A composite of marrow mesenchymal stem cells (MSCs) and porous hydroxyapatite (HA) has bone-forming capability. To promote the capability, we added recombinant human bone morphogenetic protein-2 (BMP) to the composite. The bone formation was assessed by rat subcutaneous implantation of 4 different kinds of implants, i.e., HA alone, BMP/HA composites, MSCs/HA composites, and the composites containing BMP (MSCs/BMP/HA). Both HA and the BMP/HA composites did not show bone formation at any time after implantation. The MSCs/HA composites showed moderate bone formation at 4 weeks and extensive bone formation at 8 weeks. The MSCs/BMP/HA composites showed obvious bone formation together with active osteoblasts at 2 weeks and more bone formation at 4 and 8 weeks. The MSCs/BMP/HA composites demonstrated high alkaline phosphatase and osteocalcin expression at both the protein and gene levels. These results indicate that the combination of MSCs, porous HA, and BMP synergistically enhances osteogenic potential and provides a rational basis for their clinical application in bone reconstruction surgery.

A composite of marrow mesenchymal stem cells and porous hydroxyapatite (HA) has in vivo osteogenic potential. To investigate factors enhancing the osteogenic potential of marrow/HA composites, we prepared a bone morphogenetic protein (BMP) fraction from the 4M guanidine extract of bovine bone by heparin-sepharose affinity chromatography. Marrow/HA composites or composites containing marrow mesenchymal stem cells, BMP, and HA (marrow/BMP/HA composites) were implanted subcutaneously in 7-week-old male Fischer rats. BMP/HA composites and HA alone were also implanted. The implants were harvested after 2, 4, or 8 weeks and were prepared for histological and biochemical studies. Histological examination showed obvious de novo bone formation together with active osteoblasts at 2 weeks, as well as more extensive bone formation at 4 and 8 weeks in many pores of the marrow/ BMP/HA composites. The marrow/HA composites did not induce bone formation at 2 weeks, but there was moderate bone formation at 4 weeks. At 2 weeks, only marrow/BMP/ HA composites resulted in intensive osteogenic activity, judging from alkaline phosphatase and osteocalcin expression at both the protein and gene levels. These results indicate that the combination of marrow mesenchymal stem cells, porous HA, and BMP synergistically enhances osteogenic potential, and may provide a rational basis for their clinical application, although further in vivo experiment is needed.

We evaluated the combination effect of recombinant human bone morphogenetic protein-2 (rhBMP-2) and cultured rat bone marrow mesenchymal stem cells (MSCs) in atelopeptide type I collagen (AC) solution on osteogenesis in a diffusion chamber (DC) to develop a bone substitute having consistent osteogenic capability for clinical applications. The cultured MSCs were obtained by 10-day primary culture of fresh bone marrow cells of Fischer rats. We prepared three groups of DCs: AC solution with rhBMP-2, AC solution with cultured MSCs, and AC solution with rhBMP-2 and cultured MSCs. The prepared combined solutions were injected into DCs, which were subcutaneously implanted into the backs of syngeneic rats. DCs were harvested after 2, 4, or 8 weeks and analyzed for bone-forming capability by determining histological and osteoblastic biochemical markers. De novo bone formation was observed both inside and outside of the membrane filter of DCs in the group of AC solution with rhBMP-2 and cultured MSCs. The alkaline phosphatase activity and osteocalcin content in the group of AC solution with rhBMP-2 and cultured MSCs were significantly higher than those in the group of AC solution with cultured MSCs at any time. These findings indicate that AC aqueous solution is a useful material not only as a carrier of rhBMP-2 but also as a cell-anchorage for differentiation and proliferation of MSCs. Therefore, this study suggests that clinical repairs of bone defects are feasible using injectable AC solution with rhBMP-2 and cultured MSCs as a bone substitute.

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