collagen-GAG Scaffolds Research Articles

The role of synovial fluid constituents in the lubrication of collagen-glycosaminoglycan scaffolds for cartilage repair

Extracellular matrix (ECM)-derived scaffolds have shown promise as tissue-engineered grafts for promoting cartilage repair. However, there has been a lack of focus on fine-tuning the frictional properties of scaffolds for cartilage tissue engineering as well as understanding their interactions with synovial fluid constituents. Proteoglycan-4 (PRG4) and hyaluronan (HA) are macromolecules within synovial fluid that play key roles as boundary mode lubricants during cartilage surface interactions. The overall objective of this study was to characterize the role PRG4 and HA play in the lubricating function of collagen-glycosaminoglycan (GAG) scaffolds for cartilage repair. As a first step towards this goal, we aimed to develop a suitable in vitro friction test to establish the boundary mode lubrication parameters for collagen-GAG scaffolds articulated against glass in a phosphate buffered saline (PBS) bath. Subsequently, we sought to leverage this system to determine the effect of physiological synovial fluid lubricants, PRG4 and HA, on the frictional properties of collagen-GAG scaffolds, with scaffolds hydrated in PBS and bovine synovial fluid (bSF) serving as negative and positive controls, respectively. At all compressive strains examined (ε = 0.1–0.5), fluid depressurization within hydrated collagen-GAG scaffolds was >99% complete at ½ minute. The coefficient of friction was stable at all compressive strains (ranging from a low 0.103 ± 0.010 at ε = 0.3 up to 0.121 ± 0.015 at ε = 0.4) and indicative of boundary-mode conditions. Immunohistochemistry demonstrated that PRG4 from recombinant human (rh) and bovine sources adsorbed to collagen-GAG scaffolds and the coefficient of friction for scaffolds immersed in rhPRG4 (0.067 ± 0.027) and normal bSF (0.056 ± 0.020) solution decreased compared to PBS (0.118 ± 0.21, both p < 0.05, at ε = 0.2). The ability of the adsorbed rhPRG4 to reduce friction on the scaffolds indicates that its incorporation within collagen-GAG biomaterials may enhance their lubricating ability as potential tissue-engineered cartilage replacements. To conclude, this study reports the development of an in vitro friction test capable of characterizing the coefficient of friction of ECM-derived scaffolds tested in a range of synovial fluid lubricants and demonstrates frictional properties as a potential design parameter for implants and materials for soft tissue replacement.

Three-dimensional characterization of collagen remodeling in cell-seeded collagen scaffolds via polarization second harmonic generation.

In this study, we use non-linear imaging microscopy to characterize the structural properties of porous collagen-GAG scaffolds (CGS) seeded with human umbilical vein endothelial cells (HUVECs), as well as human mesenchymal stem cells (hMSCs), a co-culture previously reported to form vessel-like structures inside CGS. The evolution of the resulting tissue construct was monitored over 10 days via simultaneous two- and three-photon excited fluorescence microscopy. Time-lapsed 2- and 3-photon excited fluorescence imaging was utilized to monitor the temporal evolution of the vascular-like structures up to 100 µm inside the scaffold up to 10 days post-seeding. 3D polarization-dependent second harmonic generation (PSHG) was utilized to monitor collagen-based scaffold remodeling and determine collagen fibril orientation up to 200 µm inside the scaffold. We demonstrate that polarization-dependent second harmonic generation can provide a novel way to quantify the reorganization of the collagen architecture in CGS simultaneously with key biomechanical interactions between seeded cells and CGS that regulate the formation of vessel-like structures inside 3D tissue constructs. A comparison between samples at different days in vitro revealed that gradually, the scaffolds developed an orthogonal net-like architecture, previously found in real skin.

The lubricating effect of iPS-reprogrammed fibroblasts on collagen-GAG scaffolds for cartilage repair applications

Tissue engineering products, like collagen-glycosaminoglycan scaffolds, have been successfully applied to chondrogenic defects. Inducible Pluripotent Stem cell (iPS) technology allows reprograming of somatic cells into an embryonic-like state, allowing for redifferentiation. We postulated that a fibroblast cell line (BJ cells – ‘pre-iPSF’) cycled through iPS reprogramming and redifferentiated into fibroblasts (post-iPSF) could lubricate collagen-glycosaminoglycan scaffolds; fibroblasts are known to produce lubricating molecules (e.g., lubricin) in the synovium. Herein, we quantified the coefficient of friction (CoF) of collagen-glycosaminoglycan scaffolds seeded with post-iPSF; tested whether cell-free scaffolds made of post-iPSF derived extracellular matrix had reduced friction vs. pre-iPSF; and assessed lubricin quantity as a possible protein responsible for lubrication. Post-iPSF seeded CG had 6- to 10-fold lower CoF versus pre-iPSF. Scaffolds consisting of a collagen and pre-/post-iPSF extracellular matrix blend outperformed these cell-seeded scaffolds (~5-fold lower CoF), yielding excellent CoF values close to synovial fluid. Staining revealed an increased presence of lubricin within post-iPSF scaffolds (confirmed by western blotting) and on the surface of iPSF-seeded collagen-glycosaminoglycan scaffolds. Interestingly, when primary cells from patient biopsy-derived fibroblasts were used, iPS reprogramming did not further reduce the already low CoF of these cells and no lubricin expression was found. We conclude that iPS reprogramming activates lubricating properties in iPS-derived cells in a source cell-specific manner. Additionally, lubricin appears to play a lubricating role, yet other proteins also contribute to lubrication. This work constitutes an important step for understanding post-iPSF lubrication of scaffolds and its potential for cartilage tissue engineering.

Functionalising Collagen-Based Scaffolds With Platelet-Rich Plasma for Enhanced Skin Wound Healing Potential.

Porous collagen-glycosaminoglycan (collagen-GAG) scaffolds have shown promising clinical results for wound healing; however, these scaffolds do not replace the dermal and epidermal layer simultaneously and rely on local endogenous signaling to direct healing. Functionalizing collagen-GAG scaffolds with signaling factors, and/or additional matrix molecules, could help overcome these challenges. An ideal candidate for this is platelet-rich plasma (PRP) as it is a natural reservoir of growth factors, can be activated to form a fibrin gel, and is available intraoperatively. We tested the factors released from PRP (PRPr) and found that at specific concentrations, PRPr enhanced cell proliferation and migration and induced angiogenesis to a greater extent than fetal bovine serum (FBS) controls. This motivated us to develop a strategy to successfully incorporate PRP homogeneously within the pores of the collagen-GAG scaffolds. The composite scaffold released key growth factors for wound healing (FGF, TGFβ) and vascularization (VEGF, PDGF) for up to 14 days. In addition, the composite scaffold had enhanced mechanical properties (when compared to PRP gel alone), while providing a continuous upper surface of extracellular matrix (ECM) for keratinocyte seeding. The levels of the factors released from the composite scaffold were sufficient to sustain proliferation of key cells involved in wound healing, including human endothelial cells, mesenchymal stromal cells, fibroblasts, and keratinocytes; even in the absence of FBS supplementation. In functional in vitro and in vivo vascularization assays, our composite scaffold demonstrated increased angiogenic and vascularization potential, which is known to lead to enhanced wound healing. Upon pro-inflammatory induction, macrophages released lower levels of the pro-inflammatory marker MIP-1α when treated with PRPr; and released higher levels of the anti-inflammatory marker IL1-ra upon both pro- and anti-inflammatory induction when treated with the composite scaffold. Finally, our composite scaffold supported a co-culture system of human fibroblasts and keratinocytes that resulted in an epidermal-like layer, with keratinocytes constrained to the surface of the scaffold; by contrast, keratinocytes were observed infiltrating the PRP-free scaffold. This novel composite scaffold has the potential for rapid translation to the clinic by isolating PRP from a patient intraoperatively and combining it with regulatory approved scaffolds to enhance wound repair.

The influence of cyclic tensile strain on multi-compartment collagen-GAG scaffolds for tendon-bone junction repair

ABSTRACTBackground: Orthopedic injuries often occur at the interface between soft tissues and bone. The tendon-bone junction (TBJ) is a classic example of such an interface. Current clinical strategies for TBJ injuries prioritize mechanical reattachment over regeneration of the native interface, resulting in poor outcomes. The need to promote regenerative healing of spatially-graded tissues inspires our effort to develop new tissue engineering technologies that replicate features of the spatially-graded extracellular matrix and strain profiles across the native TBJ.Materials and Methods: We recently described a biphasic collagen-glycosaminoglycan (CG) scaffold containing distinct compartment with divergent mineral content and structural alignment (isotropic vs. anisotropic) linked by a continuous interface zone to mimic structural and compositional features of the native TBJ.Results: Here, we report application of cyclic tensile strain (CTS) to the scaffold via a bioreactor leads to non-uniform strain profiles across the spatially-graded scaffold. Further, combinations of CTS and matrix structural features promote rapid, spatially-distinct differentiation profiles of human bone marrow-derived mesenchymal stem cells (MSCs) down multiple osteotendinous lineages. CTS preferentially upregulates MSC activity and tenogenic differentiation in the anisotropic region of the scaffold. This work demonstrates a tissue engineering approach that couples instructive biomaterials with cyclic tensile stimuli to promote regenerative healing of orthopedic interfaces.

Incorporating β-cyclodextrin into collagen scaffolds to sequester growth factors and modulate mesenchymal stem cell activity

The development of biomaterials for a range of tissue engineering applications increasingly requires control over the bioavailability of biomolecular cues such as growth factors in order to promote desired cell responses. While efforts have predominantly concentrated on covalently-bound or freely-diffusible incorporation of biomolecules in porous, three-dimensional biomaterials, opportunities exist to exploit transient interactions to concentrate growth factor activity over desired time frames. Here, we report the incorporation of β-cyclodextrin into a model collagen-GAG scaffold as a means to exploit the passive sequestration and release of growth factors via guest-host interactions to control mesenchymal stem cell differentiation. Collagen-GAG scaffolds that incorporate β-cyclodextrin show improved sequestration as well as extended retention and release of TGF-β1. We further show extended retention and release of TGF-β1 and BMP-2 from β-cyclodextrin modified scaffolds was sufficient to influence the metabolic activity and proliferation of mesenchymal stem cells as well as differential activation of Smad 2/3 and Smad 1/5/8 pathways associated with differential osteo-chondral differentiation. Further, gene expression analysis showed TGF-β1 release from β-cyclodextrin CG scaffolds promoted early chondrogenic-specific differentiation. Ultimately, this work establishes a novel method for the incorporation and display of growth factors within CG scaffolds via supramolecular interactions. Such a design framework offers opportunities to selectively alter the bioavailability of multiple biomolecules within a three-dimensional collagen-GAG scaffold to enhance cell activity for a range of musculoskeletal regenerative medicine applications. Statement of SignificanceWe describe the incorporation of β-cyclodextrin into a model CG-scaffold under development for musculoskeletal tissue engineering applications. We show β-cyclodextrin modified scaffolds promote the sequestration of soluble TGF-β1 and BMP-2 via guest-host interactions, leading to extended retention and release. Further, β-cyclodextrin modified CG scaffolds promote TGF-β1 or BMP-2 specific Smad signaling pathway activation associated with divergent osseous versus chondrogenic differentiation pathways in mesenchymal stem cells.

The influence of pore size and stiffness on tenocyte bioactivity and transcriptomic stability in collagen-GAG scaffolds

Orthopedic injuries, particularly those involving tendons and ligaments, are some of the most commonly treated musculoskeletal ailments, but are associated with high costs and poor outcomes. A significant barrier in the design of biomaterials for tendon tissue engineering is the rapid de-differentiation observed for primary tenocytes once removed from the tendon body. Herein, we evaluate the use of an anisotropic collagen-glycosaminoglycan (CG) scaffold as a tendon regeneration platform. We report the effects of structural properties of the scaffold (pore size, collagen fiber crosslinking density) on resultant tenocyte bioactivity, viability, and gene expression. In doing so we address a standing hypothesis that scaffold anisotropy and strut flexural rigidity (stiffness) co-regulate long-term maintenance of a tenocyte phenotype. We report changes in equine tenocyte specific gene expression profiles and bioactivity across a homologous series of anisotropic collagen scaffolds with defined changes in pore size and crosslinking density. Anisotropic scaffolds with higher crosslinking densities and smaller pore sizes were more able to resist cell-mediated contraction forces, promote increased tenocyte metabolic activity, and maintain and increase expression of tenogenic gene expression profiles. These results suggest that control over scaffold strut flexural rigidity via crosslinking and porosity provides an ideal framework to resolve structure-function maps relating the influence of scaffold anisotropy, stiffness, and nutrient biotransport on tenocyte-mediated scaffold remodeling and long-term phenotype maintenance.

The effect of glycosaminoglycan content on polyethylenimine-based gene delivery within three-dimensional collagen-GAG scaffolds.

The design of biomaterials for increasingly complex tissue engineering applications often requires exogenous presentation of biomolecular signals. Integration of gene delivery vectors with a biomaterial scaffold offers the potential to bypass the use of expensive and relatively inefficient growth factor supplementation strategies to augment cell behavior. However, integration of cationic polymer based gene delivery vectors within three-dimensional biomaterials, particularly matrices which can carry significant surface charge, remains poorly explored. We examined the potential of polyethylenimine (PEI) as a gene delivery vector for three-dimensional collagen-glycosaminoglycan (CG) scaffolds under development for tendon repair. While acetylated versions of PEI have demonstrated improved transfection efficiency in 2D culture assays, we investigated translation of this effect to a 3D biomaterial that contains significant electrostatic charge. A reporter gene was used to examine the impact of polymer modification, polymer:DNA ratio, and the degree of sulfation of the biomaterial microenvironment on gene delivery in vitro. We observed highest transgene expression in acetylated and unmodified PEI at distinct polymer:DNA ratios; notably, the enhancement often seen in two-dimensional culture for acetylated PEI did not fully translate to three-dimensional scaffolds. We also found highly sulfated heparin-based CG scaffolds showed enhanced initial luciferase expression but not prolonged activity. While PEI constructs significantly reduced tenocyte metabolic health during the period of transfection, heparin-based CG scaffolds showed the greatest recovery in tenocyte metabolic health over the full 2 week culture. These results suggest that the electrostatic environment of three-dimensional biomaterials may be an important design criterion for cationic polymer-based gene delivery.

Composite Growth Factor Supplementation Strategies to Enhance Tenocyte Bioactivity in Aligned Collagen-GAG Scaffolds

Biomolecular environments encountered in vivo are complex and dynamic, with combinations of biomolecules presented in both freely diffusible (liquid-phase) and sequestered (bound to the extracellular matrix) states. Strategies for integrating multiple biomolecular signals into a biomimetic scaffold provide a platform to simultaneously control multiple cell activities, such as motility, proliferation, phenotype, and regenerative potential. Here we describe an investigation elucidating the influence of the dose and mode of presentation (soluble, sequestered) of five biomolecules (stromal cell-derived factor 1α [SDF-1α], platelet-derived growth factor BB [PDGF-BB], insulin-like growth factor 1 [IGF-1], basic fibroblast growth factor [bFGF], and growth/differentiation factor 5 [GDF-5]) on the recruitment, proliferation, collagen synthesis, and genomic stability of equine tenocytes within an anisotropic collagen-GAG scaffold for tendon regeneration applications. Critically, we found that single factors led to a dose-dependent trade-off between driving tenocyte proliferation (PDGF-BB, IGF-1) versus maintenance of a tenocyte phenotype (GDF-5, bFGF). We identified supplementation schemes using factor pairs (IGF-1, GDF-5) to rescue the tenocyte phenotype and gene expression profiles while simultaneously driving proliferation. These results suggest coincident application of multi-biomolecule cocktails has a significant value in regenerative medicine applications where control of cell proliferation and phenotype are required. Finally, we demonstrated an immobilization strategy that allows efficient sequestration of bioactive levels of these factors within the scaffold network. We showed sequestration can lead to a greater sustained bioactivity than soluble supplementation, making this approach particularly amenable to in vivo translation where diffusive loss is a concern and continuous biomolecule supplementation is not feasible.

The promotion of HL-1 cardiomyocyte beating using anisotropic collagen-GAG scaffolds

Biomaterials for myocardial tissue engineering must balance structural, mechanical and bioactivity concerns. This work describes the interaction between HL-1 cardiomyocytes and a series of geometrically anisotropic collagen-GAG (CG) scaffolds with aligned tracks of ellipsoidal pores designed to mimic elements of the native geometric anisotropy of cardiac tissue. Here we report the role scaffold geometric anisotropy and pore size plays in directing cardiomyocyte bioactivity. Notably, HL-1 cardiomyocytes showed good proliferation and metabolic activity in all variants out to 14 days in culture. Critically, HL-1s exhibited significantly elevated 3D alignment and earlier spontaneous beating within anisotropic CG scaffolds relative to isotropic scaffold controls. This spontaneous beating occurred at significantly higher instances for larger pore size anisotropic variants. Gene expression and immunohistochemical analyses for key cardiac marker (α-myosin heavy chain, connexin-43) suggest that the isotropic and anisotropic scaffolds support expression of key transcriptomic markers of cardiomyocyte phenotype as well as the formation of gap junctions and elongated, aligned cell morphologies. Collectively, these results suggest that a geometrically anisotropic scaffold with sufficiently large pore size (>150 μm) provides a suitable microenvironment to induce cardiomyocyte alignment, beating, and bioactivity for cardiac tissue engineering applications.

Influence of flow rate and scaffold pore size on cell behavior during mechanical stimulation in a flow perfusion bioreactor

Mechanically stimulating cell-seeded scaffolds by flow-perfusion is one approach utilized for developing clinically applicable bone graft substitutes. A key challenge is determining the magnitude of stimuli to apply that enhances cell differentiation but minimizes cell detachment from the scaffold. In this study, we employed a combined computational modeling and experimental approach to examine how the scaffold mean pore size influences cell attachment morphology and subsequently impacts upon cell deformation and detachment when subjected to fluid-flow. Cell detachment from osteoblast-seeded collagen-GAG scaffolds was evaluated experimentally across a range of scaffold pore sizes subjected to different flow rates and exposure times in a perfusion bioreactor. Cell detachment was found to be proportional to flow rate and inversely proportional to pore size. Using this data, a theoretical model was derived that accurately predicted cell detachment as a function of mean shear stress, mean pore size, and time. Computational modeling of cell deformation in response to fluid flow showed the percentage of cells exceeding a critical threshold of deformation correlated with cell detachment experimentally and the majority of these cells were of a bridging morphology (cells stretched across pores). These findings will help researchers optimize the mean pore size of scaffolds and perfusion bioreactor operating conditions to manage cell detachment when mechanically simulating cells via flow perfusion.

The influence of collagen–glycosaminoglycan scaffold relative density and microstructural anisotropy on tenocyte bioactivity and transcriptomic stability

Biomaterials for orthopedic tissue engineering must balance mechanical and bioactivity concerns. This work describes the fabrication of a homologous series of anisotropic collagen–GAG (CG) scaffolds with aligned tracks of ellipsoidal pores but increasing relative densities (ρ∗/ρs), and we report the role scaffold relative density plays in directing tenocyte bioactivity. Scaffold permeability and mechanical properties, both in tension and compression, were significantly influenced by relative density in a manner predicted by cellular solids models. Equine tenocytes showed greater levels of attachment, metabolic activity, soluble collagen synthesis, and alignment as well as less cell-mediated scaffold contraction in anisotropic CG scaffolds of increasing relative density. Notably, the lowest density scaffolds experienced significant cell-mediated contraction with associated decreases in tenocyte number as well as loss of microstructural integrity, aligned contact guidance cues, and preferential tenocyte orientation over a 14 day culture period. Gene expression analyses suggested tenocyte de-differentiation in the lowest density scaffold while indicating that the highest density scaffold supported significant increases in COMP (4-fold), tenascin-C (3-fold), and scleraxis (15-fold) expression as well as significant decreases in MMP-1 (9-fold) and MMP-13 (13-fold) expression on day 14. These results suggest that anisotropic scaffold relative density can help to modulate the maintenance of a more tendon-like microenvironment and aid long-term tenocyte transcriptomic stability. Overall, this work demonstrates that relative density is a critical scaffold parameter, not only for insuring mechanical competence, but also for directing cell transcriptomic stability and behavior.

The effect of anisotropic collagen-GAG scaffolds and growth factor supplementation on tendon cell recruitment, alignment, and metabolic activity

Current surgical and tissue engineering approaches for treating tendon injuries have shown limited success, suggesting the need for new biomaterial strategies. Here we describe the development of an anisotropic collagen-glycosaminoglycan (CG) scaffold and use of growth factor supplementation strategies to create a 3D platform for tendon tissue engineering. We fabricated cylindrical CG scaffolds with aligned tracks of ellipsoidal pores that mimic the native physiology of tendon by incorporating a directional solidification step into a conventional lyophilization strategy. By modifying the freezing temperature, we created a homologous series of aligned CG scaffolds with constant relative density and degree of anisotropy but a range of pore sizes (55–243 μm). Equine tendon cells showed greater levels of attachment, metabolic activity, and alignment as well as less cell-mediated scaffold contraction, when cultured in anisotropic scaffolds compared to an isotropic CG scaffold control. The anisotropic CG scaffolds also provided critical contact guidance cues for cell alignment. While tendon cells were randomly oriented in the isotropic control scaffold and the transverse (unaligned) plane of the anisotropic scaffolds, significant cell alignment was observed in the direction of the contact guidance cues in the longitudinal plane of the anisotropic scaffolds. Scaffold pore size was found to significantly influence tendon cell viability, proliferation, penetration into the scaffold, and metabolic activity in a manner predicted by cellular solids arguments. Finally, the addition of the growth factors PDGF-BB and IGF-1 to aligned CG scaffolds was found to enhance tendon cell motility, viability, and metabolic activity in dose-dependent manners. This work suggests a composite strategy for developing bioactive, 3D material systems for tendon tissue engineering.

The generation of biomolecular patterns in highly porous collagen-GAG scaffolds using direct photolithography

The extracellular matrix (ECM) is a complex organization of structural proteins found within tissues and organs. Heterogeneous tissues with spatially and temporally modulated properties play an important role in organism physiology. Here we present a benzophenone (BP) based direct, photolithographic approach to spatially pattern solution phase biomolecules within collagen-GAG (CG) scaffolds and demonstrate creation of a wide range of patterns composed of multiple biomolecular species in a manner independent from scaffold fabrication steps. We demonstrate the ability to immobilize biomolecules at surface densities of up to 1000 ligands per square micron on the scaffold strut surface and to depths limited by the penetration depth of the excitation source into the scaffold structure. Importantly, while BP photopatterning does further crosslink the CG scaffold, evidenced by increased mechanical properties and collagen crystallinity, it does not affect scaffold microstructural or compositional properties or negatively influence cell adhesion, viability, or proliferation. We show that covalently photoimmobilized fibronectin within a CG scaffold significantly increases the speed of MC3T3-E1 cell attachment relative to the bare CG scaffold or non-specifically adsorbed fibronectin, suggesting that this approach can be used to improve scaffold bioactivity. Our findings, on the whole, establish the use of direct, BP photolithography as a methodology for covalently incorporating activity-improving biochemical cues within 3D collagen biomaterial scaffolds with spatial control over biomolecular deposition.

Towards in vitro vascularisation of collagen-GAG scaffolds

Collagen-glycosaminoglycan scaffolds that have been used clinically for skin regeneration have also shown significant promise for other applications in tissue engineering. However, regeneration of thicker tissues with the aid of implanted biomaterials is likely to depend on, or be accelerated by, the ability to establish rapid vascularisation of the implant. The present study aims to establish a nascent vascular network in vitro within a CG scaffold as a first step towards that goal. Mesenchymal stem cells (MSCs) were chosen as primary vasculogenic candidate cells and a culture medium that promoted maximal network formation on Matrigel by these cells was selected. MSCs seeded in the CG scaffold formed networks of cord-like structures after one to two weeks in the presence of the vasculogenic medium; similar structures were formed by aortic endothelial cells (ECs) cultured for comparison. Gene expression analysis suggested that the MSCs began to adopt an endothelial phenotype, with RNA for PECAM and VCAM rising while that for alpha-smooth muscle actin fell. However there was no increase in Tie-2 and vWF expression. Addition of smooth muscle cells (SMCs) as a potential perivascular stabilising component did not have a noticeable effect on MSC-derived networks, although it enhanced EC-derived structures.

Cross‐linking affects cellular condensation and chondrogenesis in type II collagen‐GAG scaffolds seeded with bone marrow‐derived mesenchymal stem cells

The formation of cartilaginous tissue by chondroprogenitor cells, whether in vivo or in vitro, appears to require a critical initial stage of "condensation" in which intercellular space is reduced through an aggregation of cells, leading to development of cell-to-cell junctions followed by chondrocytic differentiation. The objective of this study was to investigate the association of aggregation (condensation) of mesenchymal stem cell (MSCs) and chondrogenesis in vitro. Previous work with chondrocytes indicated that the cross-link density and related cell-mediated contraction of collagen scaffolds significantly affects cartilaginous tissue formation within the cell-seeded construct. Based on this finding, we hypothesized that the cell-aggregating effect of the contraction of MSC-seeded collagen scaffolds of lower cross-link density favors chondrogenesis; scaffolds of higher cross-link density, which resist cell-mediated contraction, would demonstrate a lower cell number density (i.e., subcritical packing density) and less cartilage formation. Type II collagen-GAG scaffolds, chemically cross-linked to achieve a range of cross-link densities, were seeded with caprine MSCs and cultured for 4 weeks. Constructs with low cross-link densities experienced cell-mediated contraction, increased cell number densities, and a greater degree of chondrogenesis (indicated by the chondrocytic morphology of cells, and synthesis of GAG and type II collagen) compared to more highly cross-linked scaffolds that resisted cellular contraction. These results provide a foundation for further investigation of the mechanisms by which condensation of mesenchymal cells induces chondrogenesis in this in vitro model, and may inform cross-linking protocols for collagen scaffolds for use in cartilage tissue engineering.

Biologically active collagen-based scaffolds: advances in processing and characterization

A small number of type I collagen-glycosaminoglycan scaffolds (collagen-GAG scaffolds; CGSs) have unusual biological activity consisting primarily in inducing partial regeneration of organs in the adult mammal. Two of these are currently in use in a variety of clinical settings. CGSs appear to induce regeneration by blocking the adult healing response, following trauma, consisting of wound contraction and scar formation. Several structural determinants of biological activity have been identified, including ligands for binding of fibroblasts to the collagen surface, the mean pore size (which affects ligand density) and the degradation rate (which affects the duration of the wound contraction-blocking activity by the scaffold). Processing variables that affect these determinants include the kinetics of swelling of collagen fibres in acetic acid, freezing of the collagen-GAG suspension and cross-linking of the freeze-dried scaffold. Recent developments in the processing of CGSs include fabrication of scaffolds that are paucidisperse in pore size, scaffolds with gradients in physicochemical properties (and therefore biological activity) and scaffolds that incorporate a mineral component. Advances in the characterization of the pore structure of CGSs have been made using confocal and nonlinear optical microscopy (NLOM). The mechanical behaviour of CGSs, as well as the resistance to degradative enzymes, have been studied. Following seeding with cells (typically fibroblasts), contractile forces in the range 26-450 nN per cell are generated by the cells, leading to buckling of scaffold struts. Ongoing studies of cell-seeded CGSs with NLOM have shown an advantage over the use of confocal microscopy due to the ability of the former method to image the CGS surfaces without staining (which alters its surface ligands), reduced cell photodamage, reduced fluorophore photobleaching and the ability to image deeper inside the scaffold.

Cell contraction forces in scaffolds with varying pore size and cell density

The contractile behavior of cells is relevant in understanding wound healing and scar formation. In tissue engineering, inhibition of the cell contractile response is critical for the regeneration of physiologically normal tissue rather than scar tissue. Previous studies have measured the contractile response of cells in a variety of conditions (e.g. on two-dimensional solid substrates, on free-floating tissue engineering scaffolds and on scaffolds under some constraint in a cell force monitor). Tissue engineering scaffolds behave mechanically like open-cell elastomeric foams: between strains of about 10 and 90%, cells progressively buckle struts in the scaffold. The contractile force required for an individual cell to buckle a strut within a scaffold has been estimated based on the strut dimensions (radius, r, and length, l) and the strut modulus, Es. Since the buckling force varies, according to Euler's law, with r4/l2, and the relative density of the scaffold varies as (r/l)2, the cell contractile force associated with strut buckling is expected to vary with the square of the pore size for scaffolds of constant relative density. As the cell density increases, the force per cell to achieve a given strain in the scaffold is expected to decrease. Here we model the contractile response of fibroblasts by analyzing the response of a single tetrakaidecahedron to forces applied to individual struts (simulating cell contractile forces) using finite element analysis. We model tetrakaidecahedra of different strut lengths, corresponding to different scaffold pore sizes, and of varying numbers of loaded struts, corresponding to varying cell densities. We compare our numerical model with the results of free-floating contraction experiments of normal human dermal fibroblasts (NHDF) in collagen-GAG scaffolds of varying pore size and with varying cell densities.

Design of a multiphase osteochondral scaffold III: Fabrication of layered scaffolds with continuous interfaces

There is a need to improve current treatments for articular cartilage injuries. This article is the third in a series describing the design and development of an osteochondral scaffold based on collagen-glycosaminoglycan and calcium phosphate technologies for regenerative repair of articular cartilage defects. The previous articles in this series described methods for producing porous, three-dimensional mineralized collagen-GAG (CGCaP) scaffolds whose composition can be reproducibly varied to mimic the composition of subchondral bone, and pore microstructure and mineral phase can be modified. This article describes a method, "liquid-phase cosynthesis," that enables the production of porous, layered scaffolds that mimic the composition and structure of articular cartilage on one side, subchondral bone on the other side, and the continuous, gradual or "soft" interface between these tissues: the tidemark of articular joints. This design enables the layered scaffolds to be inserted into the subchondral bone at an osteochondral defect site without the need for sutures, glue, or screws, with a highly interconnected porous network throughout the entire osteochondral defect. Moreover, the differential moduli of the osseous and cartilaginous compartments enable these layered scaffolds to exhibit compressive deformation behavior that mimics the behavior observed in natural articular joints.

Tissue‐Engineered Skin Containing Mesenchymal Stem Cells Improves Burn Wounds

There is increasing evidence showing that adult stem cells are useful for tissue regeneration. Bone marrow mesenchymal stem cells (MSCs) are self-renewing and are potent in differentiating into multiple cells and tissues. To investigate the practicability of repairing burn wounds with tissue-engineered (TE) skin combined with bone MSCs, we established a burn wound model in the porcine skin. With a controlling temperature and time of the burning device to obtain different degrees of burn wounds, a deep dermal partial thickness burn was introduced to the porcine skin using a heated-brass contact injury at 100 degrees C for 20 s. Collagen-GAG scaffolds were utilized as the matrix; MSCs separated from pigs were seeded on them to form the skin equivalent. When grafted to the burn wounds, the TE skin containing MSCs showed better healing and keratinization, less wound contraction, and more vascularization. Grafts proliferated well and contributed to the neo-tissues. These data suggest that TE skin containing MSCs in a burn defect can accelerate wound healing and receive satisfactory effects.