Cyclic Tensile Stretch Stimulates the Release of Reactive Oxygen Species from Osteoblast-like Cells

It is known that the excessive generation of reactive oxygen species (ROS) is a significant factor in tissue injury observed in many disease states. To determine whether extreme levels of mechanical stress applied to osteoblasts enhances ROS synthesis, we loaded cyclic tensile stretch on osteoblast-like HT-3 cells. Cyclic tensile stretch loaded on these cells clearly enhanced ROS synthesis in a time- and magnitude-dependent fashion. Cyclic tensile stretch also enhanced superoxide dismutase (SOD) activity. The disruption of microfilaments with cytochalasin D abolished the stress-induced ROS synthesis. Rotenone, an inhibitor of the mitochondrial electron transport chain, enhanced stress-induced ROS synthesis. These data suggest that actin filament and mitochondria are involved in this action.     

Mineralization and metabolic response in serially passaged adult rat bone cells

Summary Cell populations derived from adult rat bone were grown in cell culture and characterized with respect to their morphology and response to hormones. The cells were isolated from adult rat calvaria by mechanical rather than enzymatic methods. Cultures were initiated in modified BGJb medium supplemented with fetal bovine serum. These cultures and several cloned populations derived from them retained the ability to mineralize in vitro even after extended serial passage. Cultures derived from an osteoblast-enriched population showed an initial positive cAMP response to PTH and PGE₂, but not to TCT. The PTH and PGE₂ responses diminished with serial passage. The PTH response was no longer measurable at passage 6, and the PGE₂ response was not evident in passage 11. In one clone, the PGE₂ response persisted through passage 16. Adult rat skin fibroblasts cultured similarly did not respond to PTH or TCT, but still had a significant PGE₂ response through passage 21. The cultured cells formed multiple layers with localized areas of higher cell density. Mineral plaques with major diameters as great as 0.75 mm were evident in the areas of greater cell density. Less extensive mineral deposits were present throughout the culture. The mineral plaques consisted of apatite-like crystals deposited on an organic matrix. Matrix vesicles and mineralized spherules appeared to be associated with initial mineral deposition. The spherules apparently coalesced to form more complex mineralized structures. A limited amount of mineralization also was observed in rat skin fibroblast cultures.     

Effect of strain on bone nodule formation by rat osteogenic cells in vitro

The purpose of this study was to assess in vitro bone nodule formation by cells exposed to a range of microstrain, at a sub-optimal oscillation frequency for bone formation. Fetal rat calvarial cells experienced a Flexercell regimen within either FLEX I (deformable) or FLEX II (non-deformable) substrates. Cells in FLEX I plates were exposed to growth medium only; those in FLEX II plates were exposed to either growth medium only, or growth medium + 10(-7) M IGF-1. Cell numbers were assessed from 1 to 6 days. Other cells were exposed to the Flexercell regimen (-2 kPa, 0.05 Hz) for 1-3 (Group 1), 3-6 (Group 2), 1-9 (Group 3) or 10-15 (Group 4) days and were maintained, at other times, under standard conditions. After 21 days, nodules were counted within each well and within the compression, <999, 1000-4900, 5000-9999, 10,000-14,999 and 15,000-25,000 microstrain regions of the FLEX I membrane. Cyclic deformation inhibited cell numbers from 1 to 6 days, compared to control or IGF-1 groups (P<0.001). The number of nodules in Groups 2 and 4 were greater than Groups 1 or 3 (P<0.001), but not different from control or IGF-1 groups. Compression or tensile microstrain significantly affected nodule formation in all groups, with Group 4 producing more nodules than other groups in most microstrain regions. Thus, the number of bone nodules produced by osteogenic cell cultures exposed to cyclic deformation was significantly affected by the timing of initiation and the characteristics and magnitude of the deformation regimen.     

Evidence supporting a role of glucocorticoids in short-term bone loss in burned children

Children burned 40% total body surface area suffer acute bone loss. The reason(s) for this is uncertain. In order to determine whether high endogenous glucocorticoid production can contribute to the bone loss, we sequentially studied a total of 14 pediatric burn patients for bone histomorphometry; 7 of these patients and 4 controls were studied for characteristics of corticosteroid-induced bone loss, including decreased osteoblasts and down-regulation of the glucocorticoid receptor in bone. We then studied 4 of the burn patients and three controls for a decrease in markers of osteoblast differentiation, another feature of glucocorticoid toxicity. Bone biopsies were taken from each of the 14 burn patients a mean of 3 weeks post-burn. Histomorphometry was performed on one specimen (n=7) and either glucocorticoid and mineralocorticoid receptor, collagen and alkaline phosphatase expression by RT-PCR (n=7) or marrow stromal cell culture (n=4) on the other. Patients were permitted a maximum of two biopsies for study. One biopsy was obtained intra-operatively from normal subjects during elective iliac crest alveolar bone grafting and compared with burn specimens for glucocorticoid receptors and marrow stromal cell culture. A 24-h urine specimen was obtained for free cortisol (n=7). Histomorphometry revealed low osteoblast and osteoid surfaces and few detectable osteoblasts. Resorptive surfaces were also reduced. Glucocorticoid receptor mRNA (GR) was not decreased; however, there was a trend toward inverse relationships between urine free cortisol and GR and type-1 collagen mRNA, r=–0.61 and –0.64, respectively, and a significantly lower mRNA for type-1 collagen in bone in burn vs control patients by the median test, ²=7.6 (p<0.01). Markers of osteoblast differentiation, core-binding factor (cbf)a1, bone morphogenetic protein (BMP)-2, type-I collagen, and alkaline phosphatase were reduced in burn cell cultures compared with controls (p<0.05). The eightfold elevation of urinary free cortisol excretion, low osteoblast number, decreased resorptive surface, and reduced markers of osteoblast differentiation are all consistent with an acute glucocorticoid effect on bone.     

Absence of mouse pleiotrophin does not affect bone formation in vivo

Pleiotrophin (Ptn) is an extracellular matrix protein that regulates hippocampal synaptic plasticity and learning behavior in vivo. Since the overexpression of Ptn in transgenic mice leads to increased bone formation, we analyzed whether a deficiency in Ptn expression would have a negative effect on bone remodeling. Bones from Ptn-deficient mice and wild-type littermates were analyzed using radiography, μCT imaging and undecalcified histology. Biomechanical stability was determined in a three-point-bending assay. Cellular activities were assessed using dynamic histomorphometry and the determination of urinary collagen degradation products. Skeletons of Ptn-deficient mice have no gross abnormalities, displayed a normal size, and showed no differences in growth plate organization compared to wild-type littermates. There were no obvious differences in bone mass as determined by radiographic and μCT imaging. The absence of a bone remodeling phenotype in Ptn-deficient mice was further confirmed using static histomorphometry and biomechanical testing. Finally, the number, morphology, and function of osteoclasts, osteoblasts, and osteocytes were not altered in Ptn-deficient mice compared to wild-type littermates. The complete skeletal analysis of Ptn-deficient mice presented here demonstrates that the lack of Ptn in mice does not affect bone formation in vivo. Therefore, Ptn does not play a significant role in normal bone physiology.     

<Emphasis Type="Italic">In-Situ</Emphasis> Visualization and Quantification of Mineralization of Cultured Osteogenetic Cells

An osteoblastic cell line (HOS cells) produces a prominent osteoid matrix with mineralization. Fibroblasts, on the other hand, do not exhibit this mineralization. To evaluate the degree of mineralization, we added calcein to the culture medium and then observed the culture wells by using an image analyzer. The calcein uptake into the cell/matrix layer was detected in the HOS cells but not in the fibroblasts. The calcein uptake was also quantified in situ by using an image analyzer, which revealed high levels in the HOS cells, which correlated well with the calcium content of the mineralized matrix. Rat marrow cells were also cultured in media containing calcein, fetal bovine serum, -glycerophosphate, L-ascorbic acid 2-phosphate, and with or without dexamethasone. With the dexamethasone, the cells exhibited osteogenic differentiation that resulted in mineralized matrix formation after about 10 days. The matrix formation coincided with the appearance of calcein uptake into the cell/matrix layer, with the amount of calcein uptake increasing with time. By contrast, the culture without the dexamethasone did not exhibit matrix formation and the calcein uptake was negligible. In the case of both HOS cell and rat marrow cell cultures in vitro, calcein did not affect expressions of their alkaline phosphatase activity or osteocalcin production. Furthermore, histologic observation revealed that rat marrow cells subcultured with calcein could show osteogenic ability after in vivo implantation. These results suggest that the current method of detecting calcein uptake in a culture allows the monitoring of the osteogenic capacity of cultured cells, as well as the measurement of the amount of mineralization produced by the osteogenic cells. Given that osteogenic cultured cells/mineralized matrices are used in bone reconstruction surgery, the in situ monitoring method is invaluable in that it allows us to evaluate the osteogenic capacity of in vitro constructs.     

Osteogenic evaluation of glutaraldehyde crosslinked gelatin composite with fetal rat calvarial culture model

The cytotoxicity of the synthetic bone substitute composed of tricalcium phosphate and glutaraldehyde crosslinked gelatin (GTG) were evaluated by osteoblast cell culture. In a previous study, the GTG composites were soaked in distilled water for 1, 2, 4, 7, 14, 28, and 42 days, and then the solutions (or extracts) were cocultured with osteoblasts to evaluate the cytotoxicity of GTG composites by alive cell counting. In this study, the extracts were cocultured with the osteoblasts; thereafter, the concentration of transforming growth factor-beta (TGF-beta1) and prostaglandin E2 (PGE2) in the medium was analyzed to strictly reflect the biological effects of GTG composites on the growth of osteoblasts. In order to investigate the osteoconductive potential of the GTG composites on new bone formation in a relative short term, a model of neonatal rat calvarial organ culture was designed prior to animal experiments. Three experimental materials of 4, 8, and 12% GTG composites were evaluated by fetal rat calvarial organ culture for their ability for bone regeneration. Deproteinized bovine and porcine cancellous bone matrixes were used as the controlled materials. All the organ culture units were maintained in cultured medium for 5 weeks. Following the culture period, the morphology of tissue was observed under an optical microscope, and the quantitative evaluation of the new generation bone was determined by using a semiautomatic histomorphometeric method. Except in the initial 4 days, the concentration of TGF-beta1 of 4% and 8% GTG composites was higher than that of the blank group for all the other experimental time periods. The PGE2 concentration for 4% and 8% GTG composites was lower than that of the blank group. It revealed that the 4% and 8% GTG composites would not lead to inflammation and would promote osteoblast growth. The morphology and activity of the osteoblasts were not transformed or changed by the 2 GTG composites. For the 12% GTG composite, the performance of the in vitro condition was inferior to the blank group and the other 2 GTG composites. Although the concentration of TGF-beta1 and PGE2 was gradually back to normal after 14 days, the morphology of the osteoblasts was abnormal with features such as contracted cytoplast structures. The osteoblast was damaged perhaps in the initial stage. We suggested that the 4% and 8% GTG composites should be soaked in distilled water at least for 4 days before medical applications. The 12% GTG composite and the composites with a concentration of glutaraldehyde solution higher than 12% were not recommended as a medical prostheses in any condition. The fetal rat calvaria culture also showed the same results with the analysis of TGF-beta1 and PGE2. From the study, we could predict the results of animal experiments in the future.     

黄芪多糖的提取及对体外培养成骨细胞成骨能力的影响

目的：了解黄芪多糖对体外培养成骨细胞增殖、分化能力的影响。方法：采用水煮醇沉法提取黄芪多糖,用ＴＬＣ进行定性,用比色法定量,用含高低两种剂量（０．５ｍ ｇ／ｍ ｌ,５．０ｍ ｇ／ｍ ｌ）的黄芪多糖的培养液体外培养成骨细胞,ＭＴＴ法及ＡＬＰ比活性测定观察成骨细胞增殖率及细胞活性变化。结果：低浓度黄芪多糖（０．５ｍ ｇ／ｍ ｌ）及高浓度黄芪多糖（５．０ｍ ｇ／ｍ ｌ）短期时（２ 天）促进成骨细胞增殖;高浓度（５．０ｍ ｇ／ｍ ｌ）长期（４ 天）抑制成骨细胞增殖,降低其活性。结论：黄芪多糖对成骨细胞增殖、活性有双向调节作用     

二甲胺四环素促进骨髓基质细胞增殖作用的研究

为弄清二甲胺四环素增加摘除卵巢的老龄大鼠骨量作用的细胞分子基础,采用细胞增殖曲线的绘制和碱性磷酸酶测定方法,了解二甲胺四环素对成骨细胞和骨髓基质细胞的影响,结果：二甲胺四环素在体外刺激骨髓基质细胞和成骨细胞增殖的作用,但抑制细胞的分化。