Osteoblast and osteoclast precursors in primary cultures of calvarial bone cells (original) (raw)
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Morphologic characterization of osteoblast-like cell cultures isolated from newborn rat calvaria
Calcified Tissue International, 1990
Two methods for harvesting osteoblastlike cell populations from newborn (10 days) rat calvaria were compared. The first one consisted in culturing the periosteum-free bones and then trypsinizing the cells on the bone surface. The second one involved the migration of the osteoblasts on glass fragments before trypsinization. Since the plating efficiency, the proportion of alkaline phosphatasepositive cells, the population doubling time, and the calcium deposition were more adequate, the second method was used to further characterize the behavior of the cultures. During the first week of culture, the cells featured shapes similar to those observed in vivo on the surface of periosteum-free calvaria. They formed multilayers and, in the presence of ascorbic acid, synthetized an organic matrix containing exclusively type I collagen. Later, small amounts of type III collagen appeared. The cells were embedded in the matrix and progressively acquired the morphologic phenotype of osteocyte-like cells. The matrix mineralized in the presence of 13-glycerophosphate. The technique of dropinoculation (high concentration of cells in a small volume of medium) promoted the multilayer formation and the achievement of large mineralized plates (about 1 cm 2) in 3 weeks of culture.
Journal of Applied Oral Science, 2007
he objective of the present study was to characterize bone cells grown in two culture media, and to determine the effective concentration of OP-1 on the growth of osteo-1 cells. Subcultured rat bone cells (osteo-1) were grown in alpha-modified Eagle's minimal essential medium (α-MEM) and Dulbecco's modified Eagle's medium (DMEM) and total protein content, alkaline phosphatase activity and the formation of mineralized nodules were evaluated after 7, 14 and 21 days. Cells were exposed to different concentrations of rhOP-1 for 1, 3, 5 and 7 days and compared with an untreated control. Osteo-1 cells presented a significant increase in alkaline phosphatase activity and calcium deposits were observed at 21 days. Cells treated with 10 and 20 ng/mL rhOP-1 for 24 h showed a significant increase in cell viability when compared to control. Osteo-1 cells cultured on DMEM demonstrated an osteoblastic phenotype as indicated by high alkaline phosphatase activity and the presence of calcified nodules. The results suggest that low concentrations of OP-1 may promote an osteogenic effect on osteo-1 cells.
Journal of Bone and Mineral Metabolism, 2020
Introduction The aim of this study was to evaluate the in vitro osteogenic potential of osteoblasts from neural crest-derived frontal bone (OB-NC) and mesoderm-derived parietal bone (OB-MS) and the bone formation induced by them when injected into calvarial defects. Materials and methods Calvarial bones were collected from newborn Wistar rats (3-day old) and characterized as frontal and parietal prior to OB-NC and OB-MS harvesting. The cells were cultured, and several parameters of osteoblast differentiation were evaluated. These cells, or PBS without cells (control), were locally injected into 5-mm rat calvarial defects (5 × 10 6 cells/defect) and after 4 weeks bone formation was evaluated by morphometric and histological analyses. Results The characterization of frontal and parietal bones assured the different embryonic origin of both cell populations, OB-NC and OB-MS. The OB-NC presented higher proliferation while the OB-MS presented higher alkaline phosphatase (ALP) activity, extracellular matrix mineralization and gene expression of runt-related transcription factor 2, Alp, bone sialoprotein and osteocalcin revealing their high osteogenic potential. µCT analysis indicated that there was higher amount of bone formation in defects injected with both OB-NC and OB-MS compared to the control. Moreover, the bone tissue formed by both cells displayed the same histological characteristics. Conclusions Despite the distinct in vitro osteogenic potential, OB-NC and OB-MS induced similar bone repair in a rat calvarial defect model. Thus, osteoblasts, irrespective of their in vitro osteogenic potential linked to embryonic origins, seem to be suitable for cell-based therapies aiming to repair bone defects.
Generation of rodent and human osteoblasts
BoneKEy Reports, 2014
This paper describes the isolation, culture and staining of primary osteoblasts from neonatal rodents and human samples. The calvaria and long-bone assays allow direct measurement of bone matrix deposition and mineralisation, as well as producing osteoblasts at defined stages of differentiation for molecular and histological analysis. Culture of human osteoblasts enables cell function to be investigated in targeted patient groups. The described methods will provide a step-by-step guide of what to expect at each stage of the culture and highlight the varied tissue culture conditions required to successfully grow osteoblasts from different sources. A special focus of this paper is the methods used for analysis of bone mineralisation and how to ensure that nonspecific mineral deposition or staining is not quantified.
Formation of bone tissue in culture from isolated bone cells
The Journal of Cell Biology
A system is described for the formation of bone tissue in culture from isolated rat bone cells. The isolated bone cells were obtained from embryonic rat calvarium and periosteum or from traumatized, lifted periosteum of young rats. The cells were cultured for a period of up to 8 wk, during which time the morphological, biochemical, and functional properties of the cultures were studied. Formation of bone tissue by these isolated bone cells was shown, in that the cells demonstrated osteoblastic morphology in light and electron microscopy, the collagen formed was similar to bone collagen, there was mineralization specific for bone, and the cells reacted to the hormone calcitonin by increased calcium ion uptake. Calcification of the fine structure of the cells and the matrix is described. Three stages in the calcification process were observed by electron microscopy. It is concluded that these bone cells growing in vitro are able to function in a way similar to such cells in vivo. This...
Calvarial bone cells: The search for stem cells Jorge E. Villanueva PhD Dissertation University of Southern California 0 (1993) Dispersed calvarial bone cells from rat fetuses were cultured suspended in agarose and some 30% of them showed deposition of metachromic extracellular matrix. Anchorage independence was a requirement for calvarial cells to express type II collagen Cells recovered from densities 1.04 g percoll/ml or higher were able to mature like chondrocytes. This chondrogenic potential in agarose was affected by subcultivation in monolayers: fresh calvarial cells after 1-2 doubling population in monolayer produced type II collagen less than 15% of that synthesized by freshly suspended and after 3-4 doubling population in monolayer he type II collagen synthesis abolished. Also, monolayer sub-cultivation impaired the ability of calvarial cells to produce bone when implanted in rats while inside demineralized bone matrix (DBM) chambers. Moreover, both in vivo and in vitro chondrogenic potential of these cells were inversely depending upon the cell concentration. Bone development is tightly associated with vascularization. Endothelial cells enhanced bone formation by calvarial cells when implanted together in Millipore diffusion chambers. In chambers with calvarial cells alone cartilage was often seen. The calcium deposition was 70 times higher in chambers containing cell mixture than chambers containing either endothelial or calvarial cells alone. DBM-powder did not mimic similar effect on calvarial cell osteogenesis. It is not well known how angiogenesis may promote osteogenesis and a critical step is to define these events occurring between endothelial and bone cells under controlled genotype backgrounds. A cationic liposome was used for the introduction of E1A-gene to isolated rat endothelial and calvarial cell lines. Because of a low growth rate of the former cells, an improved media was designed to success in transforming these cells. Geneticin resistant and E1A-oncoprotein producing endothelial cell lines were established. In addition, a collection of drug-resistant calvaria cell lines were isolated of which morphological appearance, although similar to primary calvaria cell cultures, was very stable after extensive growth. Finally, the design of an alternative approach for the characterization of bone stem cells is proposed by using transient immortalization with a temperature sensitive oncogenic agent and scrutinizing among others the ability to mature like chondrocytes. (Copies available exclusively from Micrographics Department, Doheny Library, USC, Los Angeles, CA, 90089-0182.) Keywords: Biological sciences, Molecular biology, bone cells
Journal of Histochemistry and Cytochemistry, 1997
We investigated the expression of osteocalcin (OC), bone sialoprotein (BSP), osteonectin (ON), and alkaline phosphatase (ALP) during cell differentiation and bone nodule formation by fetal rat calvaria cells, using immunofluorescent and immunogold techniques at light and electron microscopic levels. Six hours after plating all proteins were expressed in calvaria cells. However, expression was not detected during the proliferation phase after plating. Cell morphological modifications were observed in osteoblastic cells expressing ALP, OC, and BSP, but not ON. During the matrix formation phase, all proteins were expressed with various intensities and OC was limited to differentiated osteoblastic cells. EM observations demonstrated that BSP was selectively associated with clusters of needle-like crystals, but not with collagen fibers, in mineralization foci and in the mineralized matrix. OC was localized intracellularly and in all the extracellular compartments, and was concentrated at the mineralization front. ON was distributed uniformly throughout the osteoid and mineralized matrix, which was intensely labeled. The results show that the expression of bone matrix proteins during differentiation of calvaria cells and nodule formation in vitro duplicate what is observed during osteogenesis in vivo.
American Journal of Anatomy, 1983
Taking advantage of recently developed methods for osteoblast isolation, we used these cells to study bone morphogenesis in syngeneic and allogeneic intramuscular transplants.Syngeneic osteoblasts from fetal rat calvaria produced small islands of bone by the third day after transplantation. These islands increased in size and began to fuse after about 14 days. At the surface of the woven bone laid down first, lamellar bone developed. The amount of this bone increased, and in 56‐day‐old transplants solid blocks of bone were present. Osteoclasts were scarce, and the woven bone remained unresorbed. Bone marrow was absent. The structure of bone in transplants differed from that of mature calvarial bones in which only remnants of woven bone remained and bone marrow was well developed. The scarcity of osteoclasts in transplants could be caused by their relative paucity among the injected cells, since these cells responded strongly to added parathyroid hormone by increased production of cy...
Development and characterization of a porcine model to study osteoclast differentiation and activity
Bone, 1996
The study of osteoclast differentiation, function, and fate has been hampered by the lack of nonavian, nonrodent models in which biochemical and molecular studies can be conducted. The present study was undertaken to determine if osteoclasts could be generated from porcine bone marrow ceils. Bone marrow from the long bones of neonatal female pigs was enriched for mononuclear cells and cultured in the presence or absence of 1,25-(OH)zD 3, rhIL-11, or PGE 2. A confluent layer of stromal cells was observed after 4-8 days in culture and multinucleated giant cells formed after 6-10 days of culture. The multinucleated cells stained positively for tartrateresistant acid phosphatase and formed resorption lacunae when exposed to bovine cortical bone slices. When examined by transmission electron microscopy, abundant mitochondria, perinuclear Golgi complexes, numerous variably sized vacuoles, prominent rough endoplasmic reticulum, and free polysomes were observed in the mutlinucleated cells. Stimulation of the in vitro generated osteoclasts with 10-s mol/L salmon calcitonin resulted in a three to fivefold increase in cAMP production and in cell retraction. Although the osteoclasts formed in the presence or absence of 1,25-(OH)2D3, 10-50-fold more osteoclasts were observed in the cultures treated with 1,25-(OH)zD 3 in comparison to cultures without 1,25-(OH)2D 3. Osteoclast differentiation was also stimulated by rhIL-11 and PGEz; although, the number of cells generated in 6-7 days was significantly less than the number obtained with 1,25-(OH)2D 3 treatment. In addition, these multinucleated cells expressed high levels of Src kinase activity and responded to bafilomycin A~, an inhibitor of the vacuolar type H÷-ATPase, treatment with a decrease in osteoclastic bone resorption. In summary, the porcine cells possess the major distinguishing characteristics of osteoclasts and provide an alternative mammalian model to study osteoclast differentiation and resorptive activity.
Journal of Biomedical Materials Research Part A, 2008
Bone morphogenetic proteins (BMPs) are factors that promote osteoblastic differentiation and osteogenesis. The aim of this study was to examine the behavior of neonatal rat calvarial osteoblast cells cultured on different concentrations of BMP graft materials. Fifty thousand cells per milliliter were seeded and cultured on graft materials for 24 and 48 h. Different concentrations of BMPs (combination of BMPs numbered from 1 to 14) were supplemented to the medium. To evaluate cellular proliferation and differentiation, specimens were examined for DNA synthesis, alkaline phosphatase (ALP) activity, cell numbers, and viability of the cells. Further, transforming growth factor-b 1 (TGF-b 1 ) and lactate dehydrogenase (LDH) levels were investigated. Morphological appearance of the specimens at 24 and 48 h of incubation was evaluated using scanning electron microcopy. Evaluations of DNA synthesis, cell count, and cell viability data revealed that a significant difference existed at 24 and 48 h (p < 0.05). The TGF-b 1 and ALP analysis showed only a significant difference between the groups at the end of 24 h (p < 0.05). Regarding the lactate dehydrogenase activity there was not any significant difference at 24 and 48 h (p > 0.05). No morphological differences were observed in cell morphology on BMP graft material and the control group. These results indicate that BMPs have an inductive effect on osteoblast differentiation and a possible inhibitory effect in the early phases of cell proliferation.