Characterization of cells with osteogenic potential from human marrow.

Studies using animal tissue suggest that bone marrow contains cells with the potential to differentiate into cartilage and bone. We report the extension of these studies to include human marrow. Bone marrow from male and female donors of various ages was obtained either from the femoral head or as aspirates from the iliac crest, and introduced into culture. Culture-adherent cells were expanded, subcultured, and then tested for bone and cartilage differentiation potential utilizing two different in vivo assays in nude mice. One assay involved subcutaneous implantation of porous calcium phosphate ceramics loaded with cultured, marrow-derived, mesenchymal cells; the other involved peritoneal implantation of diffusion chambers, also inoculated with cultured, marrow-derived, mesenchymal cells. Histological evaluation showed bone formation in ceramics implanted with cultured, marrow-derived, mesenchymal cells originating from both the femoral head and the iliac crest. Immunocytochemical analysis indicates that the bone is derived from the implanted human cells and not from the cells of the rodent host. No cartilage was observed in any of these ceramic grafts. In contrast, aliquots from the same preparations of cultured, marrow-derived, mesenchymal cells failed to form bone or cartilage in diffusion chambers. These data suggest that human marrow contains cells with osteogenic potential, which can be enriched and expanded in culture. Our findings also suggest that subcutaneous implantation of these cells in porous calcium phosphate ceramics may be a more sensitive in vivo assay than diffusion chambers for measuring their osteogenic lineage potential.

The origin of bone formed in composite grafts of porous calcium phosphate ceramic loaded with marrow cells.

When porous calcium phosphate ceramic is combined with marrow cells and grafted either heterotopically or orthotopically, bone forms inside the pores on the surface of the ceramic beginning at three weeks after implantation. The question remains as to whether the newly formed bone is derived from host or donor cells. To study the origin of bone cells formed in these composite grafts of marrow cells and ceramic, quail marrow cells from long bones were introduced into ceramics and the composites were implanted into subcutaneous pouches of immunologically nonreactive athymic nude mice. The ceramics were recovered at two to 84 days following surgery, fixed, decalcified, embedded, sectioned, and examined for the location of a quail-specific nucleolar marker and the binding of a specific antiserum against quail cells. Our observations indicate that ceramic-associated osteogenesis is a biphasic phenomenon: an early phase, the first three to four weeks after implantation, in which donor cells are largely responsible for the observed osteogenesis, and a second phase, eight to 12 weeks postsurgery, in which host cell actions predominate. During the second stage, the ceramic pores begin to show the formation of marrow of host origin, and the mesenchymal marrow component appears to be osteogenic because the bone formed during this late postgrafting stage contains osteocytes of host and donor origin. The second phase therefore results in chimeric bone composed of quail and mouse. These studies clearly document the donor origin of the initial bone formation and indicate that marrow contains progenitor cells capable of forming de novo bone.

Osteogenic potential of culture-expanded rat marrow cells as assayed in vivo with porous calcium phosphate ceramic.

It has been established that, when whole marrow is introduced into porous calcium phosphate ceramic, bone forms on the walls of the pores. To extend earlier studies, bone marrow cells derived from the femora of inbred rats were introduced into tissue culture and the adherent cells cultivated, mitotically expanded, passaged, harvested, placed in small cubes of porous calcium phosphate ceramics and grafted into subcutaneous sites of syngeneic rats. Marrow-derived, cultured mesenchymal cells introduced into ceramics showed strong osteogenic potential, with bone forming in the pore regions of ceramics as early as 2 wk after implantation. Osteogenesis could be observed after the eighteenth passage. With increasing passage number, the initiation of osteogenesis and the apparent rate of bone formation declined and the course of osteogenesis was delayed. In the future, it may be possible to culture marrow cells as a source for reparative cells for implantation back into autologous in vivo sites.

[Ectopic bone formation by composite graft of culture-expanded rat marrow cells and porous calcium phosphate ceramicmic].

Bone marrow cells derived from the femora of inbred rats were introduced into tissue culture. The adherent cells were cultivated, mitotically expanded, passaged, harvested, then placed in small cubes of porous calcium phosphate ceramics and finally grafted into subcutaneous sites of syngeneic rats. Primary marrow-derived cultured cells introduced into ceramics showed strong osteogenic potential with bone forming in the pore regions of ceramics as early as 2 weeks after in vivo implantation. Osteogenesis could be observed after 18th passage (over 36 population doublings). With increasing passage number, the initiation of osteogenesis and the apparent rate of bone formation declined and the course of osteogenesis was delayed. These data indicate that porous ceramic provides an excellent delivery vehicle for cells which are capable of osteogenic expression. In the future, it is possible to culture marrow cells as a source for reparative cells for implantation back into autologous in vivo sites.

[Ectopic bone formation by composite graft of culture-expanded human marrow cells and porous calcium phosphate ceramic].

Human bone marrow cells derived from three different sources; (1) cancellous bone of the femur; (2) cancellous bone of the rib from cadavers; (3) aspirate from the iliac bone were introduced into tissue culture. The adherent cells were cultivated, mitotically expanded, passaged, harvested then placed in small cubes of porous calcium phosphate ceramics and finally grafted into subcutaneous sites of athymic mouse or athymic rat. The ceramics loaded with cultured marrow cells from cancellous bone of femur showed strong osteogenic potential with bone forming in the pore regions of ceramics as early as two weeks after in vivo implantation. Osteogenesis could be observed after seventh passage (Five times passaged, frozen, stored, thawed, and replated twice). Although the aspirated marrow cells cultured also exhibited bone formation, the osteogenesis initiated later and the bone formation rate was lower. The ceramics loaded with fresh red blood cells separated from the aspirated marrow showed no evidence of bone formation. Moreover, osteogenic cells were found to have an ability to proliferate selectively in tissue culture.

The osteogenic potential of culture-expanded rat marrow mesenchymal cells assayed in vivo in calcium phosphate ceramic blocks.

When whole marrow is introduced into porous calcium phosphate ceramic, bone forms on the walls of the pores. As an extension of earlier studies, bone marrow cells derived from the femora of inbred rats were introduced into tissue culture, and the adherent cells were cultivated, mitotically expanded, subcultured, harvested, placed in small cubes of porous calcium phosphate ceramic, and grafted into subcutaneous sites of syngeneic rats. Primary marrow-derived, cultured mesenchymal cells introduced into ceramic showed strong osteogenic potential, with bone forming in the pore regions of ceramic as early as two weeks after in vivo implantation; cartilage was observed infrequently in pores that appeared to be avascular. Osteogenesis could be observed after the 18th subculture (over 36 population doublings) when the cells were tested in ceramic at subcutaneous sites, whereas chondrogenesis was observed with only the first and second subcultured cells in the ceramic delivery vehicle. With increasing numbers of subcultures, the initiation of osteogenesis and the apparent rate of bone formation declined, and the course of osteogenesis was delayed. Cultured, marrow-derived mesenchymal cells, even after the 21st subculture (over 40 population doublings), exhibited a positive histochemical reaction for alkaline phosphatase. However, the in vivo osteogenic potential of these cells was not correlated with their alkaline phosphatase activity. The implantation of cell pellets or the injection of cell suspensions of fresh or cultured, adherent marrow cells never produced bone or cartilage in heterotopic sites. These data indicate that porous ceramic provides an excellent delivery vehicle for cells that are capable of osteogenic expression and suggest that the composite graft of marrow-derived mesenchymal cells and porous ceramic may be useful for repair of massive bone defects. It may be possible to culture marrow mesenchymal cells as a source for reparative cells for implantation back into autogeneic sites.

Experimental study of vascularized tibiofibula graft in inbred rats: a preliminary report.

An experimental study of vascularized tibiofibula grafts in inbred rats was performed. Roentgenologic and histologic changes of the grafted bone in the first seven postoperative weeks were especially investigated. After preliminary experiments on the vascular anatomy of the lower limbs of rats, tibiofibular vascularized grafts with femoral artery and vein were utilized in Fischer strain F-344 rats. The rate of bony union in the vascularized graft group was superior to that in the nonvascularized control groups. Fluorochrome-labeling studies of the grafted bone at the mid-diaphysis showed active periosteal new bone formation, following the vascularized graft. In contrast, normal tibial bone growth at the mid-diaphysis was mainly endosteal. However, both vascularized graft and normal bone demonstrated evidence of a "drift phenomenon" in the direction of growth. Since the life cycle of the rat is very short, compared with other laboratory animals, this experimental model may be useful in investigating the postoperative course of vascularized bone grafts with a short follow-up period.