will help us to better explain these behavioral changes.

Rapidly developing tissue engineering treatment methods address the need to develop three dimensional (3D) porous scaffolds with biofunctionality to promote tissue ingrowth and regulate cell behaviours. Current biofunctionalization strategies rely on wet chemistry to immobilize biomolecules onto the internal complicated porous networks of the 3D porous scaffolds. However, wet chemistry approaches are limited with complicated and time-consuming process, and toxic residues. This project successfully surface-engineered three-dimensional (3D) porous tissue engineering scaffolds using plasma immersion ion implantation (PIII) technology in a customized plasma reactor. The surface-biofunctionalized scaffolds were validated by mesenchymal stem cell (MSC) expansion. PIII is a surface engineering technology that can embed highly reactive radicals in polymeric substrates surface via energetic ion bombardment. Through these radicals, biomolecules can be covalently bonded to the plasma-treated substrates and provide high level bio-functionality for tissue engineering
scaffolds. The PIII modification is typically low cost, versatile, and environmentally friendly compared to wet-chemistry-based covalent immobilization processes.¹ However, conventional plasma treatment strategies are not suitable for uniform surface treatment of porous scaffolds, because the directional approach of these methods relies on diffusion of reactive species into the pores, which has a limited penetration depth.² Therefore, there is a strong demand for a new plasma-based method suitable for homogenous plasma activation of porous 3D scaffolds. Here we report the development of a new approach in which a high voltage electrode surrounds the polymeric scaffolds for their homogenous PIII activation. Voltages in the range of 4- 8 kV and working pressures in the range of 1-3 Torr were tuned to optimize the PIII activation high impact PS scaffolds with pore sizes of 300 μm. Using micro-Fourier-transform infrared spectroscopy (micro-FTIR), the homogeneous surface modification of the PIII-treated scaffolds was confirmed by evaluating the relative concentrations of R-OH groups generated throughout the scaffolds by the interaction between the embedded radicals and oxygen in the atmosphere. FGF2 with an optimized concentration were covalently immobilized to partially cover the surfaces of the scaffolds, promoting both MSC adhesion and MSC proliferation. These results show that porous network of the scaffolds can be PIII-treated with using this new strategy, providing an excellent bio-functional platform for MSC expansion.

REFERENCES
[1] Wong KU, Biomimetic Culture Strategies for the Clinical Expansion of Mesenchymal Stromal Cells. ACS Biomaterials Science & Engineering. 2021
[2] Zelzer M, “Influence of the plasma sheath on plasma polymer deposition in advance of a mask and down pores”, The Journal of Physical Chemistry B, 113(25):8487-94 2009.

In this study, physical properties and biocompatibility of polymer/calcium phosphate cement (CPC) bone substitutes are improved with incorporation of fullerenol (Ful) which is a cutting-edge carbon nanomaterial due to its biocompatibility and specific physical and chemical features. The main characteristic of Ful is that it is a powerful scavenger for radical oxygen species (ROS) and some studies suggest that it enhances osteogenic differentiation via scavenging ROS and upregulating the expression of osteogenic markers. However, the data presenting the effect of Ful on the characteristics of bone substitutes is scarce. CPCs are the most dominantly used clinical bone substitutes since they possess a similar structure to natural bone and can easily be applied due to their moldability. CPCs are commercially available in pure form and in a compound with polymers because their drawbacks including low degradability, macroporosity and wash-out resistance can be addressed via degradable polymers, such as carboxymethyl cellulose (CMC) and gelatin (Gel). Then finally, Ful is incorporated to the bone substitutes.

In this study, tetracalcium phosphate (TTCP)/dicalcium phosphate dihydrate (DCPD) particles were ball-milled to obtain particles with diameters of less than 20 µm. The powder phase of CPC consisted of TTCP and DCPD in equivalent mass was used to produce Ca-deficient hydroxyapatite. The liquid phase of control CPC was 0.4 M Na₂HPO₄ solution with a pH of 7.4. CMC, Gel and Ful were homogenized in Na₂HPO₄ solution to synthesize the liquid phase of other CPC groups. The specimens were named control, CMC1/Gel1.5, Ful0.02, Ful0.04, Ful0.1. Gum-like consistency was obtained by mixing powder and liquid in 55.56/44.44 ratio by weight, respectively. Particles and CPCs were characterized by scanning electron microscopy (SEM) and X-ray diffraction (XRD). Setting time of specimens were measured by Gilmore test. Moreover, compressive strength and modulus were measured after their incubation at 37°C in phosphate buffered saline (PBS) for 24h. pH change was also analyzed after the incubation at 37°C in PBS. Finally, % cell viability was measured via Alamar Blue reduction.

The results so far show the beneficial effect of Ful on setting time and compressive strength of CPCs, and % cell viability on CPCs. According to the results of Gilmore test, 0.1 wt/v% Ful significantly decreased the initial setting time of CMC1/Gel1.5 CPCs from 20.78 minutes to 16.18 minutes (p<0.05) and final setting time from 27.94 minutes to 18.19 minutes (p<0.01). XRD analysis of CPCs at different time intervals reveals that total reaction of Ful containing CPCs took less than 5 hours since all TTCP and DCPD were consumed, and the only phase was apatite at 5^th hour. Compression test shows that 0.1 wt/v% Ful significantly increased the compressive strength of control CPCs to 3.68 MPa from 2.14 MPa (p<0.05) and modulus of control CPCs to 213.09 MPa from 152.08 MPa (p<0.05). pH measurements show that 6.9 was the lowest pH for the groups at the end of the first week. After the first week, the pH of the CPCs raised and remained constant between 7.0-7.4 for one month. Finally, pre-results of Alamar Blue assay demonstrates that Ful significantly increased the cell adhesion on CPCs. % cell viability on CMC1-Gel1.5 CPCs enhanced with the addition of 0.02 wt/v% Ful (p<0.05) and 0.04 wt/v% Ful (p<0.05) in 24h. The results of this study are considered to enlighten the studies on CPCs which are the main elements of orthopedic, maxillofacial, and craniofacial applications.

Bone regeneration is crucial to repair critical sized bone defects that jeopardize the quality of life. Bone tissue is regenerated by intramembranous and endochondral ossification physiologically, however, a critical sized bone defect without remaining bone pieces or periosteum can be challenging to repair on its own. Allo- and auto-grafts have been developed to enhance bone regeneration. However, the risk of infection, the need of secondary surgery, and high cost limit the use of such grafts. In clinical practice, cranial defects are often caused by surgery to release the intracranial pressure, and the defect is subcutaneously covered by non-degradable implants. Biocompatible and biodegradable biomaterials can repair critical sized bone defects and degrade while the bone tissue is regenerated. Protein-based hydrogel scaffolds are suitable for critical size cranial defects regeneration due to their tunable and pliable physical properties as well as their osteoinductivity. We hypothesized that sequential mineralization process can enhance the mechanical properties of the hydrogel scaffolds while rendering the scaffolds more bioactive, osteoinductive, and osteoconductive.
In this work, we developed sequentially mineralized gelatin-based (GelMA) scaffolds to obtain mechanically stable and biologically active three-dimensional (3D) gels that allow for osteogenesis both in vitro and in vivo. The GelMA hydrogels were mineralized for 10 cycles in which one cycle is defined as an incubation period in calcium chloride dihydrate that is followed by incubation in sodium phosphate dibasic dihydrate. The mineralized scaffolds were characterized by Dynamic Mechanical Analysis (DMA) and Scanning Electron Microscopy (SEM), Thermogravimetric analysis (TGA), and Fourier Transform Infrared (FTIR). Pre-osteoblast cells were seeded on the scaffolds for 14 days and the expression of osteogenic differentiation maker genes were determined by quantitative reverse transcription polymerase chain reaction (RT-qPCR). The scaffolds were then used to repair critical size cranial defects in a rat model.
The mineralized GelMA hydrogel scaffolds have shown outstanding tunability and pliability in physical and biological properties for bone regeneration. The mechanical properties significantly enhanced for the mineralized hydrogels (125 kPa) compared with the pristine hydrogels (4.25 kPa). The 3D scaffolds induced osteogenic differentiation of pre-osteoblasts without the use of osteogenic growth medium or growth factors. The critical sized cranial defects in a rat model were repaired after 12 weeks by the mineralized scaffolds (55.4 mm³ regenerated bone volume) while no bone regeneration was observed with the use of pristine hydrogel controls or blank control groups. Overall, the findings suggest that our mineralized hydrogel scaffolds show superior mechanical properties and indicate favorable in vivo bone regeneration through regeneration of critical sized cranial defects in a rat model.
In conclusion, sequentially mineralized hydrogels were fabricated and characterized for their chemical, physical, and biological properties. They were also evaluated for their osteoinductivity in vitro and osteoconductivity in vivo. The in vivo experiments showed that the scaffolds could simply be applied on top of the dura without causing an increase in cranial pressure. The implants were easily accepted by the host, highly vascularized, and biodegradable. We anticipate that our mineralized hydrogels will be useful for regeneration of mineralized tissues in new biomedical applications.

In the quest for the next generation of functional biomaterials and new solutions in health-related research, investigators have sought inspiration from nature by developing better performing bioderived materials (e.g. chitosan, polydopamine) and architectures (e.g. nanoporosity), reproducing naturally occurring micro and nanostructures to control cellular response at the material-host tissue interface. In this context, our team has focused on understanding the effects on cells of poly(dopamine), an adhesive polymer derived from mussels, as a multifunctional layer for direct cueing to osteoblastic and mesenchymal stem cells. In addition, we have employed anodization to create reproducible patterns of nanotubes on titanium surfaces, demonstrating their role in controlling osteoblastic and mesenchymal stem cell activities. In parallel, our work has also contributed to the development of collagen-, agarose- and chitosan-based materials for applications in neuronal tissue engineering and disease modelling. For example, we focused on the creation of micro-engineered chitosan substrates for neuronal guidance and on 3D scaffolds to support neuronal adhesion and network formation.

Cells encapsulated hydrogels are a promising material system that immobilizes and protect the cells in porous matrices. Hydrogel allows nutrient and oxygen exchanges between the cells and the matrices, which suitable for tissue engineering and cell therapy. Alginate is a natural anionic polymer that has been used in biomedical applications due to its excellent in vivo compatibility and low toxicity. Alginate hydrogels have been utilized for microfibers and implant scaffolds. However, an important limitation of the alginate hydrogels is the gradual degradation in a physiological environment that greatly affects the cell-cell interaction and tissue formation. In this study, microfluidic aqueous two-phase system (ATPS) was employed to fabricate the cell encapsulated hydrogel microfibers. In comparison with other techniques, the ATPS is a less complex and straightforward method to creates continuous microfibers. The microfibers then formed into a bulk mass and called hydrogel microfibers. In the material development, the concentration of the alginate-Dulbecco's Buffered Saline (DPBS) mixture was varied. The corresponding structure, morphology, swelling behavior, elastic modulus, and degradation of the alginate hydrogel microfibers were characterized to determine the optimal concentration. Fibroblast was selected in the biological study. Alginate lyase-loaded poly(lactide-co-glycolide) PLGA nanoparticles (NPs) were fabricated by a double emulsion/solvent evaporation technique. The NPs were then incorporated into the cell-laden alginate to accelerate the degradation and promote cell proliferation. NPs loaded and pristine hydrogel microfibers were studied for comparison. Cell viability, cell toxicity, and cell proliferation in the hydrogel microfibers were tested and observed at the day 1, 3, 7, and 14. Various characterization methods include SEM, DLS, AFM, degree of swelling, degradation, assays include cell proliferation, MTT, cell viability, and micro BCA were used.

In this talk, controls of the laminar flow ATPS to create continuous microfibers will be presented. The physical behavior, structure, and elastic modulus of the hydrogel microfibers will be discussed. The cell viability, toxicity, and proliferation will be reported.

Introduction: Oxygen is critical for ensuring cell survival and proliferation. Inadequate oxygen in the cellular microenvironment can trigger cell damage and hypoxia induced necrosis¹. Tissue constructs of physiologically relevant scale (in cm sizes) face diffusion limitations and are unable to translate into clinics successfully. Biomaterials that can release oxygen over time, for up to 4-5 weeks, can fulfill this oxygen demand of physiological scale tissue constructs for clinical applications². To yield biomaterials with high oxygen content, solid peroxides can be encapsulated within hydrophobic polymers. This novel approach can produce oxygen generating biomaterials that can release oxygen over a 5-week period in a controlled, sustainable manner, and augment cell viability and function².

Materials and Methods: We engineered the oxygen-generating microparticles by encapsulating calcium peroxide (CaO₂) within a hydrophobic biopolymer polycaprolactone (PCL). An emulsification technique was used to prepare composite microparticles with 2-10% (w/v) CaO₂ in PCL. The CaO₂ was added to a PCL solution and the blend of CaO₂ and PCL was micro pipetted dropwise to a Polyvinyl alcohol (PVA) solution under constant magnetic stirring at 720 rpm. This process produced CaO2- PCL composite microparticles which were 100mm in diameter. These microparticles were subsequently encapsulated in gelatin methacrylate (GelMA) hydrogels to engineer the oxygen-generating scaffolds. The oxygen release kinetics of these scaffolds were recorded, and the corresponding cellular viability, metabolic activity, cytotoxicity, and apoptosis of the encapsulated primary cardiac fibroblasts were studied. The cellular studies were performed by microencapsulating primary cardiac fibroblasts within the oxygen- generating hydrogels at a cell density of 5 x10⁶ cells/mL over a 5-week culture period under induced hypoxia.

Results and Discussion: We developed the oxygen-generating scaffolds and characterized their material properties. This was followed by measuring the oxygen release kinetics, and monitoring the cell behavior (viability, growth, and proliferation) and metabolic function in vitro. Our results revealed that the scaffolds sustained release of oxygen for up to 35 days under induced hypoxia with primary cardiac fibroblasts microencapsulated. The cellular metabolic activity over these 35 days reported an increasing trend over 14-days and continued to rise up to day 35 in culture. We achieved a precise control over the oxygen release kinetics by varying the CaO₂ concentration within PCL. The negative controls devoid of the oxygen-generating microparticles, were also tested simultaneously and they resulted in no improvement in cell growth and function.

Conclusions: Primary cardiac fibroblasts are high oxygen consuming cells, and they use oxygen at higher rates under hypoxic environments. Our results validated that the oxygen-generating microparticles enabled the maintenance of high cell viability and metabolic activity under induced hypoxia for up to 5 weeks We demonstrated that tunable oxygen release kinetics can be obtained by altering the CaO₂ concentration, PCL concentration, and the quantity of the CaO₂ -PCL microparticles within the hydrogel matrices. To customize the oxygen-release kinetics for culturing cells from different lineages having varying oxygen demands, these parameters can be easily modified. This approach could be used as a scalable, tunable platform for the development of functional off-the-shelf tissue constructs which could be used to surmount hypoxia-induced necrosis.

References: ¹Suvarnapathaki, and Camci-Unal, G. et al., 2021. Engineering calcium peroxide-based oxygen generating scaffolds for tissue survival. Biomaterials Science, 9(7), pp.2519-2532.² Suvarnapathaki, S., and Camci-Unal G. et al., 2019. Breathing life into engineered tissues using oxygen-releasing biomaterials. NPG Asia Materials, 11(1), pp.1-18.

Transarterial embolization is a minimally invasive procedure to selectively deliver embolic agents using a catheter into arteries to occlude diseased or injured vasculature for therapeutic intent under real-time X-ray guidance. It can be used to treat vascular malformations, aneurysms, and hypervascular tumors. A variety of embolic agents such as coils, beads, and liquids are currently used in the clinic; though their effectiveness is limited by non target embolization, failure in coagulopathic patients, high cost, and toxicity. In this study, a decellularized cardiac extracellular matrix (ECM) based nanocomposite gel is developed to provide outstanding mechanical stability, catheter injectability, retrievability, antibacterial properties, and biological activity to prevent recanalization. The malleable and shear-thinning nature of the nanocomposites gel allows the formation of an impenetrable solid cast to fill various vessel geometries and sizes, avoiding recurrent bleeding, and provides the versatility that cannot be achieved by clinically used agents. The embolic efficacy of gel is shown in a porcine survival model of embolization in the iliac artery and the renal artery. The ECM based nanocomposite promotes arterial vessel wall remodeling and a fibroinflammatory response while undergoing significant biodegradation such that only 25% of the embolic material remains at 14 days. With its unprecedented proregenerative, antibacterial properties coupled with favorable mechanical properties, and its outstanding performance in anticoagulated blood, the bioactive ECM based nanocomposite gel enables wide tunability providing a platform technology for the next-generation in vivo embolic agents to treat a broad range of vascular diseases.

Repair of wounds and surgical incisions is facilitated by primary intention with devices including sutures and staples. However, lack of immediate tissue approximation, high potential for scarring, including in visible areas of the body, propensity for tissue trauma and infection, and long procedure times necessitate new approaches for tissue repair. Light-activated tissue sealing is an emerging strategy that facilitates rapid fluid-tight approximation of ruptured tissues, but the lack of effective biomaterials compromises efficacy. I will discuss our advances in the generation, characterization and evaluation of laser-activated polypeptide biosealants and nanofibers in which, molecular or nanoparticle chromophores are embedded within natural polypeptide matrices and fibers. Irradiation of these biosealants and nanofibers with near infrared light facilitated a photothermal response, which, in turn, engendered rapid, fluid-tight sealing and accelerated repair of soft tissues both ex vivo and in live animals. I will also discuss a new approach in which biomaterials alone can be used for simultaneous photothermal conversion of non-ionizing light as well as concomitant tissue sealing, thus dispensing the need of nanoparticles or dyes. In addition to acute wounds, slow-healing and chronic wounds, including in diabetic and obese patients, place an enormous burden on the healthcare system. Advanced treatments, including biologicals, have shown promise but have largely not succeeded in intractable pathologies likely because of poorer stabilities. I will describe our new findings on the delivery of immune-modulating bioactives molecules and polypeptide biomaterials (e.g. silk) in combination with growth factor nanoparticles with an eye towards modulating different stages of tissue repair, leading to accelerate healing and repair. We describe evaluation of these new combination treatments, including using temporal delivery strategies in immunocompetent, and obese, diabetic mice. Taken together, our studies demonstrate that polypeptide biomaterials, in concert with delivery of light, show strong translational promise for accelerating wound healing, and efficacious tissue sealing and repair.

Introduction: Zinc (Zn) has been recently proposed as a novel biodegradable metal thanks to its essential physiological and biological roles and its promising in vivo degradation rate [1, 2]. Compared to Mg alloys and Fe alloys, the degradation behavior of Zn alloys is more likely in line with clinical demand. One of the main factors limiting the extensive clinical application of Zn and its alloys is their lower mechanical strength [3]. While it can be improved through alloying with different elements (such as Mg, Ca, Mn, Cu, or Li), and plastic deformation processing techniques [34], low mechanical strength still remains an important challenge, and more effort is required to obtain a series of alloys with reproducible mechanical strength and ductility.
Methods: A series of Zn-0.5X and Zn-3X alloys (X = Cr, Fe, Sr, Zr, V, at.%) were prepared and extruded from Φ28 mm down to Φ10 mm cylinder. Mechanical property was measured at a strain rate of 0.04/min using a universal material test machine (Instron 5969, USA) according to ASTM-E8M-09 and ASTM E9-89a (2000) standards. For the cell viability test, extract media was prepared by incubating samples in the corresponding cell culture media at a ratio of 1.25 mL/cm² for 3 days. Afterward, the collected extract solution was diluted with culture media to specific concentrations of 25%. The cell viability of human endothelial cells (EA.hy926, ATCC CRL-2922, US) was measured with the MTT assay. A rabbit abdominal aorta model was used for in vivo implantation. Different wire samples with dimensions of Φ0.25 × 20 mm were implanted in the abdominal aorta of each rabbit under angiography.
Results & Discussion: All the alloys showed improved mechanical strengths when compared to pure Zn, especially the Zn-0.5Cr, 0.5Zr, 0.5 and 3V alloys have significantly improved mechanical strengths and elongation. The endothelial cells cultured with Zn-0.5Zr, 0.5Sr, 0.5 and 3 Fe and V alloys showed much better cell viability than pure Zn. The in vitro and in vivo degradation tests of different materials showed similar corrosion behaviors with pure Zn, which is helpful for their potential clinical applications.
Conclusions: Collectively, these data clearly illustrate the different mechanical properties and cell viability on these Zn based materials, which could potentially provide various choices for the tissue regeneration.
References:
1. Bowen PK, Drelich J, Goldman J. Zinc exhibits ideal physiological corrosion behavior for bioabsorbable stents[J]. Advanced materials, 2013, 25(18):2577-2582.
2. Su, Y., Cockerill, I., Wang, Y., et. al. Zinc-based biomaterials for regeneration and therapy[J]. Trends in biotechnology, 2019, 37(4): 428-441.
3. Bowen PK, Shearier ER, Zhao S, et. al. Biodegradable metals for cardiovascular stents: from clinical concerns to recent Zn Alloys[J]. Advanced healthcare materials, 2016, 5(10):1121-40.

Introduction: Additive manufacturing (also called 3D printing) techniques have been utilized and developed in the field of orthopedic implants, and various materials, fabrication methods, geometry design were studied. When an implant aims to regenerate bone, the porous structure will be beneficial for mass transfer and cell migration, leading to enhanced angiogenesis and new bone growth. Even though many bioabsorbable materials, including polymers and ceramics, have been applied in 3D-printing scaffolds, their mechanical strength is still considered insufficient, especially in the case of load-bearing applications. Bioabsorbable metals, such as magnesium and its alloys, have shown great potential as the next generation of biomaterials, thanks to their decent mechanical properties, biodegradation, and biocompatibility. Here, we use additive manufacturing to fabricate porous magnesium scaffolds, the degradation behavior and biological response of which are studied in vivo with a rabbit femur model.
Methods and Materials: Magnesium scaffolds are composed of WE43 magnesium alloy. Titanium scaffolds made of Titan-Grade 1 are used as a benchmark group. Laser Powder Bed Fusion is facilitated for the manufacturing of different scaffolds. The scaffold is in the shape of a cylinder with both diameter and height of 4 mm. The pore size of all scaffolds is around 500 μm. Before the implantation, the compression test is performed on different scaffolds. The rabbit femur defect model is used in this study. Two circular defects with a diameter of 4mm are created by the drill in the distal femur, and then two scaffolds made of the same material are inserted into these two defects separately. Two collection time points are set to 5 and 25 weeks. The collected femur bones with scaffolds are analyzed with the Micro CT scanning. Meanwhile, the blood and internal organs, including the heart, liver, spleen, kidney, and lung, are collected for biotoxicity analysis.
Results: In terms of the compression strength and modulus, the titanium scaffold exhibit a higher value, while the value of WE43 scaffold is more close to the natural human bones. All scaffolds can properly fit into defects and hold well. No systematic toxicity is found for all scaffolds based on the analysis of blood and internal organs. The degradation rate of WE43 scaffolds is derived from the volume change in the Micro CT. For all scaffolds, the new growth of bone into the pores can be observed. Interestingly, neither the degradation behavior nor the new bone ingrowth is homogeneous for the whole scaffold. Different sections of the scaffold show various degradation tendencies due to the local physiological environment (contacted with cortical bones or the marrow cavity) and mass permeation. Furthermore, degradation behavior causes an influence on the new bone growth. Meanwhile, the geometry of scaffolds also has an impact on the local biological response.
Conclusion: Compared to titanium scaffolds, WE43 scaffolds present acceptable biological response, and their biodegradation feature exhibit great potential for bone regeneration application.

Skin aging is a multisystem degenerative process, which is characterized by the accumulation of senescent keratinocytes. The exposure of reactive oxygen species (ROS) is one of the major causes that induce growth arrest and cellular senescence in those cells. The preparation of polydopamine (PDA) intended for biomedical applications typically comprises oxidative self-polymerization of dopamine (DA) in a weak alkaline solution [1, 2]. PDA can be deposited as a biofunctional coating on cell culture substrates acting as a ROS scavenger [3]. However, the role of PDA particles, which are formed during the PDA coating formation, is not yet fully explored. This motivated us to investigate the multiple functions of PDA particles, especially its antioxidant and anti-senescence effect, in cultures of human skin epithelial keratinocytes, HaCaT cells. The PDA particles were synthesized through the self-polymerization of DA in a 50 mM Tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) solution at pH 8.8. The PDA particles were filtered through Millipore® Stericup® filter units to control the diameter in the range of 220-450 nm. The uptake of PDA particles by HaCaT cells exhibited a dose-dependent manner in control and PDA groups (0, 10, 20, 30, 40, and 50 µg/ml), while the cell viability was not affected by the PDA uptake as no loss of membrane integrity and inhibition of cell growth were observed. The PDA uptake decreased the oxidative stress provoked by ROS. It also decreased the activity of the senescence-associated β-galactosidase in HaCaT cells by ~10%, where a plateau was reached when the PDA-dosage exceeded 30 µg/ml. Conclusively, these findings demonstrate that PDA particles could effectively decrease the level of cellular senescence, highlighting its potential to be translated into a medicine strategy for preventing skin aging.

Reference
1. H. A. Lee, Y. Ma, F. Zhou, S. Hong and H. Lee, Accounts of chemical research 52 (3), 704 (2019).
2. H. A. Lee, E. Park and H. Lee, Advanced Materials 32 (35), 1907505 (2020).
3. Z. Deng, W. Wang, X. Xu, Y. Nie, Y. Liu, O. E. Gould, N. Ma and A. Lendlein, ACS Applied Materials & Interfaces 13 (9), 10748 (2021).

Neurodegenerative diseases represent a major incapacitating health problem that has been proven to be difficult to treat. If the nervous system is injured, neurons are unable to regenerate efficiently. Importantly, a variety of stem cells have been determined as a possible treatment provided that they can be induced to differentiate along neurogenic lineage. Here we present research on the factors which can determine neurogenic differentiation of Dental Pulp Stem Cells (DPSCs). DPSCs are pluripotent stem cells that have been shown to differentiate in vitro along neurogenic, odontogenic, or adipogenic lineage depending on the media in which they are cultured. We have previously shown that odontogenic differentiation of the DPSC is highly dependent on the properties of the substrate, where both chemistry and mechanics combine to determine the differentiation lineage. Here we present a study aimed at determining the optimal material's scaffold for implementing neurogenic differentiation.
Polymer films were spun cast onto HF etched flat Si wafers from solutions of 3% PVDF with Dimethylformamide (DMF), 20 mg/ml PB with toluene, 7 mg/ml P4VP with DMF, and 30 mg/ml RDP/clay with PLA in Chloroform were spun cast onto cleaved silicon wafers, and annealed at 150C overnight to remove residual solvent and stresses, as well as to sterilize the substrates. The quality of the films, as well as the surface roughness or crystallinity, was assessed using scanning force microscopy in lateral and friction modes. The cells were plated on these substrates, as well as on tissue culture plastic (TCP) in alpha MEM with 10% FBS and 1% pen strep, where they were cultured for three days. Subsequently, this media was replaced with neurobasal media and changed twice a week. The neurobasal A media was composed of 100 µg/mL penicillin, 1X B27 supplement, 100 µg/mL streptomycin, 20 ng/mL epidermal growth factor (EGF), and 40 ng/mL fibroblast growth factor (FGF). We then performed EVOS imaging to acquire cell morphology on day 0, day 7, and day 28. Moreover, the degree of neuronal differentiation was investigated using Real-Time Polymerase Chain Reaction (RT-PCR) on day 0, day 14, day 21, and day 28. The neuro markers are Nestin(early), β-Tubulin-III (intermediate), and NEFM (mature). The AFM film topography images provided a measure of surface roughness and revealed that the surface of P4VP and PB films had a smoother surface while PVDF and RDP/Clay films had rougher surface topographies. The aspect ratio of the cells was quantified on the different substrates at day 28, where it was found that the cells on the RDP clay had the largest value. RT-PCR indicated that the P4VP and RDP/clay with PLA substrate had downregulation of neuro markers Nestin and β-Tubulin-III and upregulation of NEFM which confirmed neurogenic differentiation. Unfortunately, differentiation was poor on the PB and most cells died on the PB+tio2 even though these substrates were shown to be very effective in induction of odontogenic differentiation in osteogenic media. It is interesting to note that the PB substrates had much lower moduli than the PLA/RDP substrate confirming that chemical composition was more important than the mechanical response in neurogenic differentiation. Further work is in progress using electrospun fibers of the same polymer composition to determine the role of substrate morphology.

Keywords: Dental Pulp Stem Cells; Neurogenic differentiation; P4VP; PVDF; RDP/Clay; PLA; PB; RT-PCR

As entering ageing society, cardiovascular diseases have become highly increasing treats to human life with their fatality. Several efforts using organism derived grafts (autografts, allografts, and xenografts) have been adopted to replace the malfunctioning vessels. However, the use of abovementioned grafts is challenging due to their limited sources. Thus, numerous researches have been conducted to fabricate artificial vascular grafts with non-cytotoxicity and sufficient mechanical properties to withstand blood pressure. Polymeric matrices consisting of polytetrafluoroethylene (PTFE), polyethylene terephthalate (PET), or polyurethane (PU) have been most widely utilized for vascular grafts because of their biological stability. Especially for PTFE, it has been clinically utilized to substitute vessels or bypass blood flow to healthy tissues. Generally, PTFE vascular grafts have been fabricated in the form of tube and it should be cut to fit into the surgical sites. Therefore, creation of PTFE vascular grafts with patient specific structure has been challenging. As a solution for that, we are aiming to adopt digital light processing (DLP) based 3D printing technique to fabricate artificial PTFE vascular grafts with complicated branched structure. Through incorporating photocrosslinking agents and photoinitiator to sustain structural integrity of PTFE emulsion, channel structures of PTFE were successfully produced. After sequential heat treatment to eliminate supplemented materials and sinter the PTFE particles, PTFE tubes with distinctive mesh structure were obtained. Since the purpose of this research is to apply PTFE tubes to vascular grafts, evaluation of mechanical properties (tensile property, elasticity, and suture retention ability) and biological properties using endothelial cells and fibroblasts was performed.

Epithelial keratin, a type of intermediate filament (IF) protein, is one of the key components in maintaining the stability of the cell nucleus in the epidermis of the skin, the largest organ in the human body. It absorbs water and withstands external pressure, affecting the structural stability and mechanical properties of the skin. Epidermolysis bullosa simplex (EBS) is a rare genetic skin disease related to genetic mutations in epithelial keratin K5/K14. The resulting structural defects can cause keratinocytes in the basal layer to become fragile and rupture when subjected to mechanical stress. Its pathological feature is that the skin and mucous membranes are extremely fragile, and wounds and blisters occur even under slight external force. In this study, we focused on the amino acid sequence of the wild-type human keratin K5/K14 and sequences with point mutations, beginning with a full atomistic model of the K5/K14 heterodimer and proceeding to the higher hierarchical structure of the tetramer model. For the heterodimer, the structures of the wild type and the mutants share a high degree of similarity, and the helical structure is preserved. Then, based on the heterodimer model, we considered the keratin tetramer model with ID-1 contact from previous experimental observations. Our results suggestedthat in the wild-type tetramer, the hydrogen bonds formed in the middle and contact regions provide extra stability to tetramer 2B-2B interactions during IF assembly. The probabilities of hydrogen bond formation are lower in the mutant tetramers than in the wild-type tetramer in the contact region; the point mutations do not necessarily affect the structure for dimer formation, but changes in the interactions of amino acids may affect the higher-order assembly of IFs. We observed that, the structures of the tetramers with point mutations were loosely stacked, and the mechanical properties were weaker than those of the wild-type tetramer. We further compare our results with the latest experimental measurements and discuss the relationship between the genotype of EBS disease and the atomic-level mutated structures. The atomistic model allowed us to study point mutations at the molecular level. The results can be further applied to reveal the effect of point mutations on EBS diseases.

Throughout history, human beings have faced the problems of wear and tear of the tissues that compose them, so the need arises to find an alternative to the regeneration, repair, or replace damaged tissues to continue performing their physiological functions; this has led us to the use of materials to help these procedures through the so-called Tissue Engineering, dedicate to the application of chemical and biological principles in the development of substitutes that restore, maintain or improve the function of living tissues through biomaterials. Currently, the bioglass (BG) and hydroxyapatite (HA) are object of study for their high bioactivity, osteoconductivity, biodegradability and because they possess compositional and structural similarities with bone mineral, nevertheless, these biomaterials are relatively brittle and for diminishing this property, they are embedded into polymers. The use of polylactic acid (PLA) had demonstrated a great potential for load-bearing applications when is used as a polymeric matrix with the HAp particles. On the other hand, polycaprolactone (PCL) has been also extensively applied in regenerative medicine applications, among all desirable properties as biocompatibility and flexibility because of its ester bonds, PCL also had shown that enhance the osteogenic differentiation of mesenchymal stem cells, and the incorporation of HAp particles modify its hydrophobic nature, stimulating the cell interaction. In the present study, the synthesis of BG, PLA, hydroxyapatite by sol-gel method (HAsg), and hydroxyapatite by precipitation method (HAp) were performed. Subsequently, those biomaterials were complemented with polycaprolactone (PCL) to elaborate 3D porous scaffolds by the solvent-casting/salt-leaching technique. The PCL- PLA -HAp and PCL-BG-HAsg scaffolds were designed at two weight proportions each. 1. 40%-10%-50% and 2. 40%-50%-10% respectively. The scaffolds were analyzed by infrared spectroscopy (FTIR), X-ray diffraction (XDR), and scanning electron microscopy (SEM) by observing their composition and morphology, besides, as known; the surface reactions of materials with their biological environment occur a few seconds after they are implanted in the body interacting with proteins present in the physiological environment, in this fact lies the importance of evaluating the in-vitro biological behavior of the biomaterials. Simulated body fluid (SBF) and Hank's saline solution at 37 C are the aqueous media that allow us to get closer to the understanding of the biodegradation and bioactivity mechanism of composite biomaterials since their ionic compositions are very close to those of human plasma.

Poly(2-methoxyethyl acrylate) (PMEA) is one of the well-known antithrombogenic materials [1,2]. Because of its low glass transition temperature around -30°C, PMEA is a liquid-like polymer at room temperature and thus its usage is limited to antithrombogenic coating. Therefore, solidifying PMEA is important to enhance its biomedical applications as a bulk material. In this study, thermally stable poly(2-methoxyethyl acrylate)-based polyurethane (thermoset PMEA-based PU), chemically crosslinked by covalent bonds, was synthesized through reversible-addition fragmentation transfer (RAFT) polymerization and polyaddition with diisocyanate.
In detail, to obtain thermoset PMEA-based PU, hydroxyl-terminated PMEA with 4 hydroxyl groups (PMEA-(OH)₄) was synthesized by RAFT polymerization, and PMEA-(OH)₄ was then reacted with diisocyanate to form urethane bonding acting as chemical crosslinkings in the material. In fact, PMEA-(OH)₄ synthesized by RAFT polymerization was a viscous liquid-like polymer. After the polyaddition using diisocyanate, a solid standing film of thermoset PMEA-based PU was obtained. The existence of urethane bonds was confirmed by FTIR.
Thermal and mechanical properties of thermoset PMEA-based PU were evaluated through dynamic mechanical analysis (DMA) and tensile testing. From the DMA results, it was found that the storage modulus (E’) of thermoset PMEA-based PU was higher than the loss modulus (E’’) at the temperature range between 25°C and 110°C. According to the results of tensile testing, it was also found that E’ was almost constant at ~7.0×10⁵ Pa in the same temperature range. The Young’s modulus, the fracture strain, and the tensile strength of thermoset PMEA-based PU were calculated as 574±54 kPa, 212±78%, and 581±90 kPa, respectively. It was, therefore, confirmed that the synthesized thermoset PMEA-based PU was a thermally-stable solid material.
Intermediate water is a group of water molecules interacting with PMEA molecules, largely contributing to the high antithrombogenicity of PMEA. Therefore, intermediate water was also analyzed by differential scanning calorimetry (DSC). The DSC curves of the hydrated samples of thermoset PMEA-based PU exhibited cold crystallization around -27°C, deriving from the intermediate water, whereas the DSC curves of dried samples of thermoset PMEA-based PU only showed the glass transition. The ratio of intermediate water to all water in thermoset PMEA-based PU was calculated as 2.1±0.03 wt%, high enough to display antithrombogenicity, considering the ratio of other conventional highly antithrombogenic polymers [3]. It was concluded that the newly synthesized thermoset PMEA-based PU effectively possessed intermediate water for antithrombogenicity.

References
[1] C. Sato, M. Aoki and M. Tanaka, Colloids Surf., B, 2016, 145, 586–596.
[2] K. Akamatsu, T. Furue, F. Han and S. Nakao, Sep. Purif. Technol., 2013, 102, 157–162.
[3] S. Kobayashi, M. Wakui, Y. Iwata and M. Tanaka, Biomacromolecules, 2017, 18, 4214–4223.

Considering the time- and temperature-sensitive nature of cell therapeutic products (CTPs), additional protection is important in cell therapy manufacturing where cells are exposed to harsh environmental stressors (i.e. temperature fluctuation) during logistical activities. As market for these products continue to expand rapidly, it is thus critical to look into improved methods of storing and transporting these products to hospitals while ensuring that their standards (i.e. quality, stability) are strictly met via the use of diagnostic tools. Chemically-induced cell ‘sporulation’ has been recently demonstrated by our group as a bioinspired cell protection strategy for maintaining cell viability in biofabrication, where cells are exposed to UV irradiation during photocrosslinking process. Tapping on the success of this strategy, we proposed its application as a practical solution for room-temperature shipping and short-term storage of CTPs during cell logistics. Herein, plant seed-inspired ‘sporulation’ of L929-laden alginate microspheres (AlgMS) were induced through the coating of pyrogallol (PG), a plant-derived polyphenol. Thereafter, both ‘sporulated’ and non-‘sporulated’ encapsulated cells were subjected to temperature conditions that the CTPs could potentially be exposed to during 3 days of transportation and/or short-term storage. These conditions include, (i) ET: extreme temperature increment as a result of heat accumulation in confined storage spaces (e.g. when transportation vehicles with malfunctioned reefers are parked under the sun for a while) – room temperature throughout with temperature increment from 30 to 50°C over 1h at day 2; (ii) CT: cyclic temperature simulation based on real-life temperature profile from cell logistics company when CTPs are transported across countries of different seasons – 30°C (day 1), 17°C (day 2), 27°C (day 3). After which, ‘germination’/restoration of cell metabolism was achieved by degrading the microspheres and releasing the cells via mild EDTA treatment. The ‘germinated’ cells were subsequently re-incubated for 72h before cell viability was assessed with LIVE/DEAD cell assay. Regardless of the temperature conditions, majority of cells released from PG-AlgMS remained viable (> 80%), in contrast with those released from AlgMS (< 30%). This suggested the potential of cell ‘sporulation’ in enhancing protection of encapsulated cells when temperature fluctuation is involved. As expected, when the two temperature conditions were compared, more cells managed to survive when subjected to CT (~91%) than ET (~80%) condition whereby the cells were exposed to much higher temperatures known to cause irreversible cellular damage. Although the viability of cells released from AlgMS was severely compromised (~10%) for ET, a relatively high cell viability (~72%) is still achievable when exposed to the CT condition. This thus, implied the capability of cell encapsulation alone in providing certain degree of protection that could suffice in less harsh temperature fluctuation conditions. Taking into consideration of unforeseen delays that could happen during cell logistics, longer duration room-temperature cell storage and transportation for up to 10 days was also proven to be possible with this bioinspired cell protection strategy, whereby majority of released cells remained viable (>85%). In conclusion, we have demonstrated the potential of translating ‘sporulated’ encapsulated cells into a cell storage and handling platform that can maintain the quality of CTPs despite the harsh environment that these temperature-sensitive products could be exposed to during cell logistical activities. We envisage that this cell-encapsulated microsphere platforms have great potential for short-term cell storage, cell-involved biofabrication and cell logistics.

Nanoparticle-based radio-enhancement has the prospect to improve cancer cell eradication by amplifying the damage caused by (X-ray) irradiation through ejection of secondary particles (electrons) leading to further oxidative stress. Gold nanoparticles are a natural choice because of their high atomic number and biological compatibility and are therefore most researched nanoparticles. However, due to the heterogeneous results and the lack in mechanistic understanding, these particle systems have not yet entered clinics. Recently, nanoparticles based on more exotic hafnium dioxide have shown promising results in clinical studies. There is increasing evidence that radio-enhancement efficacy does not solely dependent on high atomic numbers but involves a complex cascade of secondary reactions.
Here, we will present a comprehensive nanoparticle-based radio-enhancement study including a selection of inorganic oxide nanoparticles with comparable morphologies, sizes and surface chemistries. Liquid flame spray pyrolysis as synthesis technique allows effective scale up to meet clinical and industrial demands. We will bridge physical and chemical nanoparticle–radiation interactions with biological radio-enhancement effects in a radio resistant tumor cell line using clinically relevant exposure settings. We also show how the nanoparticles can be tailored to incorporate multimodal imaging possibilities for a theranostic approach. Our comprehensive analysis will pave the way for a more rationalized nanoparticle design and development for nanoparticle-based radio-enhancement studies.

Introduction: Autografts presently remain the gold standard for bone growth and regeneration. Associated complications such as their limited availability, increased surgical time, blood loss, and donor site morbidity has urged the exploration of other alternatives. Particularly, non-resorbable graphene-family nanomaterials combined with calcium phosphate has generated notable findings. However recently, hematene, a 2D nanomaterial of iron oxide has been discovered with rather extraordinary properties. This novel bioresorbable nanomaterial with its physicochemical, optical, magnetic, electrical, and mechanical properties shows a promising osteogenic potency for bone regeneration. Nevertheless, the bioactive potential of hematene combined with calcium phosphate (ie. monetite) remains to be explored. Here, hematene-monetite nanocomposites were synthesized for the first time and their potentials to stimulate osteogenesis were showcased.

Methods: 2D hematene was synthesized by ultrasonic exfoliation from natural iron hematite (α-Fe₂O₃) and confirmed with bright-field transmission electrical microscope (TEM). Monetite was composed of β-tricalcium phosphate (β-TCP) and monocalcium phosphate monohydrate (MPCM) and subjected to hydrothermal treatment. Monetite implants were decorated with hematene nanocomposites of different concentrations using centrifugal force. The two-dimensional morphology, crystallinity, thermal behaviour, and compressive strength of the scaffolds were confirmed by Scanning electron microscopy (SEM), X-ray diffraction (XRD), Thermogravimetric analysis (TGA), and a high-precision testing system (EZ test), respectively. Cytotoxicity and gene expression profiles of bone specific markers of RUNX2, ALP, BGLAP, and SPARC, were evaluated over 21 days within pre-osteoblastic MC3T3-E1 cells.

Results: TEM imaging confirmed the successful exfoliation of ultrathin mono- and bi-layer hematene sheets with a mean lateral size of 200nm. Nanocomposites at different hematene to monetite ratios were successfully prepared with good thermal stability and decomposition profiles. Physicochemical characterization and SEM imaging confirmed the presence and homogenous distribution of the 2D nanomaterials over the monetite implant without affecting its crystallinity. Furthermore, our findings indicate that the mechanical integrity was significantly enhanced when reinforced with hematene nanocomposites of higher concentrations compared to monetite alone. In vitro testing demonstrated that gene expression profiles of alkaline phosphatase (ALP), osteocalcin (BGLAP), osteonectin (SPARC), and RUNX2, were significantly elevated over the 21 days when compared to controls. These findings indicate that osteogenesis of MC3T3-E1 cells was accelerated by the hematene-monetite nanocomposites with and without osteoinductive agents.

Conclusions: For the first time, we showcase the osteogenic potency of hematene-monetite nanocomposites and the abilities to enhance cell proliferation and osteogenic differentiation in pre-osteoblastic MC3T3-E1 cells. We demonstrate the simplicity of obtaining ultrathin 2D hematene sheets through ultrasonic exfoliation to decorate monetite implants without affecting crystallinity or thermal stability within or between treatment conditions. Assuring both properties remain unaffected is of special importance to maintain implant consistency, as variations can vastly contribute to differences in physiochemical properties. Excitingly, these preliminary in vitro results suggest that hematene derivatives could be the next big candidate as a scaffold for bone tissue regeneration. Furthermore, the highly unique thermal behaviour of hematene derivatives prompts its potential to open further avenues for therapeutic agents. Future works include the study of this osteoinductive potential on a stem cell culture model, and potentially corroborate those results by an animal study.

Introduction: Most subdermal implants for long-acting (LA) drug delivery for contraception and HIV pre-exposure prophylaxis (PrEP) comprise non-biodegradable materials. The development of new biodegradable subdermal implants can advantageously eliminate the requirement of a second medical procedure to remove the devices at the end of use when drugs are depleted. One slowly degrading polymer, poly(ε-caprolactone) (PCL), is established as a prominent and suitable material for various biomedical applications. While the general mechanism of PCL degradation is understood and documented, we uniquely explore the biodegradation profiles of PCL in novel LA reservoir-biomedical implants for contraception and HIV PrEP, showing the influence of excipients and drug formulations on polymer degradation kinetics.
Methods: Different molecular weights of medical-grade PCL (PC-12 M_n 35 kDa, PC-17 M_n 55 kDa) were extruded into cylindrical tubes with an outer diameter of 2.5 mm and wall thicknesses of 70, 100 or 200 µm. Implants were prepared by loading excipients (castor oil, sesame oil) or drug-excipient formulations (contraceptive hormones or antiretrovirals) into an extruded tube and then enclosing the tubes via heat sealing. Unsealed empty tubes, excipient- and formulation-filled implants were sterilized via gamma irradiation at 18-24 kGy. Implants were submerged in phosphate buffered saline (PBS, pH 7.4) at 37 °C to mimic physiological conditions. Polymer properties were analyzed each month via gel permeation chromatography (GPC) and differential scanning calorimetry (DSC). In vitro exposure conditions were also varied to accelerate polymer degradation.
Results: Overall, the degradation rates were faster for PCL with a higher starting molecular weight (MW) (5 kDa/month), as compared to PCL of a lower initial MW (1.5 kDa/month). The presence of an excipient affected the rate of degradation; implants containing castor oil degraded slower than implants containing sesame oil. Implants with thinner walls were more susceptible to larger changes in MW when filled with sesame oil. Implants containing drug formulations degraded at similar rates, but underwent faster degradation than implants filled with excipient alone. Additionally, when the drug was formulated with sesame oil as opposed to castor oil, the polymer degradation rate was faster. For the same MW PCL, the degradation rates of empty tubes were independent of the wall thickness. Implants subjected to accelerated conditions yielded faster degradation kinetics. For instance, PCL MW at the 3-month timepoint under accelerated conditions was comparable to the PCL MW achieved at the 1-year timepoint under simulated physiological conditions. The development of this accelerated model can support rapid screening of degradation profiles for the new polymers.
Conclusions/Implications: In this long-term study, we explored novel biodegradable implants designed for contraception and HIV PrEP that comprise extruded tubes of medical grade PCL filled with relevant drugs. We studied the influence of excipients and drug formulations on the polymer degradation rate, in real time and under accelerated conditions. Next steps include evaluating the biodegradation profiles of the implants in preclinical studies and correlating the in vitro-in vivo degradation kinetics. An in-depth understanding of the degradation characteristics of this polymer platform will enable new implant designs for varied medical indications that might require tailored degradation timeframes.
Acknowledgements: This research is made possible by the generous support of the American people through the U.S. President’s Emergency Plan for AIDS Relief (USAID Cooperative Agreement numbers AID-OAA-A-14-00012 and AID-OAA-A-17-00011). The contents are the responsibility of the authors and do not necessarily reflect the views of USAID, PEPFAR, or the United States Government.

The objective of this study is to investigate the effect of hypochlorous acid (HOCl) on dental pulp stem cells (DPSCs). Analyzing the cytotoxicity of hypochlorous acid contributes important information on the safety and effectiveness of using this compound in root canal irrigation. In addition, examining the migration and differentiation of dental pulp stem cells in response to hypochlorous acid can provide insight into whether the solution will stimulate mineralization if leakage occurs on live cells around the root.
HOCl was mixed with carbon dioxide enriched DPSCs growth medium to make the HOCl solution in the certain concentrations. The HOCl solution were then applied to DPSCs in the incubator for 5 minutes. The viability test result shows that DPSCs cannot survive in the HOCl solutions with the concentration greater than 30%. Proliferation assay was made in 3 different concentration sets: 10%, 20% and 30% respectively. The readout indicates that all 3 concentration groups showed no significant difference in proliferation rate compare to the control group. To analyze genetic expression of the cell abnormalities after HOCl treatment, a 28-day reverse transcription polymerase chain reaction (RT-PCR) test was performed for the 3 concentrations groups as well as the control group. The cells were lysed and RNA were taken on day-0, day-14, day-21 and day-28.
Future directions would include additional trials of the different concentrations of HOCl on DPSCs migration, observing the effects of HOCl on scaffolds with DPSCs and relevant growth factors, and analyzing the long-term effects of HOCl compared to alternative root canal irrigation solutions on differentiation and dentine-pulp complex regeneration.

Introduction: Patients with aortic valve stenosis (AVS) can have fibrosis and/or calcification in valve tissue, which leads to heart failure if left untreated. Emerging clinical evidence suggests aortic valve stenosis (AVS) is a sexually dimorphic disease. Characterization of valve tissue from male and female AVS patients reveals that the aortic valve fibrosis to calcification ratio is significantly higher in women relative to men. Surgical valve replacements remain the gold standard of treatment, yet patients experience various post-surgical complications. Non-surgical approaches would be desirable, but no known biomolecular treatment exists to slow or halt progression of AVS in patients of either sex. Here, we developed an in vitro hydrogel culture platform for culture of male and female valvular interstitial cells (VICs) and characterized the sex-specific effects of microenvironmental cues on myofibroblast activation.
Materials and Methods: VICs were isolated from porcine aortic valves and seeded on soft RGD-functionalized PEG hydrogels (elastic modulus, E ~ 6 kPa) known to maintain the quiescent VIC phenotype and stiff hydrogels (E ~ 41 kPa) known to activate VICs to a myofibroblast state. Male and female VICs were cultured separately and seeded on hydrogels for 3 days. VICs were immuno-stained for the myofibroblast marker alpha smooth muscle actin (α-SMA) and imaged to quantify the percentage of cells with a-SMA stress fibers. Myofibroblast transcriptomes were analyzed using the HISAT2-Rsubread-EdgeR differential gene expression pipeline, and pathway enrichment analysis was performed in Ingenuity Pathway Analysis (IPA).
Results and Discussion: Our data suggest male and female VICs exhibit sex-specific myofibroblast responses to hydrogel substrates at day 3 in culture. We consistently observed female VICs to have elevated myofibroblast activation on soft gels that mimics the stiffness of healthy valves (E ~ 6 kPa) and stiff gels that recapitulate fibrotic valve stiffness (E ~ 40 kPa). Our transcriptomic data suggest hundreds of sex-specific gene expression differences between male and female myofibroblasts, and we identified the RhoA/ROCK signaling pathway to have increased activity in female VICs using IPA. Indeed, in vitro validations revealed female VICs to be less sensitive to RhoA/ROCK inhibition relative to male VICs on stiff hydrogels. Recognizing that sex-dependent variability in cellular phenotypes is attributed to the 15-25% of genes that escape X-chromosome inactivation (XCI) in women, we also found 28 genes differentially expressed in female myofibroblasts that are also known to escape X-chromosome inactivation. Two of these genes, BMX and STS, which encode for the BMX non-receptor tyrosine kinase and steroid sulfatase and necessary for cardiac hypertrophy and integrin expression, were validated as key genes that regulate RhoA/ROCK activity in female VICs. Using ibrutinib and irosustat to inhibit BMX and STS, respectively, we observed increased female VIC deactivation in response to RhoA/ROCK inhibition. Collectively, we suggest genes that escape X-chromosome inactivation uniquely regulate myofibroblast activation processes in female myofibroblasts.
Conclusions: Our data support the importance of (1) sex-separating cells and (2) engineering the appropriate culture microenvironment to understand how male and female cells interpret microenvironmental cues. We suggest female VICs are more susceptible to myofibroblast activation due to increased expression of key XCI genes, which may contribute to increased fibrosis found in valve leaflets from female AVS patients. We envision future in vitro disease models to appreciate the potent effects of cell sex, which may impact how diseases are understood and treated as a function of sex.