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Coprecipitates of calcium phosphate and DNA have been used in vitro for several decades for cell transfection. We evaluated the efficiency of calcium phosphate ceramics associated to plasmid DNA in the transfection of bone cells in vitro when they are grown in tissue culture. Newborn rat calvariae and tibia epiphyses were grown on an agar surface for a period of 48 h to 30 days. The hydroxyapatite (HA)-particles were loaded with a plasmid bearing a galactosidase reporter gene by incubation of the plasmid solution in PBS with the particles. One milligram of HA-particles was then placed in contact with the bone explants for 8 and 30 days. Histological sections were then performed and the galactosidase activity was revealed using an X-gal solution. At eight days, very few cells expressing the galactosidase activity were detected. By 30 days, however, the explants appeared uniformly stained blue. The staining of sections showed that the osteoblasts, chondroblasts, perichondroblasts, and perisoteal cells all expressed the lacZ gene while the number of cells stained in the control was negligible. The time dependence of the transfection suggests that transfection using ceramics is linked to the degradation of the ceramic by the cells. Furthermore, the cells are stained remote from the particles suggesting that the particles induce a coprecipitate of DNA in the explant.

The resistance of macroporous calcium phosphate ceramics to compressive strength generally is low and depends on, among other factors, porosity percentage and pore size. A compromise always is adopted between high porosity, required for a good integration, and mechanical strength, which increases with material density. We improved the strength of macroporous calcium phosphate ceramics of interconnected porosity by filling the pores with a highly soluble, self-setting calcium phosphate cement made of TCP and DCPD. Cylinders of the resulting material were implanted in sheep condyles and subjected to histological analysis after 20, 60, and 120 days. Microradiographs were made of the histological sections. The control material consisted of ceramic that had not been loaded with cement. Progressive ingrowth of bone into the ceramic pores occurred as the cement was degraded during the first implantation period. Marked degradation of the cement was apparent after 2 months, with fragmentation of the cement in most of the pores and the presence of bone tissue between the fragments. All the cement had been replaced by bone after 4 months. Some fragments of cement still were embedded in the newly formed bone. There was no significant difference between the integration of loaded and nonloaded ceramics. Filling the macroporous ceramic pores with a calcium phosphate cement significantly improved the mechanical strength of these ceramics without modifying their integration in the healing bone., (Copyright 2000 John Wiley & Sons, Inc.)

Unlabelled: Different chemical or physical methods of bone processing have been developed to decrease the antigenicity of allogeneic bone which may delay or prevent graft integration. We have developed a method based on delipidation and deproteination of the bone with a supercritical fluid and hydrogen peroxide. Cylinders of cancellous allogeneic bone treated in this way were implanted for four weeks, four months or eight months in holes drilled in sheep condyles or tibial plateau. Histological sections were then processed and analysed qualitatively and quantitatively using an image analysis software coupled to a light microscope. Measurements were made of the trabecular bone surface (BS/TS), the relative osteoid surface (OS/BS), the active osteoid surface (OS/BS), active resorption surface (Oc.S/BS) and the relative surface of newly formed bone. After four weeks, the control cylinders (non-treated allogeneic bone) had been invaded by cellular tissue composed of lymphocytes and plasmocytes surrounding remnants of the donor bone marrow tissue. The processed cylinders showed osteoid apposition at the surface of their external trabeculae. The trabecular bone and osteoid surfaces were significantly higher in the processed bone sections than in the control bone sections. After four months, most of the control material had been osteolysed and replaced by connective tissue containing lymphocyte islets, while the processed materials showed a large amount of bone synthesized at the surface of implant trabeculae which appeared fragmented and disseminated within the newly formed bone. All the histomorphometric parameters measured were significantly different from those of the control. By eight months, most of the control material had been totally osteolysed with very little bone ingrown in the implantation site. Only one control implant had been integrated. The processed cylinders were difficult to discern from the bone in which they were implanted. The parameters measured on the processed cylinders were significantly higher than those measured on the control sections., In Conclusion: the treatment applied to the bone enhanced allogeneic bone integration and could provide a new kind of tissue treatment for bone banking.

HA-ceramics used in human surgery as osteoconductive surfaces show a great variety of characteristics. Certain characteristics such as grain size, porosity, and surface area, are controlled by the sintering temperature of the slurry. We grew L-929 fibroblast cells on HA-ceramic disks that had been sintered at different temperatures ranging from 850 degrees-1350 degrees C. The cell line growth rate was lower on ceramic disks than on the culture-grade polystyrene used as a negative control. Cell growth correlated with the ceramic sintering temperature although no significant difference in the cell adhesion to the different ceramics was shown. Growth rate on ceramics sintered at low temperatures (850 degrees and 950 degrees C) was negative whereas it was positive on disks sintered at higher temperatures. When the cells were separated from the disks by a polycarbonate membrane, the growth rate was negative on those membranes in contact with low-temperature sintered disks and positive on the high-temperature sintered disks. The calcium and phosphorus concentration in the culture medium in contact with ceramics sintered below 1050 degrees C decreased during the culture period. Ceramics sintered between 1100 degrees and 1250 degrees C brought about an increase in Ca and P concentrations while ceramics sintered at higher temperatures did not induce any changes. SEM examination of the 850 degrees and 1200 degrees C sintered ceramics showed that the 850 degrees C sintered ceramics consisted of small grains with pores between them and the 1200 degrees C sintered ceramics were made of larger grains without any visible pores, thereby decreasing the surface of material in contact with the culture medium. This difference in surface area was confirmed by the fact that the amount of albumin absorbed onto the ceramic was dependent on the sintering temperature. In conclusion, the modification of the culture medium brought about by high-surfaced ceramics could influence the growth of cells with which such ceramics come in contact.