Molecular mechanisms for intrafibrillar collagen mineralization in skeletal tissues

The critical role of the self-assembled structure of collagen in skeletal mineralization is long recognized, yet the angstrom to tens of nanometers length-scale nucleation mechanism of calcium phosphate mineral (Ca–P_i) remains unclear. Here, by constructing three-dimensional structure of collagen fibril, we report direct computational evidence of intrafibrillar Ca–P_i nucleation in the collagen matrix and illustrate the crucial role of charged amino acid sidechains of collagen molecules in nucleation. The all-atom Hamiltonian replica exchange molecular dynamics simulation shows that these charged sidechains are oriented toward the fibril “hole zones” and significantly template nucleation with amorphous Ca–Pi phase, ∼1.3–1.6 nm in size, thus explaining the empirical observations that Ca–P_i nucleates principally in these regions. We also show that the low water density of about 0.70 g cm⁻³ in these zones may further benefit nucleation by lowering the enthalpic penalty for ion desolvation. This work provides insight, at the atomistic level, into the nucleation mechanism of bone crystals within a collagen matrix for understanding mineral deposition, interpreting mineralization experiments and guiding the design of new implantable materials.     

Purification of mineralized tissue-associated osteopontin

Summary A protocol to extract and purify osteopontin, a protein with cell adhesion and mineral-crystal binding properties, from mineral-associated tissues is described. This procedure provides for the complete separation of osteopontin from bone sialoprotein, a mineralized tissue-specific protein with similar properties to that of osteopontin.     

Regulatory role of DC‐derived osteopontin in systemic allergen sensitization

Osteopontin (OPN) is a secreted phosphoglycoprotein with a wide range of functions, and is involved in various pathophysiological conditions. However, the role of OPN in IgE and Th2‐associated allergic responses remains incompletely defined. The aim of this study was to elucidate the role of OPN in systemic allergen sensitization in mice. When compared with OPN^+/+ mice, significantly increased levels of OVA‐induced IgE were found in OPN^?/? mice. OPN^?/? DC demonstrated an increased capacity to enhance Th2 cytokine production in CD4⁺ T cells from sensitized OPN^+/+ mice. Furthermore, significantly reduced levels of IL‐12p70 expression were seen in LPS‐stimulated OPN^?/? DC as compared with the WT DC, and the reduction was reversible by the addition of recombinant OPN (rOPN). rOPN was able to suppress OVA‐induced IL‐13 production in the cultures of CD4 and OPN^?/? DC, but this inhibitory activity was neutralized by the addition of anti‐IL‐12 Ab. In addition, administration of rOPN in vivo suppressed OVA‐specific IgE production; however, this suppressive effect was abrogated in IL‐12‐deficient mice. These results indicate that DC‐derived OPN plays a regulatory role in the development of systemic allergen sensitization, which is mediated, at least in part, through the production of endogenous IL‐12.     

Mechanisms of promoting the differentiation and bone resorption function of osteoclasts by Staphylococcus aureus infection

Bone infection is a common and serious complication in the field of orthopedics, which frequently leads to excessive bone destruction and fracture nonunion. Staphylococcus aureus (S. aureus) infection affects bone cell function which, in turn, causes bone destruction. Bone is mainly regulated by osteoblasts and osteoclasts. Osteoclasts are the only cell type with bone resorptive function. Their over-activation is closely associated with excessive bone loss. Understanding how S. aureus changes the functional state of osteoclasts is the key to effective treatment. By reviewing the literature, this paper summarizes several mechanisms of bone destruction caused by S. aureus influencing osteoclasts, thereby stimulating new ideas for the treatment of bone infection.     

高盐饮食通过激活破骨细胞促进小鼠骨质疏松发生

目的　明确高盐饮食对小鼠破骨细胞分化的影响及其促进骨质疏松的作用。 方法　获得C57小鼠骨髓单个核细胞,在体外定向诱导破骨分化体系中比较对照组（未加入氯化钠）、低剂量组（20 mmol/L氯化钠）及高剂量组（40 mmol/L氯化钠）破骨细胞生成数量;将24只C57小鼠随机平均分为正常组（普通饲料喂养）、卵巢切除组（切除卵巢,普通饲料喂养）、高盐饮食组（切除卵巢,8%高盐饲料+生理盐水喂养）,6周后分析3组左侧股骨破骨细胞生成数量并测定血清骨吸收及骨保护指标水平;选择右侧股骨进行显微CT扫描,比较3组骨组织形态计量学参数。 结果　体外高盐状态破骨细胞生成数量明显增加;体内高盐饮食组破骨细胞生成数量明显高于其它两组,I型胶原羧基端肽和核因子κ B受体活化因子配体不同程度增加,骨保护素水平显著降低,骨体积分数、骨皮质厚度、骨小梁数量和骨小梁厚度下降尤为明显,骨表面积/骨体积、骨小梁分离度和骨小梁模式因子不同程度增高。 结论　高盐饮食可通过激活小鼠破骨细胞分化及其功能活化增强骨吸收作用,进而诱导骨质疏松发生。     

Formation of osteoclast-like cells on HA and TCP ceramics

An essential property of bone substitute materials is that they are integrated into the natural bone remodelling process, which involves the resorption by osteoclast cells and the formation by osteoblast cells. If monocyte cells adhere to a calcium phosphate surface (bone or bone substitute material), they can fuse together and form multinucleated osteoclast cells. In this study we show that osteoclast-like cells derived from a human leukoma monocytic lineage responded in a different way to tricalciumphosphate (TCP) than to hydroxyapatite (HA) ceramics. Both ceramics were degraded by resorbing cells; however, HA enhanced the formation of giant cells. The osteoclast-like cells on HA formed a more pronounced actin ring, and larger lacunas could be observed. TCP ceramics are medically used as bone substitute materials because of their high dissolution rate. On the other hand, highly soluble calcium phosphate ceramics like TCP seem to be inappropriate for osteoclast resorption because they produce a high calcium concentration in the osteoclast interface and in the environment.     

A delivery system specifically approaching bone resorption surfaces to facilitate therapeutic modulation of microRNAs in osteoclasts

Dysregulated microRNAs in osteoclasts could cause many skeletal diseases. The therapeutic manipulation of these pathogenic microRNAs necessitates novel, efficient delivery systems to facilitate microRNAs modulators targeting osteoclasts with minimal off-target effects. Bone resorption surfaces characterized by highly crystallized hydroxyapatite are dominantly occupied by osteoclasts. Considering that the eight repeating sequences of aspartate (D-Asp₈) could preferably bind to highly crystallized hydroxyapatite, we developed a targeting system by conjugating D-Asp₈ peptide with liposome for delivering microRNA modulators specifically to bone resorption surfaces and subsequently encapsulated antagomir-148a (a microRNA modulator suppressing the osteoclastogenic miR-148a), i.e. (D-Asp₈)-liposome-antagomir-148a. Our results demonstrated that D-Asp₈ could facilitate the enrichment of antagomir-148a and the subsequent down-regulation of miR-148a in osteoclasts in vivo, resulting in reduced bone resorption and attenuated deterioration of trabecular architecture in osteoporotic mice. Mechanistically, the osteoclast-targeted delivery depended on the interaction between bone resorption surfaces and D-Asp₈. No detectable liver and kidney toxicity was found in mice after single/multiple dose(s) treatment of (D-Asp₈)-liposome-antagomir-148a. These results indicated that (D-Asp₈)-liposome as a promising osteoclast-targeting delivery system could facilitate clinical translation of microRNA modulators in treating those osteoclast-dysfunction-induced skeletal diseases.     

Fibril formation pH controls intrafibrillar collagen biomineralization in vitro and in vivo

We demonstrate that intrafibrillar, homogenous collagen biomineralization can be achieved by controlling self-assembly under mildly alkaline conditions. Using dense collagen (DC) gels as an osteoid model, we modulated their fibrillogenesis environment to evaluate the effects of fibrillogenesis pH on the protein charge distribution and ultimately on biomineralization. Cationic and anionic dye staining and electron cryomicroscopy analyses established that fibrillogenesis under mildly alkaline conditions promotes the formation of electronegative charges within the protein (anionic DC gels). These charges are stable upon titration of the gel pH to physiological values. Subsequent exposure of anionic DC gels to simulated body fluid induced the intrafibrillar biomineralization of the gels, promoting a rapid, extensive formation of carbonated hydroxyapatite, and strongly impacting gel mechanical properties. The generality and significance of this approach has been addressed by implanting freshly made anionic DC gels in vivo, in a rat subcutaneous model. Subcutaneous implants showed an extensive, homogenous biomineralization as early as at day 7, indicating that anionic collagen gels rapidly self-mineralize upon contact with body fluids in a non-osseous implantation site. The control of collagen fibrillogenesis pH provides not only new interpretations to what has been called the collagen mineralization enigma by demonstrating that neat collagen can intrafibrilarly self-mineralize, but it will also set a new starting point for the use of DC gels in bone regenerative medicine, in addition as potential applications as mineralized tissue model or as slow-release delivery carriers.     

破骨细胞融合分子机制的研究进展

破骨细胞是体内介导骨吸收的主要细胞。破骨细胞融合过程包括:识别、半融合结构及融合孔形成,最后融合孔扩张、细胞内容物混合。破骨细胞融合与破骨细胞的大小及其骨吸收能力密切相关,对破骨细胞融合分子机制的研究可为骨吸收亢进性疾病的治疗寻求新的突破口。本文就国内外破骨细胞融合过程及其分子机制的最新研究进展进行综述。     

The effect of dose on rhBMP-2 signaling,delivered via collagen sponge,on osteoclast activation and in vivo bone resorption

While recombinant human bone morphogenetic protein (rhBMP)-2-based bone therapy presents potential osteoinductivity, it also leads concern due to transient osteoclast activation during early healing periods, ultimately limiting its clinical use. Therefore, we investigated in vivo and in vitro rhBMP-2 signaling which mediates early bone resorbing effect, depending on the dose, and attempted to inhibit this resorption phenomenon using NFAT inhibitor as a target molecule. High-dose of rhBMP-2 (20 μg/defect) enhanced osteoclast activation and the expression of bone resorption markers, compared to low dose (5 μg/defect) at one week after surgery in collagen sponge-delivered rat calvarial defect models. Interestingly, this trend was also observed in the expression of bone formation markers. In particular, rhBMP-2 upregulated RANKL expression, while it downregulated osteoprotegerin (OPG) expression, resulting in a dose-dependent increase in the ratio of RANKL to OPG. NFAT inhibitor (150 μm) treatment in vivo suppressed the high-dose effect of rhBMP-2 on both resorption and formation. In vitro results of rhBMP-2 signaling and NFAT inhibitor effects in rat mesenchymal stem cells showed similar trends as in vivo results. Microcomputer tomography-based evaluation at 4 weeks showed that combined treatment of NFAT inhibitor with 20 μg rhBMP-2 in vivo increased bone volume (BV) more than 20 μg rhBMP-2 alone, which showed little difference in BV compared to 5 μg of rhBMP-2. These results demonstrated that rhBMP-2 implantation concurrently signalized into enhanced osteoclastogenesis and osteoblastogenesis in vivo, dose-dependently. Ratio of RANKL/OPG might be an index for early bone resorbing activity of implanted rhBMP-2. A local cocktail treatment of NFAT inhibitor and high-dose rhBMP-2 might be an alternative to overcome early bone resorbing effects, thereby accelerating bone formation.     

骨桥蛋白在子宫内膜癌及子宫颈癌组织中的表达

目的：探讨骨桥蛋白(OPN)在子宫内膜癌及子宫颈癌组织中的表达及意义。 方法: 采用免疫组织化学SP法测定OPN在30例子宫内膜癌、20例子宫颈癌及30例正常对照组织的表达，并评价其与临床病理特征的关系。 结果: OPN在子宫内膜癌的阳性表达率为70%,显著高于正常增殖期子宫内膜（10%）及分泌期子宫内膜（50%），P<0.01; OPN在子宫颈癌的阳性表达率（55%）显著高于正常宫颈上皮(10%),P<0.01；而宫颈磷癌及宫颈腺癌组织的阳性表达率分别为53.3%及60.0%, 两者之间无统计学差异, P>0.05。OPN的阳性表达率随临床病理分期的增加及细胞分化程度降低而显著升高。 结论: OPN在子宫内膜癌及子宫颈癌组织均有显著高表达,其表达与肿瘤分期及分级密切相关。     

Electron microscope observations of centrioles in osteoclasts

Summary The osteoclast may contain multiple centrioles which are located in a single group surrounded by the Golgi complex. Each centriole exhibits the same structure as that of centrioles in other cell types. Microtubules, while present in all regions of the osteoclast are sometimes especially numerous in regions close to the centrioles.It is suggested that the centriole groups derive from a pooling of centrioles from the mononuclear precursors of the osteoclasts.This research was supported by the Danish Research Council. Grant no. 512-727, 512-819 and 521-1545.     

高免疫原性骨桥蛋白的构建、表达及鉴定

目的重组及表达高免疫原性骨桥蛋白（OPN）的融合蛋白。方法使用Lasergene软件分析OPN基因,选取出免疫原性高的区域作为目的片段。提取总RNA,应用逆转录、PCR等技术扩增OPN基因目的片段,将其分别与带有His标签的pET-32a及GST标签的pGEX-4T-1载体连接。构建表达载体,诱导表达OPN融合蛋白（分别含有His,GST—Tag）,并进行SDS—PAGE及Western blotting鉴定。结果扩增后获得一条351bp的DNA片段,经测序鉴定为目的片段序列,并实现了可溶性高表达,经Western blotting鉴定为高免疫原性OPN融合蛋白。结论采用原核表达经纯化后可获得高纯度高免疫原性的OPN融合蛋白,为进一步开发OPN诊断试剂打下基础。     

The effect of zoledronate-hydroxyapatite nanocomposites on osteoclasts and osteoblast-like cells in vitro

This study demonstrates that zoledronate containing hydroxyapatite nanocrystals (HA-ZOL) can be synthesized as a single crystalline phase up to a zoledronate content of about 7 wt% by direct synthesis in aqueous solution, at variance with what previously found for alendronate-hydroxyapatite nanocrystals (HA-AL). On increasing zoledronate incorporation, the length of the coherent crystalline domains and the crystal dimensions of hydroxyapatite decrease, whereas the specific surface area increases. Full profile fitting of the powder X-ray diffraction patterns does not indicate major structural modifications, but an increase of the hydroxyapatite unit cell, on increasing zoledronate content. These data, together with a structural similarity between hydroxyapatite and calcium zoledronate, suggest a preferential interaction between zoledronate and the hydroxyapatite faces parallel to the c-axis direction. Osteoblast-like MG-63 cells and human osteoclasts were cultured on HA-ZOL nanocrystals and as a comparison on HA-AL nanocrystals containing almost the same (about 7 wt%) bisphosphonate amount. The beneficial influence of bisphosphonates on osteoblast proliferation and differentiation is enhanced when the tests are performed in co-cultures. Similarly, the reduction of osteoclast proliferation and the increase of Caspase 3 production are dramatically enhanced in co-cultures, which highlight an even greater influence of HA-ZOL than HA-AL on osteoclast apoptosis.     

骨桥蛋白在食管鳞癌中的表达及其临床意义

目的观察骨桥蛋白（OPN）在食管鳞癌组织、癌旁组织和转移淋巴结的表达状况,探讨OPN在食管鳞癌中表达的临床意义。方法运用免疫组化S-P法检测44例食管鳞癌组织、癌旁组织和20例转移淋巴结免疫组化染色。结果OPN在食管癌组织、癌旁组织及转移淋巴结中的表达率分别为86．3％、0％和100％。癌组织中OPN主要表达于肿瘤细胞的细胞浆中。OPN的表达与肿瘤TNM分期、淋巴结转移状态有关,而与肿瘤位置、肿瘤直径、浸润深度及病理学分级无关。OPN在癌组织、癌旁组织及转移淋巴结表达强度亦存在显著性差异。结论食管鳞癌组织中OPN主要由肿瘤细胞产生。OPN与肿瘤的浸润、转移有关,反映了肿瘤的生物学特性。     

Increase of fibronectin and osteopontin in porcine hearts following ischemia and reperfusion

Following a severe ischemic injury or myocardial infarction, the extracellular matrix (ECM) of the heart is involved in pathophysiological conditions such as dilatation and cardiac dysfunction. Osteopontin (OPN) has been shown to interact with fibronectin suggesting its possible role in matrix organization, stability and wound healing. There is increased expression of OPN in several tissues in response to injury. Therefore, we tested the hypothesis that acute ischemia (2 h), followed by reperfusion (4 h) may induce early OPN and fibronectin in an isolated hemoperfused working porcine heart model. Twenty hearts were prepared and connected to a perfusion system. After 1 h of perfusion, these hearts were randomized to two groups: ten infarcted (MI, ramus circumflexus) and ten non-infarcted hearts (C). In addition, cardiac fibroblasts derived from infarcted, remote and control myocardium were investigated. In both groups, the heart rate, electrolytes, pH, blood gases, and lactate remained similar. The LVEDP and perfusion pressure of MI hearts increased significantly (P<0.05). The total fibronectin and OPN volume contents were clearly elevated in the infarct area. The matrix metalloproteinases (MMP-1 and MMP-8), fibronectin, OPN, TGF-₁ proteins and the mRNAs for fibronectin, TGF-₁, and OPN were significantly elevated in the infarct area as compared to the remote area and the non-infarcted hearts. Simultaneously, circulating carboxyterminal propeptide of type I procollagen (PICP) was released in the perfusion medium (threefold versus C). Fibroblast-like cells originating from the infarct area exhibited an enhanced OPN and fibronectin gene and protein expression compared to fibroblasts derived from control myocardium. Our data demonstrate the early appearance of the MMPs (increased collagen degrading enzymes) and PICP (a collagen synthesis marker) following ischemia and reperfusion. Moreover, OPN, fibronectin and TGF-₁ protein and gene expression are elevated after ischemia and reperfusion in the ex vivo working hemoperfused porcine heart model.This research was supported by the Deutsche Forschungsgemeinschaft (DFG) grants GR 1039/7-1 and GR 1039/7-2 to D.G.     

Multinucleated giant cells elicited around hydroxyapatite particles implanted in craniotomy defects are not osteoclasts

Background: The nature of the multinucleated giant cells (MNGC) elicited in contact with implantable biomaterials is still indecisive. Method: In Wistar rats the MNGC recruited after the implantation of hydroxyapatite (HA) particles in standardized skull defects were examined morphologically (at both the light and electron microscope levels), enzymatically (tartrate-resistant acid phosphatase and non-specific esterase), and after a challenge with salmon calcitonin. Results: The MNGC were of great size and contained abundant mitochondria, vacuoles, and vesicles throughout the cytoplasm; they were either tightly apposed to the HA surface or had long and thin processes penetrating the material. When processed for tartrate-resistant acid phosphatase, only a few cells were weakly stained. The staining was totally suppressed when samples were pretreated with cyanuric chloride in the MNGC but not in the host osteoclasts. Calcitonin induced the withdrewal of the host osteoclasts from the bone surface while the MNGC remained in contact with the HA material. Conclusion: The MNGC recruited to HA particles did not exhibit the morphologic, enzymatic and functional characteristics of the osteoclasts, and consequently must be regarded as macrophage polykaryons. © 1995 Wiley-Liss, Inc.     

Mineral chaperones: a role for fetuin-A and osteopontin in the inhibition and regression of pathologic calcification

Clinical nephrologists are well aware of the consequences of pathologic mineralization (calcification). Several studies have found a strong association between vascular and valvular mineralization and advanced or end-stage chronic kidney disease (CKD), with shorter survival times and increased morbidity. In the cardiology community, until quite recently, ectopic mineralization was considered harmless or even beneficial. Some still assume that atherosclerotic intima mineralization stabilizes atherosclerotic plaques, thus doing more good than harm. We suggest that vascular mineralization and indeed soft tissue mineralization in general may be a way in which the body deals with certain adverse situations involving local inflammation, associated tissue damage and tissue remodeling. Ectopic soft tissue mineralization resembles physiological bone mineralization in many ways. Markers of mineralizing bone also are present during soft tissue mineralization. We postulate that it may be possible to reverse soft tissue mineralization by applying selected principles of bone catabolism, namely mineral dissolution and phagocytosis. We consider putative strategies for therapeutic intervention to maximize the clearing of calcified debris particles. In particular, we discuss the roles of the plasma protein fetuin-A/alpha2HS-glycoprotein and the mineral-binding protein osteopontin in the prevention and possible regression of mineralization in disease.     

低氧和高二氧化碳作用下肺动脉高压形成过程中骨桥蛋白表达与分布的实验研究

目的： 观察慢性低O2高CO2大鼠肺动脉骨桥蛋白（OPN）及其基因的表达与分布情况，探讨OPN在肺动脉高压发病机制中的作用。方法： 48只雄性SD大鼠随机分为4组：即正常对照组（NC组），低O2高CO2 1周、2周和4周组（1HH、2HH和4HH）。采用RT-PCR法测定不同低O2高CO2 组大鼠离体肺动脉和肺组织骨桥蛋白（OPN）mRNA，免疫组织化学染色法测定肺细小动脉骨桥蛋白分布表达情况，ELISA 法定量测定肺组织匀浆OPN水平，Western blotting 法测定离体肺动脉OPN表达水平。结果： ①低O2高CO2各组大鼠mPAP和RV/LV+S均明显高于NC组（P<0.01），4组大鼠间mCAP比较无显著差异（P>0.05）；②低O2高CO2 各组大鼠肺组织和离体肺主动脉OPN mRNA表达水平显著高于对照组（均P<0.01）；③免疫组化法显示NC组大鼠肺组织OPN表达主要见于支气管、肺泡上皮细胞内，1HH组大鼠肺细小动脉内皮细胞、平滑肌细胞以及周围巨噬细胞均见有OPN的表达，其中以中膜平滑肌细胞最为明显，而2HH和4HH组大鼠OPN表达更加明显, 低O2高CO2 各组肺细小动脉OPN相对含量与对照组比较有显著差异（均P<0.01），且随缺氧时间的延长，骨桥蛋白表达也呈增高趋势；④ 低O2高CO2 各组大鼠肺组织匀浆OPN含量分别为正常对照组的1.69倍、2.28倍和2.87倍（均P<0.01）；⑤ Western blotting 分析1HH、2HH和4HH各组大鼠离体肺动脉OPN表达明显增强，与NC组比较有显著差异（均P<0.01）。结论： 慢性低O2高CO2能够刺激大鼠肺组织和肺动脉OPN及其基因表达增强，OPN可能在慢性低O2高CO2性肺动脉高压发病的病理和病理生理过程中起着重要的作用。     

子宫内膜异位症患者血清与腹腔液中骨桥蛋白的表达及意义

目的探讨骨桥蛋白(OPN)在子宫内膜异位症患者血清与腹腔液中的表达及意义。方法用酶联免疫吸附方法检测40例子宫内膜异位症患者术前血清与腹腔液中骨桥蛋白的表达情况。结果 (1)子宫内膜异位症患者术前血清OPN平均水平为(46.68±16.38)ng/ml(16.69~101.85ng/ml),明显高于对照组患者水平(P<0.05);内异症组患者的腹腔液OPN平均水平为(47.47±15.90)ng/ml(20.35~97.56ng/ml),也高于对照组患者的表达水平(P<0.05)。(2)Ⅲ~Ⅳ期内异症患者的血清与腹腔液中OPN的表达水平均高于I~Ⅱ期患者,差异具有显著性(P<0.05)。(3)内异症患者的血清与腹腔液的OPN表达呈正相关关系(r=0.669,P<0.05);对照组血清与腹腔液的0PN表达亦呈正相关关系(r=0.538,P<0.05)。结论 OPN在子宫内膜异位症患者血清与腹腔液中高表达,可能促进异位内膜黏附和侵袭,促进血管生成,在子宫内膜异位症发生发展中起重要作用,OPN在内异症的早期诊断方面有一定应用价值。