Mesenchymal stem cells (MSC) that can differentiate to various connective tissue cells may be useful for autologous cell transplantation to defects of bone, cartilage, and tendon, if MSC can be expanded in vitro. However, a short life span of MSC and a reduction in their differentiation potential in culture have limited their clinical application. The purpose of this study is to identify a growth factor(s) involved in self-renewal of MSC and the maintenance of their multilineage differentiation potential. Fibroblast growth factor-2 (FGF-2) markedly increased the growth rate and the life span of rabbit, canine, and human bone marrow MSC in monolayer cultures. This effect of FGF-2 was more prominent in low-density cultures than in high-density cultures. In addition, all MSC expanded in vitro with FGF-2, but not without FGF-2, differentiated to chondrocytes in pellet cultures. The FGF+ MSC also retained the osteogenic and adipogenic potential throughout many mitotic divisions. These findings suggest that FGFs play a crucial role in self-renewal of MSC.

As a means of improving the biological properties of materials for use as bone substitutes, functionally graded carbonate apatite containing Mg, FGMgCO3Ap, was synthesized at 60 degrees C and pH 7.4 using a gradient magnesium supply system. X-ray diffraction analysis of FGMgCO3Ap showed a poorly crystallized apatitic pattern, similar to that of human bone. ESCA analysis clearly showed the negative gradient distribution in Mg1s intensity (atomic concentration) of magnesium from the crystal surface toward the inner core. When the FGMgCO3Ap crystals were mixed with collagen, the resulting FGMgCO3Ap-collagen composite, irradiated with UV light for 4 h, retained their features in the saline solution. After washing away the nonadhesive cells, a cell adhesion assay showed that the optical density of the FGMgCO3Ap-collagen composite was higher than that of the CO3Ap-collagen composite. SEM observation showed that the osteoblast-like cells adhered well to the surface of the FGMgCO3Ap-collagen composite. Staining with hematoxylin-eosin and alizarin red confirmed the existence of a great many more cells and a thicker extracellular matrix layer on the FGMgCO3Ap-collagen composite than on the CO3Ap-collagen composite. This result demonstrated the acceleration effect of magnesium ions on osteoblast adhesion on the FGMgCO3Ap-collagen composite.

No abstract

The mechanisms by which the early limb cell Condensations and interzone mesenchyme give rise to skeletal elements and joints are poorly understood. Previous work from this laboratory has shown that the extracellular matrix protein tenascin-C is associated with articular cartilage and joint tissue development; others have shown that tenascin-C may exert its biological activities via interactions with cell surface receptors, such as syndecans. To further analyze the roles of tenascin-C and its putative receptors in skeletal development, we carried out a detailed in situ hybridization analysis of tenascin-C and syndecan-3 gene expression during development of chick limb skeletal elements and joints. We found that as the early mesenchymal condensations chondrify around day 5 (E5) of development, they become surrounded by a thick syndecan-3 rich perichondrium while tenascin-C transcripts are much fewer and restricted to diaphyseal perichondrium and developing interzones. Similar patterns were observed as distal carpal and digit condensations formed in older embryos. As the cartilaginous long bone models elongated proximo-distally and joint formation proceeded with age, we observed that syndecan-3 transcripts decrease significantly along the diaphysis and remain very abundant along the metaphysis and in the epiphyseal articular cap and interzone. Conversely, tenascin-C RNAs remain abundant along the diaphysis and begin to increase at the epiphysis and in interzone-derived tissues, such as menisci and joint capsule. By E10, the skeletal elements have well-defined morphologies, endochondral ossification has initiated in their diaphysis, and diaphyseal perichondrium has become periosteum. These developmental changes were accompanied by equally marked changes in gene expression; these included a marked increase in tenascin-C gene expression in articular cap, fragmentation of tenascin-C gene expression along the periosteum, reinitiation of syndecan-3 gene expression in periosteum, and differential gene expression in osteoprogenitor cells. The sheer complexity of the gene expression patterns documented in this study attests to the complexity of processes that bring about normal skelatogenesis. Clearly,tenascin-C and syndecan-3 appear to be closely associated with several of these processes, particularly in establishing tissue boundaries (perichondrium and periosteum) between condensations and surrounding mesenchymal cells, in regulating perichondral cell differentiation and incorporation into the growing skeletal elements, and in the genesis of epiphyseal chondrocytes and associated joint tissues. o 1995 Wiley-Liss, Inc.

Objective. To investigate the effects of hyaluronic acid (HA) on the release of proteoglycan by cultured rabbit chondrocytes.Methods. Articular cartilage chondrocytes were isolated from the knee joints of New Zealand white rabbits. Proteoglycan synthesis after incubation with HA was determined by measuring "S-sulfate incorporation. Cells incubated with HA were labeled with 3H-glucosamine and applied to a Sepharose CL-2B column. After incubation of confluent cells with "Ssulfate and then with HA in various concentrations in the presence or absence of cytokines, proteoglycan release from the cell matrix layer was measured.Results. HA ( M , 3 x lo5 to 19 x lo5), at 10 pg/ml ~ From the Department of Biochemistry, School of Dentistry, Hiroshima University, Hiroshima-city. the Departments of Biochemistry and Radiology, Faculty of Dentistry, Osaka University. Osaka, the Department of Orthopaedic Surgery, National Defense Medical College, Tokorozawa, and the Department of Pathology, Medical School, K a n a i w a University. Kanazawa, Japan.Atsushi Address reprint requests to Yukio Kato, PhD, DDS, Department of Biochemistry, School of Dentistry, Hiroshima University, 1-2-3, Kasumi, Minami-ku, Hiroshima-city. 734, Japan.Submitted for publication June 9, 1992; accepted in revised form October I , 1992.to 1 mg/ml, had little effect on the incorporation of 35S-sulfate or 3H-glucosamine into cartilage matrix proteoglycans, or on the hydrodynamic size of proteoglycan monomers, in rabbit chondrocyte cultures. However, at 1&1,000 pg/ml, HA suppressed the release of "Sproteoglycans from the cell matrix layer into the medium in the presence and absence of interleukin-1, tumor necrosis factor a, or basic fibroblast growth factor.Conclusion. These results suggest that HA is a potent inhibitor of the displacement of matrix proteoglycan into culture medium.The synthesis of proteoglycans by chondrocytes and the incorporation of these newly synthesized proteoglycans into the matrix are regulated by macromolecules in the extracellular matrix. Hyaluronic acid (HA) has been shown to cause a >50% reduction in the incorporation of "S-sulfate into proteoglycans synthesized by chick embryo chondrocytes ( l-3), adult pig chondrocytes (4), and Swarm rat chondrosarcoma cells (3, although the various studies have shown differences in the effective dose ( I ng/ml to I mg/ml). HA has also been found to inhibit chondrogenesis in vitro (6). Furthermore, HA or HA oligomers have been shown to stimulate the displacement of newly synthesized proteoglycans into the medium in chondrocyte cultures (3).These studies emphasized the importance of the inhibitory effect of HA on the accumulation of a proteoglycan matrix by developing chondrocytes. N o stimulatory effects of H A on chondrocytes or cartilage matrix have been demonstrated in chondrocyte cultures, even though cartilage contains 0.5-2.5 mg of HA

No abstract

The effects of PTH and calcitonin (CT) on the expression of mineralization-related phenotypes by chondrocytes were examined. In cultures of pelleted growth-plate chondrocytes. PTH caused 60-90% decreases in alkaline phosphatase activity, the incorporation of 45Ca into insoluble material, and the calcium content during the post-mitotic stage. These effects of PTH were dose-dependent and reversible. In contrast, CT increased alkaline phosphatase activity, 45Ca incorporation into insoluble material, and the calcium content by 1.4- to 1.8-fold. These observations suggest that PTH directly inhibits the expression of the mineralization-related phenotypes by growth-plate chondrocytes, and that CT has the opposite effects.

No abstract