Meniscus-Derived Matrix Scaffolds Promote the Integrative Repair of Meniscal Defects.

Meniscal tears have a poor healing capacity, and damage to the meniscus is associated with significant pain, disability, and progressive degenerative changes in the knee joint that lead to osteoarthritis. Therefore, strategies to promote meniscus repair and improve meniscus function are needed. The objective of this study was to generate porcine meniscus-derived matrix (MDM) scaffolds and test their effectiveness in promoting meniscus repair via migration of endogenous meniscus cells from the surrounding meniscus or exogenously seeded human bone marrow-derived mesenchymal stem cells (MSCs). Both endogenous meniscal cells and MSCs infiltrated the MDM scaffolds. In the absence of exogenous cells, the 8% MDM scaffolds promoted the integrative repair of an in vitro meniscal defect. Dehydrothermal crosslinking and concentration of the MDM influenced the biochemical content and shear strength of repair, demonstrating that the MDM can be tailored to promote tissue repair. These findings indicate that native meniscus cells can enhance meniscus healing if a scaffold is provided that promotes cellular infiltration and tissue growth. The high affinity of cells for the MDM and the ability to remodel the scaffold reveals the potential of MDM to integrate with native meniscal tissue to promote long-term repair without necessarily requiring exogenous cells.

Regulation of decellularized tissue remodeling via scaffold-mediated lentiviral delivery in anatomically-shaped osteochondral constructs.

Cartilage-derived matrix (CDM) has emerged as a promising scaffold material for tissue engineering of cartilage and bone due to its native chondroinductive capacity and its ability to support endochondral ossification. Because it consists of native tissue, CDM can undergo cellular remodeling, which can promote integration with host tissue and enables it to be degraded and replaced by neotissue over time. However, enzymatic degradation of decellularized tissues can occur unpredictably and may not allow sufficient time for mechanically competent tissue to form, especially in the harsh inflammatory environment of a diseased joint. The goal of the current study was to engineer cartilage and bone constructs with the ability to inhibit aberrant inflammatory processes caused by the cytokine interleukin-1 (IL-1), through scaffold-mediated delivery of lentiviral particles containing a doxycycline-inducible IL-1 receptor antagonist (IL-1Ra) transgene on anatomically-shaped CDM constructs. Additionally, scaffold-mediated lentiviral gene delivery was used to facilitate spatial organization of simultaneous chondrogenic and osteogenic differentiation via site-specific transduction of a single mesenchymal stem cell (MSC) population to overexpress either chondrogenic, transforming growth factor-beta 3 (TGF-ß3), or osteogenic, bone morphogenetic protein-2 (BMP-2), transgenes. Controlled induction of IL-1Ra expression protected CDM hemispheres from inflammation-mediated degradation, and supported robust bone and cartilage tissue formation even in the presence of IL-1. In the absence of inflammatory stimuli, controlled cellular remodeling was exploited as a mechanism for fusing concentric CDM hemispheres overexpressing BMP-2 and TGF-ß3 into a single bi-layered osteochondral construct. Our findings demonstrate that site-specific delivery of inducible and tunable transgenes confers spatial and temporal control over both CDM scaffold remodeling and neotissue composition. Furthermore, these constructs provide a microphysiological in vitro joint organoid model with site-specific, tunable, and inducible protein delivery systems for examining the spatiotemporal response to pro-anabolic and/or inflammatory signaling across the osteochondral interface.

Fabrication of anatomically-shaped cartilage constructs using decellularized cartilage-derived matrix scaffolds.

The native extracellular matrix of cartilage contains entrapped growth factors as well as tissue-specific epitopes for cell-matrix interactions, which make it a potentially attractive biomaterial for cartilage tissue engineering. A limitation to this approach is that the native cartilage extracellular matrix possesses a pore size of only a few nanometers, which inhibits cellular infiltration. Efforts to increase the pore size of cartilage-derived matrix (CDM) scaffolds dramatically attenuate their mechanical properties, which makes them susceptible to cell-mediated contraction. In previous studies, we have demonstrated that collagen crosslinking techniques are capable of preventing cell-mediated contraction in CDM disks. In the current study, we investigated the effects of CDM concentration and pore architecture on the ability of CDM scaffolds to resist cell-mediated contraction. Increasing CDM concentration significantly increased scaffold mechanical properties, which played an important role in preventing contraction, and only the highest CDM concentration (11% w/w) was able to retain the original scaffold dimensions. However, the increase in CDM concentration led to a concomitant decrease in porosity and pore size. Generating a temperature gradient during the freezing process resulted in unidirectional freezing, which aligned the formation of ice crystals during the freezing process and in turn produced aligned pores in CDM scaffolds. These aligned pores increased the pore size of CDM scaffolds at all CDM concentrations, and greatly facilitated infiltration by mesenchymal stem cells (MSCs). These methods were used to fabricate of anatomically-relevant CDM hemispheres. CDM hemispheres with aligned pores supported uniform MSC infiltration and matrix deposition. Furthermore, these CDM hemispheres retained their original architecture and did not contract, warp, curl, or splay throughout the entire 28-day culture period. These findings demonstrate that given the appropriate fabrication parameters, CDM scaffolds are capable of maintaining complex structures that support MSC chondrogenesis.

The effects of crosslinking of scaffolds engineered from cartilage ECM on the chondrogenic differentiation of MSCs.

Scaffolds fabricated from cartilage extracellular matrix provide a chondroinductive environment that stimulates cartilaginous matrix synthesis in a variety of cell types. A limitation of these cartilage-derived matrix (CDM) scaffolds is that they contract during in vitro culture, which unpredictably alters their shape. The current study examined the hypothesis that collagen crosslinking techniques could inhibit cell-mediated contraction of CDM scaffolds. We analyzed the effects of dehydrothermal (DHT) treatment, ultraviolet light irradiation (UV), and the chemical crosslinker carbodiimide (CAR) on scaffold contraction and chondrogenic differentiation of adult human bone marrow-derived stem cells (MSCs). Both physical and chemical crosslinking treatments retained the original scaffold dimensions. DHT and UV treatments produced significantly higher glycosaminoglycan and collagen contents than CAR crosslinked and non-crosslinked constructs. Crosslinking treatments influenced the composition of newly synthesized matrix, and DHT treatment best matched the composition of native cartilage. DHT, UV, and non-crosslinked CDM films supported cell attachment, while CAR crosslinking inhibited cell adhesion. These results affirm that collagen crosslinking treatments can prevent cell-mediated contraction of CDM scaffolds. Interestingly, crosslinking treatments influence chondrogenic differentiation. These effects seem to be mediated by modifications to cell-matrix interactions between MSCs and the CDM; however, further work is necessary to elucidate the specific mechanisms involved in this process.

Factors influencing the long-term behavior of extracellular matrix-derived scaffolds for musculoskeletal soft tissue repair.

Musculoskeletal connective tissues such as tendon, ligament, and cartilage possess a limited ability for self-repair. Tissue engineering seeks to use combinations of cells, bioactive molecules, and biomaterials to develop new treatment options for the repair or replacement of damaged tissues. The use of native extracellular matrix as scaffold material for tissue engineering has become increasingly attractive because such tissues can not only provide structural support, but also regulate cell behavior. Although demineralized bone matrix has long been recognized for its osteoinductive abilities, recent studies have identified the ability of cartilage and tendon extracellular matrices to stimulate the differentiation of mesenchymal or adipose-derived adult stem cells toward chondrogenic or tenogenic lineages, respectively. This review discusses the motivation for fabricating scaffolds from musculoskeletal tissues, the in vitro and in vivo efficacy of these tissue-derived scaffolds, and various processing techniques such as decellularization or cross-linking that can mitigate immunogenic responses, moderate the degradation profile, and enhance the mechanical properties of these constructs following long-term implantation in vivo.

Chondrogenesis of adult stem cells from adipose tissue and bone marrow: induction by growth factors and cartilage-derived matrix.

OBJECTIVES: Adipose-derived stem cells (ASCs) and bone marrow-derived mesenchymal stem cells (MSCs) are multipotent adult stem cells with potential for use in cartilage tissue engineering. We hypothesized that these cells show distinct responses to different chondrogenic culture conditions and extracellular matrices, illustrating important differences between cell types. METHODS: Human ASCs and MSCs were chondrogenically differentiated in alginate beads or a novel scaffold of reconstituted native cartilage-derived matrix with a range of growth factors, including dexamethasone, transforming growth factor beta3, and bone morphogenetic protein 6. Constructs were analyzed for gene expression and matrix synthesis. RESULTS: Chondrogenic growth factors induced a chondrocytic phenotype in both ASCs and MSCs in alginate beads or cartilage-derived matrix. MSCs demonstrated enhanced type II collagen gene expression and matrix synthesis as well as a greater propensity for the hypertrophic chondrocyte phenotype. ASCs had higher upregulation of aggrecan gene expression in response to bone morphogenetic protein 6 (857-fold), while MSCs responded more favorably to transforming growth factor beta3 (573-fold increase). CONCLUSIONS: ASCs and MSCs are distinct cell types as illustrated by their unique responses to growth factor-based chondrogenic induction. This chondrogenic induction is affected by the composition of the scaffold and the presence of serum.