Growth Behaviour of Rat Bone Marrow (RBM) Cells on RF Magnetron Sputtered Hydroxyapatite and Dicalcium Pyrophosphate Coatings

The aim of this study was to evaluate the osteogenic properties of magnetron sputtered dicalcium pyrophosphate (DCPP) and hydroxylapatite (HA) coatings. Therefore, DCPP and HA coatings were deposited on grit-blasted titanium discs. The substrates were used as-prepared or received an additional heat treatment with changed the amorphous coating structure to a crystalline structure. Subsequently, rat bone marrow stromal cells were cultured for 1-24 days on the various substrates. DNA and alkaline phosphatase activity was determined after 1, 3, 5, 8 and 12 days of incubation. Osteocalcin expression was evaluated after 8, 12, 16 and 24 days of incubation. Scanning electron microscopical analysis of cell morphology and coating characteristics was done after 8 and 16 days of incubation. All assays were done in duplicate and in each assay all specimens were present in fourfold. Results demonstrated that the cells did not proliferate and differentiate on all amorphous coatings. SEM revealed that the amorphous coatings showed significant dissolution. On the crystalline DCPP and HA coatings an increase in DNA and alkaline phosphatase activity was seen starting at day 8 of incubation. Osteocalcin expression on the crystalline coatings started to increase at day 16 of incubation. SEM showed that the growth and differentiation of the cells was associated with extensive collage fiber formation and surface mineralization in the form of globular accretions. Further, statistical testing revealed that proliferation and differentiation of the rat bone marrow stromal cells started significantly earlier on the crystalline HA coatings than on the crystalline DCPP coatings. These results demonstrate that the rat bone marrow stromal cells proliferated and differentiated only on crystalline magnetron sputtered DCPP as well as HA coatings, which warrants the further in vivo analysis of the bone healing supporting properties of these coatings.

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

Blood ◽

10.1182/blood.v77.9.1906.bloodjournal7791906 ◽

1991 ◽

Vol 77 (9) ◽

pp. 1906-1911

Author(s):

CA McCulloch ◽

M Strugurescu ◽

F Hughes ◽

AH Melcher ◽

JE Aubin

Keyword(s):

Bone Marrow ◽

Alkaline Phosphatase ◽

Phosphatase Activity ◽

Alkaline Phosphatase Activity ◽

Stromal Cells ◽

Bone Marrow Stromal Cells ◽

Marrow Stromal Cells ◽

Nodule Formation ◽

Self Renewal ◽

Rat Bone

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.

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

Blood ◽

10.1182/blood.v77.9.1906.1906 ◽

1991 ◽

Vol 77 (9) ◽

pp. 1906-1911 ◽

Cited By ~ 81

Author(s):

CA McCulloch ◽

M Strugurescu ◽

F Hughes ◽

AH Melcher ◽

JE Aubin

Keyword(s):

Bone Marrow ◽

Alkaline Phosphatase ◽

Phosphatase Activity ◽

Alkaline Phosphatase Activity ◽

Stromal Cells ◽

Bone Marrow Stromal Cells ◽

Marrow Stromal Cells ◽

Nodule Formation ◽

Self Renewal ◽

Rat Bone

Abstract 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.

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Mild Heat Shock Induces Proliferation, Alkaline Phosphatase Activity, and Mineralization in Human Bone Marrow Stromal Cells and Mg-63 Cells In Vitro

Journal of Bone and Mineral Research ◽

10.1359/jbmr.2001.16.4.731 ◽

2001 ◽

Vol 16 (4) ◽

pp. 731-741 ◽

Cited By ~ 75

Author(s):

Chaoxiang Shui ◽

Andrew Scutt

Keyword(s):

Bone Marrow ◽

Alkaline Phosphatase ◽

Heat Shock ◽

Phosphatase Activity ◽

Alkaline Phosphatase Activity ◽

Stromal Cells ◽

Bone Marrow Stromal Cells ◽

Marrow Stromal Cells ◽

Human Bone

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Direct flow cytometric quantification of alkaline phosphatase activity in rat bone marrow stromal cells.

Journal of Histochemistry & Cytochemistry ◽

10.1177/40.7.1376742 ◽

1992 ◽

Vol 40 (7) ◽

pp. 1059-1065 ◽

Cited By ~ 23

Author(s):

N Kamalia ◽

C A McCulloch ◽

H C Tenenbaum ◽

H Limeback

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Alkaline Phosphatase ◽

Alkaline Phosphatase Activity ◽

Stromal Cells ◽

Bone Marrow Stromal Cells ◽

Marrow Stromal Cells ◽

Flow Cytometric ◽

Fast Red ◽

Rat Bone

Cell preparations in cytochemistry are conventionally analyzed with transmitted light after fixation and reaction with agents such as azo-coupling dyes. With cell suspensions stained with fluorescent cytochemical dyes, cells can also be analyzed and sorted by flow cytometry. We have exploited the intense red fluorescence of Fast Red Violet LB generated in cytochemical reactions to perform flow cytometric analyses of alkaline phosphatase (AP) expression in rat bone marrow stromal cells. By modifying staining protocols of single-cell suspensions, we demonstrate that in comparison to staining with Fast Red TR, the method is specific, can distinguish among various levels of enzyme expression within the whole population, and permits enzyme kinetic studies of heterogeneous cell populations. The method was applied to study the effect of the glucocorticoid dexamethasone (Dx) on cell proliferation and AP expression. In low AP-expressing cells, Dx treatment at 10(-8) M increased the [3H]-thymidine labeling index from 3.85% to 5.24% (p less than 0.01). In contrast, high AP-expressing cells were unlabeled by [3H]-thymidine. The staining and analytical methods reported here facilitate the detection, isolation, and quantification of subpopulations of bone marrow stromal cells that express alkaline phosphatase activity. These experiments demonstrate the value of flow cytometry as an adjunct to conventional cytochemical methods.

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