Anatomically shaped tissue-engineered cartilage with tunable and inducible anticytokine delivery for biological joint resurfacing.

Biological resurfacing of entire articular surfaces represents an important but challenging strategy for treatment of cartilage degeneration that occurs in osteoarthritis. Not only does this approach require anatomically sized and functional engineered cartilage, but the inflammatory environment within an arthritic joint may also inhibit chondrogenesis and induce degradation of native and engineered cartilage. The goal of this study was to use adult stem cells to engineer anatomically shaped, functional cartilage constructs capable of tunable and inducible expression of antiinflammatory molecules, specifically IL-1 receptor antagonist (IL-1Ra). Large (22-mm-diameter) hemispherical scaffolds were fabricated from 3D woven poly(ε-caprolactone) (PCL) fibers into two different configurations and seeded with human adipose-derived stem cells (ASCs). Doxycycline (dox)-inducible lentiviral vectors containing eGFP or IL-1Ra transgenes were immobilized to the PCL to transduce ASCs upon seeding, and constructs were cultured in chondrogenic conditions for 28 d. Constructs showed biomimetic cartilage properties and uniform tissue growth while maintaining their anatomic shape throughout culture. IL-1Ra-expressing constructs produced nearly 1 µg/mL of IL-1Ra upon controlled induction with dox. Treatment with IL-1 significantly increased matrix metalloprotease activity in the conditioned media of eGFP-expressing constructs but not in IL-1Ra-expressing constructs. Our findings show that advanced textile manufacturing combined with scaffold-mediated gene delivery can be used to tissue engineer large anatomically shaped cartilage constructs that possess controlled delivery of anticytokine therapy. Importantly, these cartilage constructs have the potential to provide mechanical functionality immediately upon implantation, as they will need to replace a majority, if not the entire joint surface to restore function.

Scaffold-mediated lentiviral transduction for functional tissue engineering of cartilage.

The ability to develop tissue constructs with matrix composition and biomechanical properties that promote rapid tissue repair or regeneration remains an enduring challenge in musculoskeletal engineering. Current approaches require extensive cell manipulation ex vivo, using exogenous growth factors to drive tissue-specific differentiation, matrix accumulation, and mechanical properties, thus limiting their potential clinical utility. The ability to induce and maintain differentiation of stem cells in situ could bypass these steps and enhance the success of engineering approaches for tissue regeneration. The goal of this study was to generate a self-contained bioactive scaffold capable of mediating stem cell differentiation and formation of a cartilaginous extracellular matrix (ECM) using a lentivirus-based method. We first showed that poly-L-lysine could immobilize lentivirus to poly(ε-caprolactone) films and facilitate human mesenchymal stem cell (hMSC) transduction. We then demonstrated that scaffold-mediated gene delivery of transforming growth factor ß3 (TGF-ß3), using a 3D woven poly(ε-caprolactone) scaffold, induced robust cartilaginous ECM formation by hMSCs. Chondrogenesis induced by scaffold-mediated gene delivery was as effective as traditional differentiation protocols involving medium supplementation with TGF-ß3, as assessed by gene expression, biochemical, and biomechanical analyses. Using lentiviral vectors immobilized on a biomechanically functional scaffold, we have developed a system to achieve sustained transgene expression and ECM formation by hMSCs. This method opens new avenues in the development of bioactive implants that circumvent the need for ex vivo tissue generation by enabling the long-term goal of in situ tissue engineering.

Loss of stiffness in collagen-rich uterine fibroids after digestion with purified collagenase Clostridium histolyticum.

BACKGROUND: Uterine fibroids are a significant health problem. These common benign tumors occur in 70-80% of women before age 50 years and often cause bleeding and pain and can interfere considerably with daily life. Current treatment options are limited. Fibroids contain substantial amounts of altered and disordered collagens, which contribute to their bulk. Targeting these collagens directly presents a novel treatment approach. OBJECTIVES: We sought to test the hypothesis that a highly purified collagenase Clostridium histolyticum will digest interstitial collagen in uterine fibroids and reduce their stiffness and thereby evaluate the feasibility that this collagenase C histolyticum can be developed into an alternative treatment for fibroids. A secondary objective was to describe the collagen content of the fibroid tissue. STUDY DESIGN: Fibroid tissue cubes (1 cm3; n = 154) were cut from 17 uterine fibroids that were obtained from 7 consented subjects undergoing scheduled hysterectomies. Tissue cubes were injected with diluent, placebo, or highly purified collagenase C histolyticum (0.05, 0.1, or 0.2 mg/cube) and incubated at 37°C for 24, 48, 72, or 96 hours. At each time point, 6 noninjected control cubes were also evaluated. Tissue cubes were photographed before and after incubation. Myometrial samples (n = 21) were also evaluated. Stiffness was quantified through rheometry by measuring complex shear moduli of the tissues. Percent fibrosis was determined by computerized analysis of Masson-trichrome-stained slides. Digestion of collagen fibrils was confirmed by transmission electron microscopy. RESULTS: Fibrosis in untreated fibroids ranged from 37% to 77%, reflecting the collagen-rich nature of these tumors. After treatment with collagenase for 96 hours, fibrosis ranged from 5.3% to 2.4%. Transmission electron microscopy confirmed complete digestion of collagen fibrils. Tissue stiffness was reduced with all 3 doses of collagenase treatment and at all 4 time points. Longer incubation times with collagenase caused greater reduction in stiffness, and treated cubes lost their cuboidal shape and had gelatinous/liquefied centers. At 96 hours the stiffness in tissues treated with the lowest dose was reduced to 966 ± 106 Pascal compared with the diluent-treated control at the same time (5323 ± 903 Pascal; P < .0001; by analysis of variance with Tukey-Kramer). CONCLUSION: Uterine fibroids have a high content of collagen that can be effectively digested by highly purified collagenase C histolyticum, resulting in reduced tissue stiffness. Loss of stiffness may decrease bulk symptoms in vivo and possibly lead to shrinkage of fibroids through changed mechanotransduction, leading ultimately to reduced fibroid symptoms of pain and bleeding. Clinical trials are necessary to evaluate the safety and efficacy of collagenase C histolyticum including the rate of regrowth of fibroids. The data of this study provide a strong rationale for using this purified collagenase in clinical trials as a local treatment for women with fibroids.

Composite three-dimensional woven scaffolds with interpenetrating network hydrogels to create functional synthetic articular cartilage.

The development of synthetic biomaterials that possess mechanical properties that mimic those of native tissues remains an important challenge to the field of materials. In particular, articular cartilage is a complex nonlinear, viscoelastic, and anisotropic material that exhibits a very low coefficient of friction, allowing it to withstand millions of cycles of joint loading over decades of wear. Here we show that a three-dimensionally woven fiber scaffold that is infiltrated with an interpenetrating network hydrogel can provide a functional biomaterial that provides the load-bearing and tribological properties of native cartilage. An interpenetrating dual-network "tough-gel" consisting of alginate and polyacrylamide was infused into a porous three-dimensionally woven poly(ε-caprolactone) fiber scaffold, providing a versatile fiber-reinforced composite structure as a potential acellular or cell-based replacement for cartilage repair.

Functional tissue engineering of articular cartilage for biological joint resurfacing-The 2021 Elizabeth Winston Lanier Kappa Delta Award.

Biological resurfacing of entire articular surfaces represents a challenging strategy for the treatment of cartilage degeneration that occurs in osteoarthritis. Not only does this approach require anatomically sized and functional engineered cartilage, but the inflammatory environment within an arthritic joint may also inhibit chondrogenesis and induce degradation of native and engineered cartilage. Here, we present the culmination of multiple avenues of interdisciplinary research leading to the development and testing of bioartificial cartilage for tissue-engineered resurfacing of the hip joint. The work is based on a novel three-dimensional weaving technology that is infiltrated with specific bioinductive materials and/or genetically-engineered stem cells. A variety of design approaches have been tested in vitro, showing biomimetic cartilage-like properties as well as the capability for long-term tunable and inducible drug delivery. Importantly, these cartilage constructs have the potential to provide mechanical functionality immediately upon implantation, as they will need to replace a majority, if not the entire joint surface to restore function. To date, these approaches have shown excellent preclinical success in a variety of animal studies, including the resurfacing of a large osteochondral defect in the canine hip, and are now well-poised for clinical translation.

Biological resurfacing in a canine model of hip osteoarthritis.

Articular cartilage has unique load-bearing properties but has minimal capacity for intrinsic repair. Here, we used three-dimensional weaving, additive manufacturing, and autologous mesenchymal stem cells to create a tissue-engineered, bicomponent implant to restore hip function in a canine hip osteoarthritis model. This resorbable implant was specifically designed to function mechanically from the time of repair and to biologically integrate with native tissues for long-term restoration. A massive osteochondral lesion was created in the hip of skeletally mature hounds and repaired with the implant or left empty (control). Longitudinal outcome measures over 6 months demonstrated that the implant dogs returned to normal preoperative values of pain and function. Anatomical structure and functional biomechanical properties were also restored in the implanted dogs. Control animals never returned to normal and exhibited structurally deficient repair. This study provides clinically relevant evidence that the bicomponent implant may be a potential therapy for moderate hip osteoarthritis.

A genome-engineered bioartificial implant for autoregulated anticytokine drug delivery.

Biologic drug therapies are increasingly used for inflammatory diseases such as rheumatoid arthritis but may cause significant adverse effects when delivered continuously at high doses. We used CRISPR-Cas9 genome editing of iPSCs to create a synthetic gene circuit that senses changing levels of endogenous inflammatory cytokines to trigger a proportional therapeutic response. Cells were engineered into cartilaginous constructs that showed rapid activation and recovery in response to inflammation in vitro or in vivo. In the murine K/BxN model of inflammatory arthritis, bioengineered implants significantly mitigated disease severity as measured by joint pain, structural damage, and systemic and local inflammation. Therapeutic implants completely prevented increased pain sensitivity and bone erosions, a feat not achievable by current clinically available disease-modifying drugs. Combination tissue engineering and synthetic biology promises a range of potential applications for treating chronic diseases via custom-designed cells that express therapeutic transgenes in response to dynamically changing biological signals.

2010 Nicolas Andry Award: Multipotent adult stem cells from adipose tissue for musculoskeletal tissue engineering.

BACKGROUND: Cell-based therapies such as tissue engineering provide promising therapeutic possibilities to enhance the repair or regeneration of damaged or diseased tissues but are dependent on the availability and controlled manipulation of appropriate cell sources. QUESTIONS/PURPOSES: The goal of this study was to test the hypothesis that adult subcutaneous fat contains stem cells with multilineage potential and to determine the influence of specific soluble mediators and biomaterial scaffolds on their differentiation into musculoskeletal phenotypes. METHODS: We reviewed recent studies showing the stem-like characteristics and multipotency of adipose-derived stem cells (ASCs), and their potential application in cell-based therapies in orthopaedics. RESULTS: Under controlled conditions, ASCs show phenotypic characteristics of various cell types, including chondrocytes, osteoblasts, adipocytes, neuronal cells, or muscle cells. In particular, the chondrogenic differentiation of ASCs can be induced by low oxygen tension, growth factors such as bone morphogenetic protein-6 (BMP-6), or biomaterial scaffolds consisting of native tissue matrices derived from cartilage. Finally, focus is given to the development of a functional biomaterial scaffold that can provide ASC-based constructs with mechanical properties similar to native cartilage. CONCLUSIONS: Adipose tissue contains an abundant source of multipotent progenitor cells. These cells show cell surface marker profiles and differentiation characteristics that are similar to but distinct from other adult stem cells, such as bone marrow mesenchymal stem cells (MSCs). CLINICAL RELEVANCE: The availability of an easily accessible and reproducible cell source may greatly facilitate the development of new cell-based therapies for regenerative medicine applications in the musculoskeletal system.

Chondrogenic, hypertrophic, and osteochondral differentiation of human mesenchymal stem cells on three-dimensionally woven scaffolds.

The development of mechanically functional cartilage and bone tissue constructs of clinically relevant size, as well as their integration with native tissues, remains an important challenge for regenerative medicine. The objective of this study was to assess adult human mesenchymal stem cells (MSCs) in large, three-dimensionally woven poly(ε-caprolactone; PCL) scaffolds in proximity to viable bone, both in a nude rat subcutaneous pouch model and under simulated conditions in vitro. In Study I, various scaffold permutations-PCL alone, PCL-bone, "point-of-care" seeded MSC-PCL-bone, and chondrogenically precultured Ch-MSC-PCL-bone constructs-were implanted in a dorsal, ectopic pouch in a nude rat. After 8 weeks, only cells in the Ch-MSC-PCL constructs exhibited both chondrogenic and osteogenic gene expression profiles. Notably, although both tissue profiles were present, constructs that had been chondrogenically precultured prior to implantation showed a loss of glycosaminoglycan (GAG) as well as the presence of mineralization along with the formation of trabecula-like structures. In Study II of the study, the GAG loss and mineralization observed in Study I in vivo were recapitulated in vitro by the presence of either nearby bone or osteogenic culture medium additives but were prevented by a continued presence of chondrogenic medium additives. These data suggest conditions under which adult human stem cells in combination with polymer scaffolds synthesize functional and phenotypically distinct tissues based on the environmental conditions and highlight the potential influence that paracrine factors from adjacent bone may have on MSC fate, once implanted in vivo for chondral or osteochondral repair.

Composite scaffolds for cartilage tissue engineering.

Tissue engineering remains a promising therapeutic strategy for the repair or regeneration of diseased or damaged tissues. Previous approaches have typically focused on combining cells and bioactive molecules (e.g., growth factors, cytokines and DNA fragments) with a biomaterial scaffold that functions as a template to control the geometry of the newly formed tissue, while facilitating the attachment, proliferation, and differentiation of embedded cells. Biomaterial scaffolds also play a crucial role in determining the functional properties of engineered tissues, including biomechanical characteristics such as inhomogeneity, anisotropy, nonlinearity or viscoelasticity. While single-phase, homogeneous materials have been used extensively to create numerous types of tissue constructs, there continue to be significant challenges in the development of scaffolds that can provide the functional properties of load-bearing tissues such as articular cartilage. In an attempt to create more complex scaffolds that promote the regeneration of functional engineered tissues, composite scaffolds comprising two or more distinct materials have been developed. This paper reviews various studies on the development and testing of composite scaffolds for the tissue engineering of articular cartilage, using techniques such as embedded fibers and textiles for reinforcement, embedded solid structures, multi-layered designs, or three-dimensionally woven composite materials. In many cases, the use of composite scaffolds can provide unique biomechanical and biological properties for the development of functional tissue engineering scaffolds.

Composite Cellularized Structures Created from an Interpenetrating Polymer Network Hydrogel Reinforced by a 3D Woven Scaffold.

Biomaterial scaffolds play multiple roles in cartilage tissue engineering, including controlling architecture of newly formed tissue while facilitating growth of embedded cells and simultaneously providing functional properties to withstand the mechanical environment within the native joint. In particular, hydrogels-with high water content and desirable transport properties-while highly conducive to chondrogenesis, often lack functional mechanical properties. In this regard, interpenetrating polymer network (IPN) hydrogels can provide mechanical toughness greatly exceeding that of individual components; however, many IPN materials are not biocompatible for cell encapsulation. In this study, an agarose and poly(ethylene) glycol IPN hydrogel is seeded with human mesenchymal stem cells (MSCs). Results show high viability of MSCs within the IPN hydrogel, with improved mechanical properties compared to constructs comprised of individual components. These properties are further strengthened by integrating the hydrogel with a 3D woven structure. The resulting fiber-reinforced hydrogels display functional macroscopic mechanical properties mimicking those of native articular cartilage, while providing a local microenvironment that supports cellular viability and function. These findings suggest that a fiber-reinforced IPN hydrogel can support stem cell chondrogenesis while allowing for significantly enhanced, complex mechanical properties at multiple scales as compared to individual hydrogel or fiber components.

Genetic Engineering of Mesenchymal Stem Cells for Differential Matrix Deposition on 3D Woven Scaffolds.

Tissue engineering approaches for the repair of osteochondral defects using biomaterial scaffolds and stem cells have remained challenging due to the inherent complexities of inducing cartilage-like matrix and bone-like matrix within the same local environment. Members of the transforming growth factor ß (TGFß) family have been extensively utilized in the engineering of skeletal tissues, but have distinct effects on chondrogenic and osteogenic differentiation of progenitor cells. The goal of this study was to develop a method to direct human bone marrow-derived mesenchymal stem cells (MSCs) to deposit either mineralized matrix or a cartilaginous matrix rich in glycosaminoglycan and type II collagen within the same biochemical environment. This differential induction was performed by culturing cells on engineered three-dimensionally woven poly(ɛ-caprolactone) (PCL) scaffolds in a chondrogenic environment for cartilage-like matrix production while inhibiting TGFß3 signaling through Mothers against DPP homolog 3 (SMAD3) knockdown, in combination with overexpressing RUNX2, to achieve mineralization. The highest levels of mineral deposition and alkaline phosphatase activity were observed on scaffolds with genetically engineered MSCs and exhibited a synergistic effect in response to SMAD3 knockdown and RUNX2 expression. Meanwhile, unmodified MSCs on PCL scaffolds exhibited accumulation of an extracellular matrix rich in glycosaminoglycan and type II collagen in the same biochemical environment. This ability to derive differential matrix deposition in a single culture condition opens new avenues for developing complex tissue replacements for chondral or osteochondral defects.

Chondrogenic Differentiation Processes in Human Bone-Marrow Aspirates Seeded in Three-Dimensional-Woven Poly(ɛ-Caprolactone) Scaffolds Enhanced by Recombinant Adeno-Associated Virus-Mediated SOX9 Gene Transfer.

Combining gene therapy approaches with tissue engineering procedures is an active area of translational research for the effective treatment of articular cartilage lesions, especially to target chondrogenic progenitor cells such as those derived from the bone marrow. This study evaluated the effect of genetically modifying concentrated human mesenchymal stem cells from bone marrow to induce chondrogenesis by recombinant adeno-associated virus (rAAV) vector gene transfer of the sex-determining region Y-type high-mobility group box 9 (SOX9) factor upon seeding in three-dimensional-woven poly(ɛ-caprolactone; PCL) scaffolds that provide mechanical properties mimicking those of native articular cartilage. Prolonged, effective SOX9 expression was reported in the constructs for at least 21 days, the longest time point evaluated, leading to enhanced metabolic and chondrogenic activities relative to the control conditions (reporter lacZ gene transfer or absence of vector treatment) but without affecting the proliferative activities in the samples. The application of the rAAV SOX9 vector also prevented undesirable hypertrophic and terminal differentiation in the seeded concentrates. As bone marrow is readily accessible during surgery, such findings reveal the therapeutic potential of providing rAAV-modified marrow concentrates within three-dimensional-woven PCL scaffolds for repair of focal cartilage lesions.

Comparison of Fixation Techniques of 3D-Woven Poly(ϵ-Caprolactone) Scaffolds for Cartilage Repair in a Weightbearing Porcine Large Animal Model.

Objective To test different fixation methods of a 3-dimensionally woven poly(ϵ-caprolactone) (PCL) scaffold within chondral defects of a weightbearing large animal model. Methods Full thickness chondral defects were made in the femoral condyles of 15 adult male Yucatan mini-pigs. Two surgical approaches were compared including total arthrotomy (traditional) and a retinaculum-sparing, minimally invasive surgery (MIS) approach. Following microfracture (MFX), scaffolds were placed without fixation or were fixed with fibrin glue, suture, or subchondral anchor. Experimental endpoints were between 1 and 6 weeks. Micro-computed tomography and histology were used to assess samples. Results The MIS approach was superior as the traditional approach caused medial condyle cartilage wear. One of 13 (7.7%) of scaffolds without fixation, 4 of 11 (36.3%) fibrin scaffolds, 1 of 4 (25%) of sutured scaffolds, and 9 of 9 (100%) of anchor-fixed scaffolds remained in place. Histology demonstrated tissue filling with some overgrowth of PCL scaffolds. Conclusions Of the methods tested, the MIS approach coupled with subchondral anchor fixation provided the best scaffold retention in a mini-pig chondral defect model. This finding has implications for fixation strategies in future animal studies and potential future human use.

Repair response of the inner and outer regions of the porcine meniscus in vitro.

BACKGROUND: The menisci are essential intra-articular structures that contribute to knee function, and meniscal injury or loss is associated with joint degeneration. Tears of the outer vascularized zone have a greater potential for repair than do tears in the inner avascular region. OBJECTIVE AND HYPOTHESIS: Develop an in vitro explant model to examine the hypothesis that differences exist in the intrinsic repair response between the outer and inner region of the meniscus. STUDY DESIGN: Controlled laboratory study. METHODS: Cylindrical explants were harvested from the outer one third and inner two thirds of medial porcine menisci. To simulate a full-thickness defect, a central core was removed and reinserted immediately. Explants were cultured for 2, 4, or 6 weeks, and meniscal healing was investigated using mechanical testing, histologic analysis, and fluorescence confocal microscopy. RESULTS: Over the 6-week culture period, meniscal explants exhibited migration of cells into the repair site, followed by increased tissue formation that bridged the interface. The repair strength increased significantly over time, with no differences between the 2 regions. CONCLUSION: The findings show that explants from the avascular inner zone and vascular outer zone of the meniscus exhibit similar healing potential and repair strength in vitro. CLINICAL RELEVANCE: These findings support the hypothesis that the regional differences in meniscal repair observed clinically are owed to the additional vascular supply of the outer meniscus rather than intrinsic differences between the extracellular matrix and cells from these 2 areas.

Dedifferentiated Human Articular Chondrocytes Redifferentiate to a Cartilage-Like Tissue Phenotype in a Poly(ε-Caprolactone)/Self-Assembling Peptide Composite Scaffold.

Adult articular cartilage has a limited capacity for growth and regeneration and, with injury, new cellular or biomaterial-based therapeutic platforms are required to promote repair. Tissue engineering aims to produce cartilage-like tissues that recreate the complex mechanical and biological properties found in vivo. In this study, a unique composite scaffold was developed by infiltrating a three-dimensional (3D) woven microfiber poly (ε-caprolactone) (PCL) scaffold with the RAD16-I self-assembling nanofibers to obtain multi-scale functional and biomimetic tissue-engineered constructs. The scaffold was seeded with expanded dedifferentiated human articular chondrocytes and cultured for four weeks in control and chondrogenic growth conditions. The composite constructs were compared to control constructs obtained by culturing cells with 3D woven PCL scaffolds or RAD16-I independently. High viability and homogeneous cell distribution were observed in all three scaffolds used during the term of the culture. Moreover, gene and protein expression profiles revealed that chondrogenic markers were favored in the presence of RAD16-I peptide (PCL/RAD composite or alone) under chondrogenic induction conditions. Further, constructs displayed positive staining for toluidine blue, indicating the presence of synthesized proteoglycans. Finally, mechanical testing showed that constructs containing the PCL scaffold maintained the initial shape and viscoelastic behavior throughout the culture period, while constructs with RAD16-I scaffold alone contracted during culture time into a stiffer and compacted structure. Altogether, these results suggest that this new composite scaffold provides important mechanical requirements for a cartilage replacement, while providing a biomimetic microenvironment to re-establish the chondrogenic phenotype of human expanded articular chondrocytes.

Functional outcome measures in a surgical model of hip osteoarthritis in dogs.

BACKGROUND: The hip is one of the most common sites of osteoarthritis in the body, second only to the knee in prevalence. However, current animal models of hip osteoarthritis have not been assessed using many of the functional outcome measures used in orthopaedics, a characteristic that could increase their utility in the evaluation of therapeutic interventions. The canine hip shares similarities with the human hip, and functional outcome measures are well documented in veterinary medicine, providing a baseline for pre-clinical evaluation of therapeutic strategies for the treatment of hip osteoarthritis. The purpose of this study was to evaluate a surgical model of hip osteoarthritis in a large laboratory animal model and to evaluate functional and end-point outcome measures. METHODS: Seven dogs were subjected to partial surgical debridement of cartilage from one femoral head. Pre- and postoperative pain and functional scores, gait analysis, radiographs, accelerometry, goniometry and limb circumference were evaluated through a 20-week recovery period, followed by histological evaluation of cartilage and synovium. RESULTS: Animals developed histological and radiographic evidence of osteoarthritis, which was correlated with measurable functional impairment. For example, Mankin scores in operated limbs were positively correlated to radiographic scores but negatively correlated to range of motion, limb circumference and 20-week peak vertical force. CONCLUSIONS: This study demonstrates that multiple relevant functional outcome measures can be used successfully in a large laboratory animal model of hip osteoarthritis. These measures could be used to evaluate relative efficacy of therapeutic interventions relevant to human clinical care.

Tissue-engineered cartilage with inducible and tunable immunomodulatory properties.

The pathogenesis of osteoarthritis is mediated in part by inflammatory cytokines including interleukin-1 (IL-1), which promote degradation of articular cartilage and prevent human mesenchymal stem cell (MSC) chondrogenesis. In this study, we combined gene therapy and functional tissue engineering to develop engineered cartilage with immunomodulatory properties that allow chondrogenesis in the presence of pathologic levels of IL-1 by inducing overexpression of IL-1 receptor antagonist (IL-1Ra) in MSCs via scaffold-mediated lentiviral gene delivery. A doxycycline-inducible vector was used to transduce MSCs in monolayer or within 3D woven PCL scaffolds to enable tunable IL-1Ra production. In the presence of IL-1, IL-1Ra-expressing engineered cartilage produced cartilage-specific extracellular matrix, while resisting IL-1-induced upregulation of matrix metalloproteinases and maintaining mechanical properties similar to native articular cartilage. The ability of functional engineered cartilage to deliver tunable anti-inflammatory cytokines to the joint may enhance the long-term success of therapies for cartilage injuries or osteoarthritis.

The inhibition by interleukin 1 of MSC chondrogenesis and the development of biomechanical properties in biomimetic 3D woven PCL scaffolds.

Tissue-engineered constructs designed to treat large cartilage defects or osteoarthritic lesions may be exposed to significant mechanical loading as well as an inflammatory environment upon implantation in an injured or diseased joint. We hypothesized that a three-dimensionally (3D) woven poly(ε-caprolactone) (PCL) scaffold seeded with bone marrow-derived mesenchymal stem cells (MSCs) would provide biomimetic mechanical properties in early stages of in vitro culture as the MSCs assembled a functional, cartilaginous extracellular matrix (ECM). We also hypothesized that these properties would be maintained even in the presence of the pro-inflammatory cytokine interleukin-1 (IL-1), which is found at high levels in injured or diseased joints. MSC-seeded 3D woven scaffolds cultured in chondrogenic conditions synthesized a functional ECM rich in collagen and proteoglycan content, reaching an aggregate modulus of ~0.75 MPa within 14 days of culture. However, the presence of pathophysiologically relevant levels of IL-1 limited matrix accumulation and inhibited any increase in mechanical properties over baseline values. On the other hand, the mechanical properties of constructs cultured in chondrogenic conditions for 4 weeks prior to IL-1 exposure were protected from deleterious effects of the cytokine. These findings demonstrate that IL-1 significantly inhibits the chondrogenic development and maturation of MSC-seeded constructs; however, the overall mechanical functionality of the engineered tissue can be preserved through the use of a 3D woven scaffold designed to recreate the mechanical properties of native articular cartilage.

Functional properties of cell-seeded three-dimensionally woven poly(epsilon-caprolactone) scaffolds for cartilage tissue engineering.

Articular cartilage possesses complex mechanical properties that provide healthy joints the ability to bear repeated loads and maintain smooth articulating surfaces over an entire lifetime. In this study, we utilized a fiber-reinforced composite scaffold designed to mimic the anisotropic, nonlinear, and viscoelastic biomechanical characteristics of native cartilage as the basis for developing functional tissue-engineered constructs. Three-dimensionally woven poly(epsilon-caprolactone) (PCL) scaffolds were encapsulated with a fibrin hydrogel, seeded with human adipose-derived stem cells, and cultured for 28 days in chondrogenic culture conditions. Biomechanical testing showed that PCL-based constructs exhibited baseline compressive and shear properties similar to those of native cartilage and maintained these properties throughout the culture period, while supporting the synthesis of a collagen-rich extracellular matrix. Further, constructs displayed an equilibrium coefficient of friction similar to that of native articular cartilage (mu(eq) approximately 0.1-0.3) over the prescribed culture period. Our findings show that three-dimensionally woven PCL-fibrin composite scaffolds can be produced with cartilage-like mechanical properties, and that these engineered properties can be maintained in culture while seeded stem cells regenerate a new, functional tissue construct.