氟化钠对体外培养破骨细胞的影响

目的 :探讨氟对体外培养破骨细胞的影响。方法 :从新生兔四肢长骨中分离出破骨细胞 ,将其与盖玻片、骨片共同培养 ,于培养 6 h后加入不同浓度 (10 - 7～ 10 - 3mol/L)的氟化钠 (Na F) ,用酶组织化学染色法测定 Na F对破骨细胞的作用 ,用倒置相差显微镜和图像分析处理系统对骨吸收陷窝的数目和面积进行检测。结果 :Na F使抗酒石酸酸性磷酸酶(TRAP)染色阳性的多核细胞 ,即破骨细胞数目减少。染氟 10 - 5m ol/L、 10 - 4mol/L、 10 - 3mol/L 时 ,破骨细胞数目与对照组相比差异有显著性 (P<0 .0 5 ) ,细胞体积缩小 ,胞浆空泡增多 ,其中 10 - 3m ol/L Na F组破骨细胞改变尤为明显。与对照组相比 ,10 - 5mol/L、 10 - 4mol/L、 10 - 3mol/L Na F组骨片上骨吸收陷窝数量和面积均减少 (P<0 .0 5 ) ,而且陷窝周缘也变得模糊。结论 :在本实验浓度和时间范围内 ,Na F可直接损伤破骨细胞并抑制破骨细胞的骨吸收功能。     

基质Gla蛋白对骨量的调节作用及其机制

目的 检测基质Gla蛋白（MGP）对小鼠骨量的影响,分析其作用机制。 方法 利用腺相关病毒干涉小鼠体内MGP基因表达,microCT扫描分析检测小鼠骨量,利用抗酒石酸酸性磷酸酶（TRAP）染色检测小鼠股骨破骨细胞的数目,利用ELISA检测骨转换标志物TRAP5b水平。 结果 与对照组相比,经过MGP干扰处理的小鼠骨体积分数显著降低,骨小梁数目和骨小梁厚度也显著减少,骨小梁间隔显著增高,差异均有统计学意义（均P<0.01）。小鼠股骨切片TRAP阳性破骨细胞数目增多,血清TRAP5b水平显著增加（P<0.01）。 结论 MGP可能通过抑制破骨细胞形成和骨吸收来维持骨量。     

记忆T细胞CD45~+通过RANKL对破骨细胞的影响及调控

目的分析记忆T细胞CD45~+(CD45RO)通过核因子κβ受体活化因子配体(RANKL)对大鼠破骨细胞的影响及调控,观察其与骨质疏松发病机制相关性,旨在为未来骨质疏松的治疗提供新靶点。方法购入40只24 h内新生Wistar大鼠,使用机械分离法,取大鼠四肢长骨,经消化酶法提取破骨细胞,进行培养,后将大鼠处死经酒精浸泡后分离其股骨、肱骨及胫骨,剔除软组织与软骨骺,处理后,将大鼠随机分为A、B、C、D 4组,分别向内加入RANKL试剂,各组大鼠RANKL浓度分别为0μg/L、50μg/L、100μg/L、150μg/L,对比不同浓度RANKL大鼠模型破骨细胞数量、骨陷窝面积,同时计算破骨细胞所含细胞核数量,即核数目;测定不同RANKL浓度大鼠血清抗酒石酸酸性磷酸酶-5b(TRACP-5b)表达及CD45RO表达;并分析CD45RO表达与破骨细胞数量、骨陷窝面积及核数目的相关性。结果随着RANKL浓度增加,大鼠破骨细胞数量增加、骨陷窝面积增加、核数量增加,TRACP-5b、CD45RO表达均升高,但C组与D组比较差异无统计学意义(P> 0. 05),其他各组组间比较差异有统计学意义(P <0. 05)。经双变量Pearson相关性分析检验证实,大鼠CD45RO与破骨细胞数量、骨陷窝面积、核数目均呈正相关(r=0. 958、0. 834、0. 972,P <0. 05)。结论记忆T细胞CD45~+能够产生大量炎性因子,通过对RANKL/RANK/OPG信号传导系统的直接作用,对破骨细胞及成骨细胞产生影响,来促进骨吸收,抑制骨形成,可能参加了骨质疏松的调控,可将其作为未来骨质疏松治疗研究的新靶点。     

1α,25-(OH)_2D_3促进骨细胞形成及骨吸收活性（英文）

目的研究1α,25-二羟维生素D_3(1α,25-(OH)_2D_3)对破骨细胞(osteoclast,OC)形成及骨吸收活性的影响。方法在巨噬细胞集落刺激因子(macrophage colony stimulating factor,M-CSF)和核因子-κB受体活化因子配体(receptor activator for nuclear factor-иB ligand,RANKL)诱导破骨细胞前体细胞RAW264.7细胞分化为OC的基础上,添加不同浓度1α,25-(OH)_2D_3(10~(-7),10~(-8)和10~(-9)mol/L),通过抗酒石酸酸性磷酸酶(tartrate resistant acid phosphatase,TRAP)染色鉴定成熟OC的形成,环境扫描电镜观察牛皮质骨片吸收陷窝。结果各浓度1α,25-(OH)_2D_3均可以促进RAW264.7细胞分化为OC,并增强其骨吸收活力。其中,10~(-8)mol/L的1α,25-(OH)_2D_3诱导效果最好,所形成的OC数最多,骨吸收活性最强。结论 1α,25-(OH)_2D_3能促进OC形成,增强骨吸收活性,从而调节骨代谢。     

四种小鼠破骨细胞体外诱导培养方法的比较

[目的]比较不同的破骨细胞体外诱导培养方法,为研究外源性因素对骨代谢影响提供方法学基础。[方法]采用骨髓基质细胞和骨髓单核细胞三维共育、骨髓基质细胞培养液转移刺激骨髓单核细胞、重组核因子-κB受体激活物配基（RANKL）诱导骨髓单核细胞和RANKL诱导单核巨噬细胞株RAW264．7四种方法体外诱导破骨细胞。活细胞示踪剂CM-DiI标记RAW264．7后,用激光共聚焦显微镜扫描观察破骨细胞形成过程;抗酒石酸酸性磷酸酶（TRAP）染色法染色破骨细胞。[结果]四种方法均成功地诱导培养出由多个细胞互相融合的TRAP阳性多核破骨细胞。加入RANKL的方法诱导出的破骨细胞较大,数量也较多,方法简单。正常骨髓基质细胞诱导形成的破骨细胞形态较小,数量也相对较少,过程复杂,但可以用来研究干预因素作用于骨髓基质细胞后对破骨细胞分化的间接影响。在对照组及诱导培养体系中均可观察到一类不互相融合的TRAP阳性细胞,含1～3个核,TRAP染色多集中在胞核周围,此类细胞可能是尚未分化的破骨细胞前体。而互相融合的破骨细胞TRAP染色见于整个胞浆。[结论]各种破骨细胞体外诱导培养方法各有优势,应根据不同的实验目的选用不同的破骨细胞诱导培养方法。     

成纤维母细胞样基质细胞在人工假体无菌性松动中的作用

目的研究成纤维母细胞样基质细胞（FBSCs）在人工关节置换术后无菌性松动中的作用。方法酶消化法从人工假体周围假膜标本中分离FBSCs并作体外培养并加入骨水泥颗粒,检测FBSCs核因子KB受体激动剂配体（RANKL）和肿瘤坏死因子（TNFα）mRNA表达的变化。同时FBSCs与外周血单个核细胞共培养,加入巨噬细胞克隆集落刺激因子、骨保护素或抗TNFα中和抗体;检测破骨细胞的形成并比较其生物学活性。结果假膜组织来源的FBSCs均表达RANKL和TNFamRNA;骨水泥颗粒组FBSCs RANKL和TNFα mRNA的表达量分别是对照组的1．9倍和2．1倍（P=0．002;P=0．004）。共培养细胞培养终末时见到抗酒石酸磷酸酶染色阳性的多核细胞,同时象牙磨片上有虫噬样骨吸收陷窝的形成;OPG完全阻断共培养组抗酒石酸磷酸酶染色阳性多核细胞以及虫噬样骨吸收陷窝的形成,而TNFα中和抗体对上述现象无显著抑制作用。结论骨水泥颗粒显著增强人工假体周围假膜中FBSCs RANKL和TNF mRNA的表达;FBSCs通过表达RANKL而支持外周血单个核细胞分化为具有骨吸收活性的破骨细胞。     

鲫鱼卵唾液酸糖蛋白对小鼠破骨细胞活性的影响

目的研究鲫鱼卵唾液酸糖蛋白(Carassius auratus sialyglycoproteins,CA-SGP)对破骨细胞活性的影响。方法通过M-CSF和RANKL体外诱导小鼠骨髓造血细胞分化,建立破骨细胞模型,以MTT法检测CA-SGP对破骨细胞前体细胞及破骨细胞增殖活性的影响;采用TRAP染色和TRAP含量测定法评价其对小鼠骨髓细胞向破骨细胞分化的影响;利用酶联免疫吸附法(enzyme-linked immunosorbent assay,ELISA)检测CA-SGP对小鼠破骨细胞促炎症因子IL-6、IL-1β和TNF-α分泌的影响。结果 CA-SGP对破骨细胞前体细胞和破骨细胞的增殖活性无影响,可明显抑制小鼠骨髓造血细胞向破骨细胞的分化,且在早期抑制作用最强。ELISA试剂盒检测结果表明,CA-SGP能明显降低小鼠破骨细胞IL-6、IL-1β和TNF-α的含量。结论鲫鱼卵唾液酸糖蛋白可明显抑制小鼠骨髓造血细胞向破骨细胞的分化以及破骨细胞炎症因子的分泌。     

lα,25-(OH)_2D_3对体外培养小鼠破骨细胞形成及活性的影响

目的研究1α,25-(OH)_2D_3对体外培养小鼠破骨细胞(osteoclast,OC)分化及骨吸收的直接影响。方法采用核因子κB受体活化因子配体(receptor activator of nuclear factor-κB ligand,RANKL)和巨噬细胞集落刺激因子(macrophage colony stimulating factor,M-CSF)诱导体外培养C57BL/6J小鼠OC前体分化成OC。RTCA检测细胞增殖、抗酒石酸酸性磷酸酶染色检测OC数量、Western blot检测OC骨吸收功能相关蛋白和转录因子蛋白表达水平、免疫荧光和扫描电镜观察OC骨架和形态、骨细胞培养板分析骨吸收陷窝面积,观察10 nM 1α,25-(OH)_2D_3对OC形成及骨吸收功能的影响。结果 10nmol/L1α,25-(OH)_2D_3对细胞增殖无显著影响,但能显著抑制OC形成,抑制骨吸收功能相关蛋白和转录因子蛋白的表达,阻碍OC骨架肌动蛋白环和伪足小体的形成,减弱OC骨吸收功能。结论1α,25-(OH)_2D_3能抑制体外培养小鼠OC的形成及骨吸收功能,在钙磷代谢紊乱及骨骼疾病中对OC具有直接调控作用。[营养学报,2019,41(6):593-600]     

珊瑚羟基磷灰石人工骨与成骨-破骨细胞共育系联合培养的实验研究

目的 通过成骨细胞和破骨细胞及珊瑚羟基磷灰石人工骨共育的研究,探索骨重建的过程.方法 新西兰兔骨组织,分离成骨、破骨细胞,混合培养,建立成骨-破骨细胞-珊瑚羟基磷灰石人工骨共育体系培养,观察细胞形态等的变化.结果 共育系中成骨细胞和破骨细胞生长良好,新骨不断形成,骨重塑加快,珊瑚羟基磷灰石人工骨降解时间缩短.结论 成骨细胞、破骨细胞在珊瑚羟基磷灰石人工骨共育环境中生长良好,模拟了接近体内的环境,是一种简单、有效、可行的实验方法.     

过量维生素A摄入与骨质疏松

近年来发现过量维生素A可能是引起骨质疏松症的因素之一.成骨细胞和破骨细胞上可能都存在视黄醇受体,视黄酸抑制成骨细胞发挥功能,刺激破骨细胞形成.如果持续摄入高剂量的维生素A,骨量丢失加重骨脆性危险,最终导致骨折.     

Lycopene I--effect on osteoclasts: lycopene inhibits basal and parathyroid hormone-stimulated osteoclast formation and mineral resorption mediated by reactive oxygen species in rat bone marrow cultures

Osteoclasts have been shown to produce reactive oxygen species (ROS) that can stimulate bone resorption. We explored the hypothesis that lycopene, the antioxidant carotenoid from tomatoes, can inhibit mineral resorption by inhibiting osteoclast formation and the production of ROS. Cells from bone marrow prepared from rat femur were plated into 16-well calcium phosphate-coated Osteologic Multi-test Slides and cultured in alpha-minimal essential medium supplemented with dexamethasone, beta-glycerophosphate, and ascorbic acid. The cells were treated with varying doses of lycopene in the absence or presence of parathyroid hormone (PTH) at the start of culture and at each medium change (i.e., every 48 hours). On day 8, mineral resorption pits were quantitated. Similar, parallel experiments were carried out in 12-well plastic dishes to assess tartrate-resistant acid phosphatase (TRAP) activity. Results showed that lycopene inhibited TRAP + formation of multinucleated cells in both vehicle- and PTH-treated cultures. Osteoclasts reduced nitroblue tetrazolium (NBT) to purple-colored formazan, indicating the presence of ROS in these cells. The formazan-staining cells were decreased by treatment with 10(-5) M lycopene, indicating that lycopene inhibited the formation of ROS-secreting osteoclasts. In conclusion, we have shown that lycopene inhibits basal and PTH-stimulated osteoclastic mineral resorption and formation of TRAP + multinucleated osteoclasts, as well as the ROS produced by osteoclasts. These findings are novel and may be important in the pathogenesis, treatment, and prevention of osteoporosis.     

Direct inhibition of osteoclast formation and activity by the vitamin E isomer gamma-tocotrienol

Vitamin E homologues, specifically tocotrienols, have been shown to have favorable effects on bone. They possess properties that are indicative of anti-resorptive activity, suggesting the potential for vitamin E in preventing bone loss. To investigate the anti-resorptive activity of the various vitamin E homologues, we cultured human osteoclasts from blood-derived CD14+ cells on collagen, dentin, and calcium phosphate substrates, with some samples supplemented with vitamin E homologues in their cell culture medium. These were compared to the clinically used bisphosphonate, pamidronate. Compounds were either added at the start of culture to study effects on osteoclast formation, or at the start of osteoclastic resorption to determine their effects on activity. The alpha- and gamma-tocotrienol isomers inhibited osteoclast formation without consequent reduction in total cell number. Only gamma-tocotrienol inhibited osteoclast activity without toxicity. Gamma-tocotrienol was the most potent inhibitor of both osteoclast formation and activity and requires further investigation into its anti-resorptive effects on bone.     

Regulation of bone cell function by acid-base balance

Bone growth and turnover results from the coordinated activities of two key cell types. Bone matrix is deposited and mineralised by osteoblasts and it is resorbed by osteoclasts, multinucleate cells that excavate pits on bone surfaces. It has been known since the early 20th century that systemic acidosis causes depletion of the skeleton, an effect assumed to result from physico-chemical dissolution of bone mineral. However, our own work has shown that resorption pit formation by cultured osteoclasts was absolutely dependent on extracellular acidification; these cells are inactive at pH levels above about 7.3 and show maximum stimulation at a pH of about 6.9. Bone resorption is most sensitive to changes in H+ concentration at a pH of about 7.1 (which may be close to the interstitial pH in bone). In this region pH shifts of < 0.05 units can cause a doubling or halving of pit formation. In whole-bone cultures, chronic HCO3- acidosis results in similar stimulations of osteoclast-mediated Ca2+ release, with a negligible physico-chemical component. In vivo, severe systemic acidosis (pH change of about -0.05 to -0.20) often results from renal disease; milder chronic acidosis (pH change of about -0.02 to -0.05) can be caused by excessive protein intake, acid feeding, prolonged exercise, ageing, airway diseases or the menopause. Acidosis can also occur locally as a result of inflammation, infection, wounds, tumours or diabetic ischaemia. Cell function, including that of osteoblasts, is normally impaired by acid; the unusual stimulatory effect of acid on osteoclasts may represent a primitive 'fail-safe' that evolved with terrestrial vertebrates to correct systemic acidosis by ensuring release of alkaline bone mineral when the lungs and kidneys are unable to remove sufficient H+ equivalent. The present results suggest that even subtle chronic acidosis could be sufficient to cause appreciable bone loss over time.     

Extracellular pH regulates bone cell function

The skeletons of land vertebrates contain a massive reserve of alkaline mineral (hydroxyapatite), which is ultimately available to buffer metabolic H+ if acid-base balance is not maintained within narrow limits. The negative impact of acidosis on the skeleton has long been known but was thought to result from passive, physicochemical dissolution of bone mineral. This brief, selective review summarizes what is now known of the direct functional responses of bone cells to extracellular pH. We discovered that bone resorption by cultured osteoclasts is stimulated directly by acid. The stimulatory effect is near-maximal at pH 7.0, whereas above pH 7.4, resorption is switched off. In bone organ cultures, H+-stimulated bone mineral release is almost entirely osteoclast-mediated, with a negligible physicochemical component. Acidification is the key requirement for osteoclasts to excavate resorption pits in all species studied to date, and extracellular H+ may thus be regarded as the long-sought osteoclast activation factor. Acid-activated osteoclasts can be stimulated further by agents such as parathyroid hormone, 1,25-dihydroxycholecalciferol, and receptor activator of nuclear factor kappaB ligand. Osteoclasts may respond to pH changes via H+-sensing ion channels such as transient receptor potential vanilloid 1, a nociceptor that is also activated by capsaicin. Acidosis also exerts a powerful, reciprocal inhibitory effect on the mineralization of bone matrix by cultured osteoblasts. This is caused by increased hydroxyapatite solubility at low pH, together with selective inhibition of alkaline phosphatase, which is required for mineralization. Diets or drugs that shift acid-base balance in the alkaline direction may provide useful treatments for bone loss disorders.     

Effects of local and whole body irradiation on appearance of osteoclasts during wound healing of tooth extraction sockets in rats

We examined effects of local and whole body irradiation before tooth extraction on appearance and differentiation of osteoclasts in the alveolar bone of rat maxillary first molars. Wistar rats weighting 100 g were divided into three groups: non-irradiation group, local irradiation group, and whole body irradiation group. In the local irradiation group, a field made with lead blocks was placed over the maxillary left first molar tooth. In the whole body irradiation group, the animals were irradiated in cages. Both groups were irradiated at 8 Gy. The number of osteoclasts around the interradicular alveolar bone showed chronological changes common to non-irradiated and irradiated animals. Several osteoclasts appeared one day after tooth extraction, and the maximal peak was observed 3 days after extraction. Local irradiation had no difference from non-irradiated controls. In animals receiving whole body irradiation, tooth extraction one day after irradiation caused smaller number of osteoclasts than that 7 day after irradiation during the experimental period. Whole body-irradiated rats had small osteoclasts with only a few nuclei and narrow resorption lacunae, indicating deficiency of radioresistant osteoclast precursor cells. Injection of intact bone marrow cells to whole body-irradiated animals immediately after tooth extraction recovered to some content the number of osteoclasts. These findings suggest that bone resorption in the wound healing of alveolar socket requires radioresistant, postmitotic osteoclast precursor cells from hematopoietic organs, but not from local sources around the alveolar socket, at the initial phase of wound healing.     

Energy regulation by the skeleton

Bones of the skeleton are constantly remodeled through bone resorption by cells called osteoclasts and bone formation by cells called osteoblasts. Both cell types are under multi-hormone control. New research findings demonstrate that bone formation by osteoblasts is negatively regulated by the hormone leptin, which is secreted by adipocytes and acts through the leptin receptor in the central nervous system and ultimately through the sympathetic nervous system. Leptin deficiency leads to increased osteoblast activity and increased bone mass. Reciprocally, expression of the Esp gene, exclusive to osteoblasts, regulates glucose homeostasis and adiposity through controlling the osteoblastic secretion of the hormone-like substance osteocalcin. An undercarboxylated form of osteocalcin acts as a regulator of insulin in the pancreas and adiponectin in the adipocyte to modulate energy metabolism. Osteocalcin deficiency in knockout mice leads to decreased insulin and adiponectin secretion, insulin resistance, higher serum glucose levels and increased adiposity.     

Lead exposure inhibits fracture healing and is associated with increased chondrogenesis, delay in cartilage mineralization, and a decrease in osteoprogenitor frequency

Lead exposure continues to be a significant public health problem. In addition to acute toxicity, Pb has an extremely long half-life in bone. Individuals with past exposure develop increased blood Pb levels during periods of high bone turnover or resorption. Pb is known to affect osteoblasts, osteoclasts, and chondrocytes and has been associated with osteoporosis. However, its effects on skeletal repair have not been studied. We exposed C57/B6 mice to various concentrations of Pb acetate in their drinking water to achieve environmentally relevant blood Pb levels, measured by atomic absorption. After exposure for 6 weeks, each mouse underwent closed tibia fracture. Radiographs were followed and histologic analysis was performed at 7, 14, and 21 days. In mice exposed to low Pb concentrations, fracture healing was characterized by a delay in bridging cartilage formation, decreased collagen type II and type X expression at 7 days, a 5-fold increase in cartilage formation at day 14 associated with delayed maturation and calcification, and a persistence of cartilage at day 21. Fibrous nonunions at 21 days were prevalent in mice receiving very high Pb exposures. Pb significantly inhibited ex vivo bone nodule formation but had no effect on osteoclasts isolated from Pb-exposed animals. No significant effects on osteoclast number or activity were observed. We conclude that Pb delays fracture healing at environmentally relevant doses and induces fibrous nonunions at higher doses by inhibiting the progression of endochondral ossification.     

锌缺乏和过量对小鼠胚胎体外骨形成的影响

目的　研究锌缺乏和锌过量对骨形成的影响。方法　采用小鼠胚胎长骨体外培养 ,通过骨组织的形态图象分析及长骨肥大区软骨细胞计数和骨化区破骨细胞计数以及羟脯氨酸( Hyp)含量和碱性磷酸酶 ( AKP)的活性测定研究锌缺乏和锌过量对骨形成的影响。结果　当培养基锌浓度高至 1× 1 0 -3 mol/ L时 ,长骨总长度及面积和骨干长度及面积、羟脯氨酸的含量、肥大区软骨细胞数均低于对照组 ;破骨细胞数高于对照组 ;当培养基锌浓度为 5× 1 0 -4 mol/ L时 ,AKP活性开始降低 ,当为 1× 1 0 -3 mol/ L时 ,AKP活性降低更为明显 ,锌缺乏时 ,AKP活性也降低。结论　锌缺乏和锌过量均可影响骨的发育。AKP在锌对骨发育的研究中是一个较为敏感的指标     

Nutritional factors affecting poultry bone health

Outlined are two main current research concerns relating to skeletal disorders in poultry: (a) osteoporosis in egg-laying hens; (b) leg problems caused by rapid bone growth in broiler chickens. Surveys indicate that 30% of caged laying hens suffer at least one lifetime fracture (a severe welfare issue). Modern hybrids produce one egg per d for 50 weeks. For this period 'normal' bone turnover ceases; only medullary bone (MB) is formed, a woven bone type of limited structural value. MB is resorbed for eggshell formation alongside structural bone, leading to increased fracture risk. Avian osteoporosis is reduced by activity and genetic selection but nutrition is also important. Fluoride and vitamin K are beneficial but the timing of nutritional intervention is important. Ca, inorganic P and vitamin D must be adequate and the form of Ca is critical. Limestone fed as particulates benefits skeletal and eggshell quality. In hens fed particulate limestone compared with flour-fed hens the tibiotarsus breaking strength and radiographic density are increased at 56 weeks of age (P<0.01 and P<0.001 respectively) and the number of tartrate-resistant acid phosphatase-positive stained active osteoclasts (mean number per microscopic field) is decreased (P<0.001). In broiler (meat) chickens selection for rapid growth from approximately 50 g to 3 kg in 42 d has inadvertently produced skeletal disorders such as tibial dyschondroplasia, rickets and associated valgus-varus deformities leading to lameness. The beneficial skeletal effects during growth of increased dietary n-3 PUFA:n-6 PUFA (utilising salmon oil) have been demonstrated. Experiments simulating daylight UVB levels have produced beneficial skeletal effects in Ca- and vitamin D-deficient chicks.