Cells from fetal or neonatal skeleton can synthesize bone-like tissue in vitro In contrast, formation of bone-like tissue in vitro by cells derived from adult animals has rarely been reported and has not been achieved using cells from bone marrow We have explored development of bone-like tissue in vitro by bone marrow stromal cells Marrow stromal cells obtained from 40-43-day-old Wistar rats were grown in primary culture for 7 days and then subcultured for 20-30 days Cells were cultured in either alpha-minimal essential medium containing 15% fetal bovine serum, antibiotics, and 50 micrograms/ml ascorbic acid, or the above medium supplemented with either 10 mM Na-beta-glycerophosphate, 10(-8) M dexamethasone, or a combination of both Cultures were examined using phase-contrast microscopy, undemineralized and demineralized tissue histology, histochemistry (for alkaline phosphatase activity), immunohistochemistry (for collagen type, osteonectin, and bone Gla-protein), scanning and transmission electron microscopy, energy dispersive X-ray microanalysis, and X-ray diffraction Collagenous, mineralized nodules exhibiting morphological and ultrastructural characteristics similar to bone were formed in the cultures, but only in the presence of both beta-glycerophosphate and dexamethasone Cells associated with the nodules exhibited alkaline phosphatase activity The matrix of the nodules was composed predominantly of type-I collagen and both osteonectin and Gla-protein were present X-ray microanalysis showed the presence of Ca and P, and X-ray diffraction indicated the mineral to be hydroxyapatite The nodules were also examined for bone morphogenetic protein-like activity Paired diffusion chambers containing partly demineralized nodules and fetal muscle were implanted intraperitonealy in rats Induction of cartilage in relation to muscle was observed histologically after 40 days in the chambers This finding provided further support for the bone-like nature of the nodules The observations show that bone-like tissue can be synthesized in vitro by cells cultured from young-adult bone marrow, provided that the medium contains both beta-glycerophosphate and, particularly, dexamethasone

Bone formation in vitro by stromal cells obtained from bone marrow of young adult rats.

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.

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

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

In this study we investigated not only osteoblastic cell proliferation and differentiation on the surface of calcium metaphosphate (CMP) matrices in vitro but also bone formation by ectopic implantation of these cell-matrix constructs in athymic mice in vivo. Interconnected porous CMP matrices with pores 200 microm in size were prepared to use as scaffolds for rat-marrow stromal-cell attachment. Cell-matrix constructs were cultured in vitro, and cell proliferation and ALPase activities were monitored for 56 days. In addition to their being cultured in vitro, cell-matrix constructs were implanted into subcutaneous sites of athymic mice. In vitro these porous CMP matrices supported the proliferation of osteoblastic cells as well as their differentiation, as indicated by high ALPase activity. In vivo the transplanted marrow cells gave rise to bone tissues in the pores of the CMP matrices. A small amount of woven bone formation was detected first at 4 weeks; osteogenesis progressed vigorously with time, and thick lamellar bones that had been remodeled were observed at 12 weeks. These findings demonstrate the potential for using a porous CMP matrix as a biodegradable scaffold ex vivo along with attached marrow-derived mesenchymal cells for transplantation into a site for bone regeneration in vivo.

Tissue-engineered growth of bone by marrow cell transplantation using porous calcium metaphosphate matrices.

The development of a monoclonal antibody, OB 7.3, directed against a cell surface antigenic site on osteocytes is described.

Identification of osteocytes in osteoblast-like cell cultures using a monoclonal antibody specifically directed against osteocytes.

The effect of PSK on the depressed interferon (IF) production in tumor-bearing mice was studied. In tumor-bearing mice, in vitro IF production by spleen cells treated with polyinosinic-polycytidylic acid (poly I:C) was remarkably inhibited. However, these inhibitions were prevented by the intraperitoneal (ip) administration of PSK. Mice were inoculated intravenously (iv) with poly I:C-treated spleen cells, administered with PSK ip at 3 days after the tumor inoculation. When PSK was not given, poly I:C-treated spleen cells did not show an inhibitory effect on tumor growth. In mice given PSK ip, poly I:C-treated spleen cells exerted slightly inhibiting effects on tumor growth. These results suggest that PSK prevented such a modulation in tumor-bearing mice.

Effect of PSK on interferon production in tumor-bearing mice.

The purification of the osteoblast-like cells (2-3%) among the bone marrow cells (BMC) of C57BL/6 mice using a specific anti-osteoblast serum and a fluorescence-activated cell sorter is described. The antiserum was raised against osteoblast cells isolated from calvaria from neonatal mice. The majority of the cells of the osteoblast-enriched fraction from bone marrow showed a parathormone-induced increase in cyclic adenine monophosphate but no response to calcitonin. This is similar to the response of osteoblast cells obtained from the calvaria. Electron microscopic studies of the extracellular matrix of cultured osteoblast-like cells purified from bone marrow showed the deposition of apatite crystals within and in close apposition to the vesicles. These findings suggest that the isolated cell population was enriched in osteoblasts. Such a cell system from bone marrow might provide an experimental system for investigating the mechanism of bone formation.

Separation of osteoblast-like cells from bone marrow by fluorescence-activated cell sorting.

Cellular accumulation to the peritoneal cavity and modification of various functions of peritoneal macrophages were observed in mice injected intraperitoneally (ip) with thioglycollate medium (TG), liquid paraffin, proteose peptone and Corynebacterium parvum. The cellular composition of peritoneal exudates at 4 days after injection of irritants was almost the same in all the groups and the proportion of macrophages was increased approximately 4 times more than nontreated controls. The ability to kill Listeria monocytogenes and to generate chemiluminescence (CL) were augmented strongly in C. parvum-induced macrophages, while depressed in TG-induced macrophages. The activities of liquid paraffin- or proteose peptone-induced macrophages were almost the same as those in nontreated controls. However, the ability to phagocytose native sheep erythrocytes was greatly augmented both in C. parvum- and TG-induced macrophages. There is thus a discrepancy between bactericidal activity and phagocytic activity among macrophages induced with various irritants.

M Oguchi

Papers

Effect of PSK on interferon production in tumor-bearing mice.

Separation of osteoblast-like cells from bone marrow by fluorescence-activated cell sorting.

Relationship between bactericidal and phagocytic activities of peritoneal macrophages induced by irritants.