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PubMed2005

[Effects of OGP(10-14) and its derivative G38I on proliferation and differentiation of rat osteoblasts in vitro].

OBJECTIVE: To investigate the effects of the carboxyl end of osteogenic growth peptide (OGP)-OGP((10-14)) and its derivative G38I on the proliferation and differentiation of osteoblasts (OBs). METHODS: Osteoblasts were isolated from the calvariae of newborn SD rats and cultured to G3. OGP((10-14)) or G38I of the concentrations of 10(-15) to 10(-7) mol/L were added to culture medium for 48 hours respectively. The number of cells was counted and MTT analysis was used to examine the proliferation of the cells. The ultrastructure of cells was investigated by electron microscopy. The osteoblasts of G3 were divided into experimental groups, treated with OGP((10-14)) or G38I of the concentration of 10(-11) mol/L for 48 hours, and control group. The alkaline phosphatase activity in the culture medium was measured. The protein expression level of type-I collagen was evaluated by immunohistochemistry. The core binding factor 1 (Cbfa1) and type-I collagen mRNA level of osteoblasts were determined by RT-PCR. RESULTS: With a biphasic effect on, OGP((10-14)) and G38I stimulated the number enhancement of OBs dose-dependently at low concentration and inhibited it at high concentration. The numbers of OB were the highest (37 x 10(4)/ml +/- 7 x 10(4)/ml and 30 x 10(4)/ml +/- 5 x 10(4)/ml respectively) when treated by OGP((10-14)) or G38I of the concentration of 10(-11)mol/L. The rough endoplasm net was flourishing and the secreting vesicle was abounding in the experimental cells. There was calcium crystal in the control cells. The activity of alkaline phosphatase in the culture medium of the OGP (10(-14)) and G38I groups were higher than that in the control group (4.47 U/g and 3.82 U/g vs 2.21 U/g). The protein expression level of type-I collagen was higher and the mRNA levels of Cbfa1 and type-I collagen were higher in the OGP((10-14)) and G38I groups were increased in the experimental groups in comparison with the control group (P < 0.05, P < 0.01, and P < 0.05). CONCLUSION: They stimulated cell number enhancement dose dependently at low concentration and followed by inhibition at high concentration. Just as the native OGP, OGP((10-14)) and its derivative G38I stimulate the proliferation of osteoblasts, and improve their activity, up-regulate the Cbfa1 and type-I collagen mRNA expression levels and increase the collagen synthesis, thus promoting the differentiation and osteogenic effect of osteoblasts.

Alkaline Phosphatase↗

PubMed2005

[Effects of bushen huoxue recipe on proliferation, differentiation and mineralization of osteoblasts in rats].

OBJECTIVE: To observe the effects of bushen huoxue recipe (BHR) on cell proliferation, differentiation, maturation and mineralization in osteoblasts of rats cultured in vitro. METHODS: Osteoblasts from cranium of newborn SD rats were cultured by collagenase method. MTT, PNPP and ARS were used to observe the proliferation, activity of alkaline phosphatase (ALP) and the formation of mineral node of cultured osteoblasts effected by different concentrations of BHR at different time points (24, 48, 72 hrs after medication). RESULTS: Different concentrations of BHR could enhance the cell proliferation rate and ALP activity, the effects of BHR in high and moderate concentration were more significant in enhancing the cell proliferation rate (P < 0.01). And the number of mineral node of cultured osteoblasts treated by high and moderate concentration BHR was much more than that of the untreated osteoblasts (P < 0.01). CONCLUSION: BHR could promote the proliferation, differentiation, and mineralization of the osteoblasts.

Alkaline Phosphatase↗

PubMed2006

[Experimental study on the proliferation and function of osteoblast cell induced by pBLAST49-mVEGF gene transfection].

OBJECTIVE: To assess the effects of extra VEGF gene expression on the proliferation of osteoblast cell. METHODS: The extra VEGF gene was induced into osteoblast cell. Then the expression of extra VEGF gene was detected, and its effects on the cell proliferation curve, cell cycle, on the synthesis of type I collagen and on the secretion of osteocalcium of osteoblast cell were investigated. RESULTS: At the first two days of culture, the cell number of pBLAST49-mVEGF gene transfer group was larger than that of the control group, yet no statistically significant difference was seen between the two groups (P > 0.05); however, significant difference was observed from the 3rd day of culture (P < 0.05). The (G2/M+S)% of the 1-5 generation osteoblast cell in pBLAST49-mVEGF gene transfer group was higher than that of the control group, there was significant difference between them (P < 0.05). The concentration of osteocalcium and expression of type I collagen of the 1-5 generation osteoblast cell in pBLAST49-mVEGF gene transfer group were significantly higher than those of the control group (P < 0.05). CONCLUSION: pBLAST49-mVEGF gene transfer can improve the proliferation and function of osteoblast cell.

Animals↗

PubMed2006

[Influence of high molecular weight polyethylene on viability of osteoblasts and new bone formation].

To investigate the influence of high molecular weight polyethylene (HMWP) on the viability of osteoblasts and new bone formation in the process of fracture healing, the osteoblasts derived from adult human bone marrow were cultured in HMWP maceration extract and normal culture medium. The viability of the osteoblasts was measured by MTT assay, and the function of the osteoblasts was detected by use of alkaline phosphatase test kit. The locked double-plating (steel plate and HMWP plate) was implanted and fixed at the artificial fracture of distal femur of dogs. Specimens were gained at 3, 6, 9 and 12 weeks postoperatively, examined with macroscopy, microscope and scanning electron microscope (SEM). The results showed that HMWP did no harm to osteoblasts. There is no significant difference in activities of proliferation and alkaline phosphatase between HMWP maceration extract and normal culture medium at each observation time of at 2,4,8, and 14 dyas (P>0. 05). Bone tissue under the implanted HMWP plate manifested no absorption; the new bones formed under the HMWP plate and gradually matured as time went on. It is demonstrated in this study that HMWP has no adverse influence on the viability of osteoblasts and new bone formation and it can be used as internal fixation implant in treating fractures.

Animals↗

PubMed2006

[Expressions of Cbfal and osterix in osteoblasts on human acellular amniotic membrane].

OBJECTIVE: To study the differentiation of the human osteoblasts during the construction of the tissue engineered periosteum with the human acellular amniotic membrane (HAAM). METHODS: To construct the tissue engineered periosteum (n=60) with HAAM, the human fetal osteoblasts were used. The fetal osteoblasts were cultured for 2, 4, 6, 8, and 10 days, and then their total RNA was extracted, which were reversely transcripted to cDNA. The real-time PCR analysis was used to reveal Cbfal and Osterix, and the cycle threshold (Ct) was also measured. The simply-cultured osteoblasts were used as the control group (n=20). RESULTS: The expression of Cbfal was higher in the experimental group on the 2nd day when compared with that on the 4th, 6th, and 8th day (P < 0.05). The same result existed on the 10th day when compared with that on the 4th and 8th day. The expression of Osterix increased and was highest on the 8th day when compared with the other results (P < 0.05). Both of the 2 gene expressions were decreased in the control group when compared with those in the experimental group, but with no significant difference (P > 0.05). CONCLUSION: Cbfal and Osterix can be normally expressed by the osteoblasts after their integration with HAAM. As a scaffold, HAAM can be used to keep the osteoblast phenotype and differentiation with an osteoconductive ability. Such a cell-scaffold complex may provide a basis for the osteogenesis.

Amnion↗

PubMed2006

[Bone metabolic abnormality in diabetes: especially about osteoblast dysfunction].

As for the factor in the diabetes in which it takes part in a bone abnormal metabolism, this is given the insulin lack state, a high blood glucose state, and the complications accompanying diabetes, such as arteriosclerosis, neuropathy, and a renal dysfunction. The absolute, and/or relative insulin lack state deteriorate the function and reduce the number of osteoblast, in addition, the osteoblast dysfunction is also caused by the sorbitol accumulation brought in the osteoblast as the result of the continuation of high blood glucose state. It also prompts the osteoclastic bone absorption, and suppresses the expression of transcription factor gene that regulates the osteoblast differentiation, and resultantly, the apoptosis of the osteoblast is promoted. By these mechanisms intertwining, the differentiation and the maturity of the osteoblast is thought to be decrease and the bone formation process is obstructed.

Animals↗

PubMed1990

IL-6 is produced by osteoblasts and induces bone resorption.

To examine the possible involvement of IL-6 in bone metabolism, a mouse osteoblastic cell line (MC3T3-E1) and primary osteoblast-like cells from fetal mouse calvaria were cultured with several systemic and local bone-resorbing agents and their expression of IL-6 mRNA was determined. Local bone-resorbing agents such as IL-1 alpha, IL-1 beta, TNF-alpha, and LPS greatly induced IL-6 mRNA expression in both MC3T3-E1 cells and primary osteoblast-like cells. Parathyroid hormone slightly increased expression of IL-6 mRNA in primary osteoblast-like cells but not in MC3T3-E1 cells. Neither IL-6 nor 1 alpha,25-dihydroxyvitamin D3 increased expression of IL-6 mRNA in either of the osteoblast-like cells. In agreement with the expression of IL-6 mRNA, biologically active IL-6 was produced in response to the treatment with IL-1 alpha, TNF-alpha, and LPS in MC3T3-E1 cells. Adding IL-6 dose dependently stimulated the release of 45Ca from prelabeled fetal mouse calvaria. Simultaneously adding suboptimal concentrations of IL-6 and IL-1 alpha induced bone resorption cooperatively. In accord with the increase in the release of 45Ca by IL-6, there were three times as many osteoclasts in the bone sections of calvaria cultured with IL-6 for 5 days as in the controls. IL-6 slightly suppressed alkaline phosphatase activity and collagen synthesis in MC3T3-E1 cells. These results indicate that IL-6 is also produced by osteoblasts, preferentially in response to local bone-resorbing agents, and it induces bone resorption both alone and in concert with other bone-resorbing agents.

Alkaline Phosphatase↗

PubMed1978

Effects of colchicine on osteoblast in rat: an ultrastructural study.

The present study was an attempt to investigate the effects of colchicine on the ultrastructure of rat osteoblasts with special reference to the microtubular function in vivo. Rats were killed at intervals of 2, 4, 8, 12 and 24 hours after the colchicine injection (0.1 mg per 100 g of body weight, subcutaneously). At 2 and 4 hours after the injection, the secretory granules and small vesicles accumulated in the Golgi area of the osteoblasts. The dilated spherical cisterna of the rough endoplasmic reticulum and the large vacuoles appeared, being located in the periphery of the cell. Microtubules were rarely found in the cytoplasm. Small masses of microfilaments of 80 to 110 A in diameter were found also in the cytoplasm. According to the special staining for collagen, it was indicated that the contents of the secretory granules might be collagen-like materials. At 8 hours after the injection, the shape of the osteoblasts transformed to a round profile and the autophagic vacuoles increased in the cytoplasm. At 12 and 24 hours after the injection, the osteoblasts seemed to be destroyed by the breakdown of the plasma membrane and by the increase of the autophagic vacuoles. It is suggested that colchicine affects the secretory process of the bone matrix and the cytoskeletal system of the osteoblasts by interfering with the structure and the function of the microtubules and colchicine also interferes with the function of the plasma membrane followed by the destruction of the osteoblasts.

Animals↗

PubMed1988

[The effects of dexamethasone and expansion stress on the osteoblasts in mouse calvarial suture in vitro, with special reference to incorporation of 3H-thymidine and 3H-proline].

Behavior of sutures in response to expansion stress are thought to be in parallel with the response of bone to orthodontic tooth movement. To investigate this relation between psychologic stress and osteoblasts during orthodontic tooth movement, the effects of expansion stress and 10(-7) M dexamethasone on sutural osteoblasts of mouse calvaria were examined in vitro. The model consists of a tensile force applied to the mouse sagittal suture. Expansion stress induced the formation of osteoid in the sutural areas, and incorporation of 3H-thymidine and 3H-proline in sutural osteoblasts were found to be stimulated by expansion stress. Furthermore, labelling index of 3H-thymidine in the osteoblasts was also found to be stimulated. On the other hand, dexamethasone inhibited the incorporation, labelling index and matrix formation. Moreover, these induced stimulations in expansion stress were inhibited by 10(-7) M dexamethasone treatment. As described above, dexamethasone, which is a synthetic glucocorticoid, inhibited the incorporation of 3H-thymidine and 3H-proline, the labelling index of 3H-thymidine, and the matrix formation in sutural osteoblasts that were stimulated by expansion stress. Therefore, also during orthodontic tooth movement, increased secretion of glucocorticoid that is stimulated by psychologic stress may inhibit the proliferation, cell differentiation, and matrix formation of osteoblasts in periodontal ligaments.

Animals↗

PubMed1988

Direct functional assessment of human osteoblasts by radioautography: methodology and application in end stage renal disease.

Fragments approximately 1 mm3 of freshly obtained iliac crest cancellous bone were incubated for 30 minutes at 37 degrees C in Krebs buffer containing 3H-leucine or 3H-proline followed by radioautography. Grains per osteoblast, reflective of protein synthetic activity, were counted in normal controls and patients with end stage renal disease with or without significant aluminum storage in their bones. Compared with control the end stage renal disease patients without significant aluminum storage had more large osteoblasts with increased grain counts which were ascribed to secondary hyperparathyroidism. Cells from patients with greater than 30% surface aluminum had more small osteoblasts with grain counts intermediate between control and less than 2% aluminum, ascribed to an inhibitory effect of aluminum on the secondary hyperparathyroidism. Cell activity did not depend on location next to a tetracycline label or osteoid and it was suggested that all bone lining cells could be considered as osteoblasts. Aluminum storage was associated with impaired mineralisation in the presence of normal or increased osteoblastic protein synthetic activity suggesting that in these patients the mineralisation defect was not primarily due to an abnormality in osteoblast function.

Aluminum↗

PubMed1987

Tumor necrosis factors alpha and beta induce osteoblastic cells to stimulate osteoclastic bone resorption.

Antigen- or mitogen-stimulated leukocytes release bone-resorbing activity into culture supernatants in vitro. Among the agents likely to be present in such supernatants are monocyte-derived tumor necrosis factor (TNF-alpha) and lymphocyte-derived tumor necrosis factor (TNF-beta) (lymphotoxin), both of which have recently been shown to stimulate bone resorption in organ culture. To identify the mechanism of action of these agents, we compared bone resorption by isolated osteoclasts with bone resorption by osteoclasts cocultured with osteoblastic cells, and with bone resorption by osteoclasts incubated with supernatants from osteoblastic cells, in the presence and absence of recombinant TNF-alpha and TNF-beta. We found that neither TNF-alpha nor TNF-beta had any significant effect on bone resorption by isolated osteoclasts, but in the presence of osteoblasts the agents caused a twofold to threefold stimulation of bone resorption. A similar degree of stimulation was achieved by supernatants from osteoblasts incubated with TNF before addition to osteoclasts, compared with supernatants to which TNF were added after osteoblast incubation. These experiments suggest that TNF-alpha and TNF-beta stimulate bone resorption through a primary effect on osteoblastic cells, which are induced by TNF to produce a factor that stimulates osteoclastic resorption. Half-maximal stimulation of resorption occurred at 1.5 X 10(-10) M and 2.5 X 10(-10) M for TNF-alpha and TNF-beta, respectively. This degree of potency is comparable to that of parathyroid hormone, the major physiologic systemic regulator of bone resorption, and suggests that the TNF may exert a significant influence on osteoclastic bone resorption in vivo.

Animals↗

PubMed1995

Induction of osteoclast characteristics in cultured avian blood monocytes; modulation by osteoblasts and 1,25-(OH)2 vitamin D3.

It has been established, that the osteoclast is derived from the haemopoietic stem cell, but its exact lineage is still controversial. It is sometimes suggested, that osteoclasts and monocytes/macrophages are related cells. It has also been suggested that osteoclast differentiation is regulated by osteoblasts and 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). In the present paper we addressed the question whether avian monocytes can differentiate into osteoclasts in vitro, using an array of immunocytochemical, enzyme cytochemical and function markers. We have also determined the effects of osteoblasts, osteoblast conditioned medium and 1,25-(OH)2D3 on the expression of osteoclastic features on monocytes during culture. Monocytes developed tartrate resistant acid phosphatase (TRAcP) enzyme activity and antigens for all anti-osteoclast antibodies tested, during culture. However, they did not acquire the ability to resorb dentine and still showed phagocytosis of latex spheres. This indicates that the monocytes developed into cells resembling osteoclasts but lacking their function while retaining the function of macrophages. Osteoblast conditioned medium stimulated TRAcP enzyme activity and proliferation of monocytes in cultures. Addition of osteoblasts or osteoblast conditioned medium to monocyte cultures on dentine in the presence or absence of 1,25-(OH)2D3 did not result in the generation of genuine osteoclasts, nor in pit formation. 1,25-(OH)2D3 appeared to be cytotoxic to the avian monocytes in concentrations considered optimal for mouse osteoclast formation. These results suggest that avian monocytes do not readily differentiate into osteoclasts under in vitro conditions that stimulate osteoclast differentiation from bone marrow derived haemopoietic cells. Furthermore, labelling with anti-osteoclast antibodies and TRAcP as osteoclast-markers should be used only with great caution in the identification of osteoclasts formed in vitro.

Animals↗

PubMed1995

Chemotaxis of human osteoblasts. Effects of osteotropic growth factors.

The in vitro chemotactic response of human osteoblasts was investigated towards the following growth factors: TGF-beta, PDGFs, FGFs and IGFs. Human osteoblasts grown from trabecular bone after enzymatic digestion were studied. TGF-beta stimulated the migration of human osteoblasts in a dose-dependent manner with a four-fold increase in migrated cells at 100 pg/ml, which was the optimum concentration. PDGF-BB also stimulated migration four-fold in a dose-dependent manner with a maximum response at 10 ng/ml. PDGF-AA, IGF-I and IGF-II stimulated migration two-fold at 100 ng/ml. The results show that TGF-beta and PDGF-BB are important regulators of human osteoblast migration, but other growth factors IGF-I, IGF-II and PDGF-AA may also stimulate osteoblast migration. Our results additionally suggest that TGF-beta and PDGF-BB may participate in the recruitment of osteoblasts during bone remodeling since both TGF-beta and PDGF-BB are found in bone matrix and could be released during osteoclastic bone resorption. They furthermore support a possible use of TGF-beta and PDGF-BB in growth factor-induced osteogenesis.

Cells, Cultured↗

PubMed1994

[Inhibitory effects of cytokines on human osteoblastic cells].

We examined the effect of tumor necrosis factor alpha (TNF-alpha), interleukin 1 beta (IL-1 beta) and IL-6 on alkaline phosphatase (ALP) and osteocalcin (OC) production, calcification and calcium (Ca) release in human cultured osteoblastic cells established from human periosteum. The cells were cultured with varying concentrations of cytokines for three days. TNF-alpha and IL-1 beta significantly inhibited ALP production, decreased cellular Ca content, and significantly enhanced 45Ca release in human osteoblastic cells. IL-6, on the other hand, significantly suppressed 45Ca release from the osteoblastic cells. Any one of these cytokines did not influence the production of OC by the osteoblastic cells. The results obtained suggest that TNF-alpha and IL-1 beta may inhibit bone formation and calcification and promote bone resorption, while the effects of IL-6 on osteoblastic cells may be a little different from those of TNF-alpha or IL-1 beta. Cytokine-dependent these effects on the osteoblastic cells may be one of the mechanisms by which bone loss occurs in patients with rheumatoid arthritis.

Alkaline Phosphatase↗

PubMed1994

Circadian and circatrigintan changes in osteoblastic activity assessed by serum osteocalcin. Physiological and methodological aspects.

The author has shown the existence of reproducible circadian changes and significant variation during the circatrigintan rhythm in the osteoblastic marker, serum osteocalcin. The author find that appropriate exploratory data analyses are important and often imperative tools in the demonstration of these low frequent rhythms, and the effects of intervention. The metabolism of osteocalcin needs further investigation since it is essential to consider the kinetic of serum osteocalcin in the interpretation of data. However, based on current knowledge of the kinetics of serum osteocalcin, and the variation in other osteoblastic markers, the author conclude that the results are indicative of the existence of biological rhythms in osteoblastic activity rather than changes in metabolic clearance. The physiological aspects on the circadian rhythm are that osteoblastic activity peaks during the early night and decrease in the morning in both men and women. The circadian osteoblastic activity is relatively stable during seasons and apparantly not acutely related to sleep. Modest doses of alcohol decrease serum osteocalcin within few hours but the circadian rhythm is not different in smokers compared with non-smokers. There are evidences for endogeneous cortisol as regulator of the osteocalcin rhythm especially by mediating the morning decrease in osteocalcin. Osteocalcin synthesis is not completely blocked by glucocorticoids since the effect is blunted by administration of 1,25-(OH)2D3. Although serum osteocalcin may respond to acute doses of several other osteotrophic hormones, it is doubtfull whether endogeneous PTH, 1,25-(OH)2D3, sex hormones, or growth hormone play any role as acute regulators of the circadian osteocalcin rhythm. The circatrigintan rhythm is characterized by highest levels of serum osteocalcin during the luteal phase. There is a significant cross correlation with the follicular increase in serum estradiol but the regulation of serum osteocalcin during the ovulatory cycle remains to be settled. The results, however, support the interaction between sex hormones and bone metabolism. The practical implications are that blood sampling for determination of serum osteocalcin should be strictly standardized with regard to the time of the day and the time since last medication or intervention with factors that may affect osteoblastic activity.

Circadian Rhythm↗

PubMed1996

Human amniotic tumor that induces new bone formation in vivo produces growth-regulatory activity in vitro for osteoblasts identified as an extended form of basic fibroblast growth factor.

Tumors occasionally stimulate bone formation and cause osteoblastic metastases. Although this occurs most frequently in widespread prostate cancer, human prostate cancer cells are difficult to grow in culture without changing their phenotype, and the few available prostatic cancer lines do not increase bone formation in vivo. To identify tumor-derived osteoblast-stimulatory factors, we studied a long-established human tumor cell line derived from human amnion that has, in the past, been reported to cause bone formation in vivo when inoculated into nude mice. Tumor cells were inoculated into nude mice and induced extensive new bone formation. To characterize osteoblast growth factors produced by these tumor cells, solid tumor was isolated from the mice and extracted at neutral pH. Biological activity, assessed by stimulation of proliferation of MG-63 osteoblast-like cells, was used to monitor purification after heparin-Sepharose column chromatography, Mono-S, and C4 reverse-phase high-performance liquid chromatography. An extend amino-terminal form of basic fibroblast growth factor (FGF) was purified by its capacity to stimulate proliferation in MG-63 cells and partially sequenced. Basic FGF is also known to stimulate proliferation in MG-63 cells and other osteoblasts in vitro and bone formation in vivo. In summary, these human tumor cells stimulate new bone formation in vivo and produce an osteoblast stimulating activity in vitro, which has been identified as a form of basic FGF.

Amino Acid Sequence↗

PubMedDate not supplied

Endochondral and intramembranous fetal bone development: osteoblastic cell proliferation, and expression of alkaline phosphatase, m-twist, and histone H4.

We have previously studied the expression of alkaline phosphatase (ALP) and alpha2(I) collagen (two phenotypic markers of osteoblastic cell differentiation) during development of the rat mandible, and the spatial and temporal distribution of the respective transcripts. Our current studies utilize the rat mandible and hind foot as in vivo model systems to investigate the relationship between osteoblastic differentiation and proliferation during intramembranous and endochondral bone formation. Pregnant rats, at 15.17, and 19 days of gestation were intraperitoneally injected with various doses of [3H]-thymidine, and sacrificed at various time intervals in order to label dividing embryonic osteoblastic and preosteoblastic cells. Cross sections through the mid-body of 15-day embryos showed [3H-thymidine dose-dependent labeling of a relatively high percentage of cells in the liver (49 +/- 8% at 440 muCi) and a lower percentage of cells of the developing vertebral cartilage (29 +/- 6% at 440 muCi). ALP-positive condensed mesenchyme--consisting of mandibular preosteoblast (15 days of gestation) showed a relatively high (32 +/- 5%) level of [3H]-thymidine labeling, compared to surrounding ALP-negative loose mesenchymal cells (22 +/- 1%). Similar results were observed in the developing hind foot of 19-day embryos for ALP-positive cells (15 +/- 6%) and surrounding ALP-negative cells (13 +/- 5%). In both the hind foot and the mandible an overall decrease in labeling was observed during bone development. RNA samples from these tissues show increasing amounts of ALP mRNA, and decreasing amounts of histone H4 mRNA between days 15 and 19 of gestation. These data indicate that a general inverse correlation between osteoblastic differentiation and proliferation, similar to the correlation previously described in cultured osteogenic cells, is also present in developing bones in vivo. However, these results indicate that ALP-positive preosteoblasts, committed to the osteoblastic lineage, maintain their proliferative capacity. In an attempt to elucidate underlying molecular mechanisms, we further investigated the levels of expression of m-twist in these tissues. This member of the basic helix-loop-helix family of transcription regulators has been previously implied as playing a role in osteoblast differentiation in culture. Our results demonstrate a decrease in m-twist levels during bone development in both the mandible and the hind foot.

Alkaline Phosphatase↗

PubMed1996

[A new osteoblast cell culture system for standardized testing of biomaterials].

For in vitro testing of new biomaterials cultured fibroblasts are employed. In the case of the agar diffusion test survival of cells is involved in the presence of the material to be tested. Further statements on the biological effects of a biomaterial require the use of cell cultures adapted to the tissue concerned and the underlying problem being investigated. In the present study, an osteoblast cell culture system with which implant surfaces in contact with bone can be tested as required by the relevant standards is described. Test bodies made of titanium, polystyrene or copper were used in the conventional agar diffusion test, and were also overgrown with fibroblasts or a cell line of foetal human osteoblasts. For the agar diffusion test, the test criterion was the extent of the inhibition area on staining with neutral red, while for the overgrowth, the mean cell diameter and the number of cells was employed. The phenotype of the osteoblast cell line was determined immunohistochemically by means of alkaline phosphatase or immunohistologically by means of collagen I and osteocalcin. Calcification was demonstrated using the v. Kossa stain. In the case of the osteoblasts, a differentiation of a collagen I and alkaline phosphatase-positive phenotype over an osteocalcin-positive phenotype to an increase in calcium deposition was shown. As in the case of the agar diffusion test, direct overgrowth also revealed no cytotoxic effect for titanium and polystyrene. In contrast, a cytotoxic effect consisting in a decrease in the number of cells and also a left shift in the size distribution was observed for copper. The standard deviations of the individual tests were less for overgrowth than for the agar diffusion test. The culture system for osteoblast cells thus meets the criteria of the EN/DIN 30993-5 in terms of the quality and accuracy of the results obtained. In addition to excluding direct cytotoxicity, this test system offers a new possibility of examining the influence of the material on cell growth. Consequently, it permits a repeatable examination of proliferation and differentiation of the osteoblasts on each material surface.

Animals↗