Osteoblast Cell Morphology Research Articles

생체재료의 선택에 따른 조골세포의 형상, 증식 및 분화

생체재료의 선택에 따른 조골세포의 형상, 증식 및 분화

Image and fractal analysis of osteoblastic cells in viscous media.

The aim of the present study was to determine whether osteoblastic morphology was affected by the viscosity of culture media and whether any morphological differences could be readily quantified. A cytochemical stain was used for alkaline phosphatase and a combination of image and fractal analysis, utilising seven morphological parameters to assess the morphology of osteoblasts. Using regression analysis it was determined that as the viscosity of the culture media increased the area of the cells significantly decreased and the fractal dimension of the cell profiles significantly increased. A discriminant function analysis was used to examine whether cell populations could be classified according to culturing time or the viscosity of culturing media based on the seven morphological parameters. It was determined that the cells could be classified up to 93% correct according to the viscosity of the media they were cultured in and up to 93.5% correct according to the culturing time. This study demonstrated that viscous media affects the morphology of osteoblastic cells and that discriminant function analysis can be used to classify these cells based on their morphological parameters.

TGF-beta1 modifications in nuclear matrix proteins of osteoblasts during differentiation.

Nuclear matrix protein (NMP) composition of osteoblasts shows distinct two-dimensional gel electrophoretic profiles of labeled proteins as a function of stages of cellular differentiation. Because NMPs are involved in the control of gene expression, we examined modifications in the representation of NMPs induced by TGF-beta1 treatment of osteoblasts to gain insight into the effects of TGF-beta on development of the osteoblast phenotype. Exposure of proliferating fetal rat calvarial derived primary cells in culture to TGF-beta1 for 48 h (day 4-6) modifies osteoblast cell morphology and proliferation and blocks subsequent formation of mineralized nodules. Nuclear matrix protein profiles were very similar between control and TGF-beta-treated cultures until day 14, but subsequently differences in nuclear matrix proteins were apparent in TGF-beta-treated cultures. These findings support the concept that TGF-beta1 modifies the final stage of osteoblast mineralization and alters the composition of the osteoblast nuclear matrix as reflected by selective and TGF-beta-dependent modifications in the levels of specific nuclear matrix proteins. The specific changes induced by TGF-beta in nuclear matrix associated proteins may reflect specialized mechanisms by which TGF-beta signalling mediates the alterations in cell organization and nodule formation and/or the consequential block in extracellular mineralization.

Protein kinase C modulator effects on parathyroid hormone-induced intracellular calcium and morphologic changes in UMR 106-H5 osteoblastic cells

The effects of parathyroid hormone (PTH) on 1,4,5-inositol triphosphate (1,4,5-IP3) and intracellular free calcium (Cai2+) in osteoblasts are variable, whereas adenylate cyclase activity is consistently stimulated. Cyclic AMP is considered a mediator in the contractile effects of PTH on osteoblasts, but the regulation and role of Cai2+ remains unclear. Recent studies indicate that protein kinase C (PKC) inhibits PTH-stimulated Cai2+ increases in osteoblastic cells. Therefore, the objectives of this study were to determine the effects of PKC modulators and PTH on UMR 106-H5 rat osteoblastic cell morphology, and the relationship between cell shape and PTH-induced Cai2+ changes. In suspended cells loaded with the calcium indicator dye fura-2, pretreatment with PKC inhibitors calphostin C (100 nM x 1 h) and H-7 (30 microM x 18 h) potentiated the effects of 1 microgram/ml bPTH (1-84) on Cai2+ (83% increase over basal) by 1.4- and 1.65-fold, respectively. In comparison, PTH (10 ng-1 micrograms/ml) was without significant effect on adherent cell Cai2+ as measured by single-cell image analysis, although another in vitro bone resorbing agent, thrombin (10 U/ml), produced an acute 3-fold increase in the ratio (R) of emission (approximately lambda 510 nm) detected and optimized at lambda 348/374 nm (i.e., Ca-bound dye/free dye) in control cells. Phase-contrast microscopy revealed PKC inhibitor-treated cells changed from a spread configuration to a stellate form with retracting processes or cell rounding and a collapse of actin stress fibers. Within 1 h of PTH addition, PKC inhibitor-treated cells continually became extended/respread up to 3 h with an associated increase in actin stress fibers that was preceded by an acute 1.6-fold Cai2+ increase. In contrast, control or PKC activator-treated cells (phorbol 12,13-dibutyrate or 12-O-tetradecanoylphorbol-13-acetate; TPA) contracted/retracted within 5 min in response to PTH. A role for Cai2+ in PTH-induced cell spreading was further indicated by a contractile response to PTH when PKC-inhibitor-treated cells were loaded with the intracellular calcium chelator dimethyl BAPTA (3 microM x 30 min). PTH-induced Cai2+ increases in adherent PKC inhibitor-treated cells were also associated with a 1.8-fold 1,4,5-IP3 increase as measured by mass assay. The data suggest PKC contributes to UMR 106-H5 cell morphology and selectively regulates signal pathways activated by PTH to promote either cell contraction (cAMP) or extension (1,4,5-IP3/Cai2+).

Protein kinase C modulator effects on parathyroid hormone‐induced intracellular calcium and morphologic changes in UMR 106‐H5 osteoblastic cells

The effects of parathyroid hormone (PTH) on 1,4,5-inositol triphosphate (1,4,5-IP3) and intracellular free calcium (Cai2+) in osteoblasts are variable, whereas adenylate cyclase activity is consistently stimulated. Cyclic AMP is considered a mediator in the contractile effects of PTH on osteoblasts, but the regulation and role of Cai2+ remains unclear. Recent studies indicate that protein kinase C (PKC) inhibits PTH-stimulated Cai2+ increases in osteoblastic cells. Therefore, the objectives of this study were to determine the effects of PKC modulators and PTH on UMR 106-H5 rat osteoblastic cell morphology, and the relationship between cell shape and PTH-induced Cai2+ changes. In suspended cells loaded with the calcium indicator dye fura-2, pretreatment with PKC inhibitors calphostin C (100 nM × 1 h) and H-7 (30 μM × 18 h) potentiated the effects of 1 μg/ml bPTH(1–84) on Cai2+ (83% increase over basal) by 1.4- and 1.65-fold, respectively. In comparison, PTH (10 ng-1 μg/ml) was without significant effect on adherent cell Cai2+ as measured by single-cell image analysis, although another in vitro bone resorbing agent, thrombin (10 U/ml), produced an acute 3-fold increase in the ratio (R) of emission (∼ λ510 nm) detected and optimized at λ348/374 nm (i.e., Ca-bound dye/free dye) in control cells. Phase-contrast microscopy revealed PKC inhibitor-treated cells changed from a spread configuration to a stellate form with retracting processes or cell rounding and a collapse of actin stress fibers. Within 1 h of PTH addition, PKC inhibitor-treated cells continually became extended/respread up to 3 h with an associated increase in actin stress fibers that was preceded by an acute 1.6-fold Cai2+ increase. In contrast, control or PKC activator-treated cells (phorbol 12,13-dibutyrate or 12-O-tetradecanoylphorbol-13-acetate; TPA) contracted/retracted within 5 min in response to PTH. A role for Cai2+ in PTH-induced cell spreading was further indicated by a contractile response to PTH when PKC-inhibitor-treated cells were loaded with the intracellular calcium chelator dimethyl BAPTA (3 μM × 30 min). PTH-induced Cai2+ increases in adherent PKC inhibitor-treated cells were also associated with a 1.8-fold 1,4,5-IP3 increase as measured by mass assay. The data suggest PKC contributes to UMR 106-H5 cell morphology and selectively regulates signal pathways activated by PTH to promote either cell contraction (cAMP) or extension (1,4,5-IP3/Cai2+). J. Cell. Biochem. 65:276–285. © 1997 Wiley-Liss, Inc.