Large, stratified, and mechanically functional human cartilage grown in vitro by mesenchymal condensation

Abstract

The efforts to grow mechanically functional cartilage from human mesenchymal stem cells have not been successful. We report that clinically sized pieces of human cartilage with physiologic stratification and biomechanics can be grown in vitro by recapitulating some aspects of the developmental process of mesenchymal condensation. By exposure to transforming growth factor-β, mesenchymal stem cells were induced to condense into cellular bodies, undergo chondrogenic differentiation, and form cartilagenous tissue, in a process designed to mimic mesenchymal condensation leading into chondrogenesis. We discovered that the condensed mesenchymal cell bodies (CMBs) formed in vitro set an outer boundary after 5 d of culture, as indicated by the expression of mesenchymal condensation genes and deposition of tenascin. Before setting of boundaries, the CMBs could be fused into homogenous cellular aggregates giving rise to well-differentiated and mechanically functional cartilage. We used the mesenchymal condensation and fusion of CMBs to grow centimeter-sized, anatomically shaped pieces of human articular cartilage over 5 wk of culture. For the first time to our knowledge biomechanical properties of cartilage derived from human mesenchymal cells were comparable to native cartilage, with the Young's modulus of >800 kPa and equilibrium friction coeffcient of <0.3. We also demonstrate that CMBs have capability to form mechanically strong cartilage-cartilage interface in an in vitro cartilage defect model. The CMBs, which acted as "lego-like" blocks of neocartilage, were capable of assembling into human cartilage with physiologic-like structure and mechanical properties.

Recapitulation of physiological spatiotemporal signals promotes in vitro formation of phenotypically stable human articular cartilage

“…The stratification was evidenced by superficialzone expression of lubricin and deep-zone expression of osteopontin. We previously showed that self-assembling MSCs formed cartilage with a superficial lubricin lining after 5 wk of chondrogenic induction (10). In the present study, zonal lubricin expression was observed after implantation.…”

“…Mesenchymal condensation has been adapted into selfassembly methods to enhance cartilage formation in vitro (10,11). TGF-β and thyroxine, potent effectors of cartilage maintenance and chondrocyte terminal differentiation, have been used in chondrogenic and hypertrophic induction regimens to guide MSC differentiation (4,14,(17)(18)(19)(20)(21)(22).…”

“…RNA in situ hybridization would further clarify expression patterns. To increase the cartilage thickness, we could integrate our novel approach with a method we developed to engineer large cartilage by fusing condensed mesenchymal bodies on bone scaffolds (10). Importantly, this approach locally regulated but did not inhibit type X collagen expression.…”

“…Thus, there is an ongoing effort toward developing stem cell-based therapies, particularly with mesenchymal stem cells (MSCs)-the most attractive cell source for clinical application (9). Our group and others have shown that self-assembly methods recapitulated mesenchymal condensation and enhanced chondrogenesis of MSCs in vitro (10)(11)(12). Still, standard isotropic culture fails to recapitulate the spatiotemporal gradients present during native development (3,9,13,14).…”

A Modular Strategy to Engineer Complex Tissues and Organs

“…Timing may play a critical role in the success of tissue fusion13 as well as development of tissue‐specific phenotypes. As a result, cartilage and prevascular rings were cultured individually for varying time periods prior to stacking them for fusion into a composite tube to determine the impact of timing of fusion on resultant tissue structure and differentiation.…”