Bone morphogenetic protein (BMP) is known to require a suitable carrier to induce ectopic bone formation in vivo. Hydroxyapatite ceramics have been reported to be effective in some forms but ineffective in others as a carrier of BMP-induced bone formation. In this study we compare three geometrically different forms of hydroxyapatite to examine their functions as carriers of BMP-induced bone formation. A fraction containing all the active BMPs (BMP cocktail) was partially purified from a 4M guanidine extract from bovine bone by a three-step chromatographic procedure. The BMP cocktail was combined with each of three forms of hydroxyapatite--solid particles (SPHAP), porous particles (PPHAP), and coral-replicated porous tablets (coral-HAP)--and implanted subcutaneously into rats. Both the PPHAP and coral-HAP systems induced osteogenesis 2 weeks after implantation, as evidenced by morphological and biochemical observations. Details of the osteogenetic process were followed by double-fluorescence labeling in the coral-HAP system to confirm bone formation on the surface of hydroxyapatite. However, there was no evidence of osteogenesis or chondrogenesis in the SPHAP system. The results indicate that the geometry of the interconnected porous structure in PPHAP and coral-HAP create spaces for vasculature that lead to osteogenesis while the smooth structure and close contact of particles in SPHAP inhibit vascular formation and proliferation of mesenchymal cells, preventing bone and cartilage formation. It was concluded that the geometrical structure in hydroxyapatite ceramics that induces vasculature is crucial as a carrier for BMP-induced bone formation.
Osteocalcin (OC) is a bone-specific protein synthesized by osteoblasts that represents a good marker for osteogenic maturation. We examined whether in vitro osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells (MSCs) could be simply assessed at earlier stages by monitoring OC secretion into the conditioned medium, rather than measuring OC deposition on the extracellular matrix (ECM), using a sandwich enzyme immunoassay system involving a specific anti-rat OC monoclonal antibody. During a 16-day culture, OC was secreted into the medium of MSCs from day 8 and increased substantially until day 16. In contrast, OC deposition on the ECM was low, even at day 13, when calcium deposition was at high levels. The histological expression pattern of OC messenger RNA provided in situ evidence that osteoblastic cells appeared at the early stages of 6 to 9 days and matured over time in vitro. Furthermore, the temporal expression of osteogenesis-specific genes, such as the transcriptional factors core-binding factor 1 and osterix, followed by increases in secretory OC proved the commitment of MSCs to osteoblastic differentiation. These results revealed that biomineralization followed secretion of OC, which may reflect early osteoblastic differentiation of cultured MSCs under osteoinductive conditions. We ascertained the osteogenic differentiation capacity of cultured MSCs in a non-destructive manner by monitoring OC secretion into the culture medium and proved that secretory OC could represent a reliable marker for predicting in vivo osteogenic potential in bone tissue engineering.
Fresh marrow cells were obtained from the femora Fischer rats and cultured in a medium containing 15% fetal calf serum (FCS) to leach confluent. After trypsinization, cells were subcultured at a cell density of 100 x 10(3)/35 mm well in the presence of FCS, 10 mM beta-glycerophosphate, 82 micrograms/mL ascorbic acid phosphate, and 10(-8)M dexamethasone (Dex). Osteoblastic cells and microscopic mineralized nodules began to appear at about 1 week after the subculture, and at 2 weeks many macroscopic nodules that showed high alkaline phosphatase activity (ALP) and appearance of bone Gla protein (BGP) mRNA were evident. As demonstrated by in situ hybridization, the mRNA was manifested by cuboid-shaped cells (osteoblastic cells). X-ray diffraction (XRD) and Fourier-transform infrared spectroscopy (FTIR) showed the mineralization of fine crystals of hydroxyapatite comparable to natural rat bone mineral. In contrast to these findings, subculture done under the same conditions except for the lack of Dex did not show mineralized nodules, nor did they show the osteoblastic phenotype expression. These analyses indicate that Dex-induced mineralization using rat bone marrow cell culture is an in vitro counterpart of bone formed in vivo. Such a culture is useful for investigating materials/ osteogenic cells interactions.
Bioactive materials such as hydroxyapatite ceramics (HA) are known to show a stable interfacial bond with tissues (the bone bonding property has been especially well documented). However, the biological phenomenon on the material surface that evoke bone bonding is not well documented. To address this issue, composites of porous HA and rat marrow cells were implanted into ectopic (subcutaneous) sites. At 3 weeks after implantation, active osteoblasts lining the HA surface initiated bone formation, primarily as mature bone (lamellar type). Gene expression study using Northern blot analysis of the composites showed that alkaline phosphatase (ALP) and bone Gla protein (BGP) mRNAs began to appear at 2 weeks and the level of BGP mRNA at 4 weeks was comparable to that of cancerous bone. However, HA implantation without marrow cells did not show bone formation and neither BGP nor ALP mRNA were detected. These results indicate that the HA surface is able to support cell differentiation (osteoblastic phenotype expression of marrow stromal stem cells), as well as cell coordination resulting in mature bone formation that bonds to the HA.
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