Cultured Rat Bone Cells Research Articles

Muscle as an osteoinductive niche for local bone formation with the use of a biphasic calcium sulphate/hydroxyapatite biomaterial.

ObjectivesWe have observed clinical cases where bone is formed in the overlaying muscle covering surgically created bone defects treated with a hydroxyapatite/calcium sulphate biomaterial. Our objective was to investigate the osteoinductive potential of the biomaterial and to determine if growth factors secreted from local bone cells induce osteoblastic differentiation of muscle cells.Materials and MethodsWe seeded mouse skeletal muscle cells C2C12 on the hydroxyapatite/calcium sulphate biomaterial and the phenotype of the cells was analysed. To mimic surgical conditions with leakage of extra cellular matrix (ECM) proteins and growth factors, we cultured rat bone cells ROS 17/2.8 in a bioreactor and harvested the secreted proteins. The secretome was added to rat muscle cells L6. The phenotype of the muscle cells after treatment with the media was assessed using immunostaining and light microscopy.ResultsC2C12 cells differentiated into osteoblast-like cells expressing prominent bone markers after seeding on the biomaterial. The conditioned media of the ROS 17/2.8 contained bone morphogenetic protein-2 (BMP-2 8.4 ng/mg, standard deviation (sd) 0.8) and BMP-7 (50.6 ng/mg, sd 2.2). In vitro, this secretome induced differentiation of skeletal muscle cells L6 towards an osteogenic lineage.ConclusionExtra cellular matrix proteins and growth factors leaking from a bone cavity, along with a ceramic biomaterial, can synergistically enhance the process of ectopic ossification. The overlaying muscle acts as an osteoinductive niche, and provides the required cells for bone formation.Cite this article: D. B. Raina, A. Gupta, M. M. Petersen, W. Hettwer, M. McNally, M. Tägil, M-H. Zheng, A. Kumar, L. Lidgren. Muscle as an osteoinductive niche for local bone formation with the use of a biphasic calcium sulphate/hydroxyapatite biomaterial. Bone Joint Res 2016;5:500–511. DOI: 10.1302/2046-3758.510.BJR-2016-0133.R1.

Effects of new machinable ceramic on behavior of rat bone cells cultured in vitro.

The behavior of cultured rat bone cells growing on modified polyethylene terephthalate (mPET), glass, and machinable ceramic substrates containing enstatite (MgO, SiO2) and glass (CaO-P2O5-Al2O3) was studied. Cell attachment was measured directly on the substrates using an image analysis system. Electron microscopy observations and the MTT test revealed that cells are able to spread and proliferate on the material surface, keeping a healthy ultrastructure on all materials tested in the present study. After having colonized the surface of the materials, as shown by immunocytochemistry, the cells synthesize an osteoid-like matrix composed of osteocalcin, type I collagen, and fibronectin fibrils. The titration of alkaline phosphatase activity showed that the cells grown on the ceramic exhibit a greater osteogenic activity than those grown on controls (glass and mPET). This osteogenic activity results in a mineralization of the extracellular matrix in cultures on ceramic or plastic whereas only few calcium phosphate crystallite traces were revealed by Von Kossa staining on glass. Enstatite constitutes, therefore, an environment compatible with in vitro bone cell life.

Identification of integrin cell-substratum adhesion receptors on cultured rat bone cells.

The interactions of bone cells with their extracellular matrix is of major importance in bone development, repair, and disease. We examined the ability of rat calvarial bone cells to adhere to various matrix proteins and to define the role of integrin cell-substrate adhesion receptors in these interactions. Isolated newborn rat calvarial bone cells prelabeled with 3H-thymidine and plated on plastic wells that had been precoated with serial dilutions of various substrates showed typical dose-response adherence curves to fibronectin, fibrinogen, laminin, vitronectin, and collagen I and IV. Cell adherence to poly-D-lysine, a nonspecific cell adherent, was high at all substrate concentrations > 0.0001 micrograms/ml. A polyclonal anti-rat integrin antibody blocked cell adhesion to all substrates tested except poly-D-lysine. Isolated rat calvarial bone cells were surface labeled with 125I, extracted, and immunoprecipitated with polyclonal antibodies made against the rat integrin complex and peptides derived from the cytoplasmic domains of the alpha 2, alpha 3, and alpha 5 subunits. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (nonreduced) identified four bands representing a mixture of integrins including the alpha 1 beta 1 laminin/collagen receptor, the alpha 5 beta 1 fibronectin receptor, and the alpha V beta 3 (or possibly alpha V beta 5) vitronectin receptor. These experiments show that bone cells adhere to a wide variety of extracellular matrix proteins via specific integrins. Increased knowledge about the regulation of these receptors and the mechanisms by which they transmit information to the cell will be important for a more complete understanding of bone physiology and pathophysiology.

Morphological and immunocytochemical characterization of osteoblast cultures from long bones of neonatal rats.

The present study reports a successful culture system of rat bone cells obtained from the bare long bone fragments of neonatal Wistar rats. The morphology, histochemical and immunocytochemical properties of the cultured cells were characterized by phase contrast microscopy, transmission electron microscopy (TEM), scanning electron microscopy (SEM), histochemical staining with alkaline phosphatase (ALP), and immunocytochemical staining with the anti-osteocalcin antibodies. Under phase contrast microscopy, the cultured rat bone cells changed their morphology from being slender with relatively-short cytoplasmic processes to becoming polygonal with longer processes, and subsequently flattened. These cultured bone cells formed bone nodules after 3 weeks. Under SEM, the cultured bone cell appeared polygonal with some microvilli. The TEM showed that their cytoplasm contained abundant endoplasmic reticulum and well-developed Golgi apparatus. Positive histochemical staining of ALP was also detected as a blue coloration and granular appearance in the cultured bone cells. Immunocytochemistry with the polyclonal and monoclonal anti-osteocalcin antibodies showed the localization of osteocalcin in both the cytoplasm and nucleus. All these data lead us to consider that the cultured bone cells in our system were probably osteoblasts from growing bone tissue in vitro. This provides a convenient test system for further studies on the regulation of the growth and metabolism of rat bone cells.

Toxicity of root canal sealers on rat bone cells in primary culture

The cytotoxic effects on cultured rat bone cells of newly-developed root canal sealers and commercially available sealers were compared. Various root canal sealers were applied to cultured bone cells obtained from rat calvaria by the enzyme digestion method. Measurement of [3H]-thymidine incorporation, alkaline phosphatase activity, and calcium release were performed after 24 and 48 h. No significant difference was found in cellular DNA synthesis and alkaline phosphatase activity between cells exposed to New B-1, New B-5 and controls after exposure for 24 and 48 h. Cells in contact with Tubliseal, Diaket and AH-26 demonstrated a significant difference from controls in DNA synthesis and alkaline phosphatase activity. Calcium release at 24 h was significantly different in the cells treated with New B-1 and New B-5 than in controls. No appreciable difference was found, however, between New B-6, Sealapex and controls. At 48 h, cells treated with New B-1, New B-5 and New B-6 showed differed significantly difference from controls, but the cells exposed to Sealapex did not. The newly-developed root canal sealers had lower toxicity in vitro than five types of commercially available root canal sealers.

Sulphation of secreted phosphoprotein I (SPPI, osteopontin) is associated with mineralized tissue formation

Secreted phosphoprotein I (SPPI) is a prominent structural protein in mineralized connective tissues. Rat bone cells in culture produce several forms of SPPI that differ in post-translational modifications such as phosphorylation and sulphation. To determine the significance of protein sulphation in bone formation, the synthesis of SPPI was studied in vitro using rat bone marrow cells (RBMC) which form bone-like tissue when grown in the presence of dexamethasone (Dex) and β-glycerophosphate (β-GP). In the presence of 10 −7 M Dex SPPI expression was stimulated 4–5-fold. Radiolabelling multilayered RBMCs for 48 h with [ 35S]-methionine, Na 2[ 35SO 4], or Na 3[ 32PO 4] revealed that two major phosphorylated forms of SPPI were secreted into the culture medium: a highly phosphorylated form migrating at 44 kDa on 15% SDS-PAGE and a less phosphorylated 55 kDa form. In the mineralized tissue formed in the presence of Dex and β-GP, both forms of SPPI, in addition to proteoglycans and a 67 kDa protein, incorporated significant amounts of [ 35SO 4]. Sulphation of SPPI was not observed in the absence of mineral formation, indicating that the sulphation of SPP I is closely associated with mineralization and that it can be used as a sensitive and specific marker for the osteoblastic phenotype.

Different roles for calcium and cyclic AMP in the action of PTH: Studies in bone explants and isolated bone cells

In fetal mouse calvaria forskolin (0.1–100 μM), like PTH, stimulated cyclic AMP production in a dose-dependent way. The dose-response curve for forskolin-induced bone mineral release (24 hrs), however, demonstrated a biphasic character, showing stimulation at 0.1 μM and inhibition at 5 and 10 μM. In addition, forskolin-stimulated bone resorption reached a plateau after 48 hrs of incubation, a phenomenon which did not occur with PTH. Forskolin (0.1 μM) strongly stimulated PTH-induced cyclic AMP production in fetal mouse calvaria. However, PTH-stimulated bone resorption and PTH-induced increase in cytosolic free Ca 2+ in bone fetal rat cells were not stimulated by forskolin (0.1 μM). 9-(Tetrahydro-2-furyl) adenine (100 μM) completely blunted PTH-stimulated cyclic AMP response in fetal mouse calvaria. PTH-stimulated bone resorption was also completely inhibited, but only after 6 hrs and not after 24 hrs of incubation. With nifedipine and varabamil PTH-stimulated bone resorption was significantly inhibited after 24 hrs of incubation and not significantly after 6 hrs of incubation. A23187 (1 gmM) significantly stimulated PTH-stimulated cyclic AMP level and increased basal cytosolic free Ca 2+ concentration in cultured rat bone cells. In calvaria, however, it had no effect on either basal and PTH-stimulated cyclic AMP production or on basal and PTH-stimulated bone resorption (6 and 24 hrs). From these observations it follows that in calvaria manipulation of intracellular cyclic AMP only (partially) affects bone resorption. This observation points to a role for an additional second messenger in establishing full blown bone resorption. Some of the results are published in short elsewhere.

The effect of beryllium chloride on alkaline phosphatase activity in rat bone cells in culture

The effect of beryllium chloride on alkaline phosphatase activity in rat bone cells in culture

Triiodothyronine increases responsiveness of cultured rat bone cells to parathyroid hormone.

Osteoblast-like cells prepared from calvaria of newborn rats and grown in culture for 1 week show markedly increased ornithine decarboxylase (ODC) activity upon exposure to parathyroid hormone (PTH) for 4 h. Triiodothyronine (T3) increases ODC activity of the cultures in long-term experiments but does not stimulate cell replication. Moreover, PTH responsiveness is enhanced by T3. Thus, T3 acts directly on bone cells, and the clinical observation of bone sensitization to PTH by thyroid hormones is confirmed at the cellular level.

The role of membrane phospholipids in the induction of creatine kinase activity and DNA synthesis in cultured rat bone cells by PTH

The role of membrane phospholipids in the induction of creatine kinase activity and DNA synthesis in cultured rat bone cells by PTH

Regulation of creatine kinase activity in rat osteogenic sarcoma cell clones by parathyroid hormone, prostaglandin E2, and vitamin D metabolites.

We have previously shown that both parathyroid hormone (PTH) and prostaglandin E2 (PGE2) stimulate the activity of creatine kinase BB (CKBB) in rat bone cells in culture. Therefore, morphologically distinct rat osteogenic sarcoma cells in culture were tested for stimulation of CKBB activity by hormones that regulate skeletal tissues. PTH stimulated CKBB in the osteoblast-like clone ROS 17/2; 1 alpha,25(OH)2D3 inhibited this activity while PGE2, CT and 24R,25(OH)2D3 had no significant effect. PGE2 stimulated CKBB activity in the fibroblast-like clone ROS 24/1, which was unresponsive to PTH, CT and Vitamin D metabolites. 24R,25(OH)2D3 as well as PGE2 (but not PTH, CT or 1 alpha 25(OH)2D3) stimulated CKBB in clone ROS 25/1, suggesting that this fibroblast-like clone has some chondroblast-like character. Both PTH and PGE2 stimulated the brain type isoenzyme of CK (CKBB), although the osteogenic sarcoma cell clones contain a significant proportion of the muscle type of CK (CKMM). Thus, increased CKBB activity can serve as an additional characteristic marker for the action of steroid and polypeptide hormones and for prostaglandins.

Identification and control of collagenase secreted by fetal rat bone cells in culture

Identification and control of collagenase secreted by fetal rat bone cells in culture

Characterization of insulin-like growth factor I receptors on cultured rat bone cells: regulation of receptor concentration by glucocorticoids.

A specific receptor for insulin-like growth factor I (IGF-I) has been demonstrated in cultured fetal rat osteoblast-like bone cells. Specific binding of [125I]IGF-I to bone cells incubated at 15 C reached a steady state by 5 h. Half-maximal inhibition of [125I]IGF-I binding by unlabeled IGF-I was observed at 7 ng/ml. Multiplication-stimulating activity, insulin, and proinsulin were less effective than unlabeled IGF-I in competing for receptor occupancy. Scatchard analysis showed a curvilinear plot, with a Ka similar to that observed in human fibroblasts. Incubation of cell monolayers with glucocorticoids resulted in a concentration-dependent increase in [125I]IGF-I binding. This increase in [125I]IGF-I binding was dependent on cell density. After a 2-day exposure to dexamethasone, no increase in binding was observed in sparsely plated cells; however, an increase in binding was observed after 3 days in culture (log phase) and was maximal by 5 days (peak log phase). These data indicate that rat bone cells possess a specific receptor for IGF-I with binding characteristics similar to those reported in human fibroblasts, and that IGF-I receptor concentrations are increased by exposure to glucocorticoids. A role for glucocorticoids and IGF-I in rat bone proliferation is suggested by these findings.

Glucocorticoid Receptors and Actions in Subpopulations of Cultured Rat Bone Cells

We have previously shown that bone cells possess glucocorticoid receptors and that, in addition to being inhibitory to cell growth, glucocorticoid treatment potentiates the ability of parathyroid hormone (PTH) to stimulate cyclic AMP (cAMP) formation. This study extends those observations to specific subpopulations of bone cells and explores the mechanism of the cAMP augmentation. Subpopulations of cultured bone cells derived from 20-d-old fetal rat calvaria were enriched for "osteoblast-like" (OB) and "osteoclast-like" (OC) cells by sequential collagenase digestion. OC cells released during the first 30 min of collagenase digestion were characterized by low alkaline phosphatase activity, a cAMP response to salmon calcitonin (CT), but only a small cAMP response to bovine PTH. In contrast, OB cells released between 30 and 120 min of collagenase digestion, possessed high alkaline phosphatase activity, responded with a large cAMP rise to PTH, but exhibited no response to CT. Glucocorticoid receptors, with similar properties, were demonstrated in both populations (K(d) congruent with 5 nM, N(maximum) congruent with 400 fmol/mg cytosol protein). Dexamethasone equivalently inhibited cell growth and alkaline phosphatase activity in both populations. Dexamethasone potentiation of cAMP generation occurred after PTH but not CT stimulation. A greater enhancement of cAMP generation observed in OB cells appears to result from two glucocorticoid actions: (a) stimulation of adenylate cyclase and (b) inhibition of phosphodiesterase. Only the latter mechanism was found in OC cells. Dexamethasone-treated cells showed an increase in both sensitivity and maximal response of cAMP to PTH. The possible relationship of these actions to the mechanism of glucocorticoid-induced osteopenia is discussed.

Glucocorticoid Potentiation of the Adenosine 3′,5′- Monophosphate Response to Parathyroid Hormone in Cultured Rat Bone Cells*

We have previously demonstrated the presence of glucocorticoid receptors in bone cells and have suggested that direct inhibition of cell proliferation may contribute to glucocorticoid-induced osteoporosis. The present studies examine the issue of whether glucocorticoids might also alter bone status by interacting with parathyroid hormone (PTH) a major regulator of bone homeostasis. Since cyclic AMP (cAMP) is the putative mediator of PTH-induced inhibition of collagen synthesis as well as resorption, we assessed the ability of dexamethasone to modulate the cAMP response to PTH. Bone cells from calvaria of 20-21 day fetal rats were cultured for 6–7 days and the ability of PTH to stimulate cAMP was measured. The cells responded to both the synthetic (1–34) fragment and native PTH with a dose-dependent increase in cAMP. Dexamethasone treatment did not alter base-line levels of cAMP but did potentiate the cAMP response to PTH: 1- to 2- fold when assayed in the presence of theophylline and 2- to 4-fold in its a...