晚发型脊柱骨骺发育不良伴进行性骨关节病患者的成骨细胞增殖分化及基因表达谱改变

目的探讨晚发型脊柱骨骺发育不良伴进行性骨关节病(SEDT PA)患者WISP3基因突变所造成的软骨及周围骨组织病变和可能的发病机制。方法从SEDT PA患者及正常同龄个体的股骨头松质骨分离成骨细胞进行体外培养,对培养的成骨细胞进行细胞增殖、骨特异性碱性磷酸酶(BAP)、骨钙素、护骨素测定,利用cDNA表达芯片进行基因表达谱分析,并用免疫组织化学方法对基因芯片进行验证。比较SEDT PA患者及正常对照的成骨细胞在细胞增殖、分化及基因表达谱等方面的改变。同时采用Northern杂交检测成骨细胞的WISP3表达情况。结果与正常人成骨细胞相比,SEDT PA患者成骨细胞的增殖能力较强(0.86±0.04vs0.71±0.10),氚胸腺嘧啶(3H TDR)的掺入量明显较高(1363cpm±350cpmvs867cpm±128cpm)。SEDT PA患者的成骨细胞骨钙素的表达水平明显低于正常同龄人(3.62ng/μg蛋白±0.3ng/μg蛋白vs0.85ng/μg蛋白±0.06ng/μg蛋白),护骨素的表达明显低于正常同龄人(9.43pg/μg蛋白±0.41pg/μg蛋白vs1.98pg/μg蛋白±0.03pg/μg蛋白),而BAP的表达未发生明显改变;WISP3基因的表达极低。cDNA表达芯片分析结果发现,与正常对照比较,SEDT PA患者的成骨细胞有22个基因表达明显上调,16个基因表达明显下调,这些基因包括有成骨细胞的标记分子、细胞外基     

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.     

Rodent Osteoblastic Cells Express Voltage-Sensitive Calcium Channels Lacking a γ Subunit

Voltage-sensitive calcium channels (VSCC) open in response to external stimuli, including calcitropic hormones, that alter plasma membrane calcium (Ca²⁺) permeability. Ca²⁺ that enters the cell through these channels serves a second messenger function, eliciting cellular responses that include secretion and changes in gene expression. In osteoblasts, VSCCs serve as key regulators of Ca²⁺ permeability and are a major class of calcitropic hormone-sensitive Ca²⁺ channels present in the plasma membrane. The members of the VSCC family exist as a complex of polypeptide subunits that are comprised of a pore-forming ₁ subunit, an intracellular subunit, a dimer of disulfide-linked ₂ and subunits, and in some tissues, a subunit. Previous studies in our laboratory have shown that the major functional ₁ subunit present in osteoblasts is the _1C (Ca_V1.2). To determine the complement of auxiliary subunits present in rodent osteoblastic cells, we employed RT-PCR using a battery of subunit specific primers and appropriate tissue controls. Immunohistochemistry also was performed, using available subunit specific antibodies, to measure protein expression and localization. Cell types examined included MC3T3-E1 at various stages of differentiation, ROS 17/2.8 osteosarcoma, and primary cultures of rat calvarial osteoblasts. The results indicate that all cells expressed multiple subunit classes and ₂ dimers, but no subunits, regardless of differentiation state. We propose a structure for the functional osteoblast VSCC that consists of ₁, , ₂ subunits and is devoid of a subunit.     

<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.