Symposium SM05 : Biomaterials for Tissue Interface Regeneration

In the quest for the next generation of functional biomaterials, researchers have sought inspiration from nature by developing better performing bio-derived materials (e.g. collagen, keratin, chitosan), reproducing naturally occurring micro and nanostructures (e.g. gecko feet fibrils, nanoporosity of collagen-apatite interfaces in bone) and devising strategies that mimic naturally occurring phenomena (e.g. mussel attachment). In this context, our team has joined these efforts to employ bio-inspired structures and approaches in biomaterials research, aiming at creating platforms and interface to understand and control cellular events. In particular, we successfully reproduced a bioactive nanoporosity on titanium, the gold standard in medicine, by simple chemical (i.e. oxidative nanopatterning) and electrochemical (i.e. anodization) methods, capable of positively affecting cell activity. Noteworthily, anodization permitted not only to create semiordered nanotubular surfaces which can be tuned in terms of diameter and spacing, but also a nanometric 3-dimensional hierarchical surface that mimics the silicified cell wall (frustule) of diatoms. This ultimately demonstrates that a simple anodization process can create complex periodic structures which, to date, have only been made by more complex techniques (e.g. two photon lithography, atomic layer deposition). These surfaces were exploited to close in on the mechanisms that control how human mesenchymal stem cells respond to nanotopographical surfaces, a fundamental aspect in expanding the present knowledge of cell-surface phenomena. In particular, our team focuses on the correlation between the geometrical arrangement of nanoscale features to specific cellular functions, and on the evaluation the effects of a vertical nanotopographical gradient by exploiting such bioinspired surface. Moreover, we are currently working on biologically inspired adhesive interfaces because of their potentially beneficial applications in medicine, technology and industry not only for their capacity to act as intermediate linkers to immobilize bioactive agents onto surfaces, but also for their ability to direct influence cell behavior. In particular, we focused on understanding the effects on cells of poly(dopamine), an adhesive polymer derived from mussels, as a multifunctional layer for supporting the activity of osteoblastic and human mesenchymal stem cells. In parallel, our team has also contributed to the development of bioinspired techniques and materials for tissue engineering applications.

Transdermal drug delivery is a very popular and convenient approach for various therapies such as pain management, pregnancy prevention, smoking cessation, and immunotherapy. However, not all of pharmaceuticals can successfully pass through the skin. The stratum corneum on the surface of skin acts as the forefront of the defense mechanism. It prevents our body from the environmental substance like gastrointestinal tract and lung with the permeable epithelia regulating what enters the body. Therefore, how to enhance the efficiency of transdermal delivery system including breaking the skin barriers without serious damage is a great challenge. Microneedle is a good solution which can reach a minimal invasive and dramatically increase skin permeability. Such technologies will cause reversible microchannels in the skin.
Here, we propose 3D printing technologies to fabricate the mold of microneedles, which facilitate customization and overcome the geometries from individual differences. Silk protein and polyvinyl alcohol (PVA) are used as the materials of matrix for high mechanical strength and rapid dissolvable abilities. Microneedles also equip with mesoporous superparamagnetic iron oxide nanoparticles (SPIONs) in the tip of microneedle patch for the drug delivery system. After the insertion of the patch to the skin, the supports dissolve and leave SPIONs leading sustain drug release. Furthermore, magnetic nanoparticles are able to be triggered under high frequency magnetic field (HFMF). In this way, the drugs will release on demand and simultaneously induce the thermotherapy.

Gastrointestinal (GI) tract perforations are relatively frequent surgical emergencies, are potentially life-threatening, and can occur from several different sources. In general, treatment requires urgent surgical repair or resection and at times can lead to further complications. Currently available stents are non-absorbable, are manufactured in a narrow size range, and/or are limited to usage in locations that are accessible for endoscopic removal post-healing. The use of 3D-printed bioresorbable polymeric stents will provide patients with a stent that is tailored specifically to their geometry, will degrade with time to eliminate the need for further surgeries for stent removal post-healing, and will be usable in locations that are not endoscopically accessible. This project focuses on the characterization of polycaprolactone-polydioxanone (PCL-PDO) composites for use in a bioresorbable gastrointestinal stent. Dynamic Mechanical Analysis (DMA) tests were conducted to separately analyze the effects of composition, the filament formation process, and physiological temperature on the PCL-PDO material properties. The proposed stent design was then modelled using computer-aided design, and Finite Element Analysis (FEA) was used to simulate the effects of physiologically relevant forces on stent integrity. In-vitro studies were used to evaluate the biocompatibility of the polymer composite. PCL-PDO stents were then 3D-printed and placed in-vivo in a pig model, and histological evaluation was used to determine the safety of these stents.

Magnesium (Mg) and its alloys have showed a promising potential for medical implant applications due to their attractive biocompatibility and mechanical strength properties. Despite these promising properties, the critical challenge with Mg-based implants is rapid degradation in physiological environment that results in early loss of mechanical strength and hydrogen gas accumulation at the local site. Hydroxyapatite (HA) coatings provide a sound solution for controlling Mg degradation at the bone interface. In this paper, HA coatings with different particle sizes, namely, microHA (mHA) and nanoHA (nHA), were deposited on Mg discs and rods with two different pressures using N2 Biomedical’s proprietary deposition process called IonTite^TM. The degradation property of HA coated Mg prepared by IonTite^TM was studied in revised simulated body fluid (rSBF) for six weeks. Both mHA and nHA coatings have showed reduction of degradation rates and maintained the mechanical integrity of Mg during the 6-week immersion study in rSBF. The mHA samples deposited at high pressure (mHA_400) showed a slower corrosion rate among all group of samples. The effects of geometry (disc versus rod) on degradation rate were also investigated and HA coated Mg rods showed a slower degradation rate compared to HA coated Mg discs. In this study, we have demonstrated that HA coated Mg substrates are promising materials as bioresorabable implants for orthopedic and craniofacial applications, and confirmed the optimal IonTite^TM process conditions that could produce HA coatings on Mg with superior degradation performance.

Mg–Zn–Ca ternary alloys have attracted increasing attention for biomedical implant applications, especially for bone repair because of its biocompatibility, biodegradability and similar mechanical properties to cortical bone. The objective of this study was to characterize Mg–2Zn–0.5Ca (ZC21) intramedullary pins, determine the degradation of ZC21 in vitro, and evaluate the cytocompatibility of ZC21 with bone marrow derived mesenchymal stem cells (BMSCs) in direct culture and direct exposure culture. Direct culture is suitable to evaluate direct cell attachment on the biomaterial surfaces. Direct exposure culture is desired for investigating the response of well-established cells in the body with newly implanted biomaterials. Surface microstructure and composition of ZC21 pins were characterized before and after BMSCs culture using scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS). After 24 hours culture, the ZC21 pins showed a similar degradation behavior compared to pure Mg pins in vitro. The BMSCs adhesion density on ZC21 pins (direct contact) was significantly higher than pure Mg pins in both culture methods. The cell adhesion density around ZC21 pins (indirect contact) was similar to the cell-only positive control in both culture methods. Therefore, ZC21 pins are considered to be cytocompatible with BMSCs. Future study will focus on the improvement of the degradation properties of ZC21 pins and the evaluation of their degradation and biological performance in vivo.

Three-dimensional porous scaffolds play a vital role in tissue engineering and regenerative medicine of functionalizing as biomimetic substrates to control cellular behavior. However, most techniques used to create scaffolds depend on stochastic processes which typically create scaffolds with coarse uncontrollable pores in terms of size, structure, and interconnectivity, dramatically restraining their usage in tissue regeneration. Microfluidic techniques have great potential to make inversed opal scaffolds, based on their ability to produce microbubbles or beads with a low polydispersity index (<5%). Inversed opal scaffolds are scaffolds with uniform pore size, architecture, pore structure, porosity, and interconnectivity.
In this work, we report a significant advancement for the preparation of monodispersed microbubbles, which are increasingly used and have become a key constituent in many advanced technologies. A new device comprising of two T-junctions containing coarse capillaries and operating in series was assembled. Microbubble generation was facilitated by using bovine serum albumin solution and nitrogen as the liquid and the gas phase, respectively. The effect of operating parameters such as gas pressure and liquid flow rate on
the size of the microbubbles generated were investigated for the two T-junction systems and the results were compared with a single T-junction process. The experimental results showed that microbubbles produced via the double T-junction setup were smaller at any given gas pressure for both liquid flow rates of 100 and 200 μm studied in this work. A predictive model is developed from the experimental data, and the number of T-junctions was incorporated into this scaling model. It was demonstrated that the diameter of the monodisperse microbubbles generated can be tailored using multiple T-junctions while the operating parameters such as gas pressure and liquid flow rates were kept constant. The stability of the microbubbles produced was also examined and indicated that microbubbles produced through the double T-junction were more stable. By using a coarse capillary of 200µm, the new device made by combining more than two T-junctions can bring the size of microbubbles down to a new limit; from 180µm to 20µm based on the same operating parameters as a single T-junction. This allows a wider range of pore sizes for scaffolds. In conclusion, the device developed in this work provides a simple straightforward one-step method to make monodispersed scaffolds in a different range of pore sizes, which particularly have great potential in the generation of inversed opal scaffolds.

Biodegradable materials hold great promise for next-generation implants that will replace current permanent non-degradable implants, such as orthopedic fixation devices, cardiovascular stents, etc. The principle is that the implants degrade harmlessly in the body over time as new tissues grow, which eliminates the need for secondary surgeries and associated costs. Recent research on biodegradable polymers and metals have demonstrated their potentials for clinical translations, but there are still major challenges yet to be addressed, e.g. (1) how to regulate their degradation rate to match tissue healing rate, and (2) how to control their bioactivity to promote healing functions of desirable cells while inhibiting infectious functions of bacteria. In this presentation, our recent progress on developing magnesium (Mg) alloys as the next-generation biodegradable implant materials, and creating new surface modification strategies and biodegradable composites to regulate their degradation rate, will be discussed. Specifically, in vitro degradation of Mg-Zn-Sr alloy based intramedullary pins, their cytocompatibility with bone marrow derived mesenchymal stem cells (BMSCs), and the in vivo degradation and associated host responses in rat tibiae will be reported.

For cardiovascular applications, and especially for the fabrication of stents, alloys such as AISI316L and Co-Cr based ones, are preferred for their mechanical properties, overall resistance to corrosion and acceptable biocompatibility. High ductility and toughness are in fact required when the stent is deployed in the damaged vessel. The release of Ni is a major concern for the cytocompatibility properties of the considered materials. A way to improve the surface properties, and create a surface which effectively can both withstand the material plastic deformation and act as a barrier for toxic ion release is the use of plasma treatments. This work copes with the characterization of oxygen plasma ion immersion implanted (PIII) L605; several parameters were investigated in this study, such as for example received dose (proportional to implantation time, t) and substrate bias (U_bias).
Co-20Cr-15W-10Ni square samples after implantation were studied by angle resolved X-ray photoelectron spectroscopy (AR-XPS) was used to assess the surface chemistry. Transmission electron microscopy (TEM) provided information related to the oxidized layer thickness, structure and interface. Glowing discharge optical emission spectroscopy (GD-OES) was used to evaluate the chemical composition of the investigated layer. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) were used respectively to analyze the surface morphology and roughness. The sample were also subjected to static immersion tests. The viability of human umbilical vein endothelial cells (HUVECs) and smooth muscle cells (HUASMCs) were evaluated by resazurin test.
The implantation process modified from a chemical point of view the sample surface, forming a mixed Co and Cr oxides. Other elements that were present in traces, such as for example Ni, were no more present after implantation. The relative amount of Co and Cr changed with depth, with Cr amount increasing with increasing depth. The thickness of the layer varied, from around 25 nm for the highest U_bias and implantation time t (respectively -10 kV and 120'), to around few nanometers for lower U_bias and t. The presence of oxide nanocrystals in an amorphous matrix was detected, for almost all the investigated conditions; the formation of an oxide layer increased the corrosion potential in modified Hanks' solution, compared to that one of the electropolished samples. Surface roughness analysis was performed on different scales; R_a was in the range of few nanometers, and it did not vary relevantly for the studied parameter ranges. Static contact angle showed an increase for the oxidized samples. Cytocompatibility studies, performed by means of direct contact tests, showed an increasing viability trend of endothelial cells for oxidized samples. PIII surface treatment, in the range of the studied parameter, proved to be an efficient way to increase the corrosion potential and modify alloy surface cytocompatibility.

Thrombosis constitute the main clinical problem when blood-interacting devices are implanted in the body. Coatings with thin polymer layers represent a recognized strategy to modulate interactions between the material surface and the blood environment. To provide the implant success, the coating should limit platelets adhesion and delay the clot formation. Scientists have focused in many strategies, using chemical and physical modifications, to improving polymers hemocompatibility. One possibility is the introduction of sulfate or sulfonate groups in chitosan chain. This natural polymer has attracted attention due to its potential to act as a biomaterial, to mimic the effects promoted by heparin, one of the most used anticoagulant. Sulfur-containing chitosan, shows the ability to reduce proteins adsorption, decrease thrombogenic properties and limit clot formation. In this context, we produced two types of chitosan membranes: one with chlorossulfonic acid, to form 2,N-3,6,O-sulfated chitosan, and other with 5-Formyl-2-furansulfonic acid sodium salt (FFSA), to obtain N-sulfonated chitosan. The membranes were characterized and its effects over proteins adsorption and platelet adhesion evaluated. ¹H-¹³C NMR and FT-IR analysis confirmed the N and O substitution of chitosan when treated with chlorossulfonic acid, while FTIR and XPS analysis evidenced the sulfonation by FFSA. The 2,N-3,6,O-sulfated chitosan showed decrease in the bovine serum albumin-BSA (36.8%) and fibrinogen (20%) adsorption, and in the platelet adhesion (93.7%), which was observed by SEM images. For N-sulfonated chitosan, a more pronounced adsorption rate was observed at pH 5.0 than at pH 7.4, and the adsorption equilibrium was achieved, in both cases, after approximately 20 min. The platelet adhesion was about 50% lower than that of native chitosan. Afterwards, stainless steel surfaces, commonly used for cardiovascular applications, were coated with sulfonated chitosan, by using dopamine and PEG as anchors, and the effect of these grafted surfaces on platelet adhesion and clot formation were investigated. Surface characterization techniques evidenced that the coating formation was successful, and the sulfonated chitosan grafted sample exhibited a higher roughness and hydrophilicity, if compared to native chitosan one. Moreover, sulfonated surface limited platelet activation and the process of clot formation, thus confirming its high biological performances in blood. In conclusion, this sulfonated-modified chitosan has potential to be used as blood-interacting material.

Stem cell reprogramming and therapies have shown tremendous significance and potential in the field of regenerative medicine and tissue engineering. Therefore, developing tools to control stem cell fate is an attractive area of research for replacing damaged and diseased cells and re-establishing functional recovery for tissue/organ repair. Transcription factors (TFs) are multi-domain proteins that play a critical role in orchestrating stem cell differentiation and direct conversion. Previously, our group developed a highly tunable and versatile platform, named NanoScript, which is designed to mimic functions of TFs to bind to specific portions of the genome and regulate gene expression in a way that does not involve viral delivery. We have demonstrated that the NanoScript platform can upregulate targeted endogenous genes in a nonviral manner. Furthermore, we modified and utilized the tunable property of NanoScript to mimic differentiation-specific factors and activate related genes to induce stem cell differentiation. Apart from gene activation, we successfully demonstrated neuronal differentiation from neural stem cells through Sox-9 repression using NanoScript designed with Sox-9 specific polyamide and a repressor peptide.
Although there are various applications of NanoScript demonstrated by our group in stem cell differentiation and regenerative medicine, the lack of degradability hinders NanoScript from moving into in vivo applications and translational researches. As a result, we have designed a polymer micelle system based on polypeptides to renovate and improve the previous version of NanoScript. By carefully design and modulate the polypeptide polymer micelle structure, this newer version of NanoScript possesses small molecule, oligo-nucleotide and metal ion loading capacity. As a first demonstration, this new generation of polypeptide-based NanoScript was functionalized and designed to regulate skeletal muscle cell differentiation from adipose-derived mesenchymal stem cells (ADSCs) by targeting myogenic regulatory factors (MRFs), which play an important role in inducing myogenesis and skeletal muscle lineage commitment. This polypeptide-based NanoScript is stable in physiological environments, showing high nuclear localization rate, as well as inducing differentiation of ADSCs into mature myocytes in 1 week. As such, this peptide-based NanoScript can be a safe and powerful gene modulation tool for various applications in stem cell therapy and tissue regeneration.
References:
[1] Patel, S.; Jung, D.; Yin, P.T.; Carlton, P.; Yamamoto, M.; Bando, T.; Sugiyama, H.; Lee, K-.B.^†, “NanoScript: A Nanoparticle-Based Artificial Transcription Factor for Effective Gene Regulation”, ACS Nano, 2014, 9, 8959-8967
[2] Dardir, K.; Rathnam, C; Lee, K.-B.^†, “NanoScript: A Versatile Nanoparticle-Based Synthetic Transcription Factor for Innovative Gene Manipulation”, Biomedical Nanotechnology. Methods in Molecular Biology, 2017, 1570, 239-249

Organoids, organ-mimicking multicellular structures derived from pluripotent stem cells or organ progenitors, have recently emerged as an important system for both studies of stem cell biology and development of potential therapeutics; however, a large-scale culture of organoids and cryopreservation for whole organoids, a prerequisite for their industrial and clinical applications (such as, regenerative medicine), has remained a challenge. Current organoid culture systems relying on embedding the stem or progenitor cells in bulk extracellular matrix (ECM) hydrogels (e.g., Matrigel™) have limited surface area for mass transfer and are not suitable for large-scale productions. Here, we demonstrate a hydrogel capsule-based, scalable organoid production, and cryopreservation platform. The capsules have a core-shell structure where the core consists of Matrigel™ that supports the growth of organoids, and the alginate shell forms robust spherical capsules, enabling suspension culture in stirred bioreactors. Compared with conventional, bulk ECM hydrogels, the capsules, which could be produced continuously by a two-fluidic electrostatic co-spraying method, provide better mass transfer through both diffusion and convection. The core-shell structure of the capsules also leads to better cell recovery after cryopreservation of organoids probably through prevention of intracellular ice formation.

Osteoarthritis (OA) is a pathological condition that affects millions of people around the world, including approximately 30 million people in the US. OA is characterized by the deterioration of articular cartilage, preventing normal motion of the joint, which causes stiffness and pain. There are many promising therapeutic candidates for treating OA, however clinical outcomes are restricted by rapid clearance of the injected drug from the synovial fluid. Accordingly, the sustained and controlled release of therapeutics to the affected joints has become a major goal. The aim of this project is to develop an intra-articular injectable drug delivery system that promotes a sustained, triggered release of the OA therapeutic. The drug delivery system we developed is based on a core-shell structure where a polydopamine shell is assembled around a calcium carbonate template. This particulate system has several advantages for delivering OA therapeutics: 1) microscale dimensions enables improved retention in the synovial fluid without restricting movement (compared to smaller or larger delivery systems), 2) the polydopamine-based surface promotes adhesion (and hence retention) to the target tissue upon local administration, and 3) high drug loading capacity. The particles were synthesized and characterized using electron microscopy, flow cytometry and microelectrophoresis for zeta potential determination. The release profile of corticosteroid from the drug-loaded particles showed a prolonged release over a period of weeks. The adhesive properties of the polydopamine shells were tested by incubating the particles with cartilage explants: confocal fluorescence microscopy revealed the binding capacity of the particles to the cartilage surface. In conclusion, we demonstrate the assembly of a polydopamine-based drug delivery system which can deliver sustained quantities of corticosteroids in a spatio-temporal fashion. Ongoing and future work includes atomic force microscopy to further quantify the particle-cartilage interaction, co-culturing of particles with synovial cells (e.g. mesenchymal stem cells and synovial macrophages) to study their cytotoxicity and the effect of long-lasting drug release in vitro and in vivo work using osteoarthritic mouse model.

In recent years three dimensional cell culture systems have witnessed rapid expansion in the fields of tissue engineering and drug testing due to their ability to reproduce with more accuracy in-vivo cell microenvironments¹. Among the available models, the use of scaffold-free techniques for the formation of 3D cell constructs like spheroids or organoids has greatly advanced. Thanks to the ability of cells to secrete their own extracellular matrix and to self-organize into stable structure, organoids are now widely used to mimic several tissues such as cardiac, liver and neural tissue. Despite the push towards the use of spheroids for high-throughput toxicology and drug discovery assays, there is a definite gap in terms of available technologies for on-line monitoring of spheroids. Here we show the development of a sensing platform for the dynamic impedance monitoring of micro-tissue spheroids. Progressing from previous work where we coupled an organic electrochemical transistor (OECT) with spheroids trapped in a microcapillary², this couples the transistor with a PDMS microfluidic trap in order to assess spheroid integrity over-time. We propose a new fabrication strategy for both the OECT and the circular-shape microtrap to achieve the optimal performance for our organic impedance sensor. We have tested the validity of our device by sensing both mono- and co-culture spheroids, demonstrating the ability of the platform to discriminate spheroids based on the difference in cell number and cell type. Dynamic toxicological assays were also performed using the porogenic agent Triton X-100. Among techniques used for spheroids characterization the impedance-based system present the advantage to dynamically detect morphological changes in a label-free and minimally invasive manner.
References:
1. Edmondson, R., Broglie, J. J., Adcock, A. F. & Yang, L. Three-Dimensional Cell Culture Systems and Their Applications in Drug Discovery and Cell-Based Biosensors. ASSAY Drug Dev. Technol. 12, 207–218 (2014).
2. Huerta, M., Rivnay, J., Ramuz, M., Hama, A. & Owens, R. M. Research Update: Electrical monitoring of cysts using organic electrochemical transistors a. APL Mater. 3, 030701 (2015).

Nanostructuring materials allows to optimize their properties, by exploiting size effects. We created nanopatterns that act as surface cues [2,3], affecting cell behavior. Chemical oxidation creates nanoscale topographies [4], that allow to improve biocompatibility. Our treatment provides a differential signal, selectively inhibits fibroblast proliferation while promoting osteoblast growth [5] in vitro. Enhancing mechano-biocompatibility may occur by coating with spider silk [6-8]. Improving antibacterial properties using laser/plasma strategies and by growing graphene oxide coatings will also be discussed [9-13]. Finally, sensing and therapeutic approaches can be harnessed by exploiting emerging optical properties of nanocrystals such as Quantum Dots and upconverting nanoparticles [14-17].
[1] F. Variola et al., Small 5, 996 (2009)
[2] F. Variola et al., Biomaterials 29, 1285 (2008)
[3] F. Vetrone et al., Nanolett 9, 659 (2009)
[4] L. Richert et al., Adv Mater 20, 1488 (2008)
[5] C. Brown et al., Nanoscale 3, 3805 (2011)
[6] C. Brown et al., Nanoscale 3, 870 (2011)
[7] C. Brown et al., ACS Nano 6, 1961 (2012)
[8] C. Brown et al., J. Mater. Res. 30, 108 (2015)
[9] O. Seddiki et al., Appl Surf Sci 308, 275 (2014)
[10] M. Cloutier et al., Diam Rel Mater 48, 65 (2014)
[11] L. Cardenas et al., Nanoscale 6, 8664 (2014)
[12] M. Cloutier et al., Trends Biotechnol 33, 637 (2015)
[13] L. Cardenas et al., Nanoscale 6, 8664 (2014)
[14] Y. Huang et al., Nanoscale 7, 5178 (2015)
[15] H. Zhao et al., Small 11, 5741 (2015)
[16] C. Yan et al., Small 12, 3888 (2016)
[17] Y. Huang et al., J. Phys. Chem. B 120, 4992 (2016)

In this study a self-assembled arrays of titanium dioxide nanotube was used to investigate the adhesion, spreading and substrate interaction of osteoblast cells at the bio- nano interface.
Focused ion beam (FIB), scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), and In-situ Liquid TEM of osteoblast were used to investigate the cell proliferation, morphology and adhesion at the nanotube interfaces. Chemical analysis from the osteoblasts, osteoblast-nanotube interface, and milled areas of cell attached to nanotubes, revealed that the lipid bilayer of the cells has been grown inside the nanotubes, resulting in complete coverage and clogging of the nanotubes. The results indicated that osteoblasts spreading, adhesion and substrate interaction is higher in surfaces covered by nanotubes compared to bare surfaces of commonly used surgical pure Ti and Ti6Al4V alloys. In situ Liquid TEM studies provided novel insights into the dynamics and mechanisms of HA nucleation and biomineralization of titania nanotubes.

In the process of bone formation, osteoclast and osteoblast cells are two critical roles. The slight acidity induced in vivo by substituted chlorine in hydroxyapatite (ClHAp) stimulates osteoclasts for the process of bone resorption. However, no studies disclose the osteoblastic cell activity on ClHAp. The present study aims to improve the mechanical and biological properties of hydroxyapatite (HA)/polydopamine (DPA) coating on Ti64, and sintered HA bioceramics through chlorine substitution into HA.
The ph value induced by ClHAp/DPA coating on Ti64 in simulated body fluid (SBF) slightly increases from 7.4 to about 7.45 after day 1 and decreases slowly to 7 ~ 7.1 for 1 week. The release rate of chloride from the coating will be analyzed.
A higher ratio of substituted chlorine causes a greater relative density, surface hardness, compressive strength and better hydrophilicity of ClHA bioceramics. The ph value induced by ClHA bioceramics in SBF decreases from 7.4 to about 7.1 ~ 7.2 after immersion for 9 days. A slower release rate can be observed after immersion into SBF for 7 days.
Both the ClHAp/DPA coating on Ti64 and the sintered ClHAp bioceramics will undergo the tests of osteoblastic cell viability to find the optimal ratio of substituted chlorine.

The diseases related to bone-muscular system are the most common cause of disability and affect hundreds of millions of people worldwide. This is a major challenge for the orthopedic industry, because they should produce implants that improve the quality of life of patients, minimize the incidence of complications and reduce hospital stays. To meet this need, researchers worldwide are working to find a way to produce implants for clinical use with high capacity for osseo-integration. These studies have led to the production and development of biocompatible materials as calcium phosphates, while being studied and refine constantly various deposition techniques for this biomaterial on metal substrates, but also of a good osseo-integration, currently they are looking substances that avoid the formation and proliferation of bacteria on the surface of these implants, thus disadvantaging the development of infections associated with them.

As implant material, titanium and titanium alloys has been widely used because of its excellent mechanical properties and bio-inert character. But, the metallic nature of these elements makes them highly susceptible to corrosion and wear when exposed to a biological environment, where there is rapid release of metal ions to the system and in addition to this, the lack of osteoinductivity generate very weak fixation of the implant, accompanied by the development generated by the adhesion of bacteria in the place where the material was implanted infections. To solve these problems have been studied a variety of surface modification techniques for metallic materials, some of which are chemical, physical and biological treatment methods.

Physical treatments involve processes such as depositing thin films on the surface of a metal substrate, in this case, it is biofunctionalized and provide antibacterial activity to the implant by incorporating composite coatings of silver - calcium phosphate obtained by RF magnetron sputtering.

Recently, the concept of tissue-regenerative all-metallic implant system was introduced [1]. The system consists of two disparate biometals: conventional inert biometal (Ti alloys, SUS, etc.) and newly developed biodegradable metal (Mg, Zn, Fe, etc.). Those metallic systems spontaneously generate hydrogen peroxide which promotes angiogenesis and accelerates overall healing process. However, for the clinical application of this technique to bone fixation, long-term generation of hydrogen peroxide is essential in that the new vessel formation into the hematoma usually occurs in 7 to 14 days after the bone fracture. In addition, unlike the in-vitro study reported [1], in-vivo environment around the hydrogen peroxide releasing implant is composed of cells and extracellular matrix; fibrin, for instance, is the most dominant natural material in the hematoma. Therefore, control of the generation rate of hydrogen peroxide and understanding its diffusion characteristics in the extracellular matrix are essential to optimal design of the tissue-regenerative all-metallic implants. In this research, long-term generation of hydrogen peroxide from all-metallic implant systems are realized by delicate materials selection and engineering, and the diffusion characteristics of hydrogen peroxide in the extracellular matrix were analyzed based on the lab-on-chip technology. Also, considering the diffusion characteristics in the extracellular matrix, the response of human umbilical vein endothelial cells (HUVECs) was observed in the microfluidics chip.

The ability to convert an electrical field into a mechanical perturbation and vice versa makes piezoelectric materials fundamentally interesting objects of study as well as versatile components for industrial applications. Piezoelectric materials can serve as sensors and actuators in a range of fields covering vibration control in airplanes, ultrasound applications in marine and medical devices or pickups for musical instruments.
In recent years, the value of piezoelectric materials for biomedical applications, as for nerve and bone tissue repair, in-vivo sensors or energy harvesting components, has been unfolding. Depending on the specific application, biocompatibility and stable performance in the presence of body fluids are crucial factors determining a materials potential for the task. In some cases, it is even necessary that the living cells form a close interface with the implanted material. A piezoelectric implant that provides large, open pores allowing the ingrowth of cells and the formation of a living structure within the artificial pores can be of advantage in such cases.
In the present study, we investigate the potential of porous barium titanate-based piezoelectric ceramics for biomedical applications.
To study the biocompatibility of our ceramic materials, cell tests using human endothelial as well as osteoblast cells were conducted. Both cell types showed better viability and proliferation on the piezoelectric ceramics compared to a control group.
Porous piezoelectric ceramics were produced using the sacrificial template method. Different pore formers were employed to create pores of varying size and shape. The presence of porosity leads to a reduction of the piezoelectric performance, with the pore morphology playing a crucial role in the change of properties.
Both dense and porous samples were soaked in saline solution to mimic in-vivo conditions and the change in piezoelectric properties was recorded over the course of two weeks. The soaking procedure had only minor influence on the characteristics, highlighting the potential of barium titanate-based ceramics to be used as durable implant materials.

Cells respond to both physical and biochemical changes in their extracellular microenvironment. An ideal scaffold for tissue engineering application should mimic the microenvironment for natural tissue development and present the appropriate biochemical and topographical cues in a spatially controlled manner. Our research group is interested in studying the interfacial interactions of cells with the extracellular substrate and how to apply this knowledge to tissue engineering applications. In this presentation, strategies on engineering cell-materials interface, such as incorporating topographies on implantable device, development of disease model and generating functional cells, for different application for vascular and corneal repair will be discussed.
Small diameter vascular grafts (< 6 mm internal diameter) are used in bypass or replacement of occluded peripheral arteries. However, there is a lack of commercially available synthetic small diameter grafts with acceptable long-term patency. Enhancement of in situ endothelialization of small diameter vascular grafts is needed to improve clinical outcomes. Topographical cues may be used to affect the change by influencing the behavior of endothelial cells, such as increasing their migration and proliferation capacities. Poly(vinyl alcohol) (PVA), a biocompatible and non-thrombogenic hydrogel, is an excellent material for vascular tissue engineering, showing short term patency in a rat abdominal aorta model and in a rabbit model with multi-level peripheral arterial occlusion.
Although PVA has been shown to be inert, the lack of endothelial cell attachment on the luminal surface of the graft would impact the long-term patency. We fabricated PVA small diameter vascular graft with topography on its luminal surface. Implantation of patterned PVA grafts in rat abdominal aorta model exhibited patency and in situ endothelialization after 20 days. Chemical modification such as the incorporation of cyclic-RGD adhesive peptide or N₂ plasma treatment could further enhance the endothelial cell adhesion and functional marker expression, while these chemical modifications did not compromise the hemocompatibility. PVA vascular grafts are excellent candidates as a small diameter vascular graft by, preventing thrombosis, stimulating in situ endothelialization and supporting long-term patency.
In addition to incorporating topographical patterns in implantable vascular devices, we investigated the application of topographies to enhance human corneal endothelial cell growth. Cornea endothelium dysfunction is one of the main reasons for corneal transplantation. Tissue engineered cornea endothelium will be highly sought after due to the shortage of donor cornea grafts. Our studies show that micro-pillars enhanced and primary human cornea endothelial cell’s responses. Examples of topography-modulation for corneal tissue-engineering applications and corneal endothelial disease model will be also be discussed.

Cardiovascular diseases, including myocardial infarction (MI), remain a leading cause of mortality and morbidity worldwide, accounting for over 30% of all human deaths. MI leads to loss of cardiomyocytes (CMs) which have limited regenerative capacity as well as decreased contractility, adverse remodeling of the myocardium and heart failure. Heart transplantation or implantation of mechanical left ventricular (LV) assist device are treatment options of last resort, but are limited by inadequate number of organ donors and potential complications of the surgical procedures. For the vast majority of heart failure patients, treatment is limited to pharmacologic therapy to optimize the function of remaining viable CMs and slow the adverse myocardial remodeling.
Over the last decade, several approaches for myocardial replacement therapy (MRT) were developed, from cell-based transplantation, injectable biomaterials (i.e. cell-laden or acellular) or engineered tissue constructs. The common goal of these approaches is to maintain myocardial tissue homeostasis and to restore native-like functionalities.
In this talk, I will highlight recent studies in my lab at ASU, which have been focused on synthesis and characterization of new classes of biomaterials including photo-crosslinkable and electrically conductive hydrogels with optimized properties for cardiac tissue engineering. Specifically, we have developed photocrosslinkable gold nanorod (GNR)-incorporated gelatin methacrylate (GelMA) hydrogels with improved electrical conductivity, mechanical stiffness, and porous architecture to enhance the formation of intact cardiac tissues with enhanced cell-cell coupling and contractility. In a follow up study, we utilized soft lithography to create surface micro-topographies (50 µm microgrooves) within nanoengineered GelMA-GNR conductive hydrogels to provide integrated topographical and electrical cues and mimic native like cardiac cell functions. In another study, we utilize similar hydrogel constructs to develop micro-tissues with co-culture of CMs and cardiac fibroblasts (CFs) to assess the role of co-cultuer of the cells on the functionalities of the micro-tissues. Overall, our studies have demonstrated the formation of cardiac tissues with enhanced cellular organization and tissue-level synchrony using the synthesized hydrogels with significant potential for regeneration and repair of infarcted myocardium.

In this work, we designed a simple and low cost acid-dissolved/alkali-solidified self-sphering shaping method (AASS) to automatically fabricate three-dimension (3D) stem cell expansion microsphere scaffolds in large scale to satisfy the urgent need for stem cells in tissue engineering and clinical medicine research. We chose chitosan as the main part of microsphere scaffold, graphene oxide of 3 wt% as the strength agent, and genipin as the fluorescence generator to fabricate fluorescent chitosan (CS) / graphene oxide (GO) hybrid microspheres. The diameters of the hybrid microspheres are about 400 μm with the diameter error less than 10%, which is suitable for stem cells spreading. These hybrid microspheres with good biocompatibility can support the cells’ spread, growth and proliferation. After cultured for 5 days, the total number of cells on microspheres has almost increased fourfold. Most importantly, the microspheres can maintain the cell type. After cultured for 7 days, almost all cells still express main markers of human umbilical cord mesenchymal stem cells (HUMSCs). The hybrid microspheres can support long-time stem cell expansion because of their good mechanical strength, controllable degradability, and low expansion rate when soaked in media. Furthermore, their autofluorescence also makes observing and tracking the stem cells behavior on surface of microsphere scaffolds more convenient. This research provides a powerful method for mass-producing chitosan/graphene oxide hybrid microspheres for 3D stem cells expansion. This method is easy to be put into industrial production, and may have tremendous value in medicinal and clinical applications.

Bioartificial kidney (BAK) is a novel economic and sustainable biotechnological approach to chronic kidney disease patients. Hollow fiber membranes (HFMs) show the excellent mass-transfer properties, which result in their application firstly for dialysis, and consequently for design and development of bioartificial organs including BAK devices. In this study, the extracellular matrices (ECMs) were coated on TPGS-polyethersulfone hollow fiber mixed matrix membranes (CTP HFMs). The coated HFMs were characterized for surface morphology, surface properties, and strength. To evaluate the suitability of these HFMs for BAK devices, the hemocompatibility tests and kidney cells culture study were performed on the lumen-side and outer surface, respectively, of the developed CTP HFMs. To investigate the toxins clearance performance, as desired for hemodialysis, the separation performance including pure water permeability, solute rejection, and uremic toxins clearance was measured. The results obtained in these studies indicated that the developed CTP HFMs were found to be suitable for human blood-contact devices due to lower hemolysis (0.72 ± 0.1 %) and terminal complement complex SC5b-9 concentration (7.89 ± 1.5 ng/mL), and higher blood coagulation times measured for these HFMs than those measured for the commercial Hemoflow F6. The remarkably high growth, attachment and proliferation of kidney cells on the outer surface of CTP HFMs was observed. The results of glucose consumption and MTT cell proliferation assay supported this observation. With regard to the separation performance, ~ 5-fold higher ultrafiltration coefficient was measured for CTP HFMs than that measured for Hemoflow F6. Almost similar solute rejection profiles were recorded for CTP and Hemoflow F6 HFMs. The superior biocompatibility (including hemocompatibility) and separation performance of CTP HFMs can be attributed to the presence of TPGS as an additive, and ECMs coating in HFMs. In conclusion, the extracellular matrices-coated TP HFMs, developed in this study, are potential membrane materials for BAK devices.

Peripheral nerve injury is a common global clinical problem, frequently leading to life-long disability and significantly affecting the patients’ life quality. Many new therapeutic strategies for improving nerve repair have been developed, and the results showed that guidance conduit structures and living cells are essential for the repair of nerve gaps due to the trophic and structure support provided by the nerve scaffolds. Tissue engineered nerve scaffolds have been researched extensively as a potential alternative for peripheral nerve repair. So far, various kinds of nerve scaffolds have been used experimentally to bridge peripheral nerve gaps in various animal models. Common neural scaffolds could be classified into two categories: biodegradable synthetic polymers and naturally-derived polymers. Typically, the chitosan based nerve scaffolds made by naturally-derived chitin have been researched widely and proved totally biocompatible and biodegradable. In addition, the degradation products of chitosan could facilitate peripheral nerve regeneration, which makes chitosan pretty suitable for nerve repair. Accumulating evidences suggested that combining nerve scaffolds with functional cells represents a better repair effect for peripheral nerve defects. Herein, we report a self-folding chitosan-reduced graphene hybrid nerve conduit prepared by a facile and “green” method. The conduit was proved to possess high cytocompatiblity, and enhance the neural differentiation speed of neural stem cells. The stem cell assembled nerve conduit presented high ability for promoting peripheral nerve regeneration. The nerve stem cells integrated living artificial conduit provides a promising new strategy for peripheral nerve repair.

As a powerful biofabrication technique, 3D bioprinting has been nowadays used to develop various complex structures, but the printed constructs are always static. 4D bioprinting is a highly innovative additive manufacturing process to fabricate pre-designed, self-assembly structures with the ability to transform from one state to another over the time. However, current 3D/4D bioprinting based additive manufacturing technologies are hindered by the lack of advanced smart biomaterials as “inks”. Therefore, the main objective of our research is to develop novel biologically inspired nano or smart inks and advanced 3D/4D bioprinting techniques to fabricate the next generation of complex tissue constructs (such as vascularized tissue and neural tissue). For this purpose, we designed and synthesized innovative biologically inspired nanomaterials and smart natural materials. Through 3D/4D bioprinting in our lab, a series of biomimetic tissue scaffolds were successfully fabricated. Our results show that these bioprinted nano or smart scaffolds have not only improved mechanical properties but also excellent cytocompatibility properties for enhancing various cell growth and differentiation, thus promising for complex tissue/organ regeneration.

Migration in the surrounding microenvironment is the most essential step in the physical translocation of cancer cells from primary tumors to local and distant organs. Although cancer cells can move both randomly and directionally, most of the processes involved in the metastatic dissemination are more efficient when cellular movement is directed. Engineering tumor tissues with the capability to guide cancer cell migration will provide us a feasible in vitro platform to advance metastatic studies. This could be enabled via biomimicking the natural microenvironment of tumor tissues with high requirements of both the precise construction of physical structures and dynamic manipulations of extracellular chemical gradients in 3D cell-laden matrices, which remain critical challenges.
Chemotaxis is the most common mode of directed cell migration, which is demonstrated to be involved in each crucial step of tumor dissemination, such as invasion, angiogenesis, intravasation or extravasation. Therefore, we first manipulate chemical gradients surrounding 3D cultured tumor tissues and program migration pathways of cancer cells in this project. This is achieved by sequential 3D printing of cancer cell-laden natural hydrogels and multiplexed arrays of stimuli-responsive capsules within designed culture chambers. The former is constructed as tumor tissue, while the latter as programmably released chemotactic agents (i.e. growth factors and chemokines) which guide cancer cell migration. The capsules are comprised of an aqueous core with chemoattractant payloads, and a polymer shell functionalized with gold nanorods as photothermal reagents permitting selective photo-rupturing. They allow us to both spatially and temporally generate extracellular chemical cues, and provide a tool for post-printed modulation of cellular activities. Most critically, a fourth dimension (temporal control) will be added to 3D cultured tumor tissues, which has yet to be achieved.
Furthermore, by taking advantage of the manufacturing capabilities of 3D printing, we also integrate perfusable vessels within bioprinted architectures, as a tumor needs a dedicated nutrient supply and a hematogenous path. Aiming to present crucial steps of tumor dissemination, multiple cell types, including cancer cells, stromal cells, and endothelial cells, are incorporated. In addition to guided cancer cell migration, tumor angiogenesis is also dynamically mimicked, for in vitro models of both extravasation and intravasation. These 4D engineered models both physically and chemically reconstruct the surrounding microenvironments of living tumors, which provide a tool to: 1) lead 4D tissue engineering for dynamic mimicking of the in vivo natural microsystem, 2) further understand of the mechanism of metastatic dissemination, 3) test customized strategies of diagnosis and treatment, and 4) screen novel anti-cancer drugs.

Aligned fiber scaffolds can mimic the parallel-aligned fibrils in the extracellular matrix (ECM) and regulate many cellular behaviors. Generally, the aligned fibers are developed by a microplate micropatterned method, freeze casting, evaporation, slip casting, ice-templating, plain weaving, and fiber forming techniques. In this study, the Forcespinning® (FS) technology was used to prepare gelatin:zein protein fiber scaffolds with an average diameter of 400 ± 9.6 nm. Based on the collection process, aligned fiber scaffolds were manufactured by FS. Improved mechanical properties were observed. Cell viability, cell adhesion, proliferation, and drug release properties were measured. The cell viability was studied with human fibroblast cells and a low cytotoxic effect was observed on the cells at the highest ratio of gelatin:zein aligned fibers. The cells were aligned on the surface of the linear fiber scaffolds. Berberine chloride release was measured and sustained release rate was observed over the 15 days. Collectively, the morphological features, manipulable architecture, prolonged Berberin release and cytotoxicity of fiber scaffolds suggest that they could be useful in the tissue engineering, skin regeration, hernia repair, and drug delivery.

The recovery of chronic wounds, which present complications and require long periods for healing is still a challenge for medicine, since they are usually expensive processes and, in some cases, the failure of the treatment lead to amputation. Asymmetric membranes present interesting characteristics for wound healing, as the porous layer acts helping on the drainage of excess wound exudate and the dense layer (upper film) controls the transport of microorganisms and prevents the excessive loss of water. Literature presents natural polymers as good materials for production of structured membranes. Konjac glucomannan (KGM) is an example of such versatile polyssacharides that can be processed with partial drying and freezing to produce asymmetric membranes. Flexibility, resistance to handling, suitable elongation, water vapor transmission rate and fluid handling capacity are some of the recquirements that can be met by fine-tuning the processing parameters. In addition, they revealed to be non cytotoxic and capacity to prevent microbial invasion, being very interesting to support tissue repair.

For this project, pectin was selected as recent studies suggest that it is a good scaffold to develop hydrogels for biomedical applications; pectin is a polymer composed of linear chains of D-galacturonic acid, which upon cross-linking they form a sturdy support matrix for the active ingredient which in this case is allantoin (5-ureidohydantoin); which is known for speeding up the healing process by aiding in the release of dead cells and promoting healthy tissue renewal.
For the hydrogel development, were established the temperature, mixing velocity and time, and drying parameters as well; allantoin concentrations were set at 90% and 100%. Final hydrogels comprise 2 well-differentiated faces, one face rich in allantoin and one rich in pectin, these were characterized by swelling kinetics, FTIR spectroscopy, and contact angle. The morphology and topography were determined by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM).
FTIR spectroscopy validated that the pectin and allantoin molecular structure did not change and that the hydrogel remains physical. However swelling kinetics results showed significative differences between the 2 concentrations of allantoin, although this variance is only 10%. Z-stack analysis by CLSM and SEM images correlated this result with the allantoin distribution within the hydrogels.

Keywords: Pectin, Allantoin, Hydrogel and Wound healing.

Hydroxyapatite (HAp) is major constitute of bone and approved by FDA. Due to high biocompatibility and good biodegradability, HAp has been applied in various biofield. One-dimension (1D) HAp nanostructures possess unique advantages, such as high specific surface area, easy degradability, good plasticity and so on, and have been widely investigated.

Nanorods can be synthesized simply and have great potential in drug delivery and scaffold preparation. For drug delivery and sustained release visualization, endowed HAp fluorescence becomes a feasible method. By terbium/europium codoping, fluorescent HAp nanorods with dual color emission under 488 nm excitation was prepared, and can image cells efficiently. Further, CQD-HAp hybrid nanorods were prepared by self-assembling CQDs on the surface of HAp nanorods through a one-pot solvothermal method. The hybrid nanorods have long time fluorescent property and higher quantum yield compared with pure CQDs, which can be used for longtime cell-imaging, and also have good drug loading and delivery property to kill cancer cells.

1D HAp nanostructures with different length can be synthesized controllably from nanorods (~200nm) to short nanowires (~5μm) to ultralong nanowires (over 50μm) by regulating experiment condition. Different 1D HAp nanostructures have different cytocompatibility and osteogenic differentiation promotion. Nanorods have low promotion to stem cell osteogenic differentiation although with high cytocompatibility. Cells can spread on ultralong nanowires hardly following low viability. Short nanowires possess both cytocompatibility and osteogenic differentiation promotion. It gives us a cue that nanostructures not only material properties play an important effect on stem cells regulation. Further, 1D HAp nanostructures can be combined with other bio-material to form composite and used in tissue engineering. A new kind of HAp/PLA composite with novel characteristic was prepared by ingenious design. The composite can repair defect region in precise, and provide huge convenience in modern medicine.

Through structure design and property optimization, it can be believed that 1D HAp nanostructures with tunable length and multi-functions will have very important and wide applications in biofield.

Reference
1. Baojin Ma, et al. "One-Dimensional Hydroxyapatite Nanostructures with Tunable Length for Efficient Stem Cell Differentiation Regulation." ACS applied materials & interfaces 9.39 (2017): 33717–33727

2. Baojin Ma, et al. "Prolonged fluorescence lifetime of carbon quantum dots by combining with hydroxyapatite nanorods for bio-applications." Nanoscale 9.6 (2017): 2162-2171

3. Baojin Ma, et al. "Eu/Tb codoped spindle-shaped fluorinated hydroxyapatite nanoparticles for dual-color cell imaging." Nanoscale 8.22 (2016): 11580-11587.

4. Baojin Ma, et al. " Hydroxyapatite Nanobelt/Polylactic Acid Janus Membrane with Osteoinduction/Barrier Dual Functions for Precise Bone Defect Repair." Submitted

Biological hydrogels such as collagen and gelatin show a high potential in biology and medicine due to their strong biocompatibility and biodegradability. They are highly attractive materials for biomedical applications such as extracellular matrix components, coatings or implants. Thereby, precise adaption of structure and mechanics as well as stimuli-response is an interesting aspect of the modification of these materials. Reagent-free modification of hydrogels can be achieved by utilizing high energy electron irradiation inducing crosslinking.
We will demonstrate how crosslinking with high-energetic electrons allows fine-tuning of materials properties such as structure, mechanics and swelling. Furthermore, we will present the development of topographically and mechanically patterned hydrogel substrates as a functionalization of hydrogel-surfaces without any toxic reagents.

In this work, nanostructured Poly-(3,4-ethylenedioxythiophene) (PEDOT) were synthesized using starch/κ-carrageenan aerogels as templates. A biopolymer (i.e. κ-carrageenan) was added to the formulation as dopant agent of the conductive polymer to enhance the biocompatibility and the electrical response. The physicochemical, morphological, mechanical and electrical properties of the PEDOT and templates were characterized. An impedance model was proposed for the material, evidencing the incorporation of κ-carrageenan in the PEDOT nanostructuration process improved the electrical behavior of the PEDOT in the aerogel structure. The synergistic combination of the inherent electrical properties of the PEDOT, the advantageous features of κ-carrageenan as dopant agent and the porous morphology provided by the aerogel template resulted in electroactive PEDOT nanostructures with promising properties as extracellular matrix in biological cell culture.

Tissue engineering requires large amounts of cell spheroids [1-3]. Tissue spheroids have been actively used as 3D tumour models [4], tissue reconstruction and organ bioprinting [2]. Spheroids of adherent cells can be formed using different processes for cell clustering where they adhere to each other rather than to a substrate. The current processes of cell spheroid production involve spinner culture, NASA rotary culture and non-adhesive surfaces [5], the hanging-drop culture [6] and 3D culturing in microwells [7]. Although many techniques for tissue spheroids preparation have been reported, none of them are currently able to rapidly produce significant amounts of spheroids. Here we describe a simple and generic technique for high throughput generation of tissue spheroids based on encapsulation of dispersed adherent cells in a water-in-water Pickering emulsion stabilised by protein particles [8]. The emulsion is formed from a cell suspension in an aqueous solution of dextran (DEX), which is dispersed in an aqueous solution of polyethylene oxide (PEO) containing protein particles. The cells are trapped in the DEX drops of a stable DEX/PEO emulsion which they prefer compared with the continuous PEO phase. Further addition of more concentrated PEO phase leads to osmotically driven shrinking of the DEX drops and compresses the adherent cells into tissue spheroids which are isolated by breaking the emulsion by dilution with a culture media. We demonstrate the method by using HEK293 fibroblasts and show that the cells preserve their viability in the spheroid generation process. This work will give researchers cheap and scalable technique for rapid preparation of similarly sized spheroids of adherent cells for bio-inks for 3D organ bioprinting applications and potentially for tumour models.

[1] T. Sato, R.G. Vries, H.J. Snippert , M. Van de Wetering , N. Barker, D.E. Stange , J.H. Van Es , A. Abo, P. Kujala , P.J. Peters , H. Clevers, Nature, 2009, 459, 262.
[2] A.A.K. Das, R.F. Fakhrullin, V.N. Paunov, in Cell Surface Engineering: Fabrication of Functional Nanoshells, The Royal Society of Chemistry, 2014, pp. 162-184.
[3] V. Mironov, R. P. Visconti, V. Kasyanov, G. Forgacs, C. J. Drake and R. R. Markwald, Biomaterials, 2009, 30, 2164.
[4] J. Friedrich, R. Ebner and L. A. Kunz-Schughart, Internat. J. Rad. Biol., 2007, 83, 849.
[5]Y. Morimoto and S. Takeuchi, Biomater. Sci., 2013, 1, 257.
[6] Y. C. Tung, A. Y. Hsiao, S. G. Allen, Y. S. Torisawa, M. Ho and S. Takayama, Analyst, 2011, 136, 473.
[7] M. Kato-Negishi, Y. Tsuda, H. Onoe and S. Takeuchi, Biomaterials, 2010, 31, 8939.
[8] A.A.K. Das, B.W. Filby, D.A. Geddes, D. Legrande, V.N. Paunov, Materials Horizons, 2017, 4, 1196.

The life-threatening myocardial infraction is caused by the complete occlusion of coronary artery, leading to losses of downstream myocardium and the development of post-infarction heart failure. By seeding cardiac cells onto scaffolds and nurturing their growth in vitro, engineered tissues are expected to generate natural heart structures and functions, and be transplanted to restore infarcted hearts. In fact, the healthy natural myocardium possesses a hierarchically aligned structure with different layers, while the synchronous contractions are triggered by electrical impulses propagating along the cardiac conduction system. It is important but remains a challenge to consider both elongated and aligned morphologies and electrical signal propagation during cardiac tissue cultivation.

Here, super-aligned carbon nanotube sheets (SA-CNTs) are explored to culture cardiomyocytes, mimicking the aligned structure and electrical impulse transmission behavior of natural myocardium. The SA-CNTs not only induce an elongated and aligned cell morphology of cultured cardiomyocytes, but also provide efficient extracellular signal transmission pathways required for regular and synchronous cell contractions. Furthermore, the SA-CNTs can reduce the beat-to-beat and cell-to-cell dispersion in repolarization of cultured cells, which is essential for normal beating rhythm and potentially reduce the occurrence of arrhythmias. Finally, SA-CNTs based flexible one-piece electrodes demonstrate a multipoint pacing function. These excellent properties make SA-CNTs promising in applications in cardiac resynchronization therapy in patients with heart failure following myocardial infarctions.

Novel biomaterials provide fundamental platforms for diagnosing diseases at the cellular level, and also present opportunity to deliver therapy to unhealthy cells without damaging healthy cells. Among numerous biomaterials, magnetically-aligned collagen matrices offer advantages in fundamental biological studies, such as imaging and cell therapy, such as hyperthermia, magneto-mechanical rupture, and drug delivery. Understanding and predicting structure, as well as mechanical and magnetic properties, of these magnetically-aligned matrices all play a crucial role in successfully producing and implementing biological treatment techniques. Experimentally, highly uniform magnetic nanowires (MNWs) were fabricated using template assisted electrochemical deposition technique. After coating MNWs with NH₂-PEG-COOH, they were incubated in collagen type I with/without CDI crosslinker at different MNWs concentrations. By applying a weak magnetic field during gelation, the collagen matrices were aligned, providing a bi-directional alignment of the collagen fibrils without any extra preparation processes. Confocal reflectance microscopy and differential interference contrast (DIC) microscopy were used to visualize the structure of matrices. Atomic force microscopy (AFM) was used to image the structure and correlate this structure to the mechanical properties of each matrix measured using AFM contact resonance viscoelastic mapping and AFM nanoindentation techniques. AFM nanomechanical measurements show enhancement in mechanical properties of magnetically-aligned collagen matrices when the CDI crosslinker was used. For magnetic properties, vibrating sample magnetometry measurements were analyzed using First Order Reversal Curves (FORC), such that the magnetic properties of the matrices were quantitatively and qualitatively investigated, confirming that the strongest matrices contained uni-directional alignment of MNWs in bi-directionally aligned collagen.

Carbon-based aerogels prepared by organic sol-gel chemistry offer the ability to create a unique 3-D surface profile with control of pore distributions spanning both the nm and um size ranges The surface morphology, charge density, porosity, and bulk electric conductivity combines critical substrate/ scaffold parameters known to influence neuron attachment, proliferation, guidance, and growth noninvasively. The aim of this research was to (a) establish the methodology and processing techniques for culturing of PC 12 cells on carbon aerogels, (b) assess the integrity of the aerogel substrate after exposure to culture and fixing conditions and, (c) characterize and examine the effects of conductive carbon aerogel substrate on neurites. Here, PC 12 cells were stimulated with NGF, a growth factor essential for the survival, differentiation and functional activities of neurons in the peripheral and central nervous system. The neurite length, cell cluster size, and cell morphology and arrangement was compared between the two different surfaces (1) tissue culture plastic (control) and (2) carbon aerogels. Results were also compared with the response of PC 12 cells to polyurea crosslinked silica aerogels and will be reported in detail.

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 in order 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. It is expected that with both electrical and biochemical cues, the polyelectrolyte PBGA^-Na⁺ will give longer axon outgrowth and higher differentiation ratio compared with the neutral copolymer of poly(γ-benzyl-L-glutamate) and poly(L-glutamic acid) (PBGA).

Myocardial infarction is caused by the complete occlusion of coronary artery, leading to loss of downstream myocardium. The lost cardiomyocytes are gradually replaced by fibrous scar tissue, which usually leads to ventricular remolding and arrhythmogenic inhomogeneous electrophysiological distributions. Despite the improved therapeutic strategies, post-infarction heart failure and malignant arrhythmias remain therapeutic challenges. Cardiac engineering of patches and tissues is a promising option to restore infarcted hearts. By seeding cardiac cells onto scaffolds and nurturing their growth in vitro, engineered tissues are expected to generate natural heart structures and functions and can be transplanted to replace fibrous scars. In facts, electrophysiological heterogeneities in the infarct-related region have been identified as determinants of post-infarction arrhythmias. Thus, substantially homogenized electrophysiological properties of engineered tissues is essential for normal beating rhythm and the avoidance of the occurrence of arrhythmias.

Here, super-aligned carbon nanotube sheets (SA-CNTs) are applied to promote electrophysiological homogeneity for in vitro cultured cardiac cells. We found that SA-CNTs can interact with cells and induce the orientation of the growing CMs, and influent the expression and distribution of gap junction protein connexin-43 (CX43), which is essential for synchronous contraction of all adjoining CMs. CMs cultured on SA-CNTs show spontaneous synchronized regular beating rhythms. Which can be explained as (1) accelerated electrophysiological maturation of neonatal rat CMs and reduced beat-to-beat APD (action potential duration) dispersion enabled the cells to generate regular and stable beats; (2) improved intercellular coupling through the increased and lateralized CX43 expression allowed adjacent cells to synchronously contract; (3) reduced cell-to-cell APD dispersions prepared all cells, even those isolated far from each other, to be simultaneously excited upon electrical impulse. Furthermore, SA-CNTs provided an efficient pathway for rapid electronic propagation to all ready-to-excite cells, with stable conductivity and pacing threshold. Therefore, SA-CNT-based cardiac tissue patches achieved the synchronized contraction of all cultured cells with reduced electrophysiological heterogeneity, which provided a potential method to restore the infarcted myocardium and alleviate the electrophysiological heterogeneity in infarct-related regions.

Several types of non-degradable alloys are used for stent applications, for example AISI 316L stainless steel, Co-Cr alloys such as L605, and Nitinol. The used materials have to satisfy a series of requisites, such as for example excellent mechanical properties, suitable electrochemical resistance, and high cyto-/hemo-compatibility. The quest for an «ideal» material is still open, as for example AISI 316L and L605 are composed of elements (for example Ni) whose release in the bloodstream is known to be noxious. For this reason, a new Ti alloy containing only cytocompatible elements, Mo and Fe, was designed and produced to obtain a material with combined high mechanical resistance and high plastic deformation. The present work is a preliminary study of the surface features of this alloy and pure Ti after several treatments, to set the basis for a better understanding of the phenomena involved in enhanced biological properties.
Different kinds of surface treatments were performed on commercial pure Ti and cast Ti-9Mo-1.3Fe (TMF1.3) samples: mechanical polishing (MP), electropolishing (EP), a thermal treatment after electropolishing (TT) and oxygen plasma immersion ion implantation after electropolishing (PIII). The effect of these treatments on surface chemistry, morphology, physical properties and hemocompatibility were investigated by several techniques. X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM) and atomic force microscopy (AFM) were used, respectively, to analyze the surface chemical composition, morphology and roughness. Static water contact angle was also used to investigate the wettability. Hemocompatibility tests were performed on selected samples of the two materials, EP and PIII treated, to assess their resulting biological behavior.
Roughness, chemical composition, surface energy and biological property were all influenced by the treatment parameters. The PIII treatment was responsible for the highest roughness R_a, in both the alloys, respectively ~5 nm for pure Ti and ~ 3 nm for TMF1.3 (measured surface 1 mm²); while MP treatment resulted in smallest roughness R_a. The lowest contact angle, around 50°, was found for TMF1.3 in the PIII condition; while pure Ti in the MP condition showed the highest one (around 98°). In general, pure Ti showed higher contact angles for all the considered conditions. Mo traces were found in the EP condition of TMF1.3; the alloy oxidation covered the surface by a layer of Ti oxide. Hemocompatibility tests showed differences in terms of biological responses.

The beta phase stability of titanium alloys can be generally described by means of molybdenum equivalence (Mo_eq). In this study, Ti-4Mo-X (1, 2, 3, 4 wt.% Cr or V) alloys were prepared by means of vacuum arc remelting under argon atmosphere in order to enhance the workability of Ti-4Mo-X alloys and corrosion resistance. The corrosion properties, microstructural evolution and plastic workability were investigated following uniaxial cold rolling. The corrosion evaluation was conducted in a standard three-electrode cells in naturally aerated Ringer’s physiological solution at 37±1 ^oC. Optical micrograph showed an increase of acicular β phases after cold rolling. XRD results showed a higher intensity of β phase in the cold rolled specimen compared to as-homogenized one. The formation of β phase was responsible for the improved workability of Ti alloys. The maximum reduction ratio of the cold rolled specimens without any presence of cracks were 72.8% and 39.4% for Ti-4Mo-4Cr and Ti-4Mo-4V alloys, respectively. The corrosion properties of the alloy were relatively dependent on the cold rolling. Ti-4Mo-4Cr alloy showed the lowest corrosion current density with 6.71E10^-9/cm².

Polymethylmethacrylate (PMMA), Polycarbonate, (PC), and Polyetheretherketone (PEEK) are thermoplastic polymers with properties that have already been proved favorable for bio-medical applications. They have been applied in different medical fields such as maxillo-facial surgery, orthopedic surgery, intraocular surgery, and more, due to their mechanical properties, and osteoinductive and antimicrobial capabilities. As these polymers become important biomaterials for bone and cartilage replacement, the optimization of the manufacturing processes seems more relevant than ever. The adaptability of the 3D printing process allows for the manufacturing or building of the desired piece with the exact characteristics, shape and size for each individual, in a way that they may be built or modified very easily. The result is that biomedical application options are increased with the utilization of 3D printing techniques. In this work, we have studied the effect of heat treatment on the mechanical properties of 3D printed samples prepared with the three different polymers. By selecting the right printing temperature, as well as heat treatment process, the mechanical properties can be tailored specifically for different applications. It is critical to make comparisons between the mechanical properties of hard and soft tissues, and compare them to the ones of typical metallic, ceramic and polymeric biomaterials. Furthermore, by researching and optimizing the properties of 3D printed polymers, a comparison can be made with the implants commonly made using molding processes and consequently modulus of elasticity, hardness, and other properties can be optimized to make them suitable to be used as various biomedical devices.

A material that mimics the properties of bones was developed by optimizing the ratio of polymer composites of Polylactic acid (PLA) and poly-ε-caprolactone (PCL), and containing small amounts of titanium oxide (TiO₂). Although titanium-based alloys have commonly been used for bone replacement procedures due to their biocompatibility with the human body and their mechanical properties, stress shielding continues to be a problem. The structure of a bone has a porosity which permits the flow of nutrients, blood, oxygen and minerals, and is an issue at the time of creating bone replacements using conventional methods. PLA and PCL have been used in biomedical applications due to their biocompatibility with the human body and their mechanical properties in vivo. PLA and PCL provide strength to the artificial cancellous bone supplying the initial support, and allowing the gradual degradation desired in the human body. In this work the polymer composite materials were prepared, then the filaments used to print the 3D structures using Fused Deposition Modeling (FDM), after which their mechanical properties and biocompatibility were tested. Different characterization methods were used, such as differential scanning calorimetry (DSC), tensile testing, shore hardness, and scanning electron microscopy, and the effect of the filler was evaluated. DSC analysis yielded useful information regarding the immiscibility of the different polymers, and it was observed that the presence of PCL in the blend does not affect the melting temperature of PLA, but it increases the crystallinity of the blend. In addition, the particles of TiO₂ have improved the stability of PLA, showing no effect in the melting temperature through the enhanced interaction between PLA and the particles. The prepared composites demonstrated good biocompatibility, as cell growth was significantly higher in the PLA/PCL/TiO₂ composites than in the blend without TiO₂. The printed composites using FDM show excellent in vitro biocompatibility including cell proliferation, adhesion and osteoblast differentiation and are therefore promising candidates to be used in the field of bio-medical applications. Furthermore, PLA/PCL composites infused with TiO₂ seem to be a good option specifically for bone replacement procedures, since the mechanical properties of PLA/PCL/TiO₂ composites are similar to the cancellous bones making them a viable option for bone replacement and grafting procedures.

Nanostructured titanium dioxide has been used in several studies as a surface modifier because it presents high hardness, increased dielectric constant and great chemical stability [1,2]. Furthermore, the surface modification with nanostructured TiO₂ can improve in the biocorrosion resistance and increase the oxide bioactivity, presenting promising results in the interaction with the tissue, since the biocompatibility is determined by chemical processes that occur at the interface between the implant and the proteins of the biological fluids [3].
Chemically, the oxide surface is mainly terminated by -OH groups which can be functionalized by various biofunctional molecules, such as carboxylic acids, and other derivatives like esters, acid chlorides, carboxylate salts, and others forming self-assembled monolayers (SAMs) on the oxide surface [4]. SAMs have well-defined surface properties, such as uniformity, stability and reproducibility. Hence, this chemical functionalization with appropriate physical changes in the material surface can enhance the interaction between the oxide and the biological environment [5].
In this work, anatase and rutile TiO₂ films were grown by reactive sputtering on commercially pure (grade IV) titanium substrates. In the process, argon, oxygen and titanium, all grade VI, were used as working gases and sputtering target, respectively. The film structure was checked using X-ray diffraction. The surfaces of both anatase and rutile the films were functionalized with
3-mercaptopropionic acid (MPA) solution, in a concentration of 3 mM at a pH of 3.0, by immersion for 1 hour. After immersion, the samples were washed with Mili-Q water in order to remove non-adsorbed molecules. X-ray photoelectron analysis indicated the presence of hydroxyl groups for both pristine TiO₂ anatase and rutile surfaces. For the rutile phase, it was possible to see a contribution of sulfur attached to oxygen. Interestingly, no contribution of sulfur at all was detected for anatase phase. Further investigations are being performed in order to understand the interactions between rutile and anatase TiO₂ surfaces and the functional groups of MPA.

[1] HANAWA, T. A comprehensive review of techniques for biofunctionalization of titanium. Journal of Periodontal & Implant Science, 41, 263-272 (2011).
[2] YU, B. et al. Synthesis of Ag–TiO2 composite nano thin film for antimicrobial application. Nanotechnology, 22, 1-9 (2011).
[3] LI, Q. et al. The incorporation of daunorubicin in cancer cells through the use of titanium dioxide whiskers. Biomaterials, 30, 4708-4715 (2009).
[4] LOVE, J. C. et al. Self-assembled tonolayers of thiolates on metals as a form of nanotechnology. Chemical Reviews, 105, 1103-1169 (2005).
[5] HONG, H. et al. Cancer-Targeted Optical Imaging with Fluorescent Zinc Oxide Nanowires. Nano Letters, 11, 3744-3750 (2011).

Collagen is the main source of extracellular support, as it behaves like a binding agent in the human body. It holds the cells together and increases elasticity of the skin. The skin cannot absorb the collagen as most molecules are too big, so getting them through the outer layers of the skin is critical. Collagen microneedle patches have been developed with micro-manufacturing process (micro-molding). The collagen microneedles, varying in length from 300 to 600 μm reach different targeted layers of the skin depending on the application. As the microneedles penetrate the top most layers of the skin (Stratum Corneum), they dissolve, delivering collagen through the epidermis and dermis. Such addition of collagen directly to the epidermis and dermis layers allows collagen to be absorbed, increasing the skin’s strength and resilience, since it is one of the natural components of younger skin. This process of collagen delivery system is painless, hygienic and easy to use. The research includes the preparation of microneedles with the modification of various parameters such as concentrations, microneedle height, pressure, time, and also the optimization of the process of the microneedle patch construction. Further research will include penetration studies, rate of delivery and the effects of the collagen microneedles on the skin. There is future scope of using other drugs such as nicotine, BOTOX and many other pharmaceutical drugs along or without collagen.

Ti and its alloys have been widely used as biomedical materials and have been found application in artificial bones, dental and/or orthopedic implants, etc. because they have suitable chemical, physical, mechanical, and physiological properties. Their biological corrosion resistance to body fluids and biocompatibility with human tissues especially make them excellent implant materials. In addition, Ti is a relatively light metal that possesses good ductility, high tensile modulus and fatigue strength, and an elastic modulus similar to that of human bones. In order to improve bone-bonding ability of Ti implants, it is necessary to apply a suitable surface treatment. In this work, formation of nano-sized porous layers on Ti by asymmetric alternating current (AC) anodizing in sulfuric acid aqueous solution has been studied by using electrochemical techniques and scanning electron microscopy (SEM). When Ti is oxidized in 1 M H₂SO₄ aqueous solution for 1 min by AC electrolysis, vigorous gas evolution proceeds with the spark discharge on the Ti surface. As a result, a nano-sized porous layer cannot obtain on a Ti surface and the spark discharge gives some damage on the surface. The gases evolved on both electrodes during AC electrolysis consist of hydrogen and oxygen. These gases act as insulators which cause a breakdown phenomenon to proceed at evolved gas layers near Ti and Pt surfaces. Therefore, the spark discharge is observed on both the electrode surfaces during AC electrolysis. Applying a very high voltage between the Ti and Pt counter electrodes thus cannot be applied to obtain a nano-sized porous layer. In order to inhibit the spark discharge, the cathodic current on a Ti electrode, which corresponds to the rate of the hydrogen evolution reaction, is limited using a special electrical circuit. The electrical circuit consists of a variable resistance and two diodes. It inhibits the hydrogen evolution during AC electrolysis and therefore prevents the spark discharge on the Ti surface. Accordingly, a nano-sized porous layer is formed on the Ti surface. The formation of porous layer is dependent on electrical resistance value of the special electrical circuit. As formation of a nano-sized porous layer becomes insufficient when the electrical resistance is high, a low electrical resistance could be used to obtain a sufficient porous layer. The nano-sized porous layers thus obtained have the same morphological features as those formed by direct current (DC) anodization in 1 M H₂SO₄ solution. The porous layer obtained at low voltage by asymmetric AC electrolysis with the special electrical circuit is compared to those that are obtained by DC anodization voltage. The absorbed hydrogen near Ti surface during asymmetric AC electrolysis greatly influences the formation of a nano-sized porous layer.

There is also a growing interest in developing multifunctional hard bone tissue analogs exhibiting high biocompatibility and osteoconductivity together with therapeutic effect. Selenium (Se), in that respect, is an effective therapeutic agent with promising antioxidant and anti-carcinogenic effect when used in proper doses. Here, selenium-doped HAp (HAp:Se) particles have been synthesized by modified aqueous precipitation method using calcium (Ca(NO₃)×4H₂O) and phosphate ((NH₄)₂HPO₄) salts with sodium selenite (Na₂SeO₃). The effect of Se dopant in different amounts and calcination temperature (800-1100°C) on the physical, chemical and crystal structure of resultant HAp have been investigated in detail. Complete chemical investigation was performed with spectroscopical analyses including fourier tranform infrared (FTIR) and x-ray photoelectron spectroscopy (XPS) to elucidate the mechanism and chemical nature of Se doping in HAp. Meanwhile, x-ray diffraction (XRD) studies by rietveld refinement in combination with transmission electron microscopy(TEM) studies have conducted to elucidate the changes in the HAp crystal structure upon Se doping. A correlation between Se incorporation site in HAp crystal and Se dopant amount has been established, which critically regulates the therapeutic activity of Se.

Alginate is a polysaccharide found in the brown algae’s cell walls and widely used in the pharmaceutical, food and environmental care industry. It has excellent properties for use as biomaterial, as it is biodegradable, biocompatible and non-toxic. In the literature, several studies point alginate as a powerful healing agent. It has been proposed the use of a drug (enzyme) to be used with alginate membranes to accelerate and increase its healing power. Papain is a drug sold commercially and used for the treatment of skin lesions, but it is found as powder or as ointment, making the treatment less efficient since the contact with the drug cannot be modulated. The papain immobilization was studied in alginate membranes in order to provide a stable environment to the enzyme and increase the contact time with the skin. Two immobilization methodologies were used: by entrapment, where the enzyme was added to the alginate gel, followed by the production of membranes, and by physical diffusion, where the membranes were immersed in solution with papain and the enzyme is diffused. Assays were performed in order to analyze the enzyme stability and the release profile after immobilization, moreover a biological test in which hemolysis (blood cell disruption) was measured to evaluate the behavior of the material with the biological tissue (blood). It was found that the best method for papain immobilization on alginate matrix was by diffusion, since higher active papain content available for reaction was obtained, as well as a higher and faster release profile by the diffused membrane occurred when compared to the membrane with entrapped papain. A non-hemolytic membrane was also obtained, since no blood cells were disrupted. Based on these initial tests, it was possible to obtain a bioactive biomaterial with good properties to be applied as wound dressing.

The intersection of graphene and biology has emerged as a promising area where graphene’s physical properties may help elucidate fundamental insights into the chemistry of life.[1] However, graphene’s structure and properties are tightly coupled to synthesis and processing conditions, [2] thus influencing biomolecular interactions at graphene – cell interfaces. For example, graphene can be obtained via micromechanical cleavage and liquid assisted exfoliation of bulk graphite, Chemical Vapor Deposition (CVD) on transition metal foils, and epitaxial growth on SiC. Such processing conditions can impact crystal size, the density of structural defects, and chemical, thermal, and electrical properties.[2] In this study, we grew C2C12 cells, a pluripotent (mouse muscle) cell line, on graphene bioscaffolds with varying structure – property – processing – performance (SP3) correlations. We find that such SP3 correlations significantly influence C2C12 differentiation, myotube formation, and gene expression, suggesting that the cell – graphene interface can be engineered to control biomolecule structure and function in adherent cells.

Similar to our previous work, cells were seeded on glass (control), CVD graphene, printed graphene of increasing pass numbers (2 to 10 passes), and epitaxial graphene on SiC. Cells were maintained for one week in culture.[3] Gene expression patterns were analyzed by quantitative RT-PCR. Cell morphology and viability was assessed by cytochemistry. Differentiation was assessed by immunocytochemistry using an antibody specific for Troponin I. Samples were counterstained with phalloidin to identify actin and DAPI to identify the nuclei. Biocompatibility was determined by measuring cellular viability.

Our results indicate that the manner in which the graphene is produced and the surface and structural properties of the resulting bioscaffolds correlate to differential expression patterns of molecular markers for differentiation of pluripotent cells. Successful cellular attachment and persistent cellular viability may also vary depending on the surface characteristics of the graphene.

In conclusion, our results highlight the first study of graphene bioscaffold SP3 characteristics on biomolecular structure and function of adherent pluripotent cells, highlighting a novel tool for engineering tissue function on graphene surfaces.

Stem cells hold immense potential in tissue engineering applications due to their ability to differentiate into multiple cell types based on their cellular niche. In order for them to successfully differentiate and proliferate ex-vivo, it is imperative to design a microenvironment which is able to reasonably mimic the mechanical and biochemical native cell environment. We conduct in vitro studies to better understand the effect of different hydrogel matrices and intercellular interactions on the differentiation and proliferation of Human Bone Marrow Mesenchymal Cells (HBMSCs). We use droplet microfluidics to encapsulate HBMSCs using photocurable gelatin methacrylate (GelMA) and alginate hydrogels to study the effect of the encapsulating microhydrogel matrix on the proliferation and differentiation of HBMSCs. Further, we describe a strategy to coculture HBMSCs with Endothelial Cells (ECs), in 3D compartmentalized hydrogel microparticles in order to study the paracrine signaling pathways that exist between these cells and their effect on the differentiation potential of the HBMSCs. This system offers confinement of different cell types, isolation of paracrine from juxtacrine signaling, high surface area for transport, biocompatibility, control over cell ratios and good experimental statistics. Initial results show this platform is suitable for the long term culture of these cell types and detection of interactions between them.

Carbon materials have the characteristics of light weight, good biocompatibility, x - ray there is no, excellent biomechanical properties and can be designed. In the study， planed the structure of materials firstly, combined with three-dimension printing technology to assist in the forming of light weight, high purity and excellent mechanical properties of the carbon-based composites materials support cup, which was used in the thoracic lung resection surgery, filled the left lung space after removal, can effectively lift the heart, the effect is significant.

Extracellular Matrix (ECM) is a non-cellular scaffold that is composed of complex biopolymers. ECM has become a promising biomaterial to be applied in various fields: tissue reconstruction, drug delivery, and implantation. Although ECM material has been extensively researched for more than 50 years, it has still not been optimized for industrialization. In order to manufacture effective ECM material, decellularization is essential. ECM implantation in vivo without proper decellularization would result in adverse host responses with various antigens. Many researchers have proposed different effective decellularization processes that are mostly too long and still can be improved. In this research, the ECM decellularization process has been optimized into three days by using as source the pig's adipose tissue, and using enzymes and relatively strong chemical detergents such as trypsin, sodium deoxycholate, triton-X 100, peracetic-ethanol solution, and isopropanol. This optimized protocol is very efficient to obtain excellent an ECM product. Through the analysis, it was confirmed that in the resultant ECM material, DNA, RNA, and cellular contents have been completely removed from the adipose tissue, while maintaining the core components of ECM including collagen, elastin, laminin, and fibronectin.

Recently, Extracellular Matrix (ECM) has become extensively researched due to its outstanding biocompatible properties and low immune response during implantation. ECM refers to the complex bio-polymer, which has proven to be a superior biological material applicable in a variety of fields. In order to be utilized it in a human body, especially in medical industry, an ECM material must have all cellular contents removed and be purified adequately. Although there are many decellularization methods that has been researched and performed, these conventional methods are considered to be protracted. They generally cannot perfectly remove DNA,RNA, or cellular contents, so that it is safe to be implanted in human body. To overcome the indicated drawbacks, this study has conducted a three-day process, which preserves the unique properties of ECM. The three-day process completely removes DNA, RNA, nucleus of the cell, and cellular contents. By employing the concept of enzyme reaction kinetics, the rate of enzymes being reacted with ECM and the loss of ECM weight induced by the enzyme, can be determined. Combining this enzyme reaction kinetics with the process allows highly productive ECM decellularization. Within a decade, the ECM decellularized using this method has great potential to be used in various fields, such as, tissue engineering, medical engineering, food, and cosmetics.

In the US, 15% of diabetics develop chronic wounds, of which 12-25% result in amputations due to their non-healing status. Among the many methodologies for treating such wounds, oxygen plays a very important role through all wound healing stages from inflammation for prevention of infection to tissue remodeling for promoting collagen synthesis. As a result, pharmaceutical and medical device companies have recently placed a strong emphasis on the development of oxygen based therapies with various delivery modalities, including hyperbaric oxygen (HBO), topical oxygen (TO) and continuous diffusion of oxygen (CDO). HBO usually requires expensive, sophisticated, and bulky equipment and is time-consuming, thus limiting its suitability to only high-end clinics. TO can avoid the side effect of excessive oxygen associated HBO but still requires an external oxygen resource and thus immobilizes the patients, which also limits their utility in large population. CDO, providing a slow flow rate of oxygen for continuous delivery, has more practical approach for its portability but is still not cost-effective. Therefore, as a low-cost alternative CDO, we have developed a personalizable, foot pressure-triggered, oxygen-releasing insole that delivers oxygen specifically to foot regions with an ulcer. The insole consists of an oxygen-filled polydimethylsiloxane (PDMS) chamber composed of two layers, one to provide the interface to the foot with the selective laser-machined region targeting the ulcer position, and the other to store the oxygen providing structural support via an array of 1cm diameter pillars distributed with the spatial ratio of 12.4% mm³/mm³. Both layers are bonded together using oxygen plasma and a strong adhesive film (3M 300LSE) and filled with pure oxygen. The entire fabrication can be realized through a scalable and layer-by-layer process. The high permeability of PDMS to oxygen along with its strong but flexible mechanical properties make it an ideal material for use in foot insole applications with oxygen transport properties. The thickness of the upper layer of the insole is patterned by laser-machining to alter the surface-to-volume ratio of the chamber and tune the oxygen permeability of the material, thus enabling higher permeability in ulcer regions but lower permeability elsewhere. When a person applies pressure during normal walking or standing, the insole releases oxygen to the ulcer regions. Mechanical characterization of the insole revealed an average bond strength (peel test) of 6.85N. The O₂ release of the insole was investigated by placing it over a 12x8x9 mm³ 0.35% agarose gel (mimicking the wound bed) while the O₂ in the insole was pressurized to match the pressure exerted by a normal weight adult (between 50 to 60 kg). The oxygen was measured 4mm into the gel for one day. The average O₂ delivery rate was measured to be 0.05 ppb/min/mm² at 0.9mm thick PDMS region, 2.1 times of the non-laser-machined region.

Worldwide, cancer represents one of the most important health problems, that is why new research is being carried out to find effective treatments for this condition. Likewise, it seeks for nano-transporters to provide controlled drugs release and also be tissue-specific in order to minimize side effects in the patient, and ensure the success of cancer therapy. In the specific case for liver cancer, specific transporters can be obtained because the hepatocytes present an asialoglycoprotein receptor, which recognizes structures with galactose. In this work, we modified bovine serum albumin (BSA) with lactose by different methods in order to obtain a matrix able to be bio-recognized by the asyloglycoprotein receptor in liver cancer. The modification of BSA was analyzed by electrophoresis, infrared spectroscopy and fluorescence, as well as biorecognition tests with Riccinus comunis agglutinin I (RCA). The results showed that of the three different modifications of BSA were highly recognized by RCA I which indicates the presence of galactose. Subsequently, with the glycoproteins obtained, nanoparticles were synthesized, which were evaluated by means of dynamic light scattering (DLS), Z potential and electronic scanning spectroscopy (SEM). Biorecognition tests of glyconanoparticles were performed using cancer cells from liver (HepG2) and cells from cervical cancer (HeLa) as control. The results indicated the specific interaction of the nanoparticles with HepG2 cells, demonstrating that they could be used as transport for an antitumor drug.

Raman spectroscopy (RS) has gained popularity in recent years to characterize biological materials, and can be a valuable tool for plants. Current approach to plant health and disease diagnosis depends on chemical extraction and pathophysical screening. RS on the other hand can be used to identify key biological composition of plants, and investigate underlying ultrastructural changes without staining and complicated sample preparation.

This work presents investigation and quantification of plant health and growth status (specifically changes in cell-wall) using RS. We analyzed samples of sunflower plant at different time points of normal growth, and those with Phomopsis or dead arm stem canker disease. RS was performed on thin sections of the stem of growing plant using specialized test beds, and on dried thick lignified stem of diseased plant. The signatures were then analyzed to identify composition and underlying structural changes in cells-walls. Specifically, we focused on primary metabolites such as proteins, amino acids, polysaccharides, and monosaccharides, which are responsible for plant growth and development.

Overall, we show that RS can be a valuable tool to gain structural and chemical changes in plants under varying physiological conditions (specifically normal growth and disease) without detailed sample preparation approach.

Aerogels have diverse properties that are appealing to medical applications. Combining these properties with those of natural polymers, like polysaccharides, may increase the applicability of aerogel materials in life sciences. Chitosan (Cs) and acemannan (AC) are biocompatible and biodegradable polysaccharides that can promote wound healing and may activate the immune system, inducing a faster recognition of foreign antigens (viruses, bacteria, etc.) for their elimination. A dressing type aerogel biomaterial with distinctive features suitable for wound healing could be obtained with Cs and AC. Pursuing this goal, herein is reported the formation of Cs-AC mixed aerogels and partial characterization. Physical hydrogels were obtained from several mixtures of Cs and AC. The obtained gels were dried by supercritical CO₂ treatment. Morphological and diverse physicochemical characteristics were analyzed. The obtained aerogels were highly porous materials with mesoporous structures (10-40 nm pore size), low density (0.1–0.46 g/cm³) and high specific surface areas (150-300 m²/g). The obtained results suggest that these type of materials can provide high liquid absorption rates and enhanced interphase interaction in living tissues that could lead to a faster and efficient wound healing. The next goal of our investigation is to determine the biocompatibility and the ability of this aerogels to induce the production of pro-inflammatory and anti-inflammatory cytokines in different cell lines in vitro.

Liquid uni-directional transport on solid surface without energy input would advance a variety of applications, such as in bio-fluidic devices, self-lubrication, and highresolution printing. Inspired by the liquid uni-directional transportation on the peristome surface of Nepenthes alata, here, we fabricated a peristome-mimicking surface through high-resolution stereo-lithography and demonstrated the detailed uni-directional transportation mechanism from a micro-scaled view visualized through X-ray microscopy. Significantly, an overflow-controlled liquid uni-directional transportation mechanism is proposed and demonstrated. Unlike the canonical predictions for completely wetting liquids spreading symmetrically on a high-energy surface, liquids with varied surface tensions and viscosities can spontaneously propagate in a single preferred direction and pin in all others.
The fundamental understanding gained from this robust system enabled us to tailor advanced micro-computerized tomography scanning and stereo-lithography fabrication to mimic natural creatures and construct a wide variety of fluidic machines out of traditional materials.

The aim of this study was to prepare nanocomposite hollow fiber membrane to achieve higher hemocompatibility and uremic toxin separation performance. Nanocomposite zeolite based hollow fiber membranes (HFMs) were synthesized using polyethersulfone (P) as a base polymer, vitamin E TPGS (T) as an additive, and nano-zeolite (NZ) as a filler. The effect of different concentration of nanozeolite was seen. Such membranes would have widespread applications, but one goal was to improve outcomes in hemodialysis for kidney failure patients. The resulting nanocomposite membranes materials (called PT-NZ) were spun based on dry-wet spinning method based on phase inversion. The additive and filler helped nodular organization of the polymer into nano-sized domains with numerous pores inbetween, and improved transport properties. They also helped with presenting a more biocompatible surface to the blood and thereby improved hemocompatibility. The PT-NZ membranes were used to fabricate modules consisting of bundles of fibers, and the modules were, in turn, fabricated into mini dialysers. The ultrafiltration coefficient of such PT-NZ HFM-based module (of about 274 mL/m²-hrmm of Hg) was about 1.5-times higher than that of the commercial (F60S) membrane (about 152 mL/m²-hr-mm of Hg). The bovine serum albumin (BSA) rejection in aqueous mixtures was found 93.98 %. The toxin clearance performance of lab-scale PT-NZ HFM-based hemodialyzer with uremic toxin spiked goat blood was remarkably higher (about 5X more reduction ratio) than that of commercial F60S hollow fibers. The newly made HFMs reported here could help in decreasing the total treatment time and reducing side-reactions during dialysis for those end stage kidney disease (ESRD) patients dependent on hemodialysis. Hence, the synthesized PT-NZ HFMs can be a potential membrane material for the hemodialysis application.