绝经后骨质疏松症I型胶原代谢的变化

目的 研究I型胶原及其代谢在绝经后骨质疏松症发病中的作用。方法 双侧卵巢切除制造大鼠骨质疏松模型（OVX大鼠）；比色法测定骨中羟脯氨酸浓度，计算胶原含量；苦味酸天狼星红染色，从蛋白水平观察骨中I型胶原的分布和变化；地高辛标记的I型胶原和基质金属蛋白酶－1（MMP－1）基因探针进行原位杂交，从mRNA水平观察I型胶原和MMP－1的分布和变化。结果 OVX大鼠骨中无机盐和胶原含量均降低，I型胶原蛋白纤维变细、断裂、结构紊乱。I型胶原和MMP－1 mRNA水平均升高。结论 I型胶原合成及代谢的改变在绝经后骨质疏松发病中起重要作用。     

LMP-1 mRNA反义寡核苷酸抑制大鼠成骨细胞的分化

为深入探讨LIM矿化蛋白1（LMP－1）在成骨细胞分化中的作用，体外培养大鼠成骨细胞，在培养基中加入LMP－1反义寡核苷酸（LMP－1antisense终浓度为0．8mol／L），以阻断大鼠成骨细胞中LMP－1mRNA的表达。对照组加nonsense（终浓度为0．8mol／L）。测定细胞的碱性磷酸酶（ALP）活性、培养基中骨钙素（OC）的含量，免疫组化分析Ⅰ型胶原蛋白的表达，Northern blotting检测I型胶原mRAN的表达。结果显示：LMP－1mRNA被LMP－lantisense阻断后，ALP活性降低、OC分泌减少、I型胶原蛋白和mRNA的表达下降。上述结果表明：LMP－1mRNA被LMP－1反义寡核酸阻断后，成骨细胞分化受到明显的抑制，提示LMP－1是成骨细胞分化必不可少的正调节因子。     

绝经后骨质疏松症病人I型胶原的变化

目的 研究I型胶原合成及代谢在绝经后骨质疏松症发病中的作用。方法 以绝经后骨质疏松性骨折病人股骨头置换术弃去的股骨头为样本；采用苦味酸天狼星红一偏振光及I型胶原和基质金属蛋白酶-1(MMP—1)抗体免疫组织化学染色，从蛋白水平观察骨中I型胶原的分布和量的变化；地高辛标记的I型胶原和MMP-1cDNA基因探针进行原位杂交，从mRNA水平观察I型胶原和MMP-1的分布和量的变化；从骨中提取I型胶原测定肽链的羟基化水平，观察I型胶原羟基化程度的改变。结果 绝经后骨质疏松性骨折病人胶原含量降低，I型胶原蛋白纤维变细、断裂，结构紊乱。而细胞内I型胶原和MMP—lmRNA以及MMP—1蛋白水平均升高。I型胶原蛋白赖氨酸羟基化程度升高而脯氨酸羟基化程度不变。结论 I型胶原合成及代谢在质和量上的改变在绝经后骨质疏松症发病中起重要作用。     

丹酚酸B缓释羟基磷灰石颗粒与成骨细胞的活性

背景:前期实验制备了羟基磷灰石颗粒堆积型多孔支架,可通过调节羟基磷灰石颗粒的尺寸及微观空隙结构更好地构建修复超临界尺寸的生物陶瓷支架。如果再将一些可促骨愈合的药物负载其上,既可控制药物的局部浓度以提高利用率,增加专一性、又可降低不良反应,加快骨愈合。目的:观察载丹酚酸B缓释羟基磷灰石颗粒对体外培养成骨细胞活性的影响。方法:分离培养SD大鼠成骨细胞,将羟基磷灰石颗粒、无壳聚糖包裹的丹酚酸B缓释羟基磷灰石颗粒、壳聚糖包裹的丹酚酸B缓释羟基磷灰石颗粒分别与成骨细胞共培养,采用AlamarBlue法检测各组成骨细胞增殖活性;碱性磷酸酶活性检测各组成骨细胞分化情况;免疫细胞化学染色观察各组成骨细胞中Ⅰ型胶原和骨钙素表达。结果与结论:壳聚糖包裹的丹酚酸B缓释羟基磷灰石颗粒能显著促进SD大鼠成骨细胞的增殖,同时加强碱性磷酸酶表达,其效应持续时间高于羟基磷灰石组、无壳聚糖包裹的丹酚酸B缓释羟基磷灰石组;Ⅰ型胶原和骨钙素表达与羟基磷灰石组无差异。而无壳聚糖包裹的丹酚酸B缓释羟基磷灰石组由于药物的突释对细胞造成毒性,从第2天细胞开始皱缩以致死亡。说明壳聚糖包裹的丹酚酸B缓释羟基磷灰石颗粒在较长时间内具有促进成骨细胞增殖和分化,对成骨细胞的生物学活性无影响,并保持成骨细胞的生物学活性。     

rhBMP-7对小鼠骨髓间充质干细胞向成骨细胞分化的影响

目的:观察重组人骨形成蛋白-7(rhBMP-7)对小鼠骨髓间充质干细胞(BMSCs)向成骨细胞分化的影响。方法:采用密度梯度离心法分离骨髓,体外培养扩增,获得小鼠骨髓间充质干细胞,贴壁培养至第3代,向培养液中加入rhBMP-7,培养5 d后进行碱性磷酸酶(ALP)染色,ALP活性与骨钙素(OC)含量检测,并用RT-PCR法分析成骨细胞标志基因骨钙素(OC)与Ⅰ型胶原(Col-Ⅰ)表达情况,Western blot法检测Ⅰ型胶原(Col-Ⅰ)蛋白的分泌量。结果:rhBMP-7诱导组的骨髓间充质干细胞的ALP染色强度、ALP活性及OC含量明显升高,OC与Col-Ⅰ的mRNA表达水平以及Col-Ⅰ的蛋白表达水平均明显提高。结论:rhBMP-7可促进体外培养的小鼠BMSCs向成骨细胞方向分化。     

力学刺激对MG-63成骨样细胞增殖分化的影响

为探讨力学刺激对成骨细胞增殖分化的影响,应用根据四点弯曲原理设计的加力装置对MG-63成骨样细胞进行力学刺激,观察不同大小和作用时间的机械应变对成骨细胞增殖分化的影响。本研究中蛋白表达、碱性磷酸酶(ALP)活性测定分别采用Western blot、α-磷酸奈酚法,采用茜素红染色观察矿化结节的形成。结果发现:⑴与对照组相比,加力组增殖细胞核抗原(PCNA)蛋白表达、ALP活性均明显增加,但这种作用并不随着力刺激大小的增加而增加。⑵在适宜的力刺激(2 000μstrain)下,随着加力时间的增加,PCNA、Ⅰ型胶原(COLⅠ)蛋白表达及ALP活性逐渐增加,并且适宜的力刺激也促进了矿化结节的形成。该结果提示适宜的力学刺激能够促进成骨细胞的增殖分化。     

缺氧大鼠成骨细胞增殖、分化及基因表达

背景：研究表明,低氧会引起骨折延迟愈合或不愈合,骨密度减低,使骨质疏松、骨折等疾病的发病率升高。成骨细胞是骨形成、生长和发育主要的功能细胞。 目的：观察缺氧对体外培养成骨细胞增殖、分化及基因表达的影响。 方法：选用新生Wistar大鼠颅盖骨,使用胰酶-胶原酶序贯消化法获取成骨细胞,进行体外传代培养及鉴定。在缺氧培养下应用MTT法测定成骨细胞增殖率,对硝基苯磷酸盐法测定成骨细胞碱性磷酸酶活性,反转录-聚合酶链反应法测定成骨细胞内骨钙素及Ⅰ型胶原的表达。 结果与结论：缺氧具有抑制成骨细胞增殖,降低碱性磷酸酶活性及下调大鼠成骨细胞中Ⅰ型胶原α1、骨钙素基因表达的作用,随缺氧时间的增加作用更加明显。提示缺氧可通过抑制成骨细胞增殖、分化成熟及下调Ⅰ型胶原α1、骨钙素基因表达而降低成骨能力,从而促进骨质疏松的发生。     

极低频三角形电磁场对成骨细胞的影响

目的:观察三角形极低频电磁场对新生大鼠颅盖骨成骨细胞增殖与分化的影响.方法:采用频率为15Hz、不同强度、不同形状的三角形电磁场作用于成骨细胞,测量成骨细胞的增殖与碱性磷酸酶活性(ALP).结果:频率为15 Hz, 强度为4 mT～5 mT,三角形电磁场能够显著提高成骨细胞的增殖率和ALP的活性,而强度为8 mT时则显著抑制成骨细胞的增殖与ALP的活性;不同形状的三角形电磁场也明显影响成骨细胞的功能,其中以p为38%、85%对成骨细胞的影响最为明显.结论:电磁场的生物学效应依赖于其参数特征.     

抗菌钛合金Ti6Al4V-6Cu表面构建成骨细胞膜片的实验研究

目的:探讨大鼠颌骨成骨细胞在抗菌钛合金Ti6Al4V-6Cu表面构建细胞膜片的可行性。方法:体外培养大鼠颌骨成骨细胞,采用富含维生素C培养基在抗菌钛合金Ti6Al4V-6Cu表面构建细胞膜片（细胞膜片组）,并以单纯培养基作对照（对照组）,检测膜片形成过程中碱性磷酸酶（ALP）和成骨相关基因ALP、I型胶原（Col-1）、骨形成蛋白2（BMP-2）的表达情况。结果:采用富含维生素C的培养基连续培养可在抗菌钛合金Ti6Al4V-6Cu表面成功构建成骨细胞膜片,该细胞膜片由多层细胞构成,富含胞外基质。相对于对照组,细胞膜片组的膜片形成过程中成骨细胞ALP活性及成骨相关基因ALP、Col-1、BMP-2的表达均显著增高。结论:在抗菌钛合金Ti6Al4V-6Cu表面可成功构建成骨细胞膜片,有望与Ti6Al4V-6Cu联合应用于引导性骨再生术。     

大鼠真皮间充质干细胞的体外长期培养及胶原海绵对其生长的影响

从新生大鼠真皮细胞中分离多能干细胞，体外长期传代培养，观察细胞形态和超微结构的变化。检测细胞增殖和分化能力的改变及胶原基质对细胞生长的影响。揭示体外长期传代培养对大鼠真皮间充质干细胞生物学性状的影响，结果表明；大鼠真皮组织中的间充质干细胞体外培养至180d，传至25代的细胞仍具有干细胞的特征；形态的均一，细胞核结构原始，细胞器不发达；体外增殖迅速，保持了向成骨细胞和软骨细胞分化的潜能，胶原基质成分可促进细胞的生长，而胶原海绵支架更利于细胞的三维生长。结论是体外长期传代培养后大鼠真皮间充质干细胞仍保持良好的干细胞特性，为真皮间充质干细胞的深入研究与应用提供了实验依据。     

Modification of Ti6AL4V surfaces using collagen I, III, and fibronectin. II. Influence on osteoblast responses

Responses of osteoblastic cells are influenced by morphology and composition of the extracellular matrix, and this fact has been used to improve the biological acceptance of implants by modifying the surfaces with components of the extracellular matrix (ECM). In this study, the effect of the collagen types I and III on adhesion, proliferation, and differentiation was studied, using primary osteoblastic cells from rat calvariae. Differences in alkaline phosphatase activity (ALP) and collagen synthesis were observed between differently composed collagen coatings. An increase in collagen type III resulted in an increase in collagen synthesis and a concomitant decrease in ALP activity and Ca deposition. Initial adhesion mechanism of the cells depended on the substrate (titanium, collagen, fibronectin).     

SV-40 large-T immortalization of embryonic bone cells: establishment of osteoblastic clonal cell lines

Cells derived from embryonic rat calvariae were immortalized by retroviral delivery of cDNA for the SV-40 large T antigen and the bacterial neomycin resistance gene. After selection with G418, cells were cloned by limiting dilution and screened for expression of osteoblast characteristics. One clone (RCT-3), derived from cells collected during the third period of enzymatic digestion, showed high constitutive expression of alkaline phosphatase (ALP), synthesized type I collagen in the virtual absence of type III and exhibited a parathyroid hormone (PTH)-responsive adenylate cyclase (EC50, 10 nM). Messenger RNAs for osteonectin and osteopontin were present in RCT-3 cells and osteopontin mRNA was enhanced by 1,25 (OH)2 vitamin D3 treatment. The other cell line (RCT-1), derived from cells released during the first 10 min of digestion, expressed osteoblast features only after 3 d treatment with 1 microM retinoic acid (RA). ALP activity increased from 0.003 to 0.25 mumole/min/mg protein, there was a substantial increase in the steady-state level of type I collagen mRNA and a dose-dependent and saturable response to PTH was induced (EC50, 10 nM). Osteopontin mRNA was induced by 1,25 (OH)2D3. This study has provided two new cell lines which may be useful models for studies of differentiation-related gene expression in bone cells.     

SV-40 Large-T Immortalization of Embryonic Bone Cells: Establishment of Osteoblastic Clonal Cell Lines

Cells derived from embryonic rat calvariae were immortalized by retroviral delivery of cDNA for the SV-40 large T antigen and the bacterial neomycin resistance gene. After selection with G418, cells were cloned by limiting dilution and screened for expression of osteoblast characteristics. One clone (RCT-3), derived from cells collected during the third period of enzymatic digestion, showed high constitutive expression of alkaline phosphatase (ALP), synthesized type I collagen in the virtual absence of type III and exhibited a parathyroid hormone (PTH)-responsive adenylate cyclase (EC₅₀, 10 nM). Messenger RNAs for osteonectin and osteopontin were present in RCT-3 cells and osteopontin mRNA was enhanced by 1,25 (OH)₂ vitamin D₃ treatment.

The other cell line (RCT-1). derived from cells released during the first 10 min of digestion, expressed osteoblast features only after 3 d treatment with 1 mm retinoic acid (RA). ALP activity increased from 0.003 to 0.25 m mole/min/mg protein, there was a substantial increase in the steady-state level of type I collagen mRNA and a dose-dependent and saturable response to PTH was induced (EC₅₀, 10 nM). Osteopontin mRNA was induced by 1.25 (OH)₂D₃.

This study has provided two new cell lines which may be useful models for studies of differentiation-related gene expression in bone cells.     

Effects of coumestrol on neonatal and adult mice osteoblasts activities

Estrogen replacement therapy has been shown to reduce postmenopausal osteoporosis. In the present study, we examined the effects of the phytoestrogen coumestrol on neonatal and adult osteoblasts metabolism. Two different sources of osteoblast cells (neonatal mice calvaria and adult mice long bone) cultures were used in this study. The effects of coumestrol on the cellular activities were analyzed by the mitochondrial tetrazolium (MTT) assay, secretion of alkaline phosphatase (ALP), intracellular calcium content (Ca), and the gene expression of bone matrix protein, estrogen receptors (ER-alpha, ER-beta), and osteoprotegerin (OPG) and osteoprotegerin ligand (OPGL). The results showed that the proliferation of neonatal mice osteoblast cells was enhanced by treatment of coumestrol. In the presence of 10(-9)M coumestrol, the osteoblast proliferation attained 139.5% of the control and that the coumestrol can increase the intracellular calcium contents. Type I collagen gene expression was upregulated 167% at the 1st day's culture; ALP gene expression was upregulated 360% at the 7th day's culture; while the osteocalcin gene expression was upregulated 222% at the 14th day's culture. When adult mice osteoblasts were cultured in the presence of 10(-9)M coumestrol, the osteoblasts population increased significantly earlier and attained its maximal effect at the 21st day's culture with 207.4% of control group. The content of ER-beta and osteoprotegerin secretion by neonatal mice control cells gradually increased during osteoblasts differentiation, whereas the ER-alpha and OPGL content were decreased in this study. The cellular responses to the estradiol and counmestrol were quite different in the osteoblasts derived from different age.     

Effects of alpha-TCP and TetCP on MC3T3-E1 proliferation,differentiation and mineralization

Calcium phosphates with high solubility in water such as alpha-tricalcium phosphate (alpha-TCP) and tetracalcium phosphate (TetCP) have received considerable attention as components of bone-substitution materials. However, the osteoblast response to these materials has not yet been clearly understood. This study examined the effects of alpha-TCP and TetCP on osteoblast proliferation, differentiation and mineralization in the culture system of MC3T3-E1 cells. Cells were cultured in a differentiation medium with or without alpha-TCP or TetCP at 1 or 10 microM, and the number of cells attached to the culture plates was determined. To examine osteoblast differentiation, the alkaline phosphatase (ALP) activity was measured and the expression of osteoblastic markers analyzed by RT-PCR. In addition, mineralization was evaluated by staining the calcium deposit with Alizalin red. Culture in the presence of alpha-TCP or TetCP showed no significant influence on cell proliferation. ALP activities of the cells were enhanced by both calcium phosphates for 3d and the expression of type I collagen was promoted at 12h and 1d after incubation. Enhancement of bone-like tissue formation by the addition of alpha-TCP or TetCP at 10 microM was observed after 7d incubation and thereafter. The results of the present study indicate that alpha-TCP and TetCP promote osteogenesis by increasing collagen synthesis and calcification of the extra-cellular matrix.     

Bone tissue engineering on patterned collagen films: an in vitro study

This study aimed at guiding osteoblast cells from rat bone marrow on chemically modified and patterned collagen films to study the influence of patterns on cell guidance. The films were stabilized using different treatment methods including crosslinking with carbodiimide (EDC) and glutaraldehyde, dehydrothermal treatment (DHT), and deposition of calcium phosphate on the collagen membrane. Mesenchymal osteoprogenitor cells were differentiated into osteoblasts and cultured for 7 and 14 days on micropatterned (groove width: 27 microm, groove depth: 12 microm, ridge width: 2 microm) and macropatterned (groove width: 250 microm, groove depth: 250 microm, ridge width: 100 microm) collagen films to study the influence of pattern dimensions on osteoblast alignment and orientation. Fibrinogen was added to the patterned surfaces as a chemical cue to induce osteoblast adhesion. Cell proliferation on collagen films was determined using MTS assay. Deposition of calcium phosphate on the surface of the film increased surface hydrophilicity and roughness and allowed a good cell proliferation. Combined DHT and EDC treatment provided an intermediate wettability, and also promoted cell proliferation. Glutaraldehyde crosslinking was found to lead to the lowest cell proliferation but fibrinogen adsorption on glutaraldehyde treated film surfaces increased the cell proliferation significantly. Macropatterns were first tested for alignment and only microscopy images were enough to see that there is no specific alignment. As a result of this, micropatterned samples with the topography that affect cell alignment and guidance were used. Osteoblast phenotype expression (ALP activity) was observed to be highest in calcium phosphate deposited samples, emphasizing the effect of mineralization on osteoblast differentiation. In general ALP activity per cell was found to decrease from day 7 to day 14 of incubation. SEM and fluorescence microscopy revealed good osteoblast alignment and orientation along the axis of the patterns when micropatterned films were used. This study shows that it is possible to prepare cell carriers suitable for tissue engineering through choice of appropriate surface topography and surface chemistry. Presence of chemical cues and micropatterns on the surface enhance cell orientation and bone formation.     

Tissue transglutaminase (TG2) activity regulates osteoblast differentiation and mineralization in the SAOS-2 cell line

Tissue transglutaminase (type II, TG2) has long been postulated to directly promote skeletal matrix calcification and play an important role in ossification. However, limited information is available on the expression, function and modulating mechanism of TG2 during osteoblast differentiation and mineralization. To address these issues, we cultured the well-established human osteosarcoma cell line SAOS-2 with osteo-inductive conditioned medium and set up three time points (culture days 4, 7, and 14) to represent different stages of SAOS-2 differentiation. Osteoblast markers, mineralization, as well as TG2 expression and activity, were then assayed in each stage. Furthermore, we inhibited TG activity with cystamine and then checked SAOS-2 differentiation and mineralization in each stage. The results showed that during the progression of osteoblast differentiation SAOS-2 cells presented significantly high levels of osteocalcin (OC) mRNA, bone morphogenetic protein-2 (BMP-2) and collagen I, significantly high alkaline phosphatase (ALP) activity, and the increased formation of calcified matrix. With the same tendency, TG2 expression and activity were up-regulated. Furthermore, inhibition of TG activity resulted in a significant decrease of OC, collagen I, and BMP-2 mRNA and of ALP activity and mineralization. This study demonstrated that TG2 is involved in osteoblast differentiation and may play a role in the initiation and regulation of the mineralization processes. Moreover, the modulating effects of TG2 on osteoblasts may be related to BMP-2.     

Influence of recovering collagen with bioactive glass on osteoblast behavior

Bioactive ceramics have interesting properties from the biological standpoint, but their effects on cellular events remain partially unknown. In the current work, we investigated cellular viability, proliferation, and metabolic activity of rat primary osteoblasts in contact with four different samples: type I collagen, bioactive glass-coated collagen (GC), and both samples submitted to immersion for 5 days in a simulated body fluid. The bioactive glass coating was obtained from a sol-gel process. The cell viability, the alkaline phosphate, the collagen secretion, and the nitric oxide production by osteoblast were measured after 72 h of incubation in the presence of the samples. The GC that was immersed for 5 days in a simulated body fluid solution showed an increase in osteoblast viability and proliferation when it was compared with control and the other samples.     

Abnormal Differentiation in MC3T3-E1 Preosteoblasts Expressing a Dominant-Negative Type I Collagen Mutation

To examine the effects that an organizing extracellular matrix might have on osteoblast precursors, we created MC3T3-E1 cell lines that stably incorporated a plasmid that expressed proαl(I) collagen chains having a truncated triple helical domain. Cells that had incorporated the proαl(I) expression plasmid (pMG155) efficiently secreted molecules with shortened prood(I) chains into culture media. Electron micrographs indicated that expression of the minigene dramatically interferes with normal type I collagen fibril architecture. The turnover of newly deposited collagenous matrix as measured by ³[H]-hydroxyproline release was 29% after a 14 day chase in cells expressing the mini-gene compared to essentially no turnover in control cultures. MC3T3-E1 cells in culture normally demonstrate a time dependent reduction of cell division followed by an increase in osteoblast characteristics. Cell number was consistently 20–25% higher than control in MC3T3-E1 cultures expressing the truncated prooαl(I) gene but ALP activity was only 45% of control. Secretion and steady state mRNA levels for osteocalcin were over fivefold higher than control cultures but expression of other extracellular matrix components was not changed. These findings demonstrate that osteoblasts require a normally structured collagenous matrix for inhibition of cellular proliferation and subsequent upregulation of ALP. However, in the presence of rapid turnover of osteoblast matrix, the gene for osteocalcin may be upregulated in response to local signals.