High-density cell systems incorporating polymer microspheres as microenvironmental regulators in engineered cartilage tissues.

To address the significant clinical need for tissue-engineered therapies for the repair and regeneration of articular cartilage, many systems have recently been developed using bioactive polymer microspheres as regulators of the chondrogenic microenvironment within high-density cell cultures. In this review, we highlight various densely cellular systems utilizing polymer microspheres as three-dimensional (3D) structural elements within developing engineered cartilage tissue, carriers for cell expansion and delivery, vehicles for spatiotemporally controlled growth factor delivery, and directors of cell behavior via regulation of cell-biomaterial interactions. The diverse systems described herein represent a shift from the more traditional tissue engineering approach of combining cells and growth factors within a biomaterial scaffold, to the design of modular systems that rely on the assembly of cells and bioactive polymer microspheres as building blocks to guide the creation of articular cartilage. Cell-based assembly of 3D microsphere-incorporated structures represents a promising avenue for the future of tissue engineering.

Spatiotemporal regulation of chondrogenic differentiation with controlled delivery of transforming growth factor-ß1 from gelatin microspheres in mesenchymal stem cell aggregates.

The precise spatial and temporal presentation of growth factors is critical for cartilage development, during which tightly controlled patterns of signals direct cell behavior and differentiation. Recently, chondrogenic culture of human mesenchymal stem cells (hMSCs) has been improved through the addition of polymer microspheres capable of releasing growth factors directly to cells within cellular aggregates, eliminating the need for culture in transforming growth factor-ß1 (TGF-ß1)-containing medium. However, the influence of specific patterns of spatiotemporal growth factor presentation on chondrogenesis within microsphere-incorporated cell systems is unclear. In this study, we examined the effects of altering the chondrogenic microenvironment within hMSC aggregates through varying microsphere amount, growth factor concentration per microsphere, and polymer degradation time. Cartilage formation was evaluated in terms of DNA, glycosaminoglycan, and type II collagen in hMSCs from three donors. Chondrogenesis equivalent to or greater than that of aggregates cultured in medium containing TGF-ß1 was achieved in some conditions, with varied differentiation based on the specific conditions of microsphere incorporation. A more spatially distributed delivery of TGF-ß1 from a larger mass of fast-degrading microspheres improved differentiation by comparison with delivery from a smaller mass of microspheres with a higher TGF-ß1 concentration per microsphere, although the total amount of growth factor per aggregate was the same. Results also indicated that the rate and degree of chondrogenesis varied on a donor-to-donor basis. Overall, this study elucidates the effects of varied conditions of TGF-ß1-loaded microsphere incorporation on hMSC chondrogenesis, demonstrating that both spatiotemporal growth factor presentation and donor variability influence chondrogenic differentiation within microsphere-incorporated cellular constructs.

Engineered cartilage via self-assembled hMSC sheets with incorporated biodegradable gelatin microspheres releasing transforming growth factor-ß1.

Self-assembling cell sheets have shown great potential for use in cartilage tissue engineering applications, as they provide an advantageous environment for the chondrogenic induction of human mesenchymal stem cells (hMSCs). We have engineered a system of self-assembled, microsphere-incorporated hMSC sheets capable of forming cartilage in the presence of exogenous transforming growth factor ß1 (TGF-ß1) or with TGF-ß1 released from incorporated microspheres. Gelatin microspheres with two different degrees of crosslinking were used to enable different cell-mediated microsphere degradation rates. Biochemical assays, histological and immunohistochemical analyses, and biomechanical testing were performed to determine biochemical composition, structure, and equilibrium modulus in unconfined compression after 3 weeks of culture. The inclusion of microspheres with or without loaded TGF-ß1 significantly increased sheet thickness and compressive equilibrium modulus, and enabled more uniform matrix deposition by comparison to control sheets without microspheres. Sheets incorporated with fast-degrading microspheres containing TGF-ß1 produced significantly more GAG and GAG per DNA than all other groups tested and stained more intensely for type II collagen. These findings demonstrate improved cartilage formation in microsphere-incorporated cell sheets, and describe a tailorable system for the chondrogenic induction of hMSCs without necessitating culture in growth factor-containing medium.