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For tissue engineering of autologous cartilage, cell expansion is needed to obtain the cell numbers required. Standard expansion media contain bovine serum. This has several disadvantages, that is, the risk of transmitting diseases and serum-batch variations. The aim of this study was to find a serum-free medium with at least the same potential to expand cell numbers as serum-containing media. Ear chondrocytes of three young children were expanded in either serum-containing medium (SCM; DMEM with 10% fetal calf serum) or serum-free medium (SFM; DMEM with ITS+) supplemented with 5 or 100 ng/mL fibroblast growth factor-2 (FGF2). To promote cell adherence onto the culture flask, the serum-free conditions were cultured with 10% serum for 1 day after each trypsinization. After the fourth passage, the chondrocytes were encapsuled in alginate beads and redifferentiated in a SFM (DMEM with ITS+, hydrocortisone, and L-ascorbic acid) supplemented with 10 ng/mL IGF-I and 10 ng/mL TGFbeta-2. Results showed that expansion in SFM with 100 ng/mL FGF2 was comparable to expansion in SCM. Redifferentiation with SFM with IGF-I and TGFbeta-2 showed high collagen type II expression and high GAG/DNA production regardless of which expansion medium had been used. However, chondrocytes expanded in SFM with 100 ng/mL FGF2 resulted in less positive cells for collagen type I and 11-fibrau (a fibroblast membrane marker). The present study shows that it is possible to use serum-free medium for tissue engineering of cartilage. Expansion of immature ear chondrocytes in SFM supplemented with high-concentration FGF2 resulted in high cell numbers, which in addition had better redifferentiation capacity than cells expanded in medium with 10% serum.

Item does not contain fulltext The aim of this study was to compare the ability of hard tissue regeneration of four types of stem cells or precursors under both in vitro and in vivo situations. Primary cultures of rat bone marrow, rat dental pulp, human bone marrow, and human dental pulp cells were seeded onto a porous ceramic scaffold material, and then either cultured in an osteogenic medium or subcutaneously implanted into nude mice. For cell culture, samples were collected at weeks 0, 1, 3, and 5. Results were analyzed by measuring cell proliferation rate and alkaline phosphatase activity, scanning electron microscopy, and real-time PCR. Samples from the implantation study were retrieved after 5 and 10 weeks and evaluated by histology and real-time PCR. The results indicated that in vitro abundant cell growth and mineralization of extracellular matrix was observed for all types of cells. However, in vivo matured bone formation was found only in the samples seeded with rat bone marrow stromal cells. Real-time PCR suggested that the expression of Runx2 and the expression osteocalcin were important for the differentiation of bone marrow stromal cells, while dentin sialophosphoprotein contributed to the odontogenic differentiation. In conclusion, the limited hard tissue regeneration ability of dental pulp stromal cells questions their practical application for complete tooth regeneration. Repeated cell passaging may explain the reduction of the osteogenic ability of both bone- and dentinal-derived stem cells. Therefore, it is essential to develop new cell culture methods to harvest the desired cell numbers while not obliterating the osteogenic potential.

Reconstruction of large mucosal defects of the floor of the mouth is typically performed with keratinizing skin. Drawbacks include donor site defects and hair bearing of the flaps. Cultured mucosal substitutes (CMSs) have been developed for clinical use to replace keratinizing skin. Acellular dermis is often used as a dermal carrier for autologous cells, because it reduces wound contraction and is easier for the surgeon to handle than, for example, collagen gels. A major problem of CMSs using acellular dermis is variation in epidermal quality. To improve the quality of the CMSs, human fibroblasts were incorporated into the acellular dermis and seeded with human keratinocytes. To study the role of the fibroblasts in epidermal morphology and basement membrane formation, CMSs were stained for differentiation markers beta1 integrin, cytokeratin 10, and involucrin after 1 and 2 weeks in culture. Basement membrane formation was analyzed using laminin 5 and collagen IV and VII staining; proliferation was analyzed using Ki-67 staining. The epidermises of fibroblast-containing CMSs matured faster into a well-organized epithelium than did those that did not contain CMSs. A 52.7% increase in basal cells, a 53.5% increase in mitosis index, and a 78.0% increase in keratinocyte cell layers were observed. Addition of fibroblasts reduced culturing time and enhanced proliferation, maturation, and quality of the epidermis.