Symposium BM04 : Biomaterials for Regenerative Engineering

Peripheral nerve injuries commonly result in lifelong disability. Even the most advanced surgical procedures and pharmaceutical treatments have limited ability to improve clinical outcomes. Intraoperative electrical stimulation performed at the site of nerve repair is a well-established treatment that can accelerate and improve overall rates of functional recovery. Clinical utilization of electrical stimulation has, however, been limited to the intraoperative period, wherein injured tissue is physically accessible. This talk describes bioresorbable electronic materials and devices that allow for non-pharmacologic neuroregenerative therapy via prolonged post-operative electrical stimulation throughout the healing process, enabling substantially improved outcomes in nerve regeneration and functional recovery compared to the existing intraoperative mode. An essential characteristic of these implantable systems is that they undergo complete dissolution and elimination from the body via natural biochemical processes over timescales matched to operational requirements and without adverse biological effects. The result thereby eliminates the need for secondary surgical extraction and associated risks to the patient and to site of the nerve repair. This type of bioresorbable technology represents a new vehicle for the delivery of non-pharmacologic bioelectric and neuroregenerative therapies in a variety of clinical settings, and a significant paradigm shift in the treatment of critical nerve injuries with limited potential for sensorimotor recovery.

Recent advances in human pluripotent stem cell (hPSC) biology enable derivation of essentially any cell type in the human body. However, limitations related to cell maturation, vascularization, cellular fidelity and inter-organ communication still remain.
Here, biological wire (Biowire) technology will be described, developed to specifically enhance maturation levels of hPSC based cardiac tissues, by controlling tissue geometry and electrical field stimulation regime (Nunes et al Nature Methods 2013). We will describe new applications of the Biowire technology in engineering a specifically atrial and specifically ventricular cardiac tissues, safety testing of small molecule kinase inhibitors, potential new cancer drugs, and modelling of left ventricular hypertrophy using patient derived cells.

For probing of more complex physiological questions, dependent on the flow of culture media or blood, incorporation of vasculature is required, most commonly performed in organ-on-a-chip devices. Current organ-on-a-chip devices are limited by the presence of non-physiological materials such as glass and drug-absorbing PDMS as well as the necessity for specialized equipment such as vacuum lines and fluid pumps that inherently limit their throughput. An overview of two new technologies, AngioChip (Zhang et al Nature Materials 2016) and inVADE (Lai et al Advanced Functional Materials 2017) will be presented, that overcome the noted limitations and enable engineering of vascularized liver, vascularized heart tissues and studies of cancer metastasis. These platforms enable facile operation and imaging in a set-up resembling a 96-well plate. Using polymer engineering, we were able to marry two seemingly opposing criteria in these platforms, permeability and mechanical stability, to engineer vasculature suitable for biological discovery and direct surgical anastomosis to the host vasculature.
Finally, to enable minimally invasive delivery of engineered tissues into the body, a new shape-memory scaffold was developed that enables delivery of fully functional tissues on the heart, liver and aorta through a keyhole surgery (Montgomery et al Nature Materials 2017).

Biological scaffolds derived from decellularized tissues function as tissue remodeling templates during bone regeneration. This isolated extracellular matrix (ECM) provides a structural framework that regulates adherence, migration, proliferation, and differentiation of bone residing cells and those in surrounding tissues. Nonetheless, decellularization protocols, like the ones used in isolation of demineralized bone matrix (DBM), require use of acids and other harsh chemicals that render osteoinductive proteins in DBM denatured. Also, lack of vascular cues in DBM presents another impediment to bone healing, and results in non-unions from poor vascularization of the regenerating tissue. To address these limitations, we used tissue engineering approach and tested the regenerative capacity of decellularized ECMs derived from sequential cultures of fibroblasts and endothelial cells grown on polycaprolactone (PCL) fibers. We hypothesized that this vascular ECM would enhance osteoblast proliferation, differentiation, and matrix deposition in vitro. The bottom-up strategy eliminated competition between cell types with varying proliferation rates and allowed for ECM remodeling. ECMs from decellularized cultures were evaluated via methylene and Coomasie blue stains, and their protein and DNA content was quantified. Staining also revealed that endothelial cells grown on fibroblast ECM (Fibro/Endo) form networks resembling capillaries. These structures stained positively for endothelial markers CD31 and vWF. Analysis of SEM images indicated changes in morphology and preferential attachment of endothelial cells to PCL fibers laden with fibroblast ECM. Osteoblasts grown on this Fibro/Endo ECM yielded higher proliferation rates at each time point during 28 day culture. Significantly higher ALP activity in these cultures suggest better capacity of osteoblasts to form bone tissue and higher degree of differentiation. The color area and intensity of Alizarin Red staining of Fibro/Endo cultures revealed uniform and greater calcium deposits than in cultures with only single type of ECM. Relative expression of osteoclacein and osteopontin between culture conditions was compared via immunostaining. Successive culture/decellularization cycles enriched the ECM product, which in turn significantly enhanced proliferation and differentiation of osteoblasts in vitro. Thus, addition of vascular ECM cues to biological scaffolds might lead to improved bone healing rate in vivo. The use of this hybrid ECM can expanded to other combinations with additional cell types, which might further improve regeneration of tissues.

Acknowledgements:
The work was partly supported by the Assistant Secretary of Defense for Health Affairs, through the Peer Reviewed Medical Research Program under Award No. W81XWH-16-1-0132, and the National Institute of Biomedical Imaging and Bioengineering of the National Institutes of Health (award number R01EB020640).

Injectable self-setting bone pastes are a user-friendly bone void filler in comparison to dense, porous and granular ones, because pastes are applied for minimal invasive surgeries and are shaped easily to fit bone defects. However, surgeons desire biodegradable bone paste strongly because present bone pastes are very low biodegradable and brittle and a risk to be a cause of secondary bone fracture. Presently available biodegradable bone void fillers in Japan are β-tricalcium phosphates, carbonated apatite and hydroxyapatite/collagen bone-like nanocomposite (HAp/Col). The HAp/Col demonstrates good viscoelasticity and excellent bioresorbability with bone formation ability by incorporating into bone remodeling process. We focused on the HAp/Col as a base material for novel injectable bone cement. In the previous report, the HAp/Col biodegradable self-setting pastes were prepared by a mixing of the HAp/Col powder and aqueous solution of (3-glycidoxypropyl)trimethoxysilane (GPTMS), which is a setting agent by cross-linking of collagen and forming siloxane network by self-condensation. The HAp/Col-GPTMS bone pastes implanted into porcine tibia were resorbed and replaced by newly formed bone within 12 weeks. In this study, the pastes were implanted in the rat tibia and the biological tissue reaction and the absorption behavior of the pastes up to 4 weeks after transplantation were investigated in detail.
The HAp/Col (80/20 in mass ratio) powder at 100 μm or less in particle size was prepared by ball-milling of the HAp/Col compact prepared by a uniaxial pressing after synthesis by the simultaneous titration method. Aqueous solutions of GPTMS as 1.0 and 10 % in volume were prepared by mixing of GPTMS in distilled water followed by 1-hour hydrolyzation of GPTMS. They were mixed at the powder/liquid ratio of 1.00 g/cm³, optimal for the anti-washout property, for 3 min and molded into cylindrical shape with a 2.0 mm in diameter and 2.0 mm in height. The shaped pastes were then incubated for 72 h to be hardened. The hardened pastes were then implanted in 2.0-mm hole defect of proximal SD rat tibia (male, 8 weeks old). At 1, 2 and 4 weeks after the implantation, bioresorption behavior and biological tissue responses of the pastes were investigated by the X-ray µ-computed tomography (µ-CT) and histological observations. All animal tests were authorized by NIMS animal committee (49-2016-3).
The µ-CT observations demonstrated the residual volume of the pastes tended to decrease with the implantation period. The residual volume of the paste with 1.0-% GPTMS significantly decreased between 2 and 4 weeks that is considered to be an osteoclastic resorption. Although no drastically decrease was observed for the paste with 10-% GPTMS, it is expected to be substituted completely with newly formed bone deduced from the results of pig test. In addition, the results of histological observations will be presented on a podium.

Coronary artery disease is the leading cause of death in the United States, with over 700,000 myocardial infarctions (MI) occurring each year. While effective strategies have been developed to reduce mortality rates from the acute event, there remain significant challenges to preventing the formation of fibrous scar tissue, which leads to reduced cardiac function and eventual heart failure. Here, we demonstrate the fabrication of hyaluronic acid (HA)-based microrods with tunable size, shape, and stiffness for the attenuation of chronic cardiac fibrosis. These microrods modify the mechanical properties of the tissue microenvironment and modulate the fibrotic phenotype through mechanotransduction pathways. Additionally, HA is a naturally occurring material that is bioresorbable and exhibits several wound healing properties, making it a promising material for this microtopography-based approach. We show that fibroblasts closely interact with these microrods in vitro and in vivo, leading to dramatic changes in proliferation, collagen expression and fibrotic phenotype. NIH-3T3s and primary NVRFs attached to HA microrods and often conform to the rod geometry. When grown in the presence of HA microrods, fibroblasts have reduced expression of collagen I and aSMA, suggesting that HA microrods may be able to attenuate the conversion of fibroblasts to the active myofibroblast that is responsible for the overproduction of scar tissue. When injected into the myocardium of a rat infarct model, HA microrods improved cardiac function at 6 weeks post infarct. We have previously shown that PEG-based microrods are able to decrease the loss of cardiac function caused by MI, increasing the % change in EF from -11% to -3%. Impressively, HA-based microrods are able to increase EF by nearly +10%, suggesting that the rods are not only able to reduce deterioration of function, but restore function to the injured heart as well. This may be due to HA being bioresorbable and able to elicit additional biochemical effects through HA signaling pathways as elucidated on our studies. We also show demonstrate that the biochemical conjugation of the microrods with angiogenic peptides can further enhance the ability of this material to promote tissue regeneration. Such materials based approaches can be used in a variety of disease settings including vascular and musculoskeletal, opening up new therapeutic approaches for tissue regeneration.

Regenerative Engineering strategies have unprecedented potential to restore function to large musculoskeletal injuries. The talk will be used to describe methods developed together with collaborators to treat critical-sized segmental bone defects and volumetric muscle loss. Our lab has developed promising 3D-printing scaffold technologies that combine poly-ε-caprolactone (PCL) with decellularized bone (DCB) matrix to make composite PCL-DCB scaffolds. We have demonstrated the osteoinductive properties of PCL-DCB scaffolds by assessing the de novo bone formation when seeded with adipose-derived stromal/stem cells and stromal vascular fraction (SVF) and implanted in murine critical-sized cranial defects. Murine models of critical-sized bone defects are also useful in understanding the role of vascularization in bone regeneration. We have recently demonstrated its use in the application of a novel multimodality imaging platform for acquiring in vivo images of microvascular architecture, microvascular blood flow and tracer/cell tracking via intrinsic optical signaling, laser speckle contrast and fluorescence imaging. We are currently using these techniques to explore the impact of rapid, early-stage revascularization on bone healing. In considering translation to humans, however, the complex loading regimens in the craniofacial skeleton make the design of clinically applicable scaffolds inherently more complex than those used to treat murine calvariae. To address this, we are developing algorithms for designing scaffolds with heterogenous porous structures tailored to the expected physiologic loads.
To treat volumetric muscle loss, our team is developing a novel biomaterial-based approach to overcome scarring and induce regeneration of densely vascularized muscle in non-healing, skeletal muscle defects. In particular, we have demonstrated the capacity of novel electrospun fibrin hydrogel scaffolds seeded with myoblasts to regenerate the structure and function of damaged muscle. Myoblast-seeded scaffolds enabled remarkable muscle regeneration with high myofiber and vascular densities after 2 and 4 weeks, mimicking that of native skeletal muscle (while acellular scaffolds lacked muscle regeneration). Both myoblast-seeded and acellular scaffolds fully recovered muscle contractile function to uninjured values after 2 and 4 weeks. In ongoing studies, we are evaluating the potential for tuning the myogenic impact by varying the mechanical properties of the scaffolds. I will also describe our ongoing work on modulating the biochemical characteristics of the scaffold to stimulate neural infiltration and the formation of functional neuromuscular junctions within regenerated skeletal muscle tissues.

Due to disease, degeneration, trauma, and aging, bone loss occurs in the body. Although there have been remarkable improvements in development of functional bone scaffolds, it remains difficult to fabricate porous and biocompatible constructs in physiologically relevant sizes (cm-scale). Herein we developed biomineralized origami-inspired paper scaffolds in three-dimensions (3D). To our knowledge, this work is the first demonstration that paper can be used as a 3D construct to induce template-guided mineralization by osteoblasts.
In this work, we used the principles of origami to fabricate free-standing paper scaffolds in cm-scale. Because paper is an extremely flexible material that can easily be cut, creased, and folded to form 3D structures, the scaffolds were easily fabricated in a variety of different geometries. This feature can potentially be useful in generation of constructs for patient-specific applications especially for patients who have defects of irregular sizes and shapes. After sterilizing the constructs, they were seeded with osteoblasts in a collagen matrix. The samples were cultured up to 21 days and mineralization was evaluated using various assays including colorimetric assays, immunocytochemistry, high-resolution imaging (SEM), and micro-computed tomography (micro-CT). We also performed in vivo subcutaneous implantation experiments in a rat model.
In this project, we generated paper scaffolds in different shapes, sizes, and configurations (mm-cm scale). Due to its porous structure, paper allowed for transport of oxygen and nutrients across its thickness. Paper scaffolds supported a homogenous distribution of cells within their 3D structures. In our experiments, proliferation of osteoblasts increased until day three and then decreased. Hydroxyapatite content of the samples indicated that there was a progressive increase in the amount of hydroxyapatite in the paper scaffolds over 21 day of culture period. We used SEM to visualize the deposition of mineral clusters, and EDAX to calculate the ratio of calcium to phosphate. Our in vivo experiments demonstrated that paper scaffolds did not cause inflammation. The paper implants integrated with the existing tissue strongly and rapidly vascularized.
To sum up, we have shown that origami-inspired tissue engineering is useful for template-guided mineralization. We obtained partially mineralized scaffolds in various 3D geometries. The osteoblasts deposited calcium phosphate in these scaffolds and induced template-guided mineralization. Our approach used paper, a readily available material, as the cell culture scaffold. Paper has great potential to tackle the limitations of traditional scaffolds including cost, availability, accessibility, porosity, flexibility, and ease of fabrication. In the future, paper-based scaffolds could potentially guide and accelerate bone repair using patient specific cells.

Extracellular matrix (ECM) topography, composition, and stiffness vary across different tissues in the human body. Scaffold-based regenerative strategies must emulate native ECM of the region of interest and be conducive to cell function and differentiation. Healthy soft-tissue ECM has low kPa range of stiffness. Images of decellularized soft tissue ECM (dstECM) have shown that the matrix has little backbone material, a fibrous backbone thickness of ~ 1 μm and pore sizes of 10-30 μm. Currently, ECM-mimicking scaffolds of interest are nanofiber mats, sponges, hydrogels and nanofiber-hydrogel composites. While nanofiber mats have fibrous topography, they have impenetrable pores and fail to mimic either the 3D topography or stiffness of soft tissue ECM. Hydrogels allow tunable stiffness in the kPa range for soft tissue scaffolds, however, they lack an insoluble backbone to mechanically support cells. Existing hybrid nanofiber-hydrogel scaffolds and sponges do not adequately represent the topography seen in dstECM. In this work, we overcame these limitations of current scaffolds by fabricating structures with minimal fibrous backbone and pore size very similar to dstECM using an emerging technique called cryogenic electrospinning (CE).
CE is different from traditional electrospinning in that the collector plate is maintained at less than 0°C. This promotes ice crystal growth between deposited fibers, which can be subsequently lyophilized to produce air pores. This allows scalable 3D growth, increased porosity, reduced scaffold density and kPa-range bulk stiffness. CE has mostly been explored with synthetic polymers dissolved in organic solvents, producing loosely packed nanofibers. In our work, we adopt a greener approach by electrospinning hydrogel materials and ECM proteins to emulate native ECM, with water as the solvent. The topography of our fabricated scaffolds is dramatically different from CE scaffolds reported in literature, due to material and solvent choices and has a 3D fibrous, honeycomb-like backbone, highly interconnected pores that facilitate cell penetration, with pore sizes around 20 μm, and a spongy bulk, strikingly similar to dstECM. We explored collagen, a major structural component of native ECM, and elastin, a pliable protein that will accommodate the push-pull forces of migrating cells, together with alginate and/or polyethylene glycol, hydrogel materials that will act as soft cushions, as biomaterials for CE. We expect these composite fibrous sponge scaffolds to boost growth and optimal function of stromal cells, such as mesenchymal stem cells (MSC), for therapeutic use. MSCs in fibrous sponges may recapitulate a regeneration supportive microenvironment for epithelial cells, leading to improved MSC-based treatments for salivary hypofunction in patients suffering from Sjögren’s syndrome, diabetes, or side effects of radiation therapy.

There is a close correlation between vascularization and bone formation in endochondral ossification as maximum extent of bone formation follows maximum levels of VEGF expression. This suggests that osteogenesis and vascularization are coupled by spatiotemporal regulation of paracrine signaling in which the invading vascular endothelial cells secrete osteogenic morphogens to stimulate cell differentiation and bone formation. The objective of this work was to develop a tissue model to investigate the effect of spatial patterning of mesenchymal stem cells and endothelial progenitor cells and spatiotemporal delivery of osteogenic and vasculogenic morphogens on vascularized osteogenesis in a 3D culture system. To achieve the objective, a 3D co-culture system was developed consisting of a cell-adhesive, degradable polyethylene glycol matrix with gelatin methacrylate-filled microchannels for patterning of human mesenchymal stem cells (MSC) and endothelial progenitor cells (EPC). MSC were encapsulated in the matrix and a combination of MSC+EPC were encapsulated in the microchannels. Self-assembled polyethylene glycol nanogels (PEG NG) were synthesized for timed delivery of BMP-2 and VEGF morphogens. The osteogenic BMP-2 was conjugated to 21-day release NG and added to the MSC-laden matrix. The vasculogenic VEGF was conjugated to 5-day release NG and added to the MSC+EPC-laden microchannels. The 3D tissue model was cultured in osteogenic-vasculogenic medium. At each time point, the tissue model was evaluated for osteogenesis and vasculogenesis by biochemical, mRNA, and protein analysis.
Groups included MSC/EPC patterned tissue model without BMP-2/VEGF (None), with dissolved BMP-2/VEGF, and with BMP2-NG/VEGF-NG. Osteogenic control group was MSC encapsulated in degradable PEG gel with BMP-2 or BMP2-NG. Vasculogenic control group was MSC+EPC encapsulated in gelatin methacrylate with VEGF or VEGF-NG. Based on the results, the extent of vascularized osteogenesis was higher in patterned cellular constructs compared to un-patterned constructs. Further, timed-release of VEGF and BMP-2 in the patterned cellular constructs significantly enhanced the extent of vascularized osteogenesis compared with the direct addition of VEGF and BMP-2. We further discovered that the spatial patterning of MSC and EPC and the spatiotemporal of BMP-2 and VEGF sharply increased the expression of vasculogenic factors bFGF and PDGF and osteogenic factor TGF-β in the tissue constructs. The results suggest that osteogenesis and vasculogenesis are coupled by localized secretion of paracrine signaling factors during bone formation.

Endodontic root canal therapy is one of the most common clinical procedures to treat infected dental pulp. This non-regenerative treatment removes the dental pulp as well as the vascular and nerve tissues and replaces them with elastomeric composites, such as gutta-percha. The resulting tooth is devitalized and fragile, which may require additional intervention within 3 years. Our self-assembling peptide hydrogels (SAPHs) aim to establish a regenerative solution to this problem by replacing the inert material used in endodontic therapy with materials that promote dental pulp regeneration. In this work, we have used solid-phase peptide synthesis to create dentinogenic self-assembling peptides that form hydrogels under physiological pH and ionic strength. Physical characterization of these hydrogels, using circular dichroism, atomic force microscopy, and scanning electron microscopy, revealed that our peptides formed β sheet nanofibers, which in turn are non-covalently crosslinked to create robust hydrogels. We demonstrated the thixotropic nature of these hydrogels through oscillatory rheometry, and further verified their injectability and in situ reassembly into strong hydrogels through in vivo subcutaneous injection studies. In both in vitro and in vivo studies, we were able to show the efficacy of our SAPHs to support and promote the proliferation of dental pulp stem cells. Additionally, in our in vivo studies we observed the infiltration of blood vessels into our hydrogels, suggesting their ability to provide a suitable environment for dental pulp regeneration. The goal of these SAPHs is to provide an improved regenerative alternative to conventional endodontic therapy.

Neurons are naturally encompassed by a network in a highly aligned manner. After spinal cord injury (SCI) in the central nerve system (CNS), the organized extracellular matrix (ECM) within the spinal cord is profoundly disrupted, which causes axonal regeneration over injury sites challenging due to lack of orientational guidance. Because of the nature of the spinal cord, bioengineered scaffold in a three-dimensional (3D) format is of great importance for the functional recoveries after injuries. Herein, we report a self-foldable 3D silk-based nanoladder scaffold to mimic the hierarchic structure of the spinal cord with no spatial constraints, comparable mechanical properties, controllable biodegradation rate and sustainable growth factor release. In this study, we fabricated a silk-based nanoladder film with the integration of two scales, micron-meter fibers, and nanoprotrusions. We have proved that micron-meter fibers can provide directional guidance to the regenerated axons, while nanoscale protrusions can serve as mechanical cues to stimulate neurite outgrowth and synapse formation. We further developed the 3D self-foldable nanoladder by coupling the hydrophobic silk nanoladder film with a hydrophilic thermal expanding hydrogel layer. By controlling the biodegradation rate of the 3D nanoladder, a sustainable release of growth factors embedded in the silk film was achieved to trigger the axonal regeneration after injuries. We further applied organotypic spinal cord tissue slices as the ex-vivo injured model to demonstrate an enhanced axonal regeneration and functional connection between two slices placed in a distance of 2-3 mm. In all, we suggest that 3D silk-based nanoladder can serve as a grafting bridge to guide axonal regenerations to desired targets for functional reconnections after SCI.

Leveraging the multifunctional nature of citrate in chemistry and inspired by its important biological roles in human tissues, a class of highly versatile and functional citrate-based biomaterials has been developed. Citric acid, historically known as an intermediate in the Krebs cycle, is a multifunctional, nontoxic, readily available, and inexpensive cornerstone monomer used in the design of citrate-based biomaterials. In addition to the convenient citrate chemistry for the syntheses of a number of versatile polymers that may be elastomeric, mechanically strong and tough, injectable, photocrosslinkable, tissue adhesive, bioimaging/biosensing-enabled, and/or electrically conductive, citric acid also presents inherent anti-bacterial, anti-clotting, angiogenic characteristics and modulates cellular energy levels leading to facilitated stem cell differentiation, which make citrate biomaterials ideal for a number of medical applications. We have attained a comprehensive new understanding of the citrate roles on osteo-phenotype progression and identified a new mechanism pertaining to the metabolic regulation of citrate to elevate cell energy status for bone formation, referred to as citrate metabonegenic regulation. This previously unexplored citrate metabonegenic regulation has allowed us to design new biomaterials to meet the dynamic biological, biochemical, and biophysical needs in bone regeneration. In this presentation, a methodology for the design of biomimetic citrate biomaterials and their applications in regenerative engineering, drug delivery, bioimaging and biosensing will be discussed with a focus on orthopedic engineering.

Modern medicine requires biomaterials combining multiple functions such as degradability, stimuli-responsivity, cell instructivity or carrier capabilities for bioactive molecules. Complex polymer network architectures are a versatile molecular design for integrating different functions in one material system. Such networks often contain physical netpoints for adjusting mechanical properties or implementing stimuli-sensitivities. For this purpose macromolecules are potentially equipped with chain segments being able to exhibit strong physical interactions.
Here amino acid based oligomeric segments are built either from L-lysine diissocyanate or from morpholindiones.
Pure oligodepsipeptides, alternating copolymers of an α-amino acid and an α-hydroxy acid, have been selected as a hydrophobic block in segmented polymers in order to achieve strong physical interactions for stabilizing nanoparticles during their formation [1] or for providing high formstability to thermoplastic elastomers. Degradable triblock copolymers having a central oligodepsipeptide block have shown great potential as transfection agent combining high transfection capability with low toxicity [1]. Depsipeptide based multiblock copolymers are suitable for creating soft actuators with excellent performance in shape stability and reversible strain [2].
L-lysine-based oligoureas are incorporated as dangling side chains or crosslinking segments in gelatine based polymer networks. In architectured gelatin-based hydrogels (ArcGel) the local elastic modulus was adjustable independently from the macroscopic compression modulus by the molar ratio of L-lysine diissocyanate to freely available amino groups in gelatin. The dynamic alteration of cellular microenvironments accommodating mesenchymal stem cells is studied during degradation. Along with the degradation-related pore growth cell migration and differentiation were followed. The potential of ArcGels for a purely material-induced regeneration was demonstrated in a critical femur defect [3] and a cranial defect [4] in rat models.

References
[1] W. Wang, T. Naolou, N. Ma, Z. Deng, X. Xu, U. Mansfeld, C. Wischke, M. Gossen, A. T. Neffe, A. Lendlein, Biomacromolecules 2017, 18, 3819-3833.
[2] W Yan, T. Rudolph, U. Noechel, O. Gould, M. Behl, K. Kratz, A. Lendlein, Macromolecules Article ASAP, DOI: 10.1021/acs.macromol.8b00322
[3] A.T. Neffe, B. F. Pierce, G. Tronci, N. Ma, E. Pittermann, T. Gebauer, O. Frank, M. Schossig, X. Xu, B. M. Willie, M. Forner, A. Ellinghaus, J. Lienau, G. N. Duda, A. Lendlein, Adv. Mater 2015, 27, 1738-1744.
[4] P. Lohmann, A. Willuweit, A.T. Neffe, S. Geisler, T. Gebauer, S. Beer, H. Coenen, H. Fischer, B. Hermanns-Sachweh, A. Lendlein, N. J. Shah, F. Kiessling, K.-J. Langen, Biomaterials 2017, 113, 158-169.

Growth factors such as bone morphogenetic protein 2 (BMP2) have been found to stimulate the odontogenic differentiation of mesenchymal stem cells (MSCs) of the dental pulp. This is particularly important for the regeneration of the dentin tissue of the tooth. However, the short half-life and poor distribution of growth factors like BMP2 may present problems with high cost and inconvenience due to the need for repeated dose injections to sustain tissue regeneration and healing. Consequently, strategies that combine slow, sustained release of BMP2 with three dimensional (3D) bioactive scaffolds are important for the differentiation of the MSCs into the odontoblast lineage to accelerate dentinogenesis. Herein, we present the results of in vitro studies of the controlled release of BMP2 from gelatin methacrylate (GelMA) microparticles to human dental pulp stem cells (hDPSCs) growing on 3D polylactic acid (PLA)/apatite composite scaffolds. First, 3D printing is utilized to fabricate PLA scaffolds, which are then surface-coated with biomineralized apatite particles synthesized via a bioinspired mineralization process. This involves alternate immersion of the PLA scaffolds in solutions of calcium nitrate and potassium phosphate dibasic to form a precipitate that is mineralized into apatite at physiological pH. The architectural, microstructural, and mechanical properties of the resulting 3D PLA/apatite composite scaffolds are then determined. Second, GelMA photopolymer is synthesized from porcine gelatin and methacrylic anhydride. After that, BMP2-laden GelMA microparticles are fabricated using an oil-in-water emulsion technique and ultraviolet photocrosslinking. Subsequently, a scaffold/microparticle hybrid construct is produced by the attachment of the BMP2-laden microparticles to the 3D PLA/apatite composite scaffolds. The microstructure of the GelMA microparticles and the release profiles of BMP2 from the microparticles are characterized prior to and following attachment to the PLA/apatite scaffolds and the differences are highlighted. Finally, the effect of the controlled release of BMP2 on hDPSC cells growing on the PLA/apatite scaffolds is investigated. This is done by assessing hDPSC cell proliferation, odontogenic differentiation, and extracellular matrix production and mineralization. The implications of the results for tooth regeneration are then discussed.

Nature inspired chemistry, and small molecules have led to the development in the field of the material science and biomedical engineering as it exhibited unique physicochemical properties. Recently, polyphenols extracted from green tea have been widely investigated, due to their intrinsic properties such as anti-inflammation and radical scavenging. Interestingly, a 1,2,3-trihydroxyphenyl group in epigallocatechin gallate (EGCG) could mimic mussel inspired chemistry through oxidative reactions, and generate tissue adhesive nature. In this paper, we report a tissue adhesive and immune modulation hydrogel inspired by the mussel chemistry and polyphenol. We conjugated tyramine (HA_T) and EGCG (HA_E) into hyaluronic acid (HA), and the hydrogel (HA_TE) was fabricated by an oxidative reaction using tyrosinase from Streptomyces avermitillis (SA_Ty). With strong oxidative nature of EGCG, the HA_TE hydrogel can be fast formed in a few seconds. We compared HA_TE hydrogel with commercial products (cyanoacrylate and fibrin glue) in the aspects of tissue adhesive and sealants. In the lap shear and burst pressure test, HA_TE exhibited the highest tissue adhesiveness regardless of wetness compared to commercial products. When HA_TE was applied as tissue adhesive into mouse wound closure, and it successfully closed wound and recovered damaged tissue. Additionally, due to EGCG naturally possesses anti-inflammation and minimize host recognition, HA_TE hydrogel produced little inflammatory cytokines in vivo that are comparable to PBS group. This demonstrates that polyphenol based hydrogel might provide a robust platform in the field of both material science and translational medicine.

Hair regenerative medicine is a new approach for the treatment of hair loss caused by aging, diseases, injury, and medical treatments. Hair follicle morphogenesis is triggered by reciprocal interactions between hair follicle germ (HFG) epithelial and mesenchymal layers. Recent studies have revealed that HFGs can be fabricated in vitro by integrating two respective aggregates of epithelial and mesenchymal cells. This approach showed promising results for hair regenerative medicine, but preparing a large number of HFGs remains challenging, particularly considering that hundreds of thousands of HFGs are necessary for a single patient. In this study, we developed a method for the large-scale preparation of HFGs in vitro via the self-organization of cells. We mixed epithelial and mesenchymal cells in a culture medium and then seeded them onto an oxygen permeable polydimethylsiloxane (PDMS) spheroid chip, which has hemispherical wells with a diameter of 1 mm and a density of 100 wells/cm². The cells initially formed a randomly distributed single aggregate, but then were spatially separated from each other and exhibited typical morphological features of a HFG after three days of culture. Interestingly, oxygen supply through the bottom of the spheroid chip was crucial for the spontaneous formation of HFGs and subsequent hair shaft generation. The generated hair follicles also entered the hair cycle through the rearrangement of follicular stem cells. Unlike previous approaches, this spontaneous HFG formation in vitro facilitated the preparation of a large number of cell aggregates (~5000 aggregates/plate). We further optimized the HFG culture conditions for human hair cells. In particular, gene expressions related to hair morphogenesis and generation were significantly increased by increasing fibroblast growth factor (FGF-2) concentration (from 0 to 100 ng/mL). Hair shaft pigmentation was observed by transplantation of HFGs including hair pigment cells. Hair shafts that were generated showed typical morphological features, such as hair cuticles and hair growth cycle. This simple HFG preparation approach may provide a promising strategy for advancing hair regenerative medicine.

In tissue engineering and bioplotting, the major limitation to building large constructs with physiological cell densities is the poor diffusion of oxygen and nutrients. Organs and many tissues have cell densities in the range 1-5x 10⁸ cells/ml, yet bioinks can only sustain up 25x10⁶ cells/ml. Oxygen concentration in culture medium is one of the major limiting factors for cell survival and its concentration is about 30 times lower than glucose. This limits the tissue models that can be printed. Furthermore, upon implantation cell survival is dependent on revascularization rate and limit the potential applications in vivo. Using oxygen releasing microparticles we designed a bioink system capable of prolonging cell survival at high cell density (2x10⁸ cells/mL), mimicking physiological organ cell densities.

Oxygen releasing microparticles (OµP) were produced by phase separation method using polycaprolactone (Mw 80000, Aldrich, USA) and calcium peroxide (Aldrich, USA). Particles size measurement was confirmed using field emission scanning electron microscope (FE-SEM, FEI Inspect F-50, USA). The bioink was prepared by mixing 10% (w/v) of OµP in a 1% alginate solution. Oxygen release was measured in 250µL of bioink crosslinked with 0.1M calcium chloride solution and immersed in 1mL PBS at 37°C using an AL300 oxygen sensor (OceanOptics via Gamble Technologies, Canada). Organ-like high density cell culture was performed by seeding a high density (4x10⁸ cells/mL) by combining 125µL CHO cells and 125µL of 2% alginate solution alone or containing 20% w/v OµP. The mixture was then extruded through a 20G needle in a 0.1M calcium chloride solution to form 20µL beads. Cells were then cultured for 48h. Cell viability was assessed using MTT.

Scanning electron microscopy revealed that the size of the microparticles ranged from 200µm to 5µm. Oxygen measurements indicated that the dissolved oxygen content of the bioink was kept at 41 ± 5% for 72h. Bioinks with organ-like cell densities without OµP had a cell viability of 27 ± 19% and 13 ± 4.8% at 24 and 48h respectively, whereas with OµP viability was significantly higher at both times; 87 ± 18% and 63 ± 20% at 24 and 48h respectively (N=9, p<0.001).

Lack of oxygen can be detrimental to cell function and survival. In tissues the diffusion of oxygen around a capillary is reported to be around 200µm. By incorporating OµP to a bioink it was possible to create organ-like density construct (2mm thick) with a high viability. This new approach to bioinks may enable printing of more complex and physiologically relevant tissue models.

The extra-cellular matrix (ECM) conveys different biochemical, mechanical and structural cues to cells. These signals are highly orchestrated in space and time. Precise instructions from the ECM dictate cell fate processes, such as proliferation, differentiation and migration. The ECM exerts mechanical forces on cells, which are sensed by cells through different mechanisms, and are translated into biological outcomes. However, these mechanisms are not well understood. One of the main limitations in deciphering this language of forces on cells has been the lack of in vitro systems, which can generate forces on cells that mimic natural stresses.
The present-day methods, which attempt to apply such forces on cells, include single cell manipulation techniques that are highly invasive and although very cell-selective, do not mimic natural stresses. Other techniques rely on the use of flexible elastomers, which better replicate natural stretches, but do not provide user-defined cell selectivity. Therefore, it remains a challenge to develop a system for manipulating cells with mechanical forces, which are precisely controlled in space and time domains.
Here we demonstrate a novel hydrogel system, which can reversibly apply precise, user-defined mechanical forces on selected cells in a cell population. Our approach comprises a smart ECM-mimic hydrogel system, which responds to a light trigger. This causes reversible local deformations of the cell growth substrate and leads to the generation of mechanical forces on cells. These forces are transient and can be controlled at a sub cellular and sub-population scale, in a wide range of time scales (up to ms), with pre-defined directionality.
Such a system for opto-mechanical stimulation of cells is an effective tool for investigating how repeated actuation of a soft hydrogel affects cells. This is experimentally demonstrated in a case study using fibroblast cells to show the proof-of-principle of the concept.
The dynamic hydrogel swelling/shrinking closely replicates the stretches experienced by soft tissues in the body during activities, such as movement, growth etc. We believe that this system bridges the gap between single cell manipulation techniques and cell sheet deformation techniques. This system shows great potential in fields of ‘mechano-diseases' and in understanding cell-ECM interactions.

Despite the progress tissue engineering has made in the development of improved biomaterials, inhibiting bacterial infection has not been a central focus to date. Infection is a leading cause of implant failures with many agencies (such as the Centers for Disease Control) predicting more deaths from bacteria than all cancers combined by 2050. Gram-negative bacteria are naturally resistant to numerous treatments and are difficult to kill due to their robust and hydrophobic outer lipopolysaccharide membrane which helps to prevent the flow of antibiotics or drugs into the cell. Moreover, due to extensive antibiotic use, gram-positive Staphylococcus aureus has evolved to a methicillin-resistant strain, which can overcome other classes of antibiotic treatments. The development of an implant capable of reducing bacterial growth (without resorting to the use of antibiotics which causes antibiotic resistant bacteria) would be an effective way to improve implant success. Recently, scientists have been investigating novel materials and techniques to meet growing orthopedic tissue engineering needs. The first step in implant infection is bacterial adhesion, which can potentially result in the formation of antibiotic resistant biofilms for some species. Bacterial adhesion, growth, and subsequent biofilm formation on surfaces are particularly resistant towards the body’s defense mechanisms and antibiotic treatments, which can cause implant rejection. Multiple substrate properties, including chemical composition, hydrophobicity, and surface roughness, are believed to be of significance in the bacterial attachment process. In this study, multiple titanium samples were etched with different concentrations of nitric acid (10N or 12N) for varying durations (60 or 90 minutes), followed by a consistent and extended heat treatment (400 for one hour) for all samples. As a comparison to these samples, which were modified through conventional acidic etching treatment, another group of titanium samples were prepared by coating with 25 nm of titanium dioxide at 200 for approximately 4 hours through an atomic layer deposition (ALD) technique. To assess the potential effect of both approaches on inhibiting bacterial adhesion, and thus conferring antibacterial properties, samples were cultured with Staphylococcus aureus and colony forming unit (CFU) assays were conducted. ALD treatment, in comparison to conventional acidic etching treatment, demonstrated reduced bacterial density. As such, ALD treatment may pose a promising way to inhibit the growth of infectious bacterial populations, on a vast variety of surfaces and materials, without the need for antibiotics.

Several diseases include cancer, skin diseases, inflammatory conditions, trauma and congenital deformalities cause ear and nose defects that require reconstuction. Due to the wide patient population that this affects, nose reconstruction creates a huge social and economic burden. Each year, 1/6000 children are born with a small or missing ear, a condition called microtia. This devastating facial disfigurement causes high physical, social and mental burden for both the child and parent. Current surgical reconstruction involves harvesting tissue from elsewhere in the body, to recreate the cartilage framework of the ear and the nose and then implanting the framework beneath the skin. These techniques cause pain, are limited by tissue availability, can fail and have potential wound-healing complications.
Several synthetic and biological materials have been considered for the reconstruction of the nose and ear but with high levels of infection, unnatural look and feel, they are not considered an acceptable alternative. Synthetic materials are promising candidates to provide the mechanicalproperties and support for the constructs. However, biological materials are useful as they have good biocompatibility and can support tissue formation, which synthetic materials often lack. The incorporation of patient's own cells within the construct can also enhance the biocompatibility of the implant material.
Additive three dimensional bio plotting, a modified form of Fused Deposition Modelling (FDM) has now allowed synthetic and biological biomaterials to be combined to create organ replacements. In addition, to being able to create more complex shapes which better mimic the native tissue, they can be manufactured specific to the patient. Bioplotting also allows the direct printing of cells with the material to create a biocompatible and functional implant.
The work described in this paper represents a new approach to ear and nose reconstruction using 3D-Bio-plotting. We have tested several combinations of biological hydrogels and synthetic polysaccharide composite materials to act as the replacement for the cartilage framework of the ear and the nose.
We describe the concepts being developed from 3D to 4D biofabrication using a high precision Bioplotter robot (Envisiontec) to ensure we are creating accurate patient focused auricular and nasal relacements. The bioplotter has proven capable of printing several materials sequentially in very precise locations with and without cells incorporated in the material. Now, in the second stage of our current programme, we are exploring suitable combinations of synthetic and biological material for nose and ear reconstruction, printing of the patient's own cells within the biological component of the material will be optimised. The ability of the cells to survive, grow and support tissue formation is driven by novel laminar( low shear) flow mixing to ensure maximum stem cell survival.

Neural tissue engineering has emerged as a potential technology to cure neural damages. Although various synthetic polymers with good biocompatibility and biodegradability are adopted as candidate materials for scaffolds, most of them require incorporation of biomolecules or conductive materials to promote the growth of long axon. Here we propose a peptide-based polyelectrolyte which is conductive and contains neurotransmitter of glutamic acid. The designed copolymer of poly(γ-benzyl-L-glutamate) and poly(L-glutamic acid) sodium salt (PBGA^-Na⁺) is electrospun into 3D scaffold with aligned fibers. Neuron-like rat phaeochromocytoma (PC12) cells are cultured on the scaffolds to evaluate cell proliferation and differentiation. The results show with both electrical and biochemical cues, the polyelectrolyte PBGA^-Na⁺ gives longer axon outgrowth and higher differentiation ratio compared with the neutral copolymer of poly(γ-benzyl-L-glutamate) and poly(L-glutamic acid) (PBGA).

In this study, Zoledronic acid (ZOL), a type of nitrogen containing bisphosphonate, was loaded on graphene oxide (GO) particles to increase the particle size of the drug-nano-carrier complex which reduces drug filtration by the kidney and consequently, increases drug circulation time and its tumor uptake. The conjugation between ZOL and GO occurs via π-π stacking and hydrogen bonding interactions, and therefore, the drug may be gradually released from GO in physiological conditions which eliminates the need to apply high doses of the drug. Loading and release profile of ZOL on GO particles was investigated by using UV-Vis spectroscopy. Samples with different concentrations of 0.025-1.25 mg/ml of ZOL were loaded on 0.2 mg/ml GO. UV analysis showed that the maximum loading happens at ZOL to GO ratio of 1:0.2. This loading was obtained when 1 mg/ml of ZOL was initially loaded on 0.2 mg/ml of GO nanoparticles. The drug and drug carrier complexes were characterized using FTIR, AFM, and UV-vis spectroscopy. Cell culture studies were carried out with MCF-7 breast cancer cells for three dosages of ZOL, ZOL-GO and GO. Cell migration was assessed using Bio-Coat cell migration chambers and cell proliferation was investigated by alamarBlue assay. Cell viability was evaluated by staining dead cells with propidium iodide (PI) and live cells with acridine orange (AO). Overall, the characterization results confirm loading of ZOL on GO nanoparticles and cell studies results show that GO conjugated ZOL complexes are promising to reduce MCF-7 breast cancer cells migration, proliferation and viability.

Bioresorbable bone adhesives have potential to revolutionize the clinical treatment of the human skeletal system, ranging from the fixation and osseointegration of permanent implants to the direct healing and fusion of bones without permanent fixation hardware.[1] With sufficient strength bioresorbable bone adhesives could ultimately become an ideal means for fixing bone fractures instead of conventional plates, nails, pins and screws used today.[2] Despite the evident clinical need, there are currently no bioresorbable bone adhesives in clinical use that can form a bond to bone in a wet environment strong enough to bear clinical loads and sustainable enough to allow fracture healing.[1]
Inspired by the sandcastle worm that creates a protective tubular shell around its body by gluing together sand grains and shell fragments underwater using a proteinaceous adhesive, we introduce a novel mineral-organic bone adhesive (aka Tetranite®) that cures in minutes in an aqueous environment and provides high bone-to-bone adhesive strength. The new bioresorbable material is measured to be more adhesive than both bioresorbable calcium phosphate and poly(methyl methacrylate) bone cements, which are standards of care in the clinic today. Osteointegration and bioresorbability of the bone adhesive are demonstrated over a 52-week period in a critically-sized distal femur defect in rabbits. Based on its unique capabilities, Tetranite is the first in a new class of biomaterials, which may spark innovative clinical treatments and revolutionize procedures in which bone regeneration or fixation is critical for treatment.

[1] Farrar, D. F. Bone adhesives for trauma surgery: A review of challenges and developments. International Journal of Adhesion and Adhesives 2012, 33, 89-97.
[2] Heiss, C.; Kraus, R.; Schluckebier, D.; Stiller, A.-C.; Wenisch, S.; Schnettler, R. Bone Adhesives in Trauma and Orthopedic Surgery. European Journal of Trauma 2006, 32, 141-148.
[3] Kirillova, A.; Kelly, C.; Von Windheim, N.; Gall, K. Bioinspired Mineral-Organic Bioresorbable Bone Adhesive. Advanced Healthcare Materials 2018, DOI: 10.1002/adhm.201800467.

In recent years, surfaces with nano/microscale topography have been widely used to control stem cell behaviour. These patterned substrates possess fascinating qualities that render them more valuable than conventional flat surfaces in many bio-applications, such as neuronal differentiation, biosensing, tissue engineering and DNA analysis. Among these, nanopillar and nanoneedle structures have been extensively investigated because they are beneficial in terms of increasing enhancing cell adhesion and growth, and ability to penetrate cells for facilitating drug/biomolecules delivery.
We have co-developed high-aspect ratio, porous silicon nanoneedles made by electrochemical wet etching for in vitro and in vivo manipulation of cell behaviour^1,2. These structures are highly biocompatible and can be used to directly interact with the cell membrane, cytoskeleton, and nucleus of primary human cells, generating distinct responses at each of these cellular compartments³.
Moreover, we have recently developed a new generation of non-porous nanoneedles using a deep reactive ion etching process. Our new system provides high chemical stability in cell culture media, making it suitable for the long-term investigation of stem cell fates and differentiation at a nanoneedle interface. Furthermore, by systematically tuning the sharpness of the nanoneedles, we could precisely probe their effect on cellular mechanotransduction. The structural effect on cell morphology, alignment, and gene-level expression was observed with scanning electron microscopy, immunofluorescence, and real-time polymerase chain reaction. Our findings provide an ideal framework for manipulating and exploiting stem cell behaviour for longer periods, as a means for understanding cell-material interfaces and differentiation capacity of stem cells. Moreover, we used focused ion beam scanning electron microscopy to determine the critical sharpness required to achieve close interaction between the surface and the cell membrane. We expect elucidating the interfaces between nanoneedles and cells to enable new applications in bioengineering, especially in the sensing and monitoring of live cell cultures via 3D-structured electronic devices.

¹ C. Chiappini et al., ACS Nano 2015, 9, 5500-5509
² C. Chiappini et al., Nat. Mater. 2015, 14, 532-539
³ C. S. Hansel et al., 2018, In revision

Early detection of cancer plays an important role in successful treatment. Therefore, there is an urgent need for more effective and less toxic biomarkers to detect cancer. Cancer cells use exosomes to survive and metastasize to other tissues. Exosomes are small vesicles released to blood by cells, and they can deliver proteomic and genetic information unique to each cell. Therefore, isolating the exosomes secreted by cancer cells can provide us valuable information about the state of a tumor. Since every cell in the body releases exosomes, separating those coming from cancer cells can be a cumbersome task. Current techniques to isolate exosomes are time consuming and costly. Hence, our aim in this study is to use magnetic nanowires (MNWs) to magnetically isolate cancer cells’ exosomes in an efficient way through a simple blood biopsy and a magnetic stand.

In this work, we have used Fe/Au segmented MNWs to separate exosomes released by osteosarcoma cancer cells. These MNWs have been functionalized with PEG, and their concentration has been optimized in order to improve their capture and internalization by the cancer cells. We have observed by TEM that most of the MNWs end up inside the lysosomes in cancer cells. Once inside the cells, these MNWs tend to be broken into smaller pieces that can be released inside the exosomes. This way, by using a magnetic stand, we can easily and efficiently separate only the cancer cells’ exosomes, since they contain segments of magnetic nanowires.

In addition, our Fe/Au segmented MNWs can also be used as customized radio frequency identification (cRFID) labels. MNWs have magnetic (Fe) and nonmagnetic (Au) segments which resemble barcodes, and their structure can be engineered (e.g., by changing the length of each segment) to produce different cRFID signatures. Hence, distinct nanowires can be attached to different types of cancer cells in order to distinguish between the exosomes derived from each type, thereby further improving the efficacy of our blood biopsies.

Electrospun fibrous scaffolds have shown great promise in promoting axon regeneration to improve repair of peripheral nerve defect. Especially, when the fibers are collected as uniaxially aligned arrays, the growth of neurites can be guided and accelerated. Despite the progress, it remains a challenge to place temporally and spatially controlled delivery of biological effectors such as growth factors from the electrospun fibrous scaffold. Such a requirement can be met by integrating electrospun fibers with a controlled release system based upon a stimuli-responsible material. We have developed a temperature-regulated system for the on-demand release of nerve growth factor to promote neurite outgrowth. The system was based upon microparticles fabricated using co-axial electrospray, with the outer solution comprised of a phase-change material (PCM) and the inner solution containing the payloads. When the temperature was kept below the melting point of the PCM, there was no release due to the extremely slow diffusion through a solid matrix. Upon increasing the temperature to slightly pass the melting point, the encapsulated payloads could be readily released from the melted PCM. By leveraging the reversibility of phase transition, the payloads were released in a pulsatile mode through on/off heating cycles. When the PCM microparticles (co-loaded with nerve growth factor and a near-infrared dye) were sandwiched between two layers of electrospun fibers, the nerve growth factor could be released on-demand upon photothermal heating with a near-infrared laser. The nerve growth factor was released with well-preserved bioactivity and stimulated the extension of neurites from spheroids of PC12 cells. By choosing different combinations of PCM, biofactor, and scaffolding material, this controlled release system can be applied to a wide variety of biomedical applications.

Oral ulcerative lesions are a painful side effect of cancer chemo and radiation therapy. The current clinical management of this condition requires multiple classes of drugs administered through topical formulations. However, these formulations need frequent dosing for optimal therapeutic effect which is inconvenient and leads to patient non- compliance. In this study, we prepare wheat germ agglutinin conjugated liposomes (WGA- liposomes) as a bioadhesive nanocarrier that can potentially encapsulate multiple classes of drugs, show fast binding to oral cells and localized sustained drug delivery. Fluorescence studies demonstrated that WGA- liposomes can rapidly bind to cells (within 1 min) and have a significantly higher binding (p<0.05) compared to the original liposomes. Studies with model drug amoxicillin encapsulated WGA- liposomes revealed sustained in vitro drug release over several days and potent antimicrobial activity against Streptococcus mutans in an oral cell- bacteria co- culture system. Fluorescence studies on liposome release showed that the WGA- liposomes stayed in oral cells for 48h after which it was cleared from cells. A significant reduction in oral cell damage in bacteria pre- infected oral cells after treatment with amoxicillin encapsulated WGA- liposomes compared to the untreated cells was observed through cell viability studies. Cell viability studies also showed that oral cells are not significantly damaged in the presence of WGA- liposomes indicating a potentially biocompatible nanocarrier. These results point to the great potential of WGA- liposomes as a drug delivery vehicle to effectively treat oral ulcerative lesions with reduced dosing frequency.

Reprogramming cellular functions through the design, fabrication and use of engineered platforms that mimic the physiological cellular environment is a major goal of cell engineering.
Indeed, it has been widely demonstrated that the use of 3D systems, compared to 2D, is crucial for a more physiological relevant study of cellular systems, since the third dimension could differently and strongly affect diverse cell functions¹. However, the precise engineering of 3D systems often results challenging, consequently limiting the control over cellular fate.
Here, we report the fabrication of 3D instructive platforms that modulate cellular behaviour in terms of cellular polarization, membrane curvature and uptake capability. By means of two-photon polymerization (2PP) technology, we processed a commercial biocompatible photoresist for fabricating a cage-like 3D structure capable to entrap cells. We then investigated the cell-material interaction and the effect of different micro-topographies (grooves) on cellular response. To evaluate the effect of such topographies on cellular membrane curvature, we took advantage of the SEM/FIB technology and ultra-thin plasticization (UTP) of cells, which gives the opportunity to directly observe cell-material sections with nanometric resolution ². We thus gathered important informations on the relations between membrane curvature and caveolae formation, known to be fundamental in endocytosis processes³. Moreover, by functionalizing our structures with fluorescent nanoparticles (NPs) we were able to observe how different topographies modulate the cellular uptake by evaluating NPs internalization with confocal microscopy.
Our results clearly show that by modulating cellular membrane curvature through specific topographical micro-features, it is possible to tune cellular membrane curvature and, thus, the cellular uptake capabilities. Such results could then give new guidelines for the design of innovative and more efficient delivery systems based on 3D scaffold-like devices.

1. Pampaloni, F., Reynaud, E. G. & Stelzer, E. H. K. DOI: 10.1038/nrm2236 NAT REV CELL BIO (2007).
2. Santoro, F. et al. DOI: 10.1021/acsnano.7b03494 ACS NANO (2017).
3. Zhao, W. et al. doi: 10.1038/nnano.2017.98. NAT NANOTECHNOL (2017).

Synthetic hydrogels, such as those formed with multifunctional poly(ethylene glycol) (PEG) or poly(vinyl alcohol) (PVA) macromers, are of great interest for a variety of biological applications. The high degree of property control that these materials afford, including mimicking the elasticity or ‘stiffness’ of tissues in the human body, makes them particularly useful as biomimetic, multidimensional culture environments for hypothesis testing in studies of disease and regeneration. Traditionally, control of synthetic hydrogel mechanical properties has been achieved with polymer concentration, molecular weight, or reactive group stoichiometry. Recently, the rate of hydrogel formation also was demonstrated as an effective handle for controlling mechanical properties. For example, the rate of hydrogel formation by oxime chemistry was controlled using pH, where the resulting differences in mechanical properties were determined to arise from differences in network heterogeneity (e.g., defects) that depended on the rate of gelation (Zander et al, Advanced Materials, 2015). Inspired by this, in this work we investigated if a rate-based approach for controlling mechanical properties could be used with photoinitiated (lithium acylphosphinate, LAP) synthetic thiol-ene hydrogels (PEG-8-norbornene, PEG-2-thiol) and the resulting defects exploited for temporal property modulation. Specifically, we established a system for hydrogel formation using different doses of visible light (455nm LED, 70-90 mW/cm², 1-10 min) to tune the mechanical properties. We confirmed dependence of hydrogel mechanical properties on factors beyond polymer concentration, including light intensity and exposure time. Elasticity, measured by dynamic mechanical analysis (DMA), indicated that, for precursor solution of the same composition, elasticity increased with both increasing light intensity and exposure time. To better understand the source of defects contributing to differences in hydrogel mechanical properties, end group conversion during hydrogel formation was monitored with magic angle spinning (MAS-NMR) and correlated with mechanical properties over the polymerization time. Through these comparisons, both reduced end group conversion and looping were determined to contribute to differences in mechanical properties observed at different rates of hydrogel formation. Control of end group conversion subsequently was exploited to stiffen hydrogels post-polymerization by covalent incorporation of a secondary thiol-ene network using photopolymerization (365nm, 10 mW/cm²). In sum, we have demonstrated the high level of property control afforded by this visible light polymerization system and the potential utility of this approach for post-polymerization modulation of material properties. This method of modulating properties is promising for studying cell response to dynamic stiffness in three-dimensional culture, with applications in the study of cancer progression and wound healing.

In-situ monitoring of pH is of great importance in biomedical fields as pH affects activities of enzyme and drug and is a symptom of certain diseases. It is known that the microenvironment of cancer cells is weakly acidic due to the secretion of lactic acid through anaerobic respiration. Therefore, pH can be an effective indicator for cancers. However, it is very difficult to use conventional litmus papers or pH-meters for measurement of local pH in cellular environments. To provide an injectable, implantable, suspendable platform of pH sensors, we suggest a microcapsule-type sensor that is composed of the pH-responsive optical sensor in the core and semipermeable polymer in the shell. As a template to produce microcapsules, monodisperse water-in-oil-in-water (W/O/W) double-emulsion droplets are prepared using a capillary microfluidic device. The innermost water phase contains molybdenum disulfide (MoS₂) nanosheets whose surfaces are grafted by pH-responsive polymers with a fluorescent group at the distal end. As the middle oil phase, a photocurable resin of polysiloxanes modified with methacrylate is used. The double-emulsion drops are irradiated by ultraviolet, which leads to the polymerization of the resin, forming a semipermeable solid shell. The pH-responsive polymer that links the MoS₂ nanosheets and fluorescent groups are designed to show a drastic conformation change in the range of pH 6.0-7.4. At physiological condition of pH 7.4, the pH-responsive polymer is collapsed so that the fluorescent groups are brought to the optical quencher of MoS₂, yielding a weak fluorescence due to the Forster resonance energy transfer (FRET). By contrast, at cancer microenvironment with pH 6.3, the pH-responsive polymer is highly extended, increasing fluorescent intensity. As the pH sensors are encapsulated by a semipermeable shell, they are free from dilution with physiological fluids and adhesion of proteins and lipids, thereby maintaining the sensing performance in a physiological environment. The microcapsule sensors can be injected, implanted, and suspended in any target volumes, which enables the in-situ monitoring of pH in the microenvironment where the microcapsules are located.

The replacement of bone tissue in surgical interventions with artificial materials is a standard procedure in current clinical practice, however, it is a substantial surgery with high patient morbidity and high healthcare costs. It is therefore desirable to develop artificial bone materials which induce controlled, guided and rapid healing to improve patient recuperation and which allow for a stable fixation between bone and implant for immediate loading.

Interest in piezoelectric ceramics for biomedical applications has risen in recent years, due to the need for biocompatible materials with active functionalities. Several studies have proposed that osteogenic regeneration may be improved with the application of electrical stimuli. Barium titanate doped with calcium and zirconium, (Ba,Ca)(Zr,Ti)O₃, are a class of lead-free piezoelectric ceramics which generate electric surface potentials under a mechanical load due to its non-centrosymmetric crystal structure. This class of piezoelectric ceramics may serve as bioactive bone replacement materials. However, the biocompatibility of BCZT is not well established.

In the present study, we investigate the suitability of BCZT as an artificial bone replacement material. Several compositions of bulk BCZT ceramics were synthesised via solid-state synthesis, including a morphotropic phase boundary composition (MPB) and several other tetragonal compositions. The MPB composition was chosen for its expected high piezoelectric performance, and the tetragonal compositions for the different stabilities in piezoelectric performance upon mechanical loading. Piezoelectric properties were determined for all compositions. The biocompatibility of the BCZT ceramics was investigated via cell proliferation and viability studies. We determined the BCZT ceramics to be compatible with human osteoblast cells and endothelial cells, thus giving encouragement into the further study of BCZT as bioactive bone replacement materials.

I. Objectives
Elastin-like polypeptide (ELP) has a variety of application in biomedical field. ELPs are composed of repeats of the pentapeptide Val-Pro-Gly-Xaa-Gly; the guest residue Xaa can be any amino acid except Pro. We have tried to improve the mechanical property of dental cement by ELP supplementation in previous studies. Specially, we found the supplementation of ELP increased the adhesion ability of composite cement to tooth. Here, the objective of this study was to investigate the adhesion effect of ELP supplementation on the tooth between the ELP-supplemented dental cement and tooth surface.

II. Materials & Methods
ELP genes either with or without octaglutamic acid termination were genetically engineered: V125 and V125-E8. Pure ELPs were gathered through a series of protein synthesis process using E.coli through gene transformation, expression, protein purification. 10 wt% ELP solutions were then prepared. The crown of human third molar without caries and restorations was horizontally sectioned to have 2mm thickness by high-speed sawing machine (Buehler). Two holes in tooth specimen were made 4.1 mm in height and 1.4 mm in diameter.
0.3 ratio in liquid/powder were prepared: cement + 60mL of either DW, V125, or V125-E8. Mixed cements as given ratio, were loaded at tooth cavities (20 cavities x 3 subgroup) and set in 37°C incubator for 7 days under 100% humidity. Push-out strength of samples were measured in universal testing machine (SHIMADZU, Japan) at a cross-head speed of 1mm/min with compressive mode. Data were analyzed using Two-way ANOVA and Bonferroni’s post-hoc test at 95% significance level.
0.3 ratio in liquid/poweder were prepared same as push-out strength test. The composite cement was loaded in rheometer (Ta instrument Co., DE) and the viscoelastic property was measured for 1 hour.
Tooth disk of 2 mm in thickness was prepared and the 1 mm cavity was prepared. Same liquid/powder ratio of composite cement was prepared and filled in the cavity. The sample was stored in 100 % humidity for 2 days and in PBS solution for 1 -2 weeks more. Vertical section through half was performed with diamond saw and polished serially from 500 grit to 2400 grit. Dried sample was gold-coated and observed by SEM (Hitachi, Tokyo, Japan).

III. Results
V125-E8 group showed the highest push out strength significantly (p<0.001). V125 group showed lower strength than V125-E8 group, but higher push-out strength than DW group (p<0.001). V125-E8 groups showed significantly less viscosity and high flow compared to other groups. V125-E8 group showed a narrower gap between composite cement and dentin and composite tag in dentinal tubule while other group showed wider gap and no tag inserted in dentinal tubule.

IV. Conclusion
ELP-supplemented dental cement has higher push-out strength than DW-mixed MTA. V125-E8 showed the highest adhesion ability to tooth. The increased adhesion ability might be due to rheologic property through low viscosity and high flowability.

Type I collagen forms fibrous viscoelastic networks that comprise a dominant fraction of the extracellular matrix (ECM). Through interactions with single cells and tissues, networks of type I collagen can be remodeled to form anisotropic networks, which are observed in biological processes as diverse as branching morphogenesis and metastatic invasion. However, replicating the structure of anisotropic collagen networks ex vivo is challenging because current collagen fiber alignment techniques are often limited to unidirectional alignment over small mm-scale areas or in thin films. Here, we adapt three-dimensional (3D) microextrusion printing as a technique to fabricate anisotropic networks of type I collagen with tunable fiber alignment and geometry. Using collagen-Matrigel and collagen-Ficoll inks, we 3D-printed collagen fiber networks with 30-40% of the fibers oriented in the printing direction. Collagen fiber alignment increased with increasing printing speed and decreasing nozzle diameter, which suggests that shear and extensional flows in the conical nozzle are responsible for collagen alignment. Molecular crowding and substratum chemistry also affected the extent of collagen fiber alignment. Changing the concentration of Matrigel in the collagen-Matrigel ink allowed the geometry of collagen fibers to be tuned. By modifying the aforementioned parameters, we 3D-printed complex patterns of collagen fiber alignment and geometry that were separated by sharp interfaces. 3D-printed networks of type I collagen have great potential for studies that assess the role of collagen fiber alignment in developmental biology and tissue engineering.

Natural antimicrobial peptides are crucial components of the host in