Characterization of a macroporous polyvinyl alcohol scaffold for the repair of focal articular cartilage defects.

Focal cartilage defects reduce the ability of articular cartilage to resist mechanical loading and provide lubrication during joint motion. The limitations in current surgical treatments have motivated the use of biocompatible scaffolds as a future treatment option. Here we describe a second generation macroporous, polyvinyl alcohol (PVA) scaffold with independently tunable morphological and mechanical properties. The compressive moduli of the PVA scaffold increased with increasing polymer concentration and applied compressive strain, with values in the range for human articular cartilage (HA > 1000 kPa, EY > 500 kPa). Scaffolds also possessed strain-dependent permeability and Poisson's ratio. The interconnected macroporous network was found to facilitate chondrocyte seeding and proliferation through the scaffold over one week in culture. Overall, these promising characteristics demonstrate the potential of this macroporous scaffold for future studies in focal cartilage defect repair.

Matrix generation within a macroporous non-degradable implant for osteochondral defects is not enhanced with partial enzymatic digestion of the surrounding tissue: evaluation in an in vivo rabbit model.

Articular cartilage defects are a significant source of pain, have limited ability to heal, and can lead to the development of osteoarthritis. However, a surgical solution is not available. To tackle this clinical problem, non-degradable implants capable of carrying mechanical load immediately after implantation and for the duration of implantation, while integrating with the host tissue, may be viable option. But integration between articular cartilage and non-degradable implants is not well studied. Our objective was to assess the in vivo performance of a novel macroporous, nondegradable, polyvinyl alcohol construct. We hypothesized that matrix generation within the implant would be enhanced with partial digestion of the edges of articular cartilage. Our hypothesis was tested by randomizing an osteochondral defect created in the trochlea of 14 New Zealand white rabbits to treatment with: (i) collagenase or (ii) saline, prior to insertion of the implant. At 1 and 3-month post-operatively, the gross morphology and histologic appearance of the implants and the surrounding tissue were assessed. At 3 months, the mechanical properties of the implant were also quantified. Overall, the hydrogel implants performed favorably; at all time-points and in all groups the implants remained well fixed, did not cause inflammation or synovitis, and did not cause extensive damage to the opposing articular cartilage. Regardless of treatment with saline or collagenase, at 1 month post-operatively implants from both groups had a contiguous interface with adjacent cartilage and were populated with chondrocyte-like cells. At 3 months fibrous encapsulation of all implants was evident, there was no difference between area of aggrecan staining in the collagenase versus saline groups, and implant modulus was similar in both groups; leading us to reject our hypothesis. In summary, a porous PVA osteochondral implant remained well fixed in a short term in vivo osteochondral defect model; however, matrix generation within the implant was not enhanced with partial digestion of adjacent articular cartilage.

Differential cross-linking and radio-protective effects of genipin on mature bovine and human patella tendons.

Gamma irradiation is a proven sterilization method, but is not widely used on allografts for anterior cruciate ligament reconstruction (e.g., patella tendon) due to radiation-induced decreases in mechanical strength. Addressing this drawback would improve the safety and supply of allografts to meet current and future demand. It was hypothesized that genipin-induced collagen cross-linking would increase the tensile modulus of patella tendon tissue such that 5 MRad gamma irradiation would not reduce the tissue mechanical strength below the original untreated values. Optimized genipin treatment increased the tensile modulus of bovine tendons by ~2.4-fold. After irradiation, genipin treated tissue did not significantly differ from native tissue, proving the hypothesis. Optimized genipin treatment of human tendons increased the tensile modulus by ~1.3-fold. After irradiation, both control and genipin-treated tissues possessed ~50-60% of their native tendon modulus, disproving the hypothesis. These results highlight possible age- and species- dependent effects of genipin cross-linking on tendon tissue. Cross-linking of human allografts may be beneficial only in younger donor tissues. Future research is warranted to better understand the mechanisms and applications of collagen cross-linking for clinical use.

Augmenting the articular cartilage-implant interface: Functionalizing with a collagen adhesion protein.

The lack of integration between implants and articular cartilage is an unsolved problem that negatively impacts the development of treatments for focal cartilage defects. Many approaches attempt to increase the number of matrix-producing cells that can migrate to the interface, which may help to reinforce the boundary over time but does not address the problems associated with an initially unstable interface. The objective of this study was to develop a bioadhesive implant to create an immediate bond with the extracellular matrix components of articular cartilage. We hypothesized that implant-bound collagen adhesion protein (CNA) would increase the interfacial strength between a poly(vinly alcohol) implant and an articular cartilage immediately after implantation, without preventing cell migration into the implant. By way of a series of in vitro immunohistochemical and mechanical experiments, we demonstrated that (i) free CNA can bind to articular cartilage, (ii) implant-bound CNA can bind to collagen type II and (iii) implants functionalized with CNA result in a fourfold increase in interfacial strength with cartilage relative to untreated implants at day zero. Of note, the interfacial strength significantly decreased after 21 days in culture, which may be an indication that the protein itself has lost its effectiveness. Our data suggest that functionalizing scaffolds with CNA may be a viable approach toward creating an initially stable interface between scaffolds and articular cartilage. Further efforts are required to ensure long-term interface stability.

A novel macroporous polyvinyl alcohol scaffold promotes chondrocyte migration and interface formation in an in vitro cartilage defect model.

Scaffold-cartilage integration is critical for the clinical success of a scaffold used for the repair of a focal cartilage defect. In this study, a macroporous polyvinyl alcohol (PVA) scaffold was found to facilitate chondrocyte infiltration and interfacial matrix formation in a juvenile bovine in vitro cartilage defect model. These results were found to depend on the press-fit between the scaffold and the cartilage, pretreatment of the cartilage with collagenase prior to scaffold insertion, and chondrocyte preseeding of the scaffold. Infiltrated and preseeded chondrocytes in the scaffold survived for 6 weeks in culture and resulted in sufficient matrix at the interface to significantly increase the interface shear strength 30-fold that compared favorably with the interface shear strength of cartilage-cartilage constructs. The ability of this macroporous PVA scaffold to form a stable interface with articular cartilage demonstrates the potential use of this scaffold design for focal cartilage defect repair.

Transient supplementation of anabolic growth factors rapidly stimulates matrix synthesis in engineered cartilage.

The purpose of the presented work is to examine the response of engineered cartilage to a transient, 2-week application of anabolic growth factors compared to continuous exposure in in vitro culture. Immature bovine chondrocytes were suspended in agarose hydrogel and cultured for 28 days (Study 1) or 42 days (Study 2) in chondrogenic media with TGF-ß1, TGF-ß3, or IGF-I either added for only the first 14 days in culture or added to the media for the entire study period. In both studies, there were no statistical differences in tissue mechanical or biochemical properties between the growth factors on day 14. In Study 1, growth factor removal led to a significant and drastic increase in Young's modulus and glycosaminoglycans content compared to continuously exposed controls on day 28. In Study 2, both TGF-ß1 and ß3 led to significantly higher mechanical properties and collagen content vs. IGF-I on day 42. These results indicate that the rapid rise in tissue properties (previously observed with TGF-ß3 only) is not dependent on the type but rather the temporal application of the anabolic growth factor. These findings shed light on possible techniques to rapidly develop engineered cartilage tissue for the future treatment of osteoarthritis.

A semi-degradable composite scaffold for articular cartilage defects.

Few options exist to replace or repair damaged articular cartilage. The optimal solution that has been suggested is a scaffold that can carry load and integrate with surrounding tissues; but such a construct has thus far been elusive. The objectives of this study were to manufacture and characterize a nondegradable hydrated scaffold. Our hypothesis was that the polymer content of the scaffold can be used to control its mechanical properties, while an internal porous network augmented with biological agents can facilitate integration with the host tissue. Using a two-step water-in-oil emulsion process a porous polyvinyl alcohol (PVA) hydrogel scaffold combined with alginate microspheres was manufactured. The scaffold had a porosity of 11-30% with pore diameters of 107-187 µm, which readily allowed for movement of cells through the scaffold. Alginate microparticles were evenly distributed through the scaffold and allowed for the slow release of biological factors. The elastic modulus (Es ) and Poisson's ratio (υ), Aggregate modulus (Ha ) and dynamic modulus (ED ) of the scaffold were significantly affected by % PVA, as it varied from 10 to 20% wt/vol. Es and υ were similar to that of articular cartilage for both polymer concentrations, while Ha and ED were similar to that of cartilage only at 20% PVA. The ability to control scaffold mechanical properties, while facilitating cellular migration suggest that this scaffold is a potentially viable candidate for the functional replacement of cartilage defects.

Passaged adult chondrocytes can form engineered cartilage with functional mechanical properties: a canine model.

It was hypothesized that previously optimized serum-free culture conditions for juvenile bovine chondrocytes could be adapted to generate engineered cartilage with physiologic mechanical properties in a preclinical, adult canine model. Primary or passaged (using growth factors) adult chondrocytes from three adult dogs were encapsulated in agarose, and cultured in serum-free media with transforming growth factor-beta3. After 28 days in culture, engineered cartilage formed by primary chondrocytes exhibited only small increases in glycosaminoglycan content. However, all passaged chondrocytes on day 28 elaborated a cartilage matrix with compressive properties and glycosaminoglycan content in the range of native adult canine cartilage values. A preliminary biocompatibility study utilizing chondral and osteochondral constructs showed no gross or histological signs of rejection, with all implanted constructs showing excellent integration with surrounding cartilage and subchondral bone. This study demonstrates that adult canine chondrocytes can form a mechanically functional, biocompatible engineered cartilage tissue under optimized culture conditions. The encouraging findings of this work highlight the potential for tissue engineering strategies using adult chondrocytes in the clinical treatment of cartilage defects.

Zonal chondrocytes seeded in a layered agarose hydrogel create engineered cartilage with depth-dependent cellular and mechanical inhomogeneity.

We hypothesized that zonal populations of chondrocytes seeded into a bilayered scaffold with initially prescribed depth-varying, compressive material properties will lead to a biomimetic cartilage tissue construct with depth-dependent cellular and compressive mechanical inhomogeneity similar to that of the native tissue. Superficial zone chondrocytes (SZCs) and middle/deep zone chondrocytes (MDZCs) were isolated and encapsulated with 2% or 3% agarose to form single-layered constructs of 2% SZC, 3% SZC, 2% MDZC; bilayered constructs of 2% SZC/2% MDZC and 3% SZC/2% MDZC; and 2% mixed chondrocyte controls. For SZCs on day 42, increased glycosaminoglycan (GAG) and collagen was found with increased agarose concentration and when layered with MDZCs. Superficial zone protein increased with agarose concentration in bilayered constructs. For MDZCs, increased GAG content and regulation of cell proliferation was observed when layered with SZCs. Bilayered constructs possessed a depth-dependent compressive modulus qualitatively similar to that of native articular cartilage, whereas controls showed a U-shaped profile with stiffer peripheral edges and softer middle region. This study is the first to create an engineered cartilage tissue with depth-varying cellular as well as mechanical inhomogeneity. Future studies will determine if replicating inhomogeneity is advantageous in clinical applications of tissue engineered cartilage.

Influence of temporary chondroitinase ABC-induced glycosaminoglycan suppression on maturation of tissue-engineered cartilage.

OBJECTIVE: A fundamental challenge of cartilage tissue engineering has been the inability to promote collagen synthesis up to native levels. In contrast, recent protocols have demonstrated that glycosaminoglycans (GAG) can be synthesized to native levels in 4-6 weeks of in vitro culture. We hypothesize that rapid GAG synthesis may be an impediment to collagen synthesis, possibly by altering transport pathways of nutrients or synthesis products. In this study, this hypothesis is tested by inducing enzymatic GAG loss in the early culture period of cartilage tissue constructs, and monitoring collagen content at various time points after cessation of enzymatic treatment. METHODS: In Study 1, to induce breakdown of proteoglycans, chondroitinase ABC (CABC, 0.002U/mL) was continuously added into the culture media for the initial 4 weeks of culture or for 2 weeks starting on day 14 of culture. In Study 2, multiple transient CABC treatments (0.15U/mL, for 2 days) were applied to the matured tissue-engineered constructs. RESULTS: Continuous and transient CABC treatments significantly increased the collagen concentration of the constructs, improving their tensile properties. The GAG content of the treated constructs recovered quickly to the pretreatment level after 2-3 weeks. CONCLUSIONS: This study demonstrates that tissue-engineered cartilage constructs with improved tensile properties can be achieved by temporarily suppressing the GAG content enzymatically.

Duty Cycle of Deformational Loading Influences the Growth of Engineered Articular Cartilage.

This study examines how variations in the duty cycle (the duration of applied loading) of deformational loading can influence the mechanical properties of tissue engineered cartilage constructs over one month in bioreactor culture. Dynamic loading was carried out with three different duty cycles: 1 h on/1 h off for a total of 3 h loading/day, 3 h continuous loading, or 6 h of continuous loading per day, with all loading performed 5 days/week. All loaded groups showed significant increases in Young's modulus after one month (vs. free swelling controls), but only loading for a continuous 3 and 6 h showed significant increases in dynamic modulus by this time point. Histological analysis showed that dynamic loading can increase cartilage oligomeric matrix protein (COMP) and collagen types II and IX, as well as prevent the formation of a fibrous capsule around the construct. Type II and IX collagen deposition increased with increased with duration of applied loading. These results point to the efficacy of dynamic deformational loading in the mechanical preconditioning of engineered articular cartilage constructs. Furthermore, these results highlight the ability to dictate mechanical properties with variations in mechanical input parameters, and the possible importance of other cartilage matrix molecules, such as COMP, in establishing the functional material properties of engineered constructs.

Culture duration modulates collagen hydrolysate-induced tissue remodeling in chondrocyte-seeded agarose hydrogels.

Media supplementation with collagen hydrolysate was hypothesized to increase the collagen content in engineered cartilage. By d28, hydrolysate at 0.5 mg/mL increased type II collagen content and 1 mg/mL increased mechanical properties, total collagen content, and type II collagen content over controls. By d42, however, controls possessed the highest GAG content and compressive Young's modulus. Real-time PCR found that 1 mg/mL increased type II collagen gene expression in d0 constructs, but increased MMP expression with no effect on type II collagen on d28. A 10 mg/mL concentration produced the lowest tissue properties, the lowest type II collagen gene expression on d0, and the highest MMP gene expression on d28. These results indicate that the duration of culture modulates the response of chondrocytes to collagen hydrolysate in 3D culture, transforming the response from positive to negative. Therefore, collagen hydrolysate as a media supplement is not a viable long-term method to improve the collagen content of engineered cartilage tissue.

Osmotic loading of spherical gels: a biomimetic study of hindered transport in the cell protoplasm.

Osmotic loading of cells has been used to investigate their physicochemical properties as well as their biosynthetic activities. The classical Kedem-Katchalsky framework for analyzing cell response to osmotic loading, which models the cell as a fluid-filled membrane, does not generally account for the possibility of partial volume recovery in response to loading with a permeating osmolyte, as observed in some experiments. The cell may be more accurately represented as a hydrated gel surrounded by a semi-permeable membrane, with the gel and membrane potentially exhibiting different properties. To help assess whether this more elaborate model of the cell is justified, this study investigates the response of spherical gels to osmotic loading, both from experiments and theory. The spherical gel is described using the framework of mixture theory. In the experimental component of the study alginate is used as the model gel, and is osmotically loaded with dextran solutions of various concentrations and molecular weight, to verify the predictions from the theoretical analysis. Results show that the mixture framework can accurately predict the transient and equilibrium response of alginate gels to osmotic loading with dextran solutions. It is found that the partition coefficient of dextran in alginate regulates the equilibrium volume response and can explain partial volume recovery based on passive transport mechanisms. The validation of this theoretical framework facilitates future investigations of the role of the protoplasm in the response of cells to osmotic loading.

Dynamic deformational loading results in selective application of mechanical stimulation in a layered, tissue-engineered cartilage construct.

The application of dynamic physiologic loading to a bilayered chondrocyte-seeded agarose construct with a 2% (wt/vol) top layer and 3% (wt/vol) bottom layer was hypothesized to (1) improve overall construct properties and (2) result in a tissue that mimics the mechanical inhomogeneity of native cartilage. Dynamic loading over the 28 day culture period was found to significantly increase bulk mechanical and biochemical properties versus free-swelling culture. The initial depth-distribution of the compressive Young's modulus (EY) reflected the intrinsic properties of the gel in each layer and a similar trend to the native tissue, with a softer 2% gel layer and a much stiffer 3% gel layer. After 28 days in culture, free-swelling conditions maintained this general trend while loaded constructs possessed a reverse profile, with significant increases in EY observed only in the 2% gel. Histological analysis revealed preferential matrix formation in the 2% agarose layer, with matrix localized more pericellularly in the 3% agarose layer. Finite element modeling revealed that, prior to significant matrix elaboration, the 2% layer experiences increased mechanical stimuli (fluid flow and compressive strain) during loading that may enhance chondrocyte stimulation and nutrient transport in that layer, consistent with experimental observations. From these results, we conclude that due to the limitations in 3% agarose, the use of this type of bilayered construct to construct depth-dependent inhomogeneity similar to the native tissue is not likely to be successful under long-term culture conditions. Our study underscores the importance of other physical properties of the scaffold that may have a greater influence on interconnected tissue formation than intrinsic scaffold stiffness.

Spatial and temporal development of chondrocyte-seeded agarose constructs in free-swelling and dynamically loaded cultures.

Dynamic deformational loading has been shown to significantly increase the development of material properties of chondrocyte-seeded agarose hydrogels, however little is known about the spatial development of the material properties within these constructs. In this study, a technique that combines video microscopy and optimized digital image correlation, was applied to assess the spatial development of material properties in tissue-engineered cartilage constructs cultured in free-swelling and dynamically-loaded conditions (3h/day, 5 days/week, and maintained in free-swelling conditions when not being loaded) over a 6-week period. Although homogeneous at day 0, both free-swelling and dynamically loaded samples progressively developed stiffer outer edges and a softer central region. The distribution of GAGs and collagens were shown to mimic this profile. These results indicate that although dynamic loading augments the development of bulk properties in these samples, possibly by overcoming some of the diffusion limitation and nutrient transport issues, the overall profile of construct properties in the axial direction remains qualitatively the same as in free-swelling culture conditions. Poisson's ratio of these constructs increased over time in culture with increased fixed charged density contributed by the GAGs, but this increase was significantly less in dynamically loaded samples by day 42. Polarized light microscopy of Picrosirius Red labeled samples, at an angle perpendicular to the direction of loading, suggests that these differences in Poisson's ratio may be due to improved organization of collagen network in the dynamically loaded samples.

A layered agarose approach to fabricate depth-dependent inhomogeneity in chondrocyte-seeded constructs.

Inspired by the depth-dependent inhomogeneity of articular cartilage, it was hypothesized that a novel layered agarose technique, using a 2% (wt/vol) top and a 3% (wt/vol) bottom layer, would create an inhomogenous tissue construct with distinct material properties in conjoined regions. The biochemical and mechanical development of these constructs was observed alongside uniform 2% and 3% constructs. Initially, uniform 3% agarose disks had the highest bulk Young's modulus (E(Y) approximately 28 kPa) of all groups. After 28 days of culture in 20% FBS-containing media, however, uniform 2% chondrocyte-seeded constructs achieved the highest Young's modulus compared to bilayered and 3% agarose disks. Though all three groups contained similar GAG content ( approximately 1.5% ww), uniform 2% agarose disks on day 28 possessed the highest collagen content ( approximately 1% ww). Unlike in either homogeneous construct type, microscopic analysis of axial strain fields in bilayered constructs in response to applied static compression revealed two mechanically disparate regions on day 0: a softer 2% layer and a stiffer 3% layer. With time in culture, this inhomogeneity became less distinct, as indicated by increased continuity in both the local displacement field and local E(Y), and depended on the level of FBS supplementation of the feed media, with lower FBS concentrations (10%) more closely maintaining the original distinction of material properties. These results shed positive light on a layered agarose technique for the production of inhomogeneous bilayered chondrocyte-seeded agarose constructs with applications for investigations of chondrocyte mechanotransduction and for possible use in the tissue engineering of inhomogeneous articular cartilage constructs.

Functional tissue engineering of chondral and osteochondral constructs.

Due to the prevalence of osteoarthritis (OA) and damage to articular cartilage, coupled with the poor intrinsic healing capacity of this avascular connective tissue, there is a great demand for an articular cartilage substitute. As the bearing material of diarthrodial joints, articular cartilage has remarkable functional properties that have been difficult to reproduce in tissue-engineered constructs. We have previously demonstrated that by using a functional tissue engineering approach that incorporates mechanical loading into the long-term culture environment, one can enhance the development of mechanical properties in chondrocyte-seeded agarose constructs. As these gel constructs begin to achieve material properties similar to that of the native tissue, however, new challenges arise, including integration of the construct with the underlying native bone. To address this issue, we have developed a technique for producing gel constructs integrated into an underlying bony substrate. These osteochondral constructs develop cartilage-like extracellular matrix and material properties over time in free swelling culture. In this study, as a preliminary to loading such osteochondral constructs, finite element modeling (FEM) was used to predict the spatial and temporal stress, strain, and fluid flow fields within constructs subjected to dynamic deformational loading. The results of these models suggest that while chondral ("gel alone") constructs see a largely homogenous field of mechanical signals, osteochondral ("gel bone") constructs see a largely inhomogeneous distribution of mechanical signals. Such inhomogeneity in the mechanical environment may aid in the development of inhomogeneity in the engineered osteochondral constructs. Together with experimental observations, we anticipate that such modeling efforts will provide direction for our efforts aimed at the optimization of applied physical forces for the functional tissue engineering of an osteochondral articular cartilage substitute.