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.

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Connective tissue cells responding to wounding of the periodontal ligament of the lower first molar in mice were studied using the techniques of radioautography and grain counting. Animals were given an intraperitoneal injection of 2 uCi 1g 3H‐Tdr 1 hr before being killed at either 30, 72 or 120 hr after wounding. The ligament in 1 um plastic sections was divided into compartments on the basis of distance from the wound, and the relative number of labelled cells in each compartment was assessed at 30, 72 and 120 hours after wounding. The distance of each labelled cell from the closest blood vessel was also measured at each time to detect relative movement of labelled cells away from blood vessels. In a Parallel experiment, haematogenous progenitors of macrophages were depleted by irradiating the animals with 800rads prior to woundilng to determine if mistakes in identification between fibroblasts and marophages could significantly affect the results. The ultrastructural characteristics of 150 of the 3H‐Tdr labelled cells was examined in thin sections of wounded periodontal ligament prepared for elctron microcope radioautography, The majority of cells lebelled 30 hours after wounding were confirmed to be paravascular, and most of them were found to be located within 200 μ of the wound margin. Some of these cells appreared to have divided a number of times between 30 and 72 hours after wounding, and to have migrated into the wound between 70 and 120 hours after wounding. Examination of the irradiated material and the electron microscope radio‐autographs suggested that significant numbers of macrophages had not been included in the counts of labelled cells. The elctron microscope radioautographs also suggested that cells which exhibited different degrees of cytodifferentiation had incorporated 3H‐Tdr.

Endosteal spaces of alveolar bone communicate with the periodontal ligament and may contribute to its cell populations. We examined cell proliferation and migration in endosteal spaces and in the periodontal ligament contiguous with these spaces. Radioautographs of mouse mandibular molar were prepared from animals pulse-injected with 3H-Tdr and sacrificed in groups of 22 mice each at 1 h, 1, 3, and 7 d after labeling. Cell counts, labeling indices, grain counts, and progenitor cell ratios were determined. The data indicate that endosteal spaces are enriched with 3H-Tdr-labeled progenitor cells whose progeny rapidly migrate out of the compartment. The periodontal ligament contiguous with the endosteal spaces exhibited 5 times as many labeled cells as other sites in this tissue. Thickened areas of cementum were coincident with the openings of endosteal spaces in over 64% of observations. The data are consistent with the hypothesis that cells migrate from endosteal spaces into the periodontal ligament and there express the phenotype for osteoblasts or cementoblasts.

The number of cell nuclei per mm2 and the volume density of cell nuclei and blood vessels in the periodontal ligament at different levels of the mesial root of the first mandibular molar of the adult mouse and in different areas of the ligament at each level have been examined in the light microscope. Significantly higher numbers of cell nuclei per mm2 were observed adjacent to bone, cementum, and blood vessels than in the avascular body of the ligament at all levels and on all aspects of the root. This distribution of number of cell nuclei per mm2 was constant over 4 1/2 months of aging and a doubling of body weight. The volume density of cell nuclei was significantly higher in cells adjacent to bone and cementum and in gingival connective tissue than in both the vicinity of blood vessels and the body of the ligament. The blood vessels, which were present predominantly in the bone-related half of the ligament, were absent from the zone immediately adjacent to cementum. The labeling indexes of periodontal ligament cells were determined from autoradiographs of the mesial root of the first mandibular molar of the mouse after pulse-labeling with 3H-Tdr. Labeling indexes were highest in zones adjacent to blood vessels, and the labeling index was significantly higher in the middle of the ligament than in zones adjacent to bone and cementum, and consequently was inversely related to cell density.