scholarly journals Fetal bovine bone cells synthesize bone-specific matrix proteins.

1984 ◽  
Vol 99 (2) ◽  
pp. 607-614 ◽  
Author(s):  
S W Whitson ◽  
W Harrison ◽  
M K Dunlap ◽  
D E Bowers ◽  
L W Fisher ◽  
...  
Keyword(s):  
Type I Collagen ◽  
Bone Cells ◽  
Bone Sialoprotein ◽  
Cell System ◽  
Matrix Proteins ◽  
Type I ◽  
Bovine Bone ◽  
Isolated Cells ◽  
Noncollagenous Proteins

We isolated cells from both calvaria and the outer cortices of long bones from 3- to 5-mo bovine fetuses. The cells were identified as functional osteoblasts by indirect immunofluorescence using antibodies against three bone-specific, noncollagenous matrix proteins (osteonectin, the bone proteoglycan, and the bone sialoprotein) and against type 1 collagen. In separate experiments, confluent cultures of the cells were radiolabeled and shown to synthesize and secrete osteonectin, the bone proteoglycan and the bone sialoprotein by immunoprecipitation and fluorography of SDS polyacrylamide gels. Analysis of the radiolabeled collagens synthesized by the cultures showed that they produced predominantly (approximately 94%) type I collagen, with small amounts of types III and V collagens. In agreement with previous investigators who have employed the rodent bone cell system, we confirmed in bovine bone cells that (a) there was a typical cyclic AMP response to parathyroid hormone, (b) freshly isolated cells possessed high levels of alkaline phosphatase, which diminished during culture but returned to normal levels in mineralizing cultures, and (c) cells grown in the presence of ascorbic acid and beta-glycerophosphate rapidly produced and mineralized an extracellular matrix containing largely type I collagen. These results show that antibodies directed against bone-specific, noncollagenous proteins can be used to clearly identify bone cells in vitro.

Human Mesenchymal Stromal Cells are Mechanosensitive to Vibration Stimuli

2012 ◽  
Vol 91 (12) ◽  
pp. 1135-1140 ◽  
Author(s):  
I.S. Kim ◽  
Y.M. Song ◽  
B. Lee ◽  
S.J. Hwang
Keyword(s):  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Type I Collagen ◽  
Bone Cells ◽  
Type I ◽  
Related Factors ◽  
Matrix Mineralization ◽  
Vibration Signals ◽  
Human Mesenchymal Stromal Cells

Low-magnitude high-frequency (LMHF) vibrations have the ability to stimulate bone formation and reduce bone loss. However, the anabolic mechanisms that are mediated by vibration in human bone cells at the cellular level remain unclear. We hypothesized that human mesenchymal stromal cells (hMSCs) display direct osteoblastic responses to LMHF vibration signals. Daily exposure to vibrations increased the proliferation of hMSCs, with the highest efficiency occurring at a peak acceleration of 0.3 g and vibrations at 30 to 40 Hz. Specifically, these conditions promoted osteoblast differentiation through an increase in alkaline phosphatase activity and in vitro matrix mineralization. The effect of vibration on the expression of osteogenesis-related factors differed depending on culture method. hMSCs that underwent vibration in a monolayer culture did not exhibit any changes in the expressions of these genes, while cells in three-dimensional culture showed increased expression of type I collagen, osteoprotegerin, or VEGF, and VEGF induction appeared in 2 different hMSC lines. These results are among the first to demonstrate a dose-response effect upon LMHF stimulation, thereby demonstrating that hMSCs are mechanosensitive to LMHF vibration signals such that they could facilitate the osteogenic process.

scholarly journals Early Expression of Bone Matrix Proteins in Osteogenic Cell Cultures

2003 ◽  
Vol 51 (5) ◽  
pp. 633-641 ◽  
Author(s):  
Paulo Tambasco de Oliveira ◽  
Sylvia Francis Zalzal ◽  
Kazuharu Irie ◽  
Antonio Nanci
Keyword(s):  
Bone Matrix ◽  
Enzymatic Digestion ◽  
Matrix Proteins ◽  
Cell Labeling ◽  
Type I ◽  
Isolated Cells ◽  
Osteoblast Activity ◽  
Early Expression ◽  
Almost All

Osteogenic cells express some matrix proteins at early culture intervals. The aim of this study was to determine if, and in what proportion, cells used for plating contain bone sialoprotein (BSP) and osteopontin (OPN), two matrix proteins associated with initial events in bone formation. Their pattern of expression, as well as that of fibronectin (FN) and type I pro-collagen, was also examined at 6 hr and at 1 and 3 days. The cells were obtained by enzymatic digestion of newborn rat calvariae, and grown on glass coverslips. Cytocentrifuge preparations of isolated cells and coverslips were processed for single or dual immunolabeling with monoclonal and/or polyclonal primary antibodies, followed by fluorochrome-conjugated antibodies. The cell labeling was mainly associated with perinuclear elements. OPN was also distinctively found at peripheral cytoplasmic sites. About 31% of isolated cells were OPN-positive and 18% were BSP-positive. After 1 day, almost 50% of cells were immunoreactive for OPN and for type I pro-collagen, and still less than 20% reacted for BSP. Approximately 7% exhibited peripheral staining for OPN. Almost all cells were associated with extracellular FN. However, only 15% showed intracellular labeling. These results indicate that an important proportion of cells used for plating contain BSP and OPN, a situation that should be taken into consideration in experimental analyses of osteoblast activity in vitro.

Melatonin stimulates proliferation and type I collagen synthesis in human bone cells in vitro

1999 ◽  
Vol 27 (2) ◽  
pp. 106-110 ◽  
Author(s):  
Osamu Nakade ◽  
Hiroki Koyama ◽  
Hirohiko Ariji ◽  
Akihiro Yajima ◽  
Tohru Kaku
Keyword(s):  
Collagen Synthesis ◽  
Type I Collagen ◽  
Bone Cells ◽  
Human Bone ◽  
Type I

scholarly journals Collagen metabolism in cultured osteoblasts from osteogenesis imperfecta patients

1992 ◽  
Vol 286 (1) ◽  
pp. 73-77 ◽  
Author(s):  
M Mörike ◽  
R E Brenner ◽  
G B Bushart ◽  
W M Teller ◽  
U Vetter
Keyword(s):  
Osteogenesis Imperfecta ◽  
Type I Collagen ◽  
Collagen Type ◽  
Bone Cells ◽  
Collagen Type I ◽  
Collagen Metabolism ◽  
Type I ◽  
Type Iii ◽  
Type Iv

Collagen produced in vitro by bone cells isolated from 19 patients with different forms of osteogenesis imperfecta (OI) was analysed. Clinically, four patients were classified as OI type I, 10 patients as OI type III and five patients as OI type IV. Bone cells of 12 of the 19 OI patients produced structurally abnormal type I collagen. Electrophoretically uniformly slower migrating collagen type I alpha-chains were found in one case of OI type I, in seven cases of OI type III and in one case of OI type IV; two cultures of OI type III produced two different populations of collagen type I alpha-chains, and one culture of OI type IV showed reduction-sensitive dimer formation of alpha 1(I) chains, resulting from the inadequate incorporation of a cysteine residue into the triple helical domain of alpha 1(I). Quantitative analysis of collagen metabolism led to the distinction of two groups of cultured OI osteoblasts. In osteoblasts of OI type I, mainly production of collagen was decreased, whereas secretion, processing and pericellular accumulation of (pro)collagen type I was similar to that in control osteoblasts. In contrast, in osteoblasts of OI types III and IV, production as well as secretion, processing and pericellular accumulation of (pro)collagen type I were significantly decreased. Low levels of type I collagen were found irrespective of the presence or absence of structural abnormalities of collagen type I in all OI types.

Study on the Effects of Type I Collagen Combined with Noncollagenous Proteins on Hydroxyapatite Formation in vitro using SPM and GIXD

2009 ◽  
Vol 1238 ◽  
Author(s):  
Xiaolan Ba ◽  
Elaine DiMasi ◽  
Miriam H Rafailovich
Keyword(s):  
Type I Collagen ◽  
Early Stage ◽  
Cooperative Interaction ◽  
Type I ◽  
X Ray Diffraction ◽  
Noncollagenous Proteins ◽  
Force Microscopy ◽  
Kinetics Of

AbstractThe effects of the components of extracellular matrix on the bone formation and the kinetics of crystal growth of calcium phosphate have remained unknown. In this paper, we reported a method to investigate the role of Type I collagen and the interactions with other ECM proteins such as fibronectin and elastin during biomimic mineralization process in vitro. The early stage of mineralization was characterized by scanning probe microscopy (SPM) and shear modulation force microscopy (SMFM). The late stage of mineralization was investigated by synchrotron grazing incident x-ray diffraction (GIXD). The results demonstrate the cooperative interaction between type I collagen and noncollagenous proteins such as fibronectin or elastin could be essential for the biomineralization.

scholarly journals Adaptor protein CrkII negatively regulates osteoblast differentiation and function through JNK phosphorylation

2019 ◽  
Vol 51 (9) ◽  
pp. 1-10 ◽  
Author(s):  
Jung Ha Kim ◽  
Kabsun Kim ◽  
Inyoung Kim ◽  
Semun Seong ◽  
Kwang-Il Nam ◽  
...  
Keyword(s):  
Osteoblast Differentiation ◽  
Type I Collagen ◽  
Bone Cells ◽  
Biological Activities ◽  
Osteoclast Differentiation ◽  
Adaptor Protein ◽  
Type I ◽  
And Function

Abstract The adaptor protein CrkII is involved in several biological activities, including mitogenesis, phagocytosis, and cytoskeleton reorganization. Previously, we demonstrated that CrkII plays an important role in osteoclast differentiation and function through Rac1 activation both in vitro and in vivo. In this study, we investigated whether CrkII also regulates the differentiation and function of another type of bone cells, osteoblasts. Overexpression of CrkII in primary osteoblasts inhibited bone morphogenetic protein (BMP) 2-induced osteoblast differentiation and function, whereas knockdown of CrkII expression exerted the opposite effect. Importantly, CrkII strongly enhanced c-Jun-N-terminal kinase (JNK) phosphorylation, and the CrkII overexpression-mediated attenuation of osteoblast differentiation and function was recovered by JNK inhibitor treatment. Furthermore, transgenic mice overexpressing CrkII under control of the alpha-1 type I collagen promoter exhibited a reduced bone mass phenotype. Together, these results indicate that CrkII negatively regulates osteoblast differentiation and function through JNK phosphorylation. Given that CrkII acts as a negative and positive regulator of osteoblast and osteoclast differentiation, respectively, the regulation of CrkII expression in bone cells may help to develop new strategies to enhance bone formation and inhibit bone resorption.

Collagen fibrillogenesis in vitro: interaction of types I and V collagen

1986 ◽  
Vol 44 ◽  
pp. 210-211
Author(s):  
Arthur J. Wasserman ◽  
Kathy C. Kloos ◽  
David E. Birk
Keyword(s):  
Type I Collagen ◽  
Corneal Stroma ◽  
Minor Component ◽  
Homogeneous Distribution ◽  
Type I ◽  
Type V Collagen ◽  
Fibril Morphology ◽  
Type V ◽  
In Vitro Interaction

Type I collagen is the predominant collagen in the cornea with type V collagen being a quantitatively minor component. However, the content of type V collagen (10-20%) in the cornea is high when compared to other tissues containing predominantly type I collagen. The corneal stroma has a homogeneous distribution of these two collagens, however, immunochemical localization of type V collagen requires the disruption of type I collagen structure. This indicates that these collagens may be arranged as heterpolymeric fibrils. This arrangement may be responsible for the control of fibril diameter necessary for corneal transparency. The purpose of this work is to study the in vitro assembly of collagen type V and to determine whether the interactions of these collagens influence fibril morphology.

Development of an in vitro Model for the Study of the Response of Equine Tendon Fibroblasts to Injury and Medication

1997 ◽  
Vol 10 (01) ◽  
pp. 6-11 ◽  
Author(s):  
R. F. Rosenbusch ◽  
L. C. Booth ◽  
L. A. Dahlgren
Keyword(s):  
Electron Microscopy ◽  
Extracellular Matrix ◽  
Light Microscopy ◽  
Light And Electron Microscopy ◽  
Tendon Healing ◽  
Matrix Proteins ◽  
Isolated Cells ◽  
Superficial Digital Flexor Tendon ◽  
Vitro Model

SummaryEquine tendon fibroblasts were isolated from explants of superficial digital flexor tendon, subcultured and maintained in monolayers. The cells were characterized by light microscopy, electron microscopy and radiolabel studies for proteoglycan production. Two predominant cell morphologies were identified. The cells dedifferentiated toward a more spindle shape with repeated subcultures. Equine tendon fibroblasts were successfully cryopreserved and subsequently subcultured. The ability to produce proteoglycan was preserved.The isolated cells were identified as fibroblasts, based on their characteristic shape by light microscopy and ultrastructure and the active production of extracellular matrix proteins. Abundant rough endoplasmic reticulum and the production of extracellular matrix products demonstrated active protein production and export. Proteoglycans were measurable via liquid scintillation counting in both the cell-associated fraction and free in the supernatant. This model is currently being utilized to study the effects of polysulfated glycosaminoglycan on tendon healing. Future uses include studying the effects of other pharmaceuticals, such as hyaluronic acid, on tendon healing.A model was developed for in vitro investigations into tendon healing. Fibroblasts were isolated from equine superficial digital flexor tendons and maintained in monolayer culture. The tenocytes were characterized via light and electron microscopy. Proteoglycan production was measured, using radio-label techniques. The fibroblasts were cryopreserved and subsequently subcultured. The cells maintained their capacity for proteoglycan production, following repeated subculturing and cryopreservation.

scholarly journals Restoration of Osteogenesis by CRISPR/Cas9 Genome Editing of the Mutated COL1A1 Gene in Osteogenesis Imperfecta

2021 ◽  
Vol 10 (14) ◽  
pp. 3141
Author(s):  
Hyerin Jung ◽  
Yeri Alice Rim ◽  
Narae Park ◽  
Yoojun Nam ◽  
Ji Hyeon Ju
Keyword(s):  
Osteogenesis Imperfecta ◽  
Type I Collagen ◽  
Disease Modeling ◽  
Bone Fragility ◽  
Osteogenic Potential ◽  
Type I ◽  
Gene Correction ◽  
Col1a1 Gene ◽  
Collagen Formation

Osteogenesis imperfecta (OI) is a genetic disease characterized by bone fragility and repeated fractures. The bone fragility associated with OI is caused by a defect in collagen formation due to mutation of COL1A1 or COL1A2. Current strategies for treating OI are not curative. In this study, we generated induced pluripotent stem cells (iPSCs) from OI patient-derived blood cells harboring a mutation in the COL1A1 gene. Osteoblast (OB) differentiated from OI-iPSCs showed abnormally decreased levels of type I collagen and osteogenic differentiation ability. Gene correction of the COL1A1 gene using CRISPR/Cas9 recovered the decreased type I collagen expression in OBs differentiated from OI-iPSCs. The osteogenic potential of OI-iPSCs was also recovered by the gene correction. This study suggests a new possibility of treatment and in vitro disease modeling using patient-derived iPSCs and gene editing with CRISPR/Cas9.

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