Cooperative effects in differentiation and proliferation between PDGF-BB and matrix derived synthetic peptides in human osteoblasts
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Enhancing osteogenic capabilities of bone matrix for the treatment of fractures and segmental defects using growth factors is an active area of research. Recently, synthetic peptides like AC- 100, TP508 or p-15 corresponding to biologically active sequences of matrix proteins have been proven to stimulate bone formation. The platelet-derived growth factor (PDGF) BB has been identified as an important paracrine factor in early bone healing. We hypothesized that the combined use of PDGF-BB with synthetic peptides could result in an increase in proliferation and calcification of osteoblast-like cells. Osteoblast-like cell cultures were treated with PDGF and synthetic peptides, singly and as combinations, and compared to non-treated control cell cultures. The cultures were evaluated at days 2, 5, and 10 in terms of cell proliferation, calcification and gene expression of alkaline phosphate, collagen I and osteocalcin. Experimental findings revealed that the addition of PDGF, p-15 and TP508 and combinations of PDGF/AC-100, PDGF/p-15 and PDGF/TP508 resulted in an increase in proliferating osteoblasts, especially in the first 5 days of cultivation. Proliferation did not significantly differ between single factors and factor combinations (p > 0.05). The onset of calcification in osteoblasts occurred earlier and was more distinct compared to the corresponding control or PDGF stimulation alone. Significant difference was found for the combined use of PDGF/p-15 and PDGF/AC-100 (p < 0.05). Our findings indicate that PDGF exhibits cooperative effects with synthetic peptides in differentiation and proliferation. These cooperative effects cause a significant early calcification of osteoblast-like cells (p < 0.05). We suggest the combination of synthetic peptides and PDGF as a potential clinical approach for accelerating bone healing or coating osteosynthesis materials.Keywords:
Platelet-derived growth factor
Objective To study the expression of ERK5 on human,rat,mouse osteoblasts and the regulation of IL-6 on ERK5 expression,preliminarily investigate the effect of ERK5 on proliferation and differentiation of osteoblast.Methods The protein expression of ERK5 on osteoblasts was confirmed by western blot.ERK5 and P-ERK5 were detected when different dose of IL-6 treated osteoblast during different time.ERK5 siRNA plasmids were constructed and transfected into MG-63 cells.MTT,ALP activity and osteocalcin expression were detected after treated with 10 nmol/L IL-6.Results ERK5 was expressed in human,rat,mouse osteoblasts.The phosphorylation level of ERK5 were increased by IL-6 on time and dose dependent manner.As compared to control cells,ERK5 siRNA cells' MTT reduced,osteocalcin expression reduced(P0.05) yet the ALP activity was no significant difference.Conclusion ERK5 signal pathway is regulated by IL-6 in osteoblast,and ERK5 signal pathway participate in the proliferation and differentiation of osteoblast.
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To observe the effect of leptin on osteoblast.Human osteoblast primary culture was carried out, and the morphology and function of osteoblast were observed. The effects of different levels of leptin on osteoblast in different days were assessed by MTT colorimetry. Osteocalcin production was measured also.Human osteoblasts were fusiform in shape and were positive for alkaline phosphatase by histochemical staining, positive for osteocalcin by immunofluorescence staining, and positive by Alizarin Reds staining after mineralized upon supplementation with ascorbate and beta-glycerophosphate. On the first, second and third days, the proliferation of osteoblast, cultured with different concentrations of leptin, had no changes. The leptin-stimulated synthesis of osteocalcin of cells was found to be dose-dependent (P < 0.05), but not time-dependent (P > 0.05).The above data indicated that there were no evidences for the effects of leptin on the proliferation of human osteoblast, but leptin could enhance the function of human osteoblast.
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Abstract The current study investigated the immunomodulating and osteoblast differentiation potential of the natural compounds from Leea macrophylla (LMN). Immunomodulatory effects have been investigated by the phagocytosis of Candida albicans using polymorphonuclear neutrophil cells in the in vitro slide method. A bioactivity‐guided fractionation technique was used to evaluate the stimulating effect of L. macrophylla methanol extract on osteoblast differentiation using mouse osteoblastic cells. A low dose of LMN was found to stimulate the phagocytic effect better than a higher dose. The natural compounds from L. macrophylla have significantly increased alkaline phosphatase (ALP) and osteocalcin activities. The LMN promoted the osteoblast differentiation through upregulation of ALP, osteocalcin, and type 1 collagen in a dose‐dependent manner. These natural compounds also upregulated ALP, osteocalcin, and type 1 collagen gene expressions. The data suggest that LMN has potential anabolic sequel on bone formation and osteoblast differentiation.
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Objective: To investigate the effects of IGF-1 on bone formation of rabbit osteoblast in vitro.Methods:Osteoblasts were obtained from rabbit costal bone by explant culture technique.The second subculture was exposed to IGF-1 at variant concentrations of 0.1,1,10 and 20ng/ml for 24 and 36 hours,and the effects of IGF-1 on osteoblast proliferation was observed by MTT assay.The contents of BGP were measured by the radioimmunoassay in the culture supernatant harvested after the second subculture were exposed to IGF-1 for 72 and 108 hours.Results:Osteoblast exposed to IGF-1 for 24 and 36 hours at variant concentrations of 0.1,1,10,20ng/ml proliferated significantly compared with the control (P0.001),and significant differences were found between every two groups when osteoblast were exposed to IGF-1 at range from 0.1 to 10ng/ml(P0.05).Levels of osteocalcin in culture supernatant of osteoblast exposed to IGF-1 at variant concentrations for 72 and 108 hours was not statistically significant compared with control group (P0.05).Conclusions:IGF-1 could promote the proliferation of osteoblast,and there were dose-dependent relationship at a limited range from 0.1 to 10ng/ml.IGF-1 had no effect on osteocalcin synthesis of osteoblast.
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Abstract This study was designed to investigate whether methotrexate (MTX), used in the treatment of rheumatoid arthritis (RA), affects proliferation and differentiation of human osteoblasts in culture. The effects of MTX were assessed by analyzing markers of proliferation and differentiation of human trabecular bone–derived osteoblast-like cells cultured in the presence or absence of 1,25-dihydroxyvitamin D3 (1,25[OH]2D3). Treatment of the osteoblastic cells with MTX resulted in a strong dose-dependent inhibition of cell proliferation with half maximal response at a dose of 30 nM. MTX did not interfere with cellular alkaline phosphatase (AP) activity, the number of cells expressing cytochemical AP, or basal osteocalcin production. Addition of 1,25(OH)2D3 to the cultures caused an enhanced AP expression and osteocalcin production coinciding with a decreased osteoblast proliferation. Coincubation of 1,25(OH)2D3 with MTX in doses ≥100 nM further inhibited osteoblast growth and induced a significant stimulation of AP expression and activity, and production of osteocalcin above the values reached in the 1,25(OH)2D3 cultures. In conclusion, MTX proved to be a potent inhibitor of osteoblast proliferation but did not affect basal osteoblastic phenotypic expression. In the presence of the osteoblast differentiation-promotor, 1,25(OH)2D3, MTX further inhibited cell growth which was associated with enhanced AP activity and osteocalcin production. Thus, MTX may have profound effects on bone metabolism and remodeling by interfering with bone cell turnover.
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Abstract Excess levels of glucocorticoids are known to cause osteoporosis. It is speculated that the effect of glucocorticoids could be mediated via regulation of IGF-I. The aims of the present study were to detect and quantify the expression of IGF-I and/or IGF-II mRNA transcripts in human osteoblast-like cells and to investigate whether glucocorticoids regulate the expression of IGF-I mRNA transcripts in human osteoblast-like cells. Cultures of human osteoblast-like cells from trabecular bone were established. The IGF-IA and IGF-IB transcripts were detected in human osteoblast-like cells from seven out of nine patients while the IGF-II transcript was detected in human osteoblast-like cells from eight out of nine patients, as determined by RT-PCR assays. Human osteoblast-like cells, as well as human muscle tissue, expressed approximately 1/10 of the IGF-I mRNA levels found in liver, as determined by RNase protection solution hybridization assay. The IGF-I mRNA levels did not decrease with age in the human osteoblast-like cells and no difference was seen between males and females. However, cortisol (10 −6 mol/l) decreased IGF-I mRNA levels. In summary, the present study has shown that human osteoblast-like cells express IGF-I and IGF-II mRNA transcripts and that cortisol down-regulates the IGF-I mRNA levels, indicating that some of the inhibitory effect of glucocorticoids on bone formation in humans is mediated via a reduced autocrine/paracrine expression of IGF-I. Journal of Endocrinology (1996) 149, 397–403
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In Brief Study Design. Human osteoblast cultures were exposed to a very low intensity static magnetic fields (SMF) to investigate its effects on osteoblast growth and differentiation. Objective. Analysis of the effects of periprosthetic SMF on the growth and differentiation of human osteoblast cell cultures in vitro. Summary of Background Data. The effects of pulsed electromagnetic fields (PEMF) on cell proliferation, especially in human osteoblast-like cells is well described, whereas few data are available on the effects of SMF on osteoblast cell culture. We previously demonstrated that the proliferation of human osteoblast cultures is reduced when cells are exposed to a continuous low intensity SMF comparable to the one that occurs around metal devices (Ti spinal implant) because of the generation of electric currents between the screw (Ti6Al4V) and the rod (Ti). Methods. Primary osteoblastic cells were isolated from a human femoral head. Osteoblast cultures were exposed to SMF and alkaline phosphatase activity was evaluated in the osteoblast cell cultures at different time points. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to evaluate mRNA expression levels of osteocalcin, Runx2, and collagen I genes. Results. The SMF-treated cells showed a progressive increase in the alkaline phosphatase activity which, however, remained always lower than the one observed in the control group at each observation time (72 hours, 7 and 14 days). RT-PCR demonstrated that Runx2 and collagen I mRNA were downregulated following SMF stimulation, whereas no change in osteocalcin mRNA was observed. Conclusion. Continuous low-intensity electromagnetic field comparable to the one that generates around metal devices because of the generation of corrosion currents inhibits osteoblasts differentiation pattern and might contribute at least in part to a decrease in periprosthetic bone formation occurring in vivo. Static electromagnetic fields (SMF) occur around titanium spinal devices because of the generation of electric currents between the screw (Ti6A14V) and the rod (Ti). Electromagnetic phenomena generated around metal spinal influence osteoblast homeostasis and might, at least in part, contribute to aseptic periprosthetic bone loss. We hypothesize that periprosthetic SMF stimulation may inhibit not only osteoblast proliferation rate but also osteoblast differentiation pattern.
RUNX2
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Objective To study different concentrations of Eucommia extract effect on osteoblast proliferation and osteocalcin expression. Methods Newborn SD rats( 24 ~ 48 h,6) are isolated by enzymatic digestion to get primary neonatal rat osteoblasts,osteoblasts were identified by alkaline phosphatase staining; First each groups were added with serum-free medium without phenol red starved SD rats osteoblasts at 2h,then give five groups concentrations Eucommia extract(0mg/ml,1mg/ml,10 mg/ml,50 mg/ml,100 mg/ml) interfere in SD rat osteoblasts and cells were incubated for 48 hours,set the control group(0mg/ml),then MTT method detect cell proliferation; ibid steps,Western blot detect the expression levels of osteocalcin. Results After alkaline phosphatase staining,SD rats osteoblasts was purple,MTT method showed that the leaf extract on the proliferation of osteoblasts have a role in promoting(P 0. 05) Compared with the control group,proliferation of osteoblas tsassociated with the drug concentration,wherein the most significant concentration was 50 mg/ml(P 0. 01). Western bolt results are shown that each dosing groups of osteocalcin protein levels were significantly elevated(P 0. 05) compared with the control group,and also has a consistent concentration-dependent. Conclusion Eucommia extract can promote osteoblast proliferation through affecting the expression of osteocalcin and osteoblast proliferation has a concentration-dependent nature.
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Physiological actions of osteoblasts are disordered by gravity unloading. We investigated the possibility that the appropriate level of hypergravity could improve osteoblast functions that are susceptible to mechanical unloading. We evaluated hypergravity effects on the 1alpha,25-dihydroxyvitamin D(3) (VD)-inducible osteocalcin expression of primary rat osteoblasts. Cell culture plates were centrifuged for 24 h at 3, 6, 12, 24, and 48 g in a 37 degrees C incubator. The mRNA levels were analyzed by quantitative RT-PCR. The mRNA levels for osteocalcin and vitamin D receptor (VD-R) at 12 g were enhanced to 187% and 228% of the 1 g control, respectively. However, the excess hypergravity conversely decreased osteocalcin expression. Osteocalcin gene expression was enhanced by VD/VD-R through the vitamin D-responsive element in the promoter. The increased osteocalcin expression might reflect the augmented VD-R expression. Alternatively, Runx2, a master gene of osteoblast differentiation, might be responsible for the osteocalcin induction, since the Runx2 mRNA levels were also increased to 247% of control at 12 g. Another VD-inducible osteoblast phenotype, alkaline phosphatase, was also upregulated at 12 g and 24 g. The appropriate level of hypergravity enhanced the VD-inducible expression of osteocalcin, a typical phenotype of osteoblast differentiation. These data suggest molecular features to prevent disuse bone atrophy of long-term bed-rest patients.
Hypergravity
RUNX2
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