J. Goshima

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.

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.

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.