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We report studies of bone tissue engineering using human mesenchymal stem cells (MSCs), a protein substrate (film or scaffold; fast degrading unmodified collagen, or slowly degrading cross-linked collagen and silk), and a bioreactor (static culture, spinner flask, or perfused cartridge). MSCs were isolated from human bone marrow, characterized for the expression of cell surface markers and the ability to undergo chondrogenesis and osteogenesis in vitro, and cultured for 5 weeks. MSCs were positive for CD105/endoglin, and had a potential for chondrogenic and osteogenic differentiation. In static culture, calcium deposition was similar for MSC grown on collagen scaffolds and films. Under medium flow, MSC on collagen scaffolds deposited more calcium and had a higher alcaline phosphatase (AP) activity than MSC on collagen films. The amounts of DNA were markedly higher in constructs based on slowly degrading (modified collagen and silk) scaffolds than on fast degrading (unmodified collagen) scaffolds. In spinner flasks, medium flow around constructs resulted in the formation of bone rods within the peripheral region, that were interconnected and perpendicular to the construct surface, whereas in perfused constructs, individual bone rods oriented in the direction of fluid flow formed throughout the construct volume. These results suggest that osteogenesis in cultured MSC can be modulated by scaffold properties and flow environment.

Porous biodegradable silk scaffolds and human bone marrow derived mesenchymal stem cells (hMSCs) were used to engineer bone-like tissue in vitro. Two different scaffolds with the same microstructure were studied: collagen (to assess the effects of fast degradation) and silk with covalently bound RGD sequences (to assess the effects of enhanced cell attachment and slow degradation). The hMSCs were isolated, expanded in culture, characterized with respect to the expression of surface markers and ability for chondrogenic and osteogenic differentiation, seeded on scaffolds, and cultured for up to 4 weeks. Histological analysis and microcomputer tomography showed the development of up to 1.2-mm-long interconnected and organized bonelike trabeculae with cuboid cells on the silk-RGD scaffolds, features still present but to a lesser extent on silk scaffolds and absent on the collagen scaffolds. The X-ray diffraction pattern of the deposited bone corresponded to hydroxyapatite present in the native bone. Biochemical analysis showed increased mineralization on silk-RGD scaffolds compared with either silk or collagen scaffolds after 4 weeks. Expression of bone sialoprotein, osteopontin, and bone morphogenetic protein 2 was significantly higher for hMSCs cultured in osteogenic than control medium both after 2 and 4 weeks in culture. The results suggest that RGD-silk scaffolds are particularly suitable for autologous bone tissue engineering, presumably because of their stable macroporous structure, tailorable mechanical properties matching those of native bone, and slow degradation.

Human mesenchymal stem cells (hMSC) derived from bone marrow aspirates can form the basis for the in vitro cultivation of autologous tissue grafts and help alleviate the problems of immunorejection and disease transmission associated with the use of allografts. We explored the utility of hMSC cultured on protein scaffolds for tissue engineering of cartilage. hMSC were isolated, expanded in culture, characterized with respect to the expression of surface markers and ability for chondrogenic and osteogenic differentiation, and seeded on scaffolds. Four different scaffolds were tested, formed as a highly porous sponge made of: 1) collagen, 2) cross-linked collagen, 3) silk, and 4) RGD-coupled silk. Cell-seeded scaffolds were cultured for up to 4 weeks in either control medium (DMEM supplemented with 10% fetal bovine serum) or chondrogenic medium (control medium supplemented with chondrogenic factors). hMSC attachment, proliferation, and metabolic activity were markedly better on slowly degrading silk than on fast-degrading collagen scaffolds. In chondrogenic medium, hMSC formed cartilaginous tissues on all scaffolds, but the extent of chondrogenesis was substantially higher for hMSC cultured on silk as compared to collagen scaffolds. The deposition of glycosaminoglycan (GAG) and type II collagen and the expression of type II collagen mRNA were all higher for hMSC cultured on silk than on collagen scaffolds. Taken together, these results suggest that silk scaffolds are particularly suitable for tissue engineering of cartilage starting from hMSC, presumably due to their high porosity, slow biodegradation, and structural integrity.

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