Experimental study on bone formation potential of cryopreserved human bone marrow mesenchymal cell/hydroxyapatite complex in the presence of recombinant human bone morphogenetic protein-2.
ABSTRACT Secondary bone grafting in the alveolar cleft is one of the most important treatment modalities for patients with a cleft lip and palate. In children, however, harvesting a sufficient amount of bone graft from the donor site is difficult, and the procedure imposes a heavy burden on the patients. This problem may be resolved if autologous transplantation can be performed using a cell-hybrid type of artificial bone prepared with the patient's own bone marrow cells through a tissue engineering technique. In the current study, we examined the possibility of achieving such an autologous transplantation using cryopreserved human bone marrow cells. Human bone marrow cells were grown in culture and cryopreserved, and the cells preserved for 3 years and the cells preserved for 3 months were then thawed and recultivated. In one experiment, the recultivated cells were seeded onto a complex composed of porous hydroxyapatite and bone morphogenetic protein (BMP), and the complex was grafted subcutaneously in nude mice. In another experiment, the cells were seeded onto the porous hydroxyapatite and cultivated for 10 days before grafting in a medium supplemented with BMP. The bone formation potential of the cells was compared between these two experiments. Formation of mature bone was observed 2 weeks after transplantation in the former experiment, whereas only scarce bone formation was evident 4 weeks after transplantation in the latter experiment. It is therefore assumed that exposing the cultured human bone marrow cells to a high concentration of BMP in vivo strongly promotes bone formation.
- [show abstract] [hide abstract]
ABSTRACT: We examined the effect of rhBMP-2 (recombinant human bone morphogenetic protein-2), delivered in a porous poly(DL-lactic acid) implant, on bone formation in a critical-sized defect in the radial diaphysis in rabbits. A unilateral segmental defect, twenty millimeters long, was created in the radius in ninety-six skeletally mature New Zealand White rabbits. Forty-eight rabbits were evaluated at four weeks and forty-eight, at eight weeks. Six groups were studied at each time-period. The defect was left empty in one group (control), the defect was filled with an autogenous corticocancellous bone graft in one group, and the defect was filled with a porous poly(DL-lactic acid) implant containing zero, seventeen, thirty-five, or seventy micrograms of rhBMP-2 (one group each). Radiographs of the defects were made every two weeks. The percentage of the total area of the defect that was radiopaque was determined with use of computerized radiomorphometry, and this percentage was used as a quantitative measure of the extent of new-bone formation in the defect. There were time and dose-dependent responses to rhBMP-2 for as long as four weeks; thereafter, the effects of seventeen, thirty-five, and seventy micrograms of rhBMP-2 were independent of dose and time (p < or = 0.05). The defects that had been treated with either thirty-five or seventy micrograms of rhBMP-2 had a significantly greater (p < or = 0.05) area of radiopacity than the defects that had been treated with either zero or seventeen micrograms of rhBMP-2. No significant difference could be found between the defects treated with thirty-five or seventy micrograms of rhBMP-2 and the defects filled with an autogenous graft. Healing and bone formation were examined histologically and histomorphometrically as well. At four weeks, polarized light microscopy revealed remnants of poly(DL-lactic acid) only in the defects that had been filled with an implant containing zero micrograms of rhBMP-2. At eight weeks, regardless of the dose of rhBMP-2, poly(DL-lactic acid) was not visible on histological examination. The presence of multinucleated giant cells was the hallmark of the inflammatory response elicited by poly(DL-lactic acid). At four and eight weeks, macrophages and lymphocytes were also present. The intensity of the cellular response at four weeks suggested an inverse relationship between these cells and the dose of rhBMP-2 -- that is, there appeared to be more multinucleated giant cells in defects treated with zero micrograms of rhBMP-2 than in defects treated with seventy micrograms of rhBMP-2. At eight weeks, multinucleated giant cells were rare in the defects treated with seventeen, thirty-five, or seventy micrograms of rhBMP-2. Histomorphometric data at four and eight weeks indicated that the amount of bone formation in the defects treated with seventeen, thirty-five, or seventy micrograms of rhBMP-2 was equivalent to the amount in the defects treated with an autogenous graft and was significantly less (p < or = 0.05) in the untreated defects and the defects treated with zero micrograms of rhBMP-2 (p < or = 0.05). By eight weeks, only thirty-five and seventy micrograms of rhBMP-2 had restored cortices and marrow elements.The Journal of Bone and Joint Surgery 12/1997; 79(12):1778-90. · 3.23 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: To investigate the significance of apatite-wollastonite-containing glass ceramic (AW ceramic) surfaces and the biological apatite layer formed on these surfaces, rat marrow cell culture, which shows osteogenic differentiation, was carried out on four different culture substrata (control culture dish, two AW ceramics, each having a different surface roughness, and a ceramic on which an apatite layer was formed. A culture period of 2 weeks in the presence of beta-glycerophosphate, ascorbic acid, and dexamethasone resulted in abundant mineralized nodule formations that were positive for alkaline phosphatase (ALP) stain on all substrata. The stain on the apatite-formed AW ceramic was the most intense, the enzyme activity being about twice that of the control culture dish, which had the lowest stain and activity of the four substrata. Northern blot analysis of bone Gla protein (BGP) showed the same tendency, that is, the amount of BGP mRNA from cultured cells on the apatite-formed AW ceramics was the highest and the mRNA on the control dish was the lowest. These data indicate that the glass ceramic surface promotes osteoblastic differentiation and that the promotion can be further enhanced by the formation of a biological apatite layer on the ceramic surface.Journal of Biomedical Materials Research 12/1996; 32(3):341-8.
- [show abstract] [hide abstract]
ABSTRACT: Fibroblast colonies (clones) were obtained by explantation of bone marrow single-cell suspensions and were used to establish multicolony and single-colony derived fibroblast cultures by successive passaging of either pooled or individual colonies. When transplanted in diffusion chambers after 20-30 cell doublings in vitro, the descendants of fibroblast colony-forming cells (FCFC), whether grown from single or pooled colonies, retained the ability for bone and cartilage formation. The content of osteogenic precursors in the cultured progeny significantly outnumbered the initiating FCFC. Thus the high proliferative potential of bone marrow FCFC and their ability to serve as common precursors of bone and cartilage-forming cells makes them probable candidates for the role of osteogenic stem cells.Cell and tissue kinetics 06/1987; 20(3):263-72.