External ear reconstruction with autologous cartilage still remains one of the most difficult problems in the fields of plastic and reconstructive surgery. As the absence of tissue vascularization limits the ability to stimulate new tissue growth, relatively few surgical approaches are currently available (alloplastic implants or sculpted autologous cartilage grafts) to repair or reconstruct the auricle (or pinna) as a result of traumatic loss or congenital absence (e.g., microtia). Alternatively, tissue engineering can offer the potential to grow autogenous cartilage suitable for implantation. While tissue-engineered auricle cartilage constructs can be created, a substantial number of cells are required to generate sufficient quantities of tissue for reconstruction. Similarly, as routine cell expansion can elicit negative effects on chondrocyte function, we have developed an approach to generate large-sized engineered auricle constructs (≥3 cm(2)) directly from a small population of donor cells (20,000-40,000 cells/construct). Using rabbit donor cells, the developed bioreactor-cultivated constructs adopted structural-like characteristics similar to native auricular cartilage, including the development of distinct cartilaginous and perichondrium-like regions. Both alterations in media composition and seeding density had profound effects on the formation of engineered elastic tissue constructs in terms of cellularity, extracellular matrix accumulation, and tissue structure. Higher seeding densities and media containing sodium bicarbonate produced tissue constructs that were closer to the native tissue in terms of structure and composition. Future studies will be aimed at improving the accumulation of specific tissue constituents and determining the clinical effectiveness of this approach using a reconstructive animal model.

These findings support the previous notion that certain bisphosphonates may be useful as adjunctive therapies to potentially ameliorate progression of cartilage degeneration and improve arthritis management.

Background: The use of chondrocytes for cartilage tissue engineering is hampered by the limited number of chondrocytes that can be harvested and potential dedifferentiation during cell expansion. While stem cells is a promising approach to create a large population of differentiated cells, multiple growth factors are typically required for differentiation. Alternatively, co-culturing of stem cells with mature chondrocytes can induce differentiation. However, it is not clear which stem cell population and co-culturing method best supports chondrogenesis. While co-culture of stem cells with chondrocytes has been extensively shown to improve chondrogenesis in general, results from previous reports were convoluted by the use of 2D culture or scaffold materials, resulting in discrepancies with the comparison between direct and indirect cocultures. Methods: The purpose of this study was to investigate the extent of chondrogenic differentiation of direct and indirect co-culture of bone marrow stem cells (BMSCs) or adipose-derived stem cells (ASCs) with mature chondrocytes in 3D scaffold-free culture. For direct co-culture, cell pellets were created by centrifugation, consisting of chondrocytes alone or different proportions of stem cells to chondrocytes. For indirect co-culture, cell pellets of chondrocytes or stem cells were created individually and cultured with a separation by a trans-well membrane. Chondrogenic differentiation potential was assessed by quantification of DNA, GAG and collagen contents, as well as collagen I, collagen II and Safranin-O staining. Statistical significance was analyzed using one-way ANOVA with Tukey's post-hoc tests. Results: Direct co-culture of chondrocytes with BMSCs resulted in superior chondrogenesis compared to all other co-culture methods. Cultures with the ratio of 3:1 BMSCs to chondrocytes stained positive for chondrogenic markers and displayed a uniform deposition of cartilaginous extracellular matrix. In addition, the extent of matrix deposition in direct BMSC co-cultures were comparable to growth factor differentiated BMSCs. Conclusions: Thus, BMSCs appear to be superior to ASCs in their differentiation capacity during coculture and a direct co-culture with chondrocytes (3:1 ratio) may be a feasible strategy for cartilage tissue engineering.