REDV-Functionalized Recombinant Spider Silk for Next-Generation Coronary Artery Stent Coatings: Hemocompatible, Drug-Eluting, and Endothelial Cell-Specific Materials.

Coronary artery stents are life-saving devices, and millions of these devices are implanted annually to treat coronary heart disease. The current gold standard in treatment is drug-eluting stents, which are coated with a biodegradable polymer layer that elutes antiproliferative drugs to prevent restenosis due to neointimal hyperplasia. Stenting is commonly paired with systemic antiplatelet therapy to prevent stent thrombosis. Despite their clinical success, current stents have significant limitations including inducing local inflammation that drives hyperplasia; a lack of hemocompatibility that promotes thrombosis, increasing need for antiplatelet therapy; and limited endothelialization, which is a critical step in the healing process. In this research, we designed a novel material for use as a next-generation coating for drug-eluting stents that addresses the limitations described above. Specifically, we developed a recombinant spider silk material that is functionalized with an REDV cell-adhesive ligand, a peptide motif that promotes specific adhesion of endothelial cells in the cardiovascular environment. We illustrated that this REDV-modified spider silk variant [eADF4(C16)-REDV] is an endothelial-cell-specific material that can promote the formation of a near-confluent endothelium. We additionally performed hemocompatibility assays using human whole blood and demonstrated that spider silk materials exhibit excellent hemocompatibility under both static and flow conditions. Furthermore, we showed that the material displayed slow enzyme-mediated degradation. Finally, we illustrated the ability to load and release the clinically relevant drug everolimus from recombinant spider silk coatings in a quantity and at a rate similar to that of commercial devices. These results support the use of REDV-functionalized recombinant spider silk as a coating for drug-eluting stents.

Bridging bench to body: ex vivo models to understand articular cartilage repair.

With little to no ability to self-regenerate, human cartilage defects of the knee remain a major clinical challenge. Tissue engineering strategies include delivering specific types of cells and biomaterials to the injured cartilage for restoration of architecture and function. Pre-clinical models to test the efficacy of the therapies come with high costs and ethical issues, and imperfect prediction of performance in humans. Ex vivo models represent an alternative avenue to trial cartilage tissue engineering. Defined as viable explanted cartilage samples, ex vivo models can be cultured with a cell-laden biomaterial or tissue-engineered construct to evaluate cartilage repair. Though human and animal ex vivo models are currently used in the field, there is a need for alternative methods to assess the strength of integration, to increase throughput and manage variability and to optimise and standardise culture conditions, enhancing the utility of these models overall.

Spontaneous Orthogonal Alignment of Smooth Muscle Cells and Endothelial Cells Captures Native Blood Vessel Morphology in Tissue-Engineered Vascular Grafts.

Tissue-engineered vascular grafts (TEVGs) have emerged as a potential alternative to autologous grafts for replacing small-diameter blood vessels during bypass surgery. The axial alignment of endothelial cells (ECs) and the circumferential alignment of smooth muscle cells (SMCs) are crucial for functional native blood vessels (NBVs). However, achieving this cellular alignment in TEVGs remains a formidable challenge. In this study, TEVGs were developed using a low-cost technique that aligned ECs axially and SMCs circumferentially within hours. The TEVGs comprised an electrospun polycaprolactone (PCL) layer and a gelatin methacryloyl (GelMA) cast layer. A freezing-induced alignment technique was developed that partially aligns the electrospun fibers axially, thereby promoting rapid axial alignment of ECs. Furthermore, SMCs cultured in a GelMA layer with intermediate stiffness (5-12 kPa) surrounding a PCL tube could promote conformation of the SMCs to the curvature of the PCL tube, resulting in their spontaneous circumferential alignment. Additionally, the TEVGs demonstrated mechanical properties similar to those of NBVs, which could facilitate future translation. This approach represents a significant advance in tissue engineering, enabling the fabrication of TEVGs with appropriate mechanical properties that recapitulate key NBV cell structural features within hours using a scalable and accessible method.

Using inorganic nanoparticles to fight fungal infections in the antimicrobial resistant era.

Fungal infections pose a serious threat to human health and livelihoods. The number and variety of clinically approved antifungal drugs is very limited, and the emergence and rapid spread of resistance to these drugs means the impact of fungal infections will increase in the future unless alternatives are found. Despite the significance and major challenges associated with fungal infections, this topic receives significantly less attention than bacterial infections. A major challenge in the development of fungi-specific drugs is that both fungi and mammalian cells are eukaryotic and have significant overlap in their cellular machinery. This lack of fungi-specific drug targets makes human cells vulnerable to toxic side effects from many antifungal agents. Furthermore, antifungal drug resistance necessitates higher doses of the drugs, leading to significant human toxicity. There is an urgent need for new antifungal agents, specifically those that can limit the emergence of new resistant species. Non-drug nanomaterials have primarily been explored as antibacterial agents in recent years; however, they are also a promising source of new antifungal candidates. Thus, this article reviews current research on the use of inorganic nanoparticles as antifungal agents. We also highlight challenges facing antifungal nanoparticles and discuss possible future research opportunities in this field. STATEMENT OF SIGNIFICANCE: Fungal infections pose a growing threat to human health and livelihood. The rapid spread of resistance to current antifungal drugs has led to an urgent need to develop alternative antifungals. Nanoparticles have many properties that could make them useful antimycotic agents. To the authors' knowledge, there is no published review so far that has comprehensively summarized the current development status of antifungal inorganic nanomaterials, so we decided to fill this gap. In this review, we discussed the state-of-the-art research on antifungal inorganic nanoparticles including metal, metal oxide, transition-metal dichalcogenides, and inorganic non-metallic particle systems. Future directions for the design of inorganic nanoparticles with higher antifungal efficacy and lower toxicity are described as a guide for further development in this important area.

Improvement of Mesenchymal Stromal Cell Proliferation and Differentiation via Decellularized Extracellular Matrix on Substrates With a Range of Surface Chemistries.

Decellularized extracellular matrix (dECM) deposited by mesenchymal stromal cells (MSCs) has emerged as a promising substrate for improved expansion of MSCs. To date, essentially all studies that have produced dECM for MSC expansion have done so on tissue culture plastic or glass. However, substrate surface chemistry has a profound impact on the adsorption of proteins that mediate cell-material interactions, and different surface chemistries can cause changes in cell behavior, ECM deposition, and the in vivo response to a material. This study tested the hypothesis that substrate surface chemistry impacts the deposition of ECM and its subsequent bioactivity. This hypothesis was tested by producing glass surfaces with various surface chemistries (amine, carboxylic acid, propyl, and octyl groups) using silane chemistry. ECM was deposited by an immortalized MSC line, decellularized, and characterized through SDS-PAGE and immunofluorescence microscopy. No significant difference was observed in dECM composition or microarchitecture on the different surfaces. The decellularized surfaces were seeded with primary MSCs and their proliferation and differentiation were assessed. The presence of dECM improved the proliferation of primary MSCs by ~100% in comparison to surface chemistry controls. Additionally, the adipogenesis increased by 50-90% on all dECM surfaces in comparison to surface chemistry controls, and the osteogenesis increased by ~50% on the octyl-modified surfaces when dECM was present. However, no statistically significant differences were observed within the set of dECM surfaces or control surfaces. These results support the null hypothesis, meaning surface chemistry (over the range tested in this work) is not a key regulator of the composition or bioactivity of MSC-derived dECM. These results are significant because they provide an important insight into regenerative engineering technologies. Specifically, the utilization of dECM in stem cell manufacturing and tissue engineering applications would require the dECM to be produced on a wide variety of substrates. This work indicates that it can be produced on materials with a range of surface chemistries without undesired changes in the bioactivity of the dECM.

A review of simulation training and new 3D computer-generated synthetic organs for robotic surgery education.

We conducted a comprehensive review of surgical simulation models used in robotic surgery education. We present an assessment of the validity and cost-effectiveness of virtual and augmented reality simulation, animal, cadaver and synthetic organ models. Face, content, construct, concurrent and predictive validity criteria were applied to each simulation model. There are six major commercial simulation machines available for robot-assisted surgery. The validity of virtual reality (VR) simulation curricula for psychomotor assessment and skill acquisition for the early phase of robotic surgery training has been demonstrated. The widespread adoption of VR simulation has been limited by the high cost of these machines. Live animal and cadavers have been the accepted standard for robotic surgical simulation since it began in the early 2000s. Our review found that there is a lack of evidence in the literature to support the use of animal and cadaver for robotic surgery training. The effectiveness of these models as a training tool is limited by logistical, ethical, financial and infection control issues. The latest evolution in synthetic organ model training for robotic surgery has been driven by new 3D-printing technology. Validated and cost-effective high-fidelity procedural models exist for robotic surgery training in urology. The development of synthetic models for the other specialties is not as mature. Expansion into multiple surgical disciplines and the widespread adoption of synthetic organ models for robotic simulation training will require the ability to engineer scalability for mass production. This would enable a transition in robotic surgical education where digital and synthetic organ models could be used in place of live animals and cadaver training to achieve robotic surgery competency.

The Challenge of Cartilage Integration: Understanding a Major Barrier to Chondral Repair.

Articular cartilage defects caused by injury frequently lead to osteoarthritis, a painful and costly disease. Despite widely used surgical methods to treat articular cartilage defects and a plethora of research into regenerative strategies as treatments, long-term clinical outcomes are not satisfactory. Failure to integrate repair tissue with native cartilage is a recurring issue in surgical and tissue-engineered strategies, seeing eventual degradation of the regenerated or surrounding tissue. This review delves into the current understanding of why continuous and robust integration with native cartilage is so difficult to achieve. Both the intrinsic limitations of chondrocytes to remodel injured cartilage, and the significant challenges posed by a compromised biomechanical environment are described. Recent scaffold and cell-based techniques to repair cartilage are also discussed, and limitations of existing methods to evaluate integrative repair. In particular, the importance of evaluating the mechanical integrity of the interface between native and repair tissue is highlighted as a meaningful assessment of any strategy to repair this load-bearing tissue. Impact statement The failure to integrate grafts or biomaterials with native cartilage is a major barrier to cartilage repair. An in-depth understanding of the reasons cartilage integration remains a challenge is required to inform cartilage repair strategies. In particular, this review highlights that integration of cartilage repair strategies is frequently assessed in terms of the continuity of tissue, but not the mechanical integrity. Given the load-bearing nature of cartilage, evaluating integration in terms of interfacial strength is essential to assessing the potential success of cartilage repair methods.

Biomaterials functionalized with nanoclusters of integrin- and syndecan-binding ligands improve cell adhesion and mechanosensing under shear flow conditions.

We have engineered biomaterials that display nanoclusters of ligands that bind both integrin and syndecan-4 cell receptors. These surfaces regulate cell behaviors under static conditions including adhesion, spreading, actin stress fiber formation, and migration. The syndecan-4 receptors are also critical mediators of cellular mechanotransduction. In this contribution we assess whether this novel class of materials can regulate the response of cells to applied mechanical stimulation, using the shear stress imparted by laminar fluid flow as a model stimulus. Specifically, we assess endothelial cell detachment due to flow, cell alignment due to flow, and cell adhesion from the flowing fluid. A high degree of cell retention was observed on surfaces containing integrin-binding ligands or a mixed population of integrin- and syndecan-binding ligands. However, the presence of both ligand types was necessary for the cells to align in the direction of flow. These results imply that integrin engagement is necessary for adhesion strength, but engagement of both receptor types aids in appropriate mechanotransduction. Additionally, it was found that surfaces functionalized with both ligand types were able to scavenge a larger number of cells from flow, and to do so at a faster rate, compared to surfaces functionalized with only integrin- or syndecan-binding ligands. These results show that interfaces functionalized with both integrin- and syndecan-binding ligands regulate a significant range of biophysical cell behaviors in response to shear stress.

Microbial Transglutaminase Improves ex vivo Adhesion of Gelatin Methacryloyl Hydrogels to Human Cartilage.

Current surgical techniques to treat articular cartilage defects fail to produce a satisfactory long-term repair of the tissue. Regenerative approaches show promise in their ability to generate hyaline cartilage using biomaterials in combination with stem cells. However, the difficulty of seamlessly integrating the newly generated cartilage with the surrounding tissue remains a likely cause of long-term failure. To begin to address this integration issue, our strategy exploits a biological enzyme (microbial transglutaminase) to effect bioadhesion of a gelatin methacryloyl implant to host tissue. Mechanical characterization of the bioadhesive material shows that enzymatic crosslinking is compatible with photocrosslinking, allowing for a dual-crosslinked system with improved mechanical properties, and a slower degradation rate. Biocompatibility is illustrated with a 3D study of the metabolic activity of encapsulated human adipose derived stem cells. Furthermore, enzymatic crosslinking induced by transglutaminase is not prevented by the presence of cells, as measured by the bulk modulus of the material. Adhesion to human cartilage is demonstrated ex vivo with a significant increase in adhesive strength (5.82 ± 1.4 kPa as compared to 2.87 ± 0.9 kPa, p < 0.01) due to the addition of transglutaminase. For the first time, we have characterized a bioadhesive material composed of microbial transglutaminase and GelMA that can encapsulate cells, be photo crosslinked, and bond to host cartilage, taking a step toward the integration of regenerative implants.

Antimicrobial nanoparticle coatings for medical implants: Design challenges and prospects.

Microbial colonization, infection, and biofilm formation are major complications in the use of implants and are the predominant risk factors in implant failure. Although aseptic surgery and the administration of antimicrobial drugs may reduce the risk of infection, the systemic use of antibiotics can lead to a lack of efficacy, an increase in the risk of tissue toxicity, and the development of drug-resistant infections. To reduce implant-related infections, antimicrobial materials are increasingly being investigated and applied to implant surfaces using various methods depending on the agents and their microbicidal mechanisms. Through the development of biomaterials and nanotechnology, antimicrobial nanoparticles are becoming promising candidates for implant coatings, as their multifactorial antimicrobial mechanisms combat microbial adherence, viability, and biofilm formation. Despite their antimicrobial promise, the application of nanoparticles onto implant surfaces while retaining their antimicrobial potency faces many challenges. Herein, we review the potential and challenges associated with the design and implementation of antimicrobial nanoparticle coatings for the medical implant industry, particularly focusing on manufacturing considerations, sterilization, long-term stability, protein fouling, regulation, and safety, with a view to providing researchers the necessary tools to aid the translation of materials from the bench to the clinic.

Multifunctional Antimicrobial Polypeptide-Selenium Nanoparticles Combat Drug-Resistant Bacteria.

Antibiotic-resistant bacteria are a severe threat to human health. The World Health Organization's Global Antimicrobial Surveillance System has revealed widespread occurrence of antibiotic resistance among half a million patients across 22 countries, with Staphylococcus aureus, Escherichia coli, and Klebsiella pneumoniae being the most common resistant species. Antimicrobial nanoparticles are emerging as a promising alternative to antibiotics in the fight against antimicrobial resistance. In this work, selenium nanoparticles coated with the antimicrobial polypeptide, ε-poly-l-lysine, (Se NP-ε-PL) were synthesized and their antibacterial activity and cytotoxicity were investigated. Se NP-ε-PL exhibited significantly greater antibacterial activity against all eight bacterial species tested, including Gram-positive, Gram-negative, and drug-resistant strains, than their individual components, Se NP and ε-PL. The nanoparticles showed no toxicity toward human dermal fibroblasts at the minimum inhibitory concentrations, demonstrating a therapeutic window. Furthermore, unlike the conventional antibiotic kanamycin, Se NP-ε-PL did not readily induce resistance in E. coli or S. aureus. Specifically, S. aureus began to develop resistance to kanamycin from ∼44 generations, whereas it took ∼132 generations for resistance to develop to Se NP-ε-PL. Startlingly, E. coli was not able to develop resistance to the nanoparticles over ∼300 generations. These results indicate that the multifunctional approach of combining Se NP with ε-PL to form Se NP-ε-PL is a highly efficacious new strategy with wide-spectrum antibacterial activity, low cytotoxicity, and significant delays in development of resistance.

Enhanced Antibacterial Activity of Se Nanoparticles Upon Coating with Recombinant Spider Silk Protein eADF4(κ16).

PURPOSE: Selenium nanoparticles (Se NPs) are promising antibacterial agents to tackle the growing problem of antimicrobial resistance. The aim of this study was to fabricate Se NPs with a net positive charge to enhance their antibacterial efficacy. METHODS: Se NPs were coated with a positively charged protein - recombinant spider silk protein eADF4(κ16) - to give them a net positive surface charge. Their cytotoxicity and antibacterial activity were investigated, with negatively charged polyvinyl alcohol coated Se NPs as a control. Besides, these eADF4(κ16)-coated Se NPs were immobilized on the spider silk films, and the antibacterial activity of these films was investigated. RESULTS: Compared to the negatively charged polyvinyl alcohol coated Se NPs, the positively charged eADF4(κ16)-coated Se NPs demonstrated a much higher bactericidal efficacy against the Gram-negative bacteria E. coli, with a minimum bactericidal concentration (MBC) approximately 50 times lower than that of negatively charged Se NPs. Cytotoxicity testing showed that the eADF4(κ16)-coated Se NPs are safe to both Balb/3T3 mouse embryo fibroblasts and HaCaT human skin keratinocytes up to 31 µg/mL, which is much higher than the MBC of these particles against E. coli (8 ± 1 µg/mL). In addition, antibacterial coatings were created by immobilising the eADF4(κ16)-coated Se NPs on positively charged spider silk films and these were shown to retain good bactericidal efficacy and overcome the issue of low particle stability in culture broth. It was found that these Se NPs needed to be released from the film surface in order to exert their antibacterial effects and this release can be regulated by the surface charge of the film, such as the change of the spider silk protein used. CONCLUSION: Overall, eADF4(κ16)-coated Se NPs are promising new antibacterial agents against life-threatening bacteria.

Spider-silk inspired polymeric networks by harnessing the mechanical potential of ß-sheets through network guided assembly.

The high toughness of natural spider-silk is attributed to their unique ß-sheet secondary structures. However, the preparation of mechanically strong ß-sheet rich materials remains a significant challenge due to challenges involved in processing the polymers/proteins, and managing the assembly of the hydrophobic residues. Inspired by spider-silk, our approach effectively utilizes the superior mechanical toughness and stability afforded by localised ß-sheet domains within an amorphous network. Using a grafting-from polymerisation approach within an amorphous hydrophilic network allows for spatially controlled growth of poly(valine) and poly(valine-r-glycine) as ß-sheet forming polypeptides via N-carboxyanhydride ring opening polymerisation. The resulting continuous ß-sheet nanocrystal network exhibits improved compressive strength and stiffness over the initial network lacking ß-sheets of up to 30 MPa (300 times greater than the initial network) and 6 MPa (100 times greater than the initial network) respectively. The network demonstrates improved resistance to strong acid, base and protein denaturants over 28 days.

Personalized, Mechanically Strong, and Biodegradable Coronary Artery Stents via Melt Electrowriting.

Biodegradable coronary artery stents are sought-after alternatives to permanent stents. These devices are designed to degrade after the blood vessel heals, leaving behind a regenerated artery. The original generation of clinically available biodegradable stents required significantly thicker struts (∼150 µm) than nondegradable ones to ensure sufficient mechanical strength. However, these thicker struts proved to be a key contributor to the clinical failure of the stents. A current challenge lies in the fabrication of stents that possess both thin struts and adequate mechanical strength. In this contribution, we describe a method for the bottom-up, additive manufacturing of biodegradable composite stents with ultrathin fibers and superior mechanical properties compared to the base polymer. Specifically, we illustrate that melt electrowriting (MEW) can be used to 3D print composite structures with thin struts (60-80 µm) and a high degree of geometric complexity required for stenting applications. Additionally, this technology allows additive manufacture of personalized stents that are customized to a patient's unique anatomy and disease state. Furthermore, we illustrate that polycaprolactone-reduced graphene oxide nanocomposites have superior mechanical properties compared to original polycaprolactone without detriment to the material's cytocompatibility and that customizable stent-like structures can be fabricated from these materials with struts as thin as 60 µm, well below the target value for clinical use of 80 µm.

Development of Macroporous Chitosan Scaffolds for Eyelid Tarsus Tissue Engineering.

Background: Reconstruction of large eyelid defects remains challenging due to the lack of suitable eyelid tarsus tissue substitutes. We aimed to evaluate a novel bioengineered chitosan scaffold for use as an eyelid tarsus substitute. Methods: Three-dimensional macroporous chitosan hydrogel scaffold were produced via cryogelation with specific biomechanical properties designed to directly match characteristics of native eyelid tarsus tissue. Scaffolds were characterized by confocal microscopy and tensile mechanical testing. To optimise biocompatibility, human eyelid skin fibroblasts were cultured from biopsy-sized samples of fresh eyelid skin. Immunological and gene expression analysis including specific fibroblast-specific markers were used to determine the rate of fibroblast de-differentiation in vitro and characterize cells cultured. Eyelid skin fibroblasts were then cultured over the chitosan scaffolds and the resultant adhesion and growth of cells were characterized using immunocytochemical staining. Results: The chitosan scaffolds were shown to support the attachment and proliferation of NIH 3T3 mouse fibroblasts and human orbital skin fibroblasts in vitro. Our novel bioengineered chitosan scaffold has demonstrated biomechanical compatibility and has the ability to support human eyelid skin fibroblast growth and proliferation. Conclusions: This bioengineered tissue has the potential to be used as a tarsus substitute during eyelid reconstruction, offering the opportunity to pre-seed the patient's own cells and represents a truly personalised approach to tissue engineering.

Engineering highly effective antimicrobial selenium nanoparticles through control of particle size.

The overuse of antibiotics has induced the rapid development of antibiotic resistance in bacteria. As a result, antibiotic efficacy has become limited, and infection with multidrug-resistant bacteria is considered to be one of the largest global human health threats. Consequently, new, effective and safe antimicrobial agents need to be developed urgently. One promising candidate to address this requirement is selenium nanoparticles (Se NPs), which are made from the essential dietary trace element Se and have antimicrobial activity against Gram-positive bacteria. The size of nanomaterials can strongly affect their biophysical properties and functions; however, the effects of the size of Se NPs on their antibacterial efficacy has not been systematically investigated. Therefore, in this work, spherical Se NPs ranging from 43 to 205 nm in diameter were fabricated, and their mammalian cytotoxicity and antibacterial activity as a function of their size were systematically studied. The antibacterial activity of the Se NPs was shown to be strongly size dependent, with 81 nm Se NPs showing the maximal growth inhibition and killing effect of methicillin-sensitive and methicillin-resistant Staphylococcus aureus (MSSA and MRSA). The Se NPs were shown to have multi-modal mechanisms of action that depended on their size, including depleting internal ATP, inducing ROS production, and disrupting membrane potential. All the Se NPs were non-toxic towards mammalian cells up to 25 µg mL-1. Furthermore, the MIC value for the 81 nm particles produced in this research is 16 ± 7 µg mL-1, significantly lower than previously reported MIC values for Se NPs. This data illustrates that Se NP size is a facile yet critical and previously underappreciated parameter that can be tailored for maximal antimicrobial efficacy. We have identified that using Se NPs with a size of 81 nm and concentration of 10 µg mL-1 shows promise as a safe and efficient way to kill S. aureus without damaging mammalian cells.

Selenium nanoparticles as anti-infective implant coatings for trauma orthopedics against methicillin-resistant Staphylococcus aureus and epidermidis: in vitro and in vivo assessment.

Background: Bacterial infection is a common and serious complication in orthopedic implants following traumatic injury, which is often associated with extensive soft tissue damage and contaminated wounds. Multidrug-resistant bacteria have been found in these infected wounds, especially in patients who have multi trauma and prolonged stay in intensive care units.Purpose: The objective of this study was to develop a coating on orthopedic implants that is effective against drug-resistant bacteria. Methods and results: We applied nanoparticles (30-70nm) of the trace element selenium (Se) as a coating through surface-induced nucleation-deposition on titanium implants and investigated the antimicrobial activity against drug resistant bacteria including Methicillin-resistant Staphylococcus aureus (MRSA) and Methicillin-resistant Staphylococcus epidermidis (MRSE) in vitro and in an infected femur model in rats.The nanoparticles were shown in vitro to have antimicrobial activity at concentrations as low as 0.5ppm. The nanoparticle coatings strongly inhibited biofilm formation on the implants and reduced the number of viable bacteria in the surrounding tissue following inoculation of implants with biofilm forming doses of bacteria. Conclusion: This study shows a proof of concept for a selenium nanoparticle coatings as a potential anti-infective barrier for orthopedic medical devices in the setting of contamination with multi-resistant bacteria. It also represents one of the few (if only) in vivo assessment of selenium nanoparticle coatings on reducing antibiotic-resistant orthopedic implant infections.

Remote Control in Formation of 3D Multicellular Assemblies Using Magnetic Forces.

Cell constructs have been utilized as building blocks in tissue engineering to closely mimic the natural tissue and also overcome some of the limitations caused by two-dimensional cultures or using scaffolds. External forces can be used to enhance the cells' adhesion and interaction and thus provide better control over production of these structures compared to methods like cell seeding and migration. In this paper, we demonstrate an efficient method to generate uniform, three-dimensional cell constructs using magnetic forces. This method produced spheroids with higher densities and more symmetrical structures than the commonly used centrifugation method for production of cell spheroids. It was also shown that shape of the cell constructs could be changed readily by using different patterns of magnetic field. The application of magnetic fields to impart forces on the cells enhanced the fusion of these spheroids, which could be used to produce larger and more complicated structures for future tissue engineering applications.

Improved ex vivo expansion of mesenchymal stem cells on solubilized acellular fetal membranes.

Coatings produced from extracellular matrixes (ECMs) have emerged as promising surfaces for the improved ex vivo expansion of mesenchymal stem cells (MSCs). However, identifying a readily available source of ECM to generate these coatings is currently the bottleneck of this technology. In this study, we assessed if ECM coatings derived from decellularized fetal membranes were a suitable substrate for MSC expansion. We separated and decellularized the two main components of the fetal membranes, the amnion and the chorion. Characterization of the decellularized membranes revealed that each membrane component has a distinct composition, implying that coatings produced from these materials would have unique biological properties. The membranes were processed further to produce solubilized forms of the decellularized amniotic membrane (s-dAM) and decellularized chorionic membrane (s-dCM). On s-dAM coatings decidual MSCs (DMSC) were more proliferative than those cultured on tissue culture plastic alone or on Matrigel coatings; were smaller in size (a measure of MSC potency); exhibited greater adipogenic differentiation capacity; and improved osteogenic capacity. Additionally, long term culture studies showed late passage DMSCs (passage 8) cultured on s-dAM showed a decrease in cell diameter over three passages. These data support the use of s-dAM as a substrate for improved MSC expansion. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 232-242, 2019.

Beyond RGD; nanoclusters of syndecan- and integrin-binding ligands synergistically enhance cell/material interactions.

Biomaterials are a powerful platform for directing cellular behaviour. Herein, we employed a biomimetic strategy to synthesize a low-fouling polymer functionalized with nano-scale clusters of ligands that bind both integrin and syndecan-4 receptors, as both receptor types are critical in focal adhesion signalling and mechanotransduction. Our results demonstrate that the presence of both ligand types synergistically increases the adhesion of human umbilical vein endothelial cells (more than a two fold increase after 4 h) and increases the rate of surface endothelialization compared to surfaces functionalized with only one ligand type. Additionally, we observe that the mixed population of ligands regulates endothelial cell migration, likely due to improved focal adhesion formation as observed through confocal microscopy. Furthermore, we illustrate that only endothelial cells cultured on these mixed ligand surfaces exhibit the appropriate morphological changes - elongation and alignment in the direction of flow - when exposed to laminar shear flow, and neither of the individual ligands alone is sufficient. These results illustrate that both receptor types must be engaged for optimum cell-material interactions and are mandatory for appropriate mechanotransduction. The results presented in this manuscript will be critical for the development of next generation biomedical devices and tissue engineering scaffolds.