Long-Term Culture Performance of a Polyelectrolyte Complex Microcapsule Platform for Hyaline Cartilage Repair.

Articular cartilage (AC) tissue repair and regeneration remains an ongoing challenge. One component of the challenge is the limited ability to scale an engineered cartilage graft to clinically relevant sizes while maintaining uniform properties. In this paper, we report on the evaluation of our polyelectrolyte complex microcapsule (PECM) platform technology as a technique for generating cartilage-like spherical modules. Bone marrow-derived mesenchymal stem cells (bMSCs) or primary articular chondrocytes were encapsulated within PECMs composed of methacrylated hyaluronan, collagen I, and chitosan. The formation of cartilage-like tissue in the PECMs over a 90-day culture was characterized. The results showed that chondrocytes exhibited superior growth and matrix deposition compared to either chondrogenically-induced bMSCs or a mixed PECM culture containing both chondrocytes and bMSCs. The chondrocyte-generated matrix filled the PECM and produced substantial increases in capsule compressive strength. The PECM system thus appears to support intracapsular cartilage tissue formation and the capsule approach promotes efficient culture and handling of these micro tissues. Since previous studies have proven the feasibility of fusing such capsules into large tissue constructs, the results suggest that encapsulating primary chondrocytes in PECM modules may be a viable route toward achieving a functional articular cartilage graft.

Imaging of Chronic Kidney Disease-Mineral and Bone Disorder.

The characteristic radiological appearances of metabolic bone and soft tissue diseases in chronic renal failure are described and illustrated in the context of advancing understanding of the complex metabolic changes that occur in chronic kidney disease and its management.

Transport Analysis of Engineered Liver Tissue Fabricated Using a Capsule-Based, Modular Approach.

The bioinspired, microscale tissue engineering approach has emerged in recent years as a promising alternative to preformed scaffolds. Using this approach, complex tissues and organs can be efficiently engineered from microscale modules to replicate the intricate architecture and physiology of vascularized organs and tissues. Previously, we demonstrated assembly of a prototype, engineered liver tissue, formed by the fusion of hepatocyte-containing capsules. Here, we analyzed the effects of various controllable system parameters with the aim of predicting the operating limits of our modular tissue in high cell density, perfused cultures. Both the capsule diameter and construct height were limited by mass transfer requirements, while the shear stress on the capsule wall and the pressure drop across the packed capsule bed were dictated by the capsule diameter and permissible flow rates of the system. Our analysis predicts that capsules with a 200 µm radius can efficiently maintain hepatocytes at cell densities comparable to liver tissue. Some model predictions were validated by packed bed perfusion cultures. Flow-induced bed compaction hysteresis was tested experimentally and found to have minimal effect on flow characteristics. The effectiveness factor (η) for the overall oxygen transfer within packed beds of capsule modules was estimated to be 0.72 for all conditions. Primary hepatocytes encapsulated in the capsules exhibited normal metabolism and formed spheroids during a 7-day culture. The model predictions can be useful to study mass transfer and shear stress in high-density perfusion cultures. Overall, analysis of a perfused, capsule-based, modular tissue demonstrated the feasibility of the technology as a platform for fabrication of highly metabolic solid organs.

Scalable MSC-derived bone tissue modules: In vitro assessment of differentiation, matrix deposition, and compressive load bearing.

Enhancements to the mechanical properties of modular designs for bone tissue engineering could increase their clinical applications. In this study, bone marrow mesenchymal stem cells (MSCs) and hydroxyapatite (HAP) microgranules were encapsulated in polyelectrolyte complex membranes composed of chondroitin 4-sulfate (C4S), carboxymethyl cellulose (CMC) and chitosan. Microcapsules were formed with and without HAP microgranules, and cultured in either osteoinduction medium (Osteo) or expansion medium (Exp) to produce four microcapsule conditions: Osteo, Osteo+HAP, Exp, and Exp+HAP. Microcapsules facilitated alkaline phosphatase secretion and deposition of bone specific proteins (osteocalcin and osteopontin) by encapsulated MSCs over 28 days of osteogenic culture. SEM and micro-CT analysis showed cell-deposited mineral covering the surfaces of the HAP microgranules and interior of the microcapsule membrane. The mineralized microcapsules could be combined and fused into cylindrical constructs (4 × 5 mm, W × H), and uniaxial compression tests confirmed that microcapsule mineralization greatly enhanced the yield stresses of Osteo and Osteo+HAP fused constructs (10.4 ±â€¯4.4 MPa and 6.4 ±â€¯2.8 MPa), compared to only HAP microgranules (Exp+HAP, 0.5 ±â€¯0.3 MPa). The C4S/CMC/Chitosan microcapsules provide a platform allowing pre-mineralization of microcapsules in vitro for later assembly of larger load-bearing constructs, or for use as an injectable bone regeneration strategy. STATEMENT OF SIGNIFICANCE: Clinical translation of bone tissue engineering is limited by the difficulty of generating space filling implants that both resist compressive loading, and simultaneously deliver cells throughout the bone defect. Here, we present the design of a microcapsule system containing both stem cells capable of rebuilding bone tissue, and a mechanically tough bone-like mineral, that imparts compression resistance to the microcapsules. The microcapsules support stem cell differentiation to an osteogenic phenotype, that can mineralize the microcapsule membrane and interior. The mineralized microcapsules can be assembled into larger bone constructs, and have mechanical properties on par with trabecular bone.

Biomimetic Scaffolds in Skeletal Muscle Regeneration.

Skeletal muscle tissue has inherent capacity for regeneration in response to minor injuries. However, in the case of severe trauma, tumor ablations, or in congenital muscle defects, these myopathies can cause irreversible loss of muscle mass and function, a condition referred to as volumetric muscle loss (VML). The natural muscle repair mechanisms are overwhelmed, prompting the search for new muscle regenerative strategies, such as using biomaterials that can provide regenerative signals to either transplanted or host muscle cells. Recent studies involve the use of suitable biomaterials which may be utilized as a template to guide tissue reorganization and ultimately provide optimum micro-environmental conditions to cells. These strategies range from approaches that utilize biomaterials alone to those that combine materials with exogenous growth factors, and ex vivo cultured cells. A number of scaffold materials have been used in the development of grafts to treat VML. In this brief review, we outline the natural skeletal regeneration process, available treatments used in the clinic for muscle injury and promising tissue bioengineering and regenerative approaches for muscle loss treatment.

Encapsulation of mesenchymal stem cells in glycosaminoglycans-chitosan polyelectrolyte microcapsules using electrospraying technique: Investigating capsule morphology and cell viability.

Polyelectrolyte microcapsules are modular constructs which facilitate cell handling and assembly of cell-based tissue constructs. In this study, an electrospray (ES) encapsulation apparatus was developed for the encapsulation of mesenchymal stem cells (MSCs). Ionic complexation between glycosaminoglycans (GAGs) and chitosan formed a polyelectrolyte complex membrane at the interface. To optimize the capsules, the effect of voltage, needle size and GAG formulation on capsule size were investigated. It was observed that by increasing the voltage and decreasing the needle size, the capsule size would decrease but at voltages above 12 kV, capsule size distribution broadened significantly which yields lower circularity. Increase in GAG viscosity resulted in larger microcapsules and cell viability exhibited no significant changes during the encapsulation procedure. These results suggest that ES is a highly efficient, and scalable approach to the encapsulation of MSCs for subsequent use in bioprinting and other modular tissue engineering or regenerative medicine applications.

Structural and functional insights into RHA-P, a bacterial GH106 α-L-rhamnosidase from Novosphingobium sp. PP1Y.

α-L-Rhamnosidases (α-RHAs, EC 3.2.1.40) are glycosyl hydrolases (GHs) hydrolyzing terminal α-l-rhamnose residues from different substrates such as heteropolysaccharides, glycosylated proteins and natural flavonoids. Although the possibility to hydrolyze rhamnose from natural flavonoids has boosted the use of these enzymes in several biotechnological applications over the past decades, to date only few bacterial rhamnosidases have been fully characterized and only one crystal structure of a rhamnosidase of the GH106 family has been described. In our previous work, an α-l-rhamnosidase belonging to this family, named RHA-P, was isolated from the marine microorganism Novosphingobium sp. PP1Y. The initial biochemical characterization highlighted the biotechnological potential of RHA-P for bioconversion applications. In this work, further functional and structural characterization of the enzyme is provided. The recombinant protein was obtained fused to a C-terminal His-tag and, starting from the periplasmic fractions of induced recombinant cells of E. coli strain BL21(DE3), was purified through a single step purification protocol. Homology modeling of RHA-P in combination with a site directed mutagenesis analysis confirmed the function of residues D503, E506, E644, likely located at the catalytic site of RHA-P. In addition, a kinetic characterization of the enzyme on natural flavonoids such as naringin, rutin, hesperidin and quercitrin was performed. RHA-P showed activity on all flavonoids tested, with a catalytic efficiency comparable or even higher than other bacterial α-RHAs described in literature. The results confirm that RHA-P is able to hydrolyze both α-1,2 and α-1,6 glycosidic linkages, and suggest that the enzyme may locate different polyphenolic aromatic moities in the active site.

Metabolic Oscillations in Co-Cultures of Hepatocytes and Mesenchymal Stem Cells: Effects of Seeding Arrangement and Culture Mixing.

In vitro assembly of functional liver tissue is a prerequisite for the transplantation of tissue-engineered livers. There is an increasing demand for in vitro models that replicate complex events occurring in the liver. However, tissue engineering of implantable liver systems is currently limited by the difficulty of assembling three dimensional hepatocyte cultures of a useful size, while maintaining full cell viability. Recent reports have demonstrated that bone marrow mesenchymal stem cells (BM-MSCs) can provide a number of cues promoting hepatocyte growth and development. In this study, the effects of BM-MSCs co-culture on hepatocyte metabolism were evaluated as a function of scaffold seeding arrangement. BM-MSCs were co-cultured with hepatocytes in porous chitosan-heparin scaffolds using several seeding arrangements. The seeded scaffolds were subjected to orbital shaking to enhance mass transfer. BM-MSC-hepatocyte co-cultures exhibited higher rates of hepatocyte-specific functions, compared to hepatocyte-only cultures, regardless of the seeding arrangement. Cells formed smaller-compact spheroids in the heterotypic systems compared to mono-cultures of hepatocytes only. The spheroids exhibited reduction in size with time in all conditions except for the condition where BM-MSCs were seeded one day after seeding hepatocytes. In this condition, spheroids increased in size due to BM-MSC proliferation. Spheroid size reduction was hypothesized to be the result of cyclic shear stresses generated by the orbital shaking. Furthermore, results suggested that BM-MSC seeding onto preformed hepatocyte spheroids provide a degree of shear-protection and trophic stimuli. Overall, the results indicate that co-culturing hepatocytes with BM-MSCs enhanced their metabolic functions for the first week of culture. J. Cell. Biochem. 118: 3003-3015, 2017. © 2017 Wiley Periodicals, Inc.

Morphological and growth responses of vascular smooth muscle and endothelial cells cultured on immobilized heparin and dextran sulfate surfaces.

Heparin has shown promise as a component of various biomaterial formulations, but its variable properties and inhibitory effects on some cell types have raised interest in use of dextran sulfate as an alternative. In this study, we characterized the interactions of vascular smooth muscle (SMC) and endothelial cells (EC) with heparin and dextran sulfate immobilized onto chitosan-based films. Films were modified by blending chitosan with type I collagen and covalently attaching heparin or dextran sulfate at various levels. Cell-material interactions were evaluated by quantifying cell spreading, shape and proliferation rate. ECs proliferated well on chitosan, but the polymer was a mediocre substrate for SMC growth. Immobilizing heparin on chitosan further inhibited SMC proliferation. However, blending collagen reversed the heparin inhibition of SMC growth, resulting in a pro-proliferative effect of heparin immobilized on chitosan-collagen films. Dextran sulfate surfaces supported both SMC and EC proliferation with or without the presence of collagen. The results indicate that inhibitory effects of heparin on SMC are reversed by immobilization in the presence of collagen, and that dextran sulfate may be superior to heparin as a biomaterial additive for promoting vascular cell growth in chitosan-based scaffolds. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1725-1735, 2017.

Chitosan films with improved tensile strength and toughness from N-acetyl-cysteine mediated disulfide bonds.

To improve the mechanical properties of chitosan (Ct) materials without the use of cytotoxic crosslinkers, disulfide cross-linkable Ct was synthesized by grafting N-acetyl-cysteine (NAC) to Ct using carbodiimide chemistry. Cast films of NAC-Ct conjugates were prepared with degrees of substitution (DS) of 0%, 6%, 15%, and 20%, and the disulfide bond formation was induced by increasing the reaction media pH to 11. The tensile strength, breaking strain, elastic moduli and toughness of disulfide cross-linked polymers were analyzed by monotonic tensile testing of hydrated NAC-Ct films. Crystallinity was determined via XRD. Results demonstrated that NAC incorporation and crosslinking in chitosan produced tougher polymer films with 4-fold higher tensile strength (10 MPa) and 6-fold greater elongation (365%), but reduced crystallinity, compared to unmodified chitosan. The resilience of NAC-Ct films was evaluated by cyclic testing, and results demonstrate that increasing NAC content produced a more resilient material that dissipated less energy when deformed. These improved mechanical properties broaden chitosan's applicability towards the construction of mechanically robust implantable scaffolds for tissue regeneration.

Flow perfusion culture of MC3T3-E1 osteogenic cells on gradient calcium polyphosphate scaffolds with different pore sizes.

Calcium polyphosphate is a biodegradable bone substitute. It remains a challenge to prepare porous calcium polyphosphate with desired gradient porous structures. In this study, a modified one-step gravity sintering method was used to prepare calcium polyphosphate scaffolds with desired-gradient-pore-size distribution. The differences of porous structure, mechanical strength, and degradation rate between gradient and homogenous calcium polyphosphate scaffolds were evaluated by micro-computed tomography, scanning electron microscopy, and mechanical testing. Preosteoblastic MC3T3-E1 cells were seeded onto gradient and homogenous calcium polyphosphate scaffolds and cultured in a flow perfusion bioreactor. The distribution, proliferation, and differentiation of the MC3T3-E1 cells were compared to that of homogenous calcium polyphosphate scaffolds. Though no significant difference of cell proliferation was found between the gradient and the homogenous calcium polyphosphate scaffolds, a much higher cell differentiation and mineralization were observed in the gradient calcium polyphosphate scaffolds than that of the homogenous calcium polyphosphate scaffolds, as manifested by increased alkaline phosphatase activity (p < 0.05). The improved distribution and differentiation of cultured cells within gradient scaffolds were further supported by both (18)F-fluorine micro-positron emission tomography scanning and in vitro tetracycline labeling. We conclude that the calcium polyphosphate scaffold with gradient pore sizes enhances osteogenic cell differentiation as well as mineralization. The in vivo performance of gradient calcium polyphosphate scaffolds warrants further investigation in animal bone defect models.

Biomechanical Characterization of a Model of Noninvasive, Traumatic Anterior Cruciate Ligament Injury in the Rat.

The onset of post-traumatic osteoarthritis (PTOA) remains prevalent following traumatic joint injury such as anterior cruciate ligament (ACL) rupture, and animal models are important for studying the pathomechanisms of PTOA. Noninvasive ACL injury using the tibial compression model in the rat has not been characterized, and it may represent a more clinically relevant model than the common surgical ACL transection model. This study employed four loading profiles to induce ACL injury, in which motion capture analysis was performed, followed by quantitative joint laxity testing. High-speed, high-displacement loading repeatedly induces complete ACL injury, which causes significant increases in anterior-posterior and varus laxity. No loading protocol induced valgus laxity. Tibial internal rotation and anterior subluxation occurs up to the point of ACL failure, after which the tibia rotates externally as it subluxes over the femoral condyles. High displacement was more determinative of ACL injury compared to high speed. Low-speed protocols induced ACL avulsion from the femoral footprint whereas high-speed protocols caused either midsubstance rupture, avulsion, or a combination injury of avulsion and midsubstance rupture. This repeatable, noninvasive ACL injury protocol can be utilized in studies assessing PTOA or ACL reconstruction in the rat.

A glycosaminoglycan based, modular tissue scaffold system for rapid assembly of perfusable, high cell density, engineered tissues.

The limited ability to vascularize and perfuse thick, cell-laden tissue constructs has hindered efforts to engineer complex tissues and organs, including liver, heart and kidney. The emerging field of modular tissue engineering aims to address this limitation by fabricating constructs from the bottom up, with the objective of recreating native tissue architecture and promoting extensive vascularization. In this paper, we report the elements of a simple yet efficient method for fabricating vascularized tissue constructs by fusing biodegradable microcapsules with tunable interior environments. Parenchymal cells of various types, (i.e. trophoblasts, vascular smooth muscle cells, hepatocytes) were suspended in glycosaminoglycan (GAG) solutions (4%/1.5% chondroitin sulfate/carboxymethyl cellulose, or 1.5 wt% hyaluronan) and encapsulated by forming chitosan-GAG polyelectrolyte complex membranes around droplets of the cell suspension. The interior capsule environment could be further tuned by blending collagen with or suspending microcarriers in the GAG solution These capsule modules were seeded externally with vascular endothelial cells (VEC), and subsequently fused into tissue constructs possessing VEC-lined, inter-capsule channels. The microcapsules supported high density growth achieving clinically significant cell densities. Fusion of the endothelialized, capsules generated three dimensional constructs with an embedded network of interconnected channels that enabled long-term perfusion culture of the construct. A prototype, engineered liver tissue, formed by fusion of hepatocyte-containing capsules exhibited urea synthesis rates and albumin synthesis rates comparable to standard collagen sandwich hepatocyte cultures. The capsule based, modular approach described here has the potential to allow rapid assembly of tissue constructs with clinically significant cell densities, uniform cell distribution, and endothelialized, perfusable channels.

Chitosan fibers with improved biological and mechanical properties for tissue engineering applications.

The low mechanical properties of hydrogel materials such as chitosan hinder their broad utility for tissue engineering applications. Previous research efforts improved the mechanical properties of chitosan fiber through chemical and physical modifications; however, unfavorable toxicity effects on cells were reported. In this paper, we report the preparation of chitosan fibers with improved mechanical and biocompatibility properties. The structure-property relationships of extruded chitosan fibers were explored by varying acetic acid (AA) concentration, ammonia concentration, annealing temperature and degree of heparin crosslinking. Results showed that optimizing AA concentration to 2vol% improved fiber strength and stiffness by 2-fold. Extruding chitosan solution into 25wt% of ammonia solution reduced fiber diameters and improved fiber strength by 2-fold and stiffness by 3-fold, due to an increase in crystallinity as confirmed by XRD. Fiber annealing further reduced fiber diameter and improved fiber strength and stiffness as temperature increased. Chitosan fibers crosslinked with heparin had increased diameter but lower strength and stiffness properties and higher breaking strain values. When individual parameters were combined, further improvement in fiber mechanical properties was achieved. All mechanically improved fibers and heparin crosslinked fibers promoted valvular interstitial cells (VIC) attachment and growth over 10 day cultures. Our results demonstrate the ability to substantially improve the mechanical properties of chitosan fibers without adversely affecting their biological properties. The investigated treatments offer numerous advantages over previous physical/chemical modifications and thus are expected to expand the utility of chitosan fibers with tunable mechanical properties in various tissue engineering applications.

Nanopatterning effects on astrocyte reactivity.

An array of design strategies have been targeted toward minimizing failure of implanted microelectrodes by minimizing the chronic glial scar around the microelectrode under chronic conditions. Current approaches toward inhibiting the initiation of glial scarring range from altering the geometry, roughness, size, shape, and materials of the device. Studies have shown materials which mimic the nanotopography of the natural environment in vivo will consequently result in an improved biocompatible response. Nanofabrication of electrode arrays is being pursued in the field of neuronal electrophysiology to increase sampling capabilities. Literature shows a gap in research of nanotopography influence in the reduction of astrogliosis. The aim of this study was to determine optimal feature sizes for neural electrode fabrication, which was defined as eliciting a nonreactive astrocytic response. Nanopatterned surfaces were fabricated with nanoimprint lithography on poly(methyl methacrylate) surfaces. The rate of protein adsorption, quantity of protein adsorption, cell alignment, morphology, adhesion, proliferation, viability, and gene expression was compared between nanopatterned surfaces of different dimensions and non-nanopatterned control surfaces. Results of this study revealed that 3600 nanopatterned surfaces elicited less of a response when compared with the other patterned and non-nanopatterned surfaces. The surface instigated cell alignment along the nanopattern, less protein adsorption, less cell adhesion, proliferation and viability, inhibition of glial fibrillary acidic protein, and mitogen-activated protein kinase kinase 1 compared with all other substrates tested.

Improving the mechanical properties of chitosan-based heart valve scaffolds using chitosan fibers.

Chitosan is being widely studied for tissue engineering applications due to its biocompatibility and biodegradability. However, its use in load-bearing applications is limited due to low mechanical properties. In this study, we investigated the effectiveness of a chitosan fiber reinforcement approach to enhancing the mechanical properties of chitosan scaffolds. Chitosan fibers were fabricated using a solution extrusion and neutralization method and incorporated into porous chitosan scaffolds. The effects of fiber/scaffold mass ratio, fiber mechanical properties and fiber length on scaffold mechanical properties were studied. The results showed that incorporating fibers improved scaffold strength and stiffness in proportion to the fiber/scaffold mass ratio. A fiber-reinforced, heart valve scaffold achieved leaflet tensile strength values of 220±17 kPa, comparable to the radial values of human pulmonary valve leaflets. Additionally, the effects of 2 mm fibers were found to be up to threefold greater than 10 mm fibers at identical mass ratios. Heparin crosslinking of fibers produced a reduction in fiber strength, and thus failed to produce additional improvements to fiber-reinforced scaffold properties. Despite this reduction in fiber strength, heparin-modified fibers still improved the mechanical properties of reinforced scaffolds, but to a lesser extent than unmodified fibers. The results demonstrate that chitosan fiber reinforcement can be used to achieve porous chitosan scaffold strength approaching that of tissue, and that fiber length and mechanical properties are important parameters in defining the degree of mechanical improvement.

Covalently immobilized glycosaminoglycans enhance megakaryocyte progenitor expansion and platelet release.

Application of umbilical cord blood (UCB) transplantation in adults as a treatment post-chemotherapy is hampered due to delayed platelet recovery. A potential solution suggested is the transfusion of ex vivo expanded megakaryocytes (Mks) from hematopoietic stem cells (HSCs). Alternatively, large-scale production of platelets in vitro has also been attempted with the goal of transfusing them into patients with thrombocytopenia. Glycosaminoglycans (GAGs) have been shown to influence the proliferation and differentiation of HSCs. This study sought to examine the effects of immobilized GAGs on the expansion, apoptosis, and platelet release activity of CD41a+ Mk progenitors in vitro. Freshly isolated HSCs from UCB were cultured in serum-free media supplemented with thrombopoietin on GAG-derivatized chitosan membranes for 17 days. Controls consisted of uncoated and chitosan-coated wells. Wells were demidepopulated at periodic intervals and analyzed by flow cytometry. Heparin and dermatan sulfate surfaces significantly enhanced total cell and Mk cell expansion (p < 0.05) compared to both the controls. The apoptotic Mk fraction was significantly lower on GAG surfaces (p < 0.05) compared to the polystyrene control during the early stages of the culture (days 7 and 11). However, by day 17, the apoptotic Mk fraction was comparable on all surfaces. The cumulative number of platelets generated on dermatan sulfate and heparan sulfate surfaces was significantly higher (p < 0.05) than on both the controls. These results suggest that immobilized GAGs delay Mk apoptosis and thereby enhance Mk expansion and platelet production.

Optimization-based metabolic control analysis.

In this work, a novel optimization-based metabolic control analysis (OMCA) method is introduced for reducing data requirement for metabolic control analysis (MCA). It is postulated that using the optimal control approach, the fluxes in a metabolic network are correlated to metabolite concentrations and enzyme activities as a state-feedback control system that is optimal with respect to a homeostasis objective. It is then shown that the optimal feedback gains are directly related to the elasticity coefficients (ECs) of MCA. This approach requires determination of the relative "importance" of metabolites and fluxes for the system, which is possible with significantly reduced experimental data, as compared with typical MCA requirements. The OMCA approach is applied to a top-down control model of glycolysis in hepatocytes. It is statistically demonstrated that the OMCA model is capable of predicting the ECs observed experimentally with few exceptions. Further, an OMCA-based model reconciliation study shows that the modification of four assumed stoichiometric coefficients in the model can explain most of the discrepancies, with the exception of elasticities with respect to the NADH/NAD ratio.

Thymosin beta4 and corneal wound healing: visions of the future.

Persistent corneal epithelial defects and inflammation within the central cornea can directly distort visual acuity and may lead to permanent visual loss. Therefore, treatments with agents that enhance corneal reepithelialization and regulate the inflammatory response without the deleterious side effects of currently used agents such as corticosteroids would result in improved clinical outcome and would represent a major advance in the field. Despite much progress in the areas of corneal wound healing research, clinically available pharmacological therapies that can promote repair and limit the visual complications from persistent corneal wounds are severely limited and remains a major deficiency in the field. Prior studies from our laboratory have demonstrated the potent wound healing and anti-inflammatory effects of thymosin beta4 (Tbeta(4); Tbeta4) in numerous models of corneal injury. We are studying the mechanisms by which Tbeta(4) suppresses inflammation and promotes repair. Herein, we discuss some of our new basic scientific directions that may lead to the use of Tbeta(4) as a novel corneal wound healing and anti-inflammatory therapy.

Membrane thickness is an important variable in membrane scaffolds: Influence of chitosan membrane structure on the behavior of cells.

Cell and tissue responses to polymeric materials are orchestrated in part by the conformations of adsorbed plasma proteins. Thus, the chemical properties of a polymer membrane that govern protein adsorption behavior can play an important role in determining the biological properties of tissue engineered scaffolds derived from that polymer. In this study, we explored the role of membrane thickness as a factor influencing cell adhesion and proliferation on chitosan membranes with and without covalently attached glycosaminoglycans. Rat mesenchymal stem cells (MSCs) cultured on chitosan membranes of various thicknesses demonstrated significantly improved cell adhesion, spreading and proliferation as membrane thickness was increased. Rat hepatocytes displayed increased spreading on the substrate with increasing membrane thickness, similar to MSCs. Increased thickness reduced the overall crystallinity of the membrane, and the data indicate that the improved cellular responses were likely due to enhanced adsorption of serum vitronectin, presumably due to reduced membrane crystallinity. These results demonstrate that membrane thickness is an important design variable that can be manipulated in chitosan-based scaffolds to achieve enhanced cell spreading, proliferation and function.