龟板对大鼠骨髓间充质干细胞向成骨分化的影响

目的探讨益肾中药龟板对大鼠骨髓间充质干细胞(mesenchymal stem cells,MSCs)体外向成骨分化的影响.方法使用密度梯度法分离成年大鼠骨髓间充质干细胞,观察在培养液中添加不同浓度龟板血清条件下MSCs的成骨变化.应用形态学、碱性磷酸酶组织化学染色、Von Kossa 染色、骨钙素测定等方法观察细胞成骨活性.结果形态学表明,MSCs贴壁细胞呈集落生长,有成纤维细胞外观.龟板组碱性磷酸酶、钙化结节、骨钙素明显升高,与对照组比较具有显著性差异(P<0.05).结论 MSCs受龟板诱导高效地向成骨细胞分化,可以为体内骨组织工程提供种子细胞.     

糖皮质激素对肾病综合征患儿成骨细胞功能的影响

目的　糖皮质激素是治疗肾病综合征的首选药物。但糖皮质激素可抑制成骨细胞功能,导致骨质疏松。该研究通过检测成骨细胞不同分化阶段的生化指标:I型前胶原羧基端前肽(PICP)、骨钙素(BGP)和总碱性磷酸酶(AKP),探讨糖皮质激素对肾病综合征(NS)患儿成骨细胞功能的影响。方法　测定正常对照组(n=30),未治NS患儿(n=30)和激素治疗后NS患儿(每日泼尼松2mg/kg治疗4 ~8周,n=30)血清PICP、BGP及AKP水平。结果　未治NS患儿血清PICP165 ±56μg/L,BGP15 ±9ng/L水平明显低于正常对照组205 ±81μg/L, 19 ±12ng/L(均P<0. 05),而血清总AKP198 ±71U/L与正常对照组202 ±46U/L比较差异无显著性。激素治疗后NS患儿血清PICP85 ±56μg/L、BGP8±5ng/L、AKP104 ±59 U/L均明显低于未治NS患儿(P<0. 01)。结论　NS患儿本身存在骨合成障碍,大剂量糖皮质激素治疗可进一步抑制NS患儿的成骨细胞合成功能。     

葛根对大鼠成骨细胞增殖分化影响的血清药理学实验

目的：观察葛根含药血清对新生大鼠颅骨成骨细胞增殖分化的影响。方法：将32只SD大鼠随机分为实验组与对照组，在同等条件下制备葛根处理大鼠含药血清和对照血清，观察不同采血时间及血清添加量对成骨细胞增殖和碱性磷酸酶（ALP）的影响。结果：葛根末次灌胃后1～2小时采血的含药血清对成骨细胞增殖和ALP的作用明显（P〈0．01），随着含药血清添加量的增加，对成骨细胞增殖和ALP的作用明显增强（P〈0．01）。结论：含葛根处理大鼠血清可促进成骨细胞增殖和分化。     

Vitamin K2 promotes 1α,25(OH)2 vitamin D3-induced mineralization in human periosteal osteoblasts

The effect of vitamin K on mineralization by human periosteal osteoblasts was investigated in the absence and presence of 1,25 dihydroxyvitamin D₃ (1,25(OH)₂D₃). Vitamin K₁ and K₂, but not vitamin K₃, at 2.5 M enhanced in vitro mineralization when cells were cultured with vitamin K for 20 days after reaching confluence in vitro. Vitamin K₂ (2-methyl-3-all-trans-tetraphenyl-1,4-naphthoquinone: menatetrenone) was the most potent of these vitamin K analogs; it slightly inhibited alkaline phosphatase (ALP) activity. Human osteoblasts were mineralized and showed the enhanced ALP activity on treatment with 10^-9 M of 1,25(OH)₂D₃ for 20 or 25 days after confluence. Vitamin K₂ promoted the 1,25(OH)₂D₃-induced mineralization, but slightly inhibited the 1,25(OH)₂D₃-induced ALP activity. Moreover, vitamin K₂ enhanced the 1,25(OH)₂D₃-induced osteocalcin accumulation in the cells and the extracellular matrix (cell layer), but inhibited the osteocalcin content in the medium produced by the 1,25(OH)₂D₃ treatment. However, vitamin K₂ alone did not induce osteocalcin production in the human osteoblasts. On Northern blot analysis, osteocalcin mRNA expression on 1,25(OH)₂D₃-treated cells was enhanced by vitamin K₂ treatment, but vitamin K₂ alone did not induce osteocalcin mRNA expression. Warfarin blocked both the 1,25(OH)₂D₃-induced osteocalcin production and the accumulation in the cell layer, and also blocked the 1,25(OH)₂D₃ plus vitamin K₂-induced osteocalcin production and the accumulation in the cell layer. The 1,25(OH)₂D₃-induced mineralization promoted by vitamin K₂ was probably due to the enhanced accumulation of osteocalcin induced by vitamin K₂ in the cell layer. However, we concluded that the mineralization induced by vitamin K₂ alone was due to the accumulation of osteocalcin in bovine serum on the cell layer, since osteocalcin extracted from the cell layer was not identified by specific antiserum against human osteocalcin, which does not cross-react with bovine osteocalcin. These results suggest that the mechanism underlying the mineralization induced by vitamin K₂ in the presence of 1,25(OH)₂D₃ was different from that of vitamin K₂ alone, and that osteocalcin plays an important role in mineralization by osteoblasts in vitro.     

Dexamethasone enhances the effects of parathyroid hormone on human periodontal ligament cells in vitro

Periodontal ligament cells (PDL) are thought to play a major role in promoting periodontal regeneration. Recent studies, focused on characterizing PDL cells, have been directed at establishing their osteoblast-like properties and determining biological mediators and/or factors that induce osteoblastic cell populations in the PDL. The glucocorticoid, dexamethasone (Dex), has been shown to selectively stimulate osteoprogenitor cell proliferation and to induce osteoblastic cell differentiation in many cell systems. In the present study the ability of Dex to modulate parathyroid hormone (PTH)-stimulated cAMP synthesis in cultured human PDL cells was examined. PDL cells, obtained from premolar teeth extracted for orthodontic reasons, were cultured with Dex (0–1000 nM) for 7 days prior to PTH (1–34) stimulation. The exposure of PDL cells to Dex resulted in a dose-dependent increase in cAMP production in response to PTH stimulation. This response was seen in cells obtained from three different patients. The first significant Dex effect was seen on day 7 when compared to day 1 for 100 nM Dex. PTH (1–34) stimulation caused a dose-dependent increase in cAMP synthesis after Dex (1000 nM) treatment for 7 days. Conversely, stimulation of the cells with PTH (7–34) (0–1000 nM) did not increase cAMP production in PDL cells after Dex treatment. Forskolin- (1 M) and isoproterenol- (1 M) stimulated cAMP synthesis was not augmented by Dex treatment. Dex treatment did not alter calcitonin-(1 M) stimulated cAMP production in PDL cells. Glucocorticoid enhancement of PTH-stimulated cAMP synthesis in these cells supports the presence of an osteoblast-like population in the PDL, in vitro.     

Calcium currents in osteoblastic cells: Dependence upon cellular growth stage

Patch clamp physiological techniques were used to characterize the voltage-activated calcium currents (VACC) expressed in the plasma membrane of osteoblastic cells as a function of time in culture and proliferative state of the cell. Osteoblast-enriched preparations were isolated by collagenase digestions of newborn rat calvaria and cultured under different conditions which affected cell proliferation (i.e., low serum in the media to arrest proliferation). VACC were isolated by replacing the intracellular potassium with cesium, and adding 1 M tetrodotoxin to the bath. Under conditions that favored cell proliferation, low cell density, and media supplemented with 10% fetal calf serum (FCS), a transient calcium current was not expressed until day 3 in culture. There was a statistically significant relationship between the precentage of cells expressing this current and the time in culture. The magnitude of the current significantly increased as days in culture increased. Under the same conditions, the sustained VACC was detected after 7 or 8 days in culture. However, arresting cell proliferation after 2 days in culture by reducing the FCS concentration to 0.01% induced the expression of the sustained VACC the next day. The data suggest that the expression of VACC in the plasma membrane of rat calvarial osteoblasts depends on the time in culture and the state of proliferation of the cells. These results should prove to be valuable in studying the functional significance of VACC in osteoblastic cells and their regulation by various bone regulatory agents.     

Serum leptin levels,bone mineral density and osteoblast alkaline phosphatase activity in elderly men and women

Although primarily secreted by adipose cells, leptin, a polypeptide hormone that influences body weight, satiety and lipid metabolism, and its receptor are also expressed in human osteoblasts. Leptin plays a role in the central, hypothalamic modulation of bone formation, as well as locally within the skeleton by enhancing differentiation of bone marrow stroma into osteoblasts and inhibiting its differentiation into osteoclasts and adipocytes. The purpose of this investigation was to compare serum leptin values in 100 postmenopausal women (age 62-97) and 31 men (age 72-92) to bone mineral density (BMD) measurements made by dual X-ray absorptiometry and additionally to biochemical markers of bone resorption and formation, including crosslinked collagen N-telopeptides (NTx), aminoterminal extension procollagen propeptides (PINP) and bone-specific alkaline phosphatase (bAP). The circulating level of leptin directly correlated with body mass index (BMI) (r=0.61-0.78, P<0.001) and was modestly, but significantly and positively associated with bAP activity (r=0.24-0.33, P<0.01) in the sera of men and women after adjustment for BMD, age and BMI. The association of circulating leptin levels with bAP, a specific marker of osteoblast activity suggests that leptin levels influence osteoblast activity in vivo in elderly women and men.     

极低频电磁场对成骨细胞增殖与分化的影响

目的 研究极低频电磁场（ELFEMF）的生物学效应，探讨ELFEMF特征参数对新生大鼠成骨细胞的影响。方法 采用不同强度、频率和占空比的ELFEMF作用于大鼠颅骨成骨细胞，检测成骨细胞的增殖与分化指标。结果 ELFEMF生物效应存在强度、频率、占夺比窗口，频率为15Hz、磁感应强度为5mT、占空比为15％的ELFEMF可以显著提高成骨细胞的增殖率、降低其分化能力。结论 ELFEMF对成骨细胞的影响依赖于电磁场的特征参数，对成骨细胞的增殖与分化而言，磁感应强度5mT、频率15Hz、占空比15％可能为一组合适的参数。     

Effect of therapeutic levels of doxycycline and minocycline in the proliferation and differentiation of human bone marrow osteoblastic cells

Semi-synthetic tetracyclines (TCs) have been reported to reduce pathological bone resorption through several mechanisms, although their effect over bone physiological metabolism is not yet fully understood. The present study aims at evaluate the behaviour of osteoblastic-induced human bone marrow cells regarding proliferation and functional activity, in the presence of representative therapeutic concentrations of doxycycline and minocycline. First passage human osteoblastic bone marrow cells were cultured for 35 days in conditions known to favor osteoblastic differentiation. Doxycycline (1-25 micro g/ml) or minocycline (1-50 micro g/ml) were added continuously, with the culture medium, twice a week with every medium change. Cultures were characterised at several time points for cell proliferation and function. Present data showed that 1 micro g/ml of both tetracyclines, level representative of that attained in plasma and crevicular fluid with the standard therapeutic dosage, increased significantly the proliferation of human bone marrow osteoblastic cells without altering their specific phenotype and functional activity. Long-term exposure to these TCs induced a significant increase in the number of active osteoblastic cells that yielded a proportional amount of a normal mineralised matrix, suggesting a potential application in therapeutic approaches aiming to increase bone formation. The presence of higher levels of these agents led to a dose-dependent deleterious effect over cell culture, delaying cell proliferation and differentiation.     

Comprehensive study on the roles of released ions from biodegradable Mg–5 wt% Ca–1 wt% Zn alloy in bone regeneration

We report here the effect of micro‐environmental changes from biodegradable magnesium alloys on the activities of cells – osteoblasts, osteoclasts and macrophages – which play critical roles in each phase of the bone‐regeneration process. Despite positive bone formation effects from several in vivo studies, minimal progress has been made in identifying underlying mechanisms through in vitro studies, which are currently concentrated on osteoblastic activities. The observed in vitro and in vivo results indicated that alkaline pH and released magnesium and zinc ions derived from Mg–5 wt% Ca–1 wt% Zn alloy biodegradation promote the progress of bone formation. In contrast, alkaline pH and magnesium ions remarkably suppressed osteoclastic activities and pro‐inflammatory cytokine production, closely related to osteolysis and prosthesis failure. Findings from the present study conclude that the degradation of Mg–5 wt% Ca–1 wt% Zn alloys can promote new bone formation by simultaneously affecting the complex combination of variable cellular activities and phases. Copyright © 2016 John Wiley & Sons, Ltd.