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Biomedical subjects

A H Melcher

Publications and source records attributed to A H Melcher.

At least 19 recordsLinked to original sources
PubMed1991

Osteogenic progenitor cells in rat bone marrow stromal populations exhibit self-renewal in culture.

Marrow stromal cells are a heterogeneous population, comprising a variety of lineages including osteogenic cells. In the presence of ascorbic acid, sodium beta-glycerophosphate, and dexamethasone, rat bone marrow stromal cells form discrete nodules of mineralized, bonelike tissue. We used nodule formation by rat bone marrow stromal cells to assay for the self-renewal capacity of osteogenic progenitor cell populations. Cultures were subcultured every 5 days up to six times. Osteogenesis was assayed from second to sixth subcultures by counting the number and measuring the areas of mineralized nodules formed in cultures grown with 10(-8) mol/L dexamethasone. Nodule number and area decreased progressively between second and sixth subcultures. Alkaline phosphatase activity associated with individual cells and measured videodensitometrically decreased exponentially between the second and sixth subculture. The number of cells with alkaline phosphatase activity also decreased with progressive subculturing. The proportions of 3H-thymidine-labeled cells after continuous labeling from the beginning of the culture period showed 90% labeling for cells with alkaline phosphatase activity and fibroblastlike cells. Cultures labeled for only the first 3 days exhibited higher labeling of alkaline phosphatase-positive cells than fibroblastlike cells (P less than .05). Cultures that were flash-labeled at the end of the culture period demonstrated low labeling indices for cells with alkaline phosphatase activity and up to 10-fold higher labeling indices for fibroblastlike cells. Separate cultures treated with a cytocidal dose of high specific activity 3H-thymidine did not form nodules. These results indicate that osteogenic progenitor cells or another cell type required for nodules to develop must divide early in culture if nodule formation is to occur, and that osteoprogenitor cells express a limited capacity for self-renewal.

Alkaline Phosphatase↗
PubMed1991

Fibroblasts inhibit mineralised bone nodule formation by rat bone marrow stromal cells in vitro.

The ability of rat skin fibroblasts (RSF) and human periodontal ligament fibroblasts (HPL) to inhibit the formation of mineralised bone nodules in rat bone marrow stromal cell (BMSC) cultures was studied. Co-culture of HPL or RSF with BMSC resulted in a large reduction of bone nodule formation when compared with controls. Conditioned medium from HPL or RSF cultures inhibited bone nodule formation in a dose-dependent manner. HPL-conditioned medium depressed cell proliferation and alkaline phosphatase expression in BMSC cultures. These effects were not due to increased cytotoxicity or nutrient depletion. Inhibitory activity was recovered in a fraction of less than 1 kD following ultrafiltration and was insensitive to freeze-thawing. The inhibitory activity was blocked when HPL cultures were grown in the presence of 10(-5) M indomethacin. Dose-dependent inhibiton of bone nodule formation was also observed in cultures incubated with prostaglandins E2 (at 10(-6) M) or F2 alpha (at 10(-7) M). The results indicate that fibroblasts may inhibit osteoblast differentiation and function in part by release of soluble factors including prostaglandins.

Alkaline Phosphatase↗
PubMed1990

Effects of donor age on osteogenic cells of rat bone marrow in vitro.

The effect of donor age on the production of bone-like tissue and expression of cellular alkaline phosphatase was examined in cultures of cells obtained from rat bone marrow. Stromal cells were obtained from the bone marrow of young (5-6 weeks) and old (18 months) rats and cultured in vitro. After 28 days in first subculture, the following were quantified: (1) the total number of mineralised nodules and the size distribution of nodules and (2) the density of osteoblasts and osteocytes associated with nodules in histological sections. The doubling times of the cultures and the numbers of cells in cultures which expressed alkaline phosphatase activity were determined in separate experiments. Cells from young cultures produced three times more bone-like nodules than old cultures, although no differences were seen in the size distribution of nodules, or on osteoblast and osteocyte density. Doubling times for both groups were similar. The numbers of alkaline phosphatase (AP) positive cells was reduced by half in old cultures. These data show that this model may be useful for the study of the mechanisms of ageing on osteogenesis, and demonstrate a reduced osteogenic capacity in old cultures. The results suggest that this effect may be due to a reduction in the generation of cells of osteoblast lineage during ageing.

Aging↗
PubMed1989

Coordinated regulation of endothelial and fibroblast cell proliferation and matrix synthesis in periodontal ligament adjacent to appositional and resorptive bone surfaces.

Little is known about the remodeling of blood vessels and soft connective tissue or the proliferation of endothelial cells in the periodontal ligament (PL) of teeth undergoing physiological drift. To determine whether there is evidence for coordinated regulation of endothelial cell and fibroblast proliferation and matrix synthesis in sites within the PL adjacent to bone-appositional (A) and bone-resorptive (R) surfaces, the PL in mouse mandibular molar was subdivided into A and R sectors on the basis of 3H-proline incorporation into alveolar bone. Computer-assisted morphometry of radioautographs showed that the number and area of blood vessels were similar in A and R sectors. Proliferation of endothelial cells and fibroblasts was assessed from radioautographs prepared from mice continuously labeled with 3H-thymidine at times between 2 and 60 days. Significantly more labeled endothelial cells (P less than .001) and fibroblasts (P less than .05) were seen in the A sector. The percent of labeled endothelial cells and the percent of labeled fibroblasts increased linearly to 25 days and then formed a plateau. The rate of increase of labeled fibroblasts was higher in the A sector than in the R sector (P less than .025). In addition, 3H-proline grain counts over extracellular matrix were significantly higher in the appositional sector than in the resorptive sector (P less than .025).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Cell death and the regulation of populations of cells in the periodontal ligament.

The contribution of cell death in regulating cellular populations of periodontal ligament was studied in young adult rats. Mandibular first molar periodontium was prepared for light-microscopic radioautography after a pulse of 3H-thymidine in 6 rats and for electron microscopy in 4 rats. The labeling index for 3H-thymidine and the density of fibroblast-like cells were computed from radioautographs. The percentages of dying or dead cells and macrophages were computed from electron micrographs. The labeling index of cells within 20 microns of bone and cementum was significantly lower (p less than 0.01) than the labeling index within the body of the periodontal ligament. The patterns of cellular density and indices of death were the inverse of the labeling indices. Macrophages were plentiful (% macrophages = 3.68% +/- 0.30) and were clustered around blood vessels (mean distance from blood vessel = 2.3 microns). However, only 10% of dying or dead cells were within 10 microns of blood vessels. These data show that death of cells in the periodontal ligament may, in part, balance production of cells by mitosis. The relationships between labeling index, index of death, and cellular density suggest that cells born in the middle of the periodontal ligament may migrate to regions of high cellular density near bone and cementum, and that they may die there. Macrophages do not appear to be associated with dying cells of the periodontal ligament.

Animals↗
PubMed1988

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

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.

Aging↗
PubMed1988

Cell attachment of human gingival fibroblasts in vitro to porous-surfaced titanium alloy discs coated with collagen and platelet-derived growth factor.

The influence of biological coating, with or without the incorporation of growth factor, on the migration, attachment and orientation of human gingival fibroblasts in relation to porous-surfaced titanium alloy (Ti6AI4V) discs, was measured. Comparison was made between coating the discs with collagen and with collagen incorporating platelet-derived growth factor (PDGF); controls comprised porous-surfaced discs coated with agar or collagen containing bovine serum albumin (used as a carrier for the PDGF), uncoated porous-surfaced Ti6AI4V discs (with or without additional protein additives) exhibited significantly higher attachment indices (AI) and orientation indices (OI) compared with naked control discs (p less than 0.01); OI was also significantly higher than that of surface-demineralized root slices (p less than 0.001) on days 1, 2 and 3. Addition of PDGF to the collagen resulted in a further enhancement in OI on days 1 and 2 (p less than 0.01) over that shown by discs coated with collagen incorporating the bovine serum albumin vehicle. There was no cell attachment and consequently, no cell orientation, in relation to Ti alloy discs that had been coated with agar. These data suggest that attachment and orientation of cells following migration in relation to porous-surfaced Ti6AI4V discs can be modified by the application of biological molecules to the surface of the disc. This may have a useful application in clinical implantology.

Alloys↗
PubMed1987

Paravascular cells in endosteal spaces of alveolar bone contribute to periodontal ligament cell populations.

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.

Alveolar Process↗
PubMed1987

Effect of the surface geometry of smooth and porous-coated titanium alloy on the orientation of fibroblasts in vitro.

The migration and orientation of human gingival fibroblasts in relation to the rim of smooth-surfaced and porous-coated titanium discs placed on multilayers in vitro was investigated. Samples were examined after 6 h, 24 h, 3 days, and 7 days of culture using phase-contrast and scanning electron microscopy. The cells migrated from the multilayer onto the smooth-surfaced discs forming bridges between them, and orientated along parallel circumferential grooves in the rim of the discs. This resulted in the cellular bridges orientating at an acute angle to the rim of the disc, and adjacent cells in the multilayer orientating parallel to the rim. Cellular bridges were also formed between the porous-coated discs and the multilayer but, because the cells that migrated onto, and between, the spheres of the porous-coat showed no preferred orientation, the bridges retained their orientation at right angles to the surface of the rim. This in turn resulted in the cells of the adjacent multilayer becoming similarly orientated. These observations suggest that the geometrical configuration of the surface of implants could influence whether a capsule or an orientated fibrous attachment is developed in relation to implants in vivo.

Alloys↗
PubMed1987

Migration, attachment, and orientation of human gingival fibroblasts to root slices, naked and porous-surfaced titanium alloy discs, and Zircalloy 2 discs in vitro.

Cell migration, attachment, and orientation of cultured human gingival fibroblasts (HGF) were measured in relation to four types of specimens: Ti-6Al-4V alloy discs with either ground surfaces or porous structured surfaces, and Zr alloy discs (Zircalloy 2) with either ground surfaces or surfaces modified by being heated to form a thicker oxide cover. Surface-demineralized and non-demineralized root slices were used as controls. Initial cell attachment was measured by means of a 51Cr assay, and cell attachment and orientation following migration by an assay that measures development of the resulting refractile material in relation to the specimen. No significant difference between initial cell attachment to Ti alloy and control demineralized root slices could be detected after one hr using the 51Cr assay. However, with the second assay, cell attachment and orientation at three, seven, 10, and 14 days were significantly higher in relation to surface-demineralized root slices than to all of the metal discs and the non-demineralized root slices. While cell attachment to the surface-ground Ti alloy discs appeared to be higher than that to the porous-surfaced Ti alloy discs at three days and seven days, cell orientation to the porous-surfaced Ti alloy discs and the non-demineralized root slices at days 7, 10, and 14 was greater than that to all of the other metal discs. These data suggest that surface geometry could affect attachment and orientation of cells in vitro.

Alloys↗
PubMed1987

An ancient oath?

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Dental Implantation, Endosseous↗