A device for imposing cyclic strain to cells growing on implant alloys

The objective of this study was to develop a unique device for applying well-characterized cyclic, strain to cells growing on implant alloys. The device is based on the four-point bending principle and utilizes an electric motor, belt, and cam system to cyclically deflect a commercially pure titanium plate with cell culture wells in the middle of the plate. Analyses demonstrated that 182±3, 366±9, and 984±7 microstrain may be cyclically generated in culture areas from 0.5 to 10 Hz for up to 72 h. UMR-106 osteoblast-like cells growing on the titanium plate were subjected to these strain magnitudes at 1.5 Hz for periods of 4 or 24 h. Cells were checked for viability, total protein as a general indicator of cell number, and alkaline phosphatase activity (ALP) as an indicator of bone cell function. Cells strained at 984±7 microstrain exhibited 21%–24% more protein but 45%–49% less ALP activity than cells strained at 182±2 or 366±9 microstrain. Decreased ALP activity may indicate impaired mineralization. Results indicate the device is suitable for applying known, cyclic strain to cells growing on implant alloys and evaluating cellular responses to strain while growing on implant alloys. © 2002 Biomedical Engineering Society. PAC2002: 8780Rb, 8768+z, 8717Ee     

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.     

The role of TGF-β1/Smad3 signaling pathway and oxidative stress in the inhibition of osteoblast mineralization by copper chloride

To explore the relationship of oxidative stress and TGF-β 1/Smad3 pathway in the inhibition of osteoblast mineralization by copper chloride (CuCl₂), the osteoblasts were treated with CuCl₂ (0, 50 μM, 100 μM, 150 μM CuCl₂ 5H₂O) for 24 h. We found that Cu impaired the osteoblast structure, inhibited the glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) activities, alkaline phosphatase (ALP) content, mRNA expression of collagen I (COL-I), osteocalcin (OCN), insulin-like growth factor I (IGF-I), bone morphogenetic protein-2 (BMP-2), transforming growth factor β1 (TGF-β1) and core-binding factor α1 (Cbfα1), promoted the reactive oxygen species (ROS) production, inactivated the TGF-β1/Smad3 pathway. It indicates that the inactivated TGF-β1/Smad3 pathway leads to osteoblast impairment by CuCl₂. It will contribute to clarify the influence of CuCl₂ on the osteoblast mineralization.     

Differentiation of nerve-derived adult pluripotent stem cells into osteoblastic and endothelial cells

Background Context

Stem cell-involved tissue engineering has gained dramatic attention as a therapeutic strategy for tissue regeneration including bone repair. However, the currently available possibilities to use embryonic stem cells and induced pluripotent stem cells (iPCs) face potential ethical issues, as well as risks of malignant transformation and immune rejection. Recently identified peripheral nerve-derived adult pluripotent stem cells (NEDAPS) that quickly proliferate after exposure to bone morphogenetic protein-2 (BMP-2) or nerve trauma and exhibit many embryonic stem cell characteristics may provide an attractive source cells for a variety of regenerative therapies.

聚醚醚酮/碳纳米管/生物活性玻璃复合材料的成骨细胞相容性研究

目的:探讨新型聚醚醚酮/碳纳米管/生物活性玻璃(PEEK/CNTs/BGs)复合材料的体外成骨细胞相容性。方法:体外培养小鼠成骨细胞MC3T3-E1,通过噻唑蓝法、吖啶橙荧光染色法研究PEEK/CNTs/BGs复合材料对成骨细胞增殖的影响。应用扫描电镜(SEM)观察MC3T3-E1细胞在PEEK/CNTs/BGs复合材料表面的黏附生长情况。结果:MC3T3-E1细胞在PEEK/CNTs/BGs复合材料表面的增殖能力较聚醚醚酮和聚醚醚酮/碳纳米管(PEEK/CNTs)复合材料强。SEM实验结果表明,PEEK/CNTs/BGs复合材料表面有利于MC3T3-E1细胞黏附、铺展和生长。结论:PEEK/CNTs/BGs复合材料具有良好的成骨细胞相容性,有望作为新型骨科材料。     

Establishment and Characterization of a Simian Virus 40-Immortalized Osteoblastic Cell Line from Normal Human Bone

We established a human osteoblastic cell line immortalized by simian virus 40 (SV40) in vitro , and designated it SV-HFO. Immunocytochemically, the cells were positive for SV40 large T-antigen, vimentin and osteocalcin, but negative for keratin and epithelial membrane antigen. The cells had characteristic morphologic and ultrastructural features of osteoblasts, produced alkaline phosphatase, and synthesized osteocalcin, the levels of which were elevated by treatment of the cells with 1α,25-dihydroxyvitamin D₃. The cells proliferated and showed such osteoblastic properties even under serum-free conditions. The cells grew in soft agar, but did not form tumors when transplanted into athymic nude mice. Karyotypic analysis by the Q-banding technique showed that these cells were of human origin. The SV-HFO cell line is expected to serve as a suitable model for studying metabolism and carcinogenesis in human bone.     

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.     

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.