高纯度破骨样细胞体外培养及功能表达的研究

目的建立大量高纯度破骨样细胞体外培养的方法,并运用分子生物学技术观察体外诱导培养的破骨样细胞标志酶的基因表达。方法按照巨噬细胞集落刺激因子（M-CSF） 30 ng/mL、核因子κB受体活化因子配基（RANKL） 50 ng/mL的质量浓度对骨髓单个核细胞诱导培养6 d,利用形态学观察、抗酒石酸酸性磷酸酶（TRAP）染色、Giemsa染色、骨吸收陷窝检测以及破骨细胞标志酶基因表达的检测,对生成的破骨样细胞进行鉴定。结果实验获得的破骨样细胞中,TRAP阳性的单核破骨样细胞多见、TRAP阳性的多核破骨样细胞数量相对较少,胞核从2个到十几个不等;光镜下牙本质片上可见各种形态的骨吸收陷窝;应用RT-PCR方法证实破骨样细胞表达膜型基质金属酶（MT1-MMP）、基质金属蛋白酶-9（MMP-9）、TRAP和组织蛋白酶K（CK）4种标志酶。结论以M-CSF和RANKL作为诱导因子,对大鼠骨髓单个核细胞进行诱导培养可以获得具有典型的破骨细胞形态特征,可以表达特征性标志酶基因,且数量大、纯度高。     

体外培养鼠破骨细胞对牙体硬组织的吸收实验

目的本文旨在观察体外培养鼠破骨细胞对牙体硬组织的吸收。方法采用鼠长骨机械分离法获得破骨细胞,通过破骨细胞形态学观察及其特异性染色鉴定后,在牙本质上培养破骨细胞并用扫描电镜观察其吸收情况。结果鼠破骨细胞能在牙本质磨片上形成大小不一、形态不规则的吸收陷窝。结论长骨机械分离法可获得一定量特异性染色阳性的破骨细胞,并可在牙本质磨片上形成明显吸收陷窝。     

唑来膦酸对体外破骨细胞性骨吸收影响的研究

目的：研究唑来膦酸（zoledronicacid,ZOL）对体外培养的破骨细胞骨（osteoclastic0C）吸收的抑制作用。方法：体外培养兔破骨细胞,通过抗酒石酸酸性磷酸酶（TRAP）染色破骨细胞计数、图像分析计算骨吸收陷窝面积、吖啶橙染色计算破骨细胞凋亡比率观察不同浓度唑来膦酸对细胞影响。结果：随着唑来膦酸浓度增高,TRAP阳性破骨细胞数量减少,骨吸收陷窝数量、陷窝面积亦减少;凋亡OC数目增多,凋亡率亦升高。结论：唑来膦酸通过抑制OC活性而直接抑制OC骨吸收功能,随其浓度增加而抑制作用增强。     

牙周膜成纤维细胞对外周血单个核细胞分化的影响

目的：探讨牙周膜成纤维细胞在破骨细胞形成过程中作用;观察破骨样细胞的生长过程。方法：本实验以含有1α,25（OH）2D3和地塞米松的培养基将牙周膜成纤维细胞、单个核细胞分别进行单独或直接共培养,每3d对TRAP阳性多核破骨细胞的数量及牙本质磨片的吸收陷窝数目和面积分别进行记录、计算。结果：不同时间段间的TRAP阳性单个核细胞与TRAP阳性多核细胞数目相比较,差异具有统计学意义（P〈0.05）;同时,不同组间的吸收陷窝数目和面积比较,差异具有显著性（P〈0.001）。牙周膜成纤维细胞明显增加了共培养组TRAP阳性多核细胞数量、吸收陷窝数目和面积。然而牙周膜成纤维细胞组与单个核细胞组之间的吸收陷窝数目与吸收陷窝面积差异无统计学意义。结论：末梢血单个核细胞需在牙周膜成纤维细胞存在的条件下,才能形成多核的破骨样细胞。同时,在共培养中,可以发现破骨样细胞在体外存活的时间短暂。     

阿伦膦酸盐在不同分化阶段对破骨细胞生成及骨吸收功能的影响

目的：研究阿伦膦酸盐作用在不同分化阶段对破骨细胞生成及骨吸收功能的影响。方法：体外分离骨髓单核细胞,用30μg/L M-CSF对其预诱导3d,然后将细胞分为A、B、C、D四组。A组（对照组）,用50μg/L M-CSF＋100μg/L RANKL进行破骨细胞诱导;B、C、D组除加入上述诱导因子外,在不同时间点加入0.1μmol/L阿伦膦酸盐（ALN）,加入时间分别为：B组第0～2天加入,C组第3～4天加入,D组第5～6天加入。检测每组细胞正式诱导第6天TRAP染色情况及牙本质磨片吸收陷窝情况。结果：各组细胞均有TRAP阳性多核破骨细胞生成,并在牙本质磨片上形成吸收陷窝;但A组TRAP阳性多核细胞数目、吸收陷窝数目及陷窝面积最高,D组次之,B组最差。结论：阿伦膦酸盐在破骨细胞分化阶段作用越早,对破骨细胞生成的抑制效应越大,所形成破骨细胞骨吸收能力越差。     

VEGF对兔骨髓细胞分化形成破骨细胞的诱导作用

目的:观察血管内皮生长因子(VEGF)对兔骨髓细胞分化为破骨细胞的诱导作用。方法:应用终未浓度为5 ng/mL的人重组rhVEGF体外诱导培养兔骨髓细胞,并与骨片共同培养,分别于诱导培养后3、6、9 d用抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphatase,TRAP)染色进行破骨细胞鉴定计数和骨片吸收陷窝数目的统计。结果:诱导培养3 d,实验组与对照组的破骨细胞数和骨吸收陷窝数无显著性差异(P>0.05),而随着诱导培养时间延长,实验组两者的数量明显增加,在诱导培养6 d和9 d时,均明显高于对照组,差异有统计学意义(P<0.05)。结论:VEGF具有体外诱导骨髓细胞形成破骨细胞的作用。     

鼠破骨细胞培养过程中凋亡现象的观察研究

目的 观察研究鼠破骨细胞培养过程中的凋亡现象。方法 对机械分离法获得的鼠破骨细胞进行形态学、特异性耐酒石酸酸性磷酸酶(TRAP)染色及硬组织吸收实验鉴定。实验分为12h、24h、48h、72h组,分别在相应培养时间点行TRAP染色,计数每组阳性红染破骨细胞量。结果 机械分离法获得的鼠破骨细胞形态较大,TRAP染色见胞质呈酒红色不均匀沉淀,能形成不规则硬组织吸收陷窝。随着实验组培养时间的延长,TRAP阳性破骨细胞数量呈下降的趋势。在培养48h内,细胞数量减少不明显,当到达72h后,细胞数量明显减少。结论 鼠破骨细胞在培养过程中因细胞凋亡发生细胞数逐渐减少。     

铜离子对牙各矿化成分破骨细胞性吸收的体外调控

目的研究铜离子对破骨细胞体外吸收人牙磨片功能的影响。方法体外分离、培养兔破骨细胞，与玻片和灭活人牙磨片共同培养，加入不同浓度的铜离子。抗酒石酸酸性磷酸酶染色鉴定玻片上的破骨细胞，显微摄影分析破骨细胞吸收造成的牙磨片上的吸收陷窝，原子吸收分光光度法测定溶出的钙，并将实验组上清液中钙离子浓度与对照组相比，定义为矿化组织吸收指数，以评价破骨细胞的功能。结果体外成功分离培养出多核的、抗酒石酸酸性磷酸酶染色（＋）的破骨细胞。破骨细胞吸收牙磨片时，首先在接近牙骨质或牙本质的部位开始形成吸收陷窝；破骨细胞在牙磨片上形成的吸收陷窝与骨片相比，数量较少，体积较小，多为正圆形；吸收深度较浅，常为大面积的浅吸收。（1×10^-14）～（1×10^-4）mol／L铜离子均能抑制矿化组织吸收，实验组矿化组织吸收指数均小于1。培养第3天，1×10^-10mol／L铜离子与对照组相比，其抑制效果差异有统计学意义（P〈0．05）。培养第7天，1×10^-4mol／L、（1×10^-10）～（1×10^-12）mol／L铜离子均能显著抑制矿化组织吸收（P〈0．05），但各浓度之间相比差异无统计学意义（P〉0．05）。结论一定浓度的铜离子可以抑制破骨细胞在牙磨片上的吸收。     

腺病毒介导TWEAK基因对破骨细胞影响的体外研究

目的:研究TWEAK对成熟破骨细胞的作用。方法:体外分离培养破骨细胞;以腺病毒为载体,携带TWEAK基因,转染培养体系中的细胞;3d后计数抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphatase,TRAP)阳性细胞数7,d后测量骨吸收陷窝的面积。采用t检验分析TWEAK对破骨细胞生存时间和功能的影响。结果:转染空病毒组与空白对照组没有差异;转染TWEAK组与转染空病毒组相比,TRAP阳性细胞数增加(P<0.05),骨吸收陷窝面积增加(P<0.01)。结论:腺病毒本身对破骨细胞没有作用。TWEAK在一定程度上能延长破骨细胞的生存时间并能显著促进破骨细胞的骨吸收功能。     

不同诱导因子对大鼠破骨细胞样细胞体外形成的影响

目的：研究不同诱导因子对大鼠破骨细胞样细胞体外形成的影响。方法：建立大鼠脾细胞与成骨细胞的联合培养体系，将细胞悬液接种于预置盖玻片或牙本质片的24孔培养板内，实验组分别加入骨吸收因子1，25（OH）2D3、PTH、PGE2，对照组不加药，每3d换培养液并加药1次。结果：培养5d后实验组可见多核细胞形成，并逐渐增多，TRAP染色阳性，电镜下见牙本质片上有吸收陷窝形成，其中以1，25（OH）2D3组结果最明显。结论：体外培养大鼠破骨样细胞的形成需要骨吸收因子的诱导，1，25（OH）2D3的诱导作用最强。     

Dual regulation of osteoclast differentiation by periodontal ligament cells through RANKL stimulation and OPG inhibition

Periodontal ligament (PDL) cells play an important role in maintaining the homeostasis of periodontal tissues. However, it is not known how PDL cells contribute to osteoclastogenesis. In this study, we examined the consequences of cell-to-cell interactions between peripheral blood mononuclear cells (PBMCs) and PDL cells during osteoclastogenesis. PBMCs were co-cultured directly or indirectly with PDL cells for two to four weeks. PBMCs that were directly co-cultured with PDL cells formed significantly more resorption pits on dentin slices than did PBMCs that were cultured alone. However, soluble factor(s) produced from PDL cells inhibited the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells. Furthermore, PDL cells expressed both receptor activator nuclear factor kappa B ligand (RANKL) and osteoprotegerin (OPG) mRNA. In conclusion, PDL cells support osteoclastogenesis through cell-to-cell contact. PDL cells might regulate osteoclastogenesis by opposing mechanisms--stimulation of resorptive activity by RANKL and inhibition by OPG--thus affecting processes such as periodontitis and orthodontic tooth movement.     

Formation of osteoclast-like cells from peripheral blood of periodontitis patients occurs without supplementation of macrophage colony-stimulating factor

Aim: To determine whether peripheral blood mononuclear cells (PBMCs) from chronic periodontitis patients differ from PBMCs from matched control patients in their capacity to form osteoclast-like cells.
Material and Methods: PBMCs from 10 subjects with severe chronic periodontitis and their matched controls were cultured on plastic or on bone slices without or with macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor- κ B ligand (RANKL). The number of tartrate-resistant acid phosphatase-positive (TRACP⁺) multinucleated cells (MNCs) and bone resorption were assessed.
Results: TRACP⁺ MNCs were formed under all culture conditions, in patient and control cultures. In periodontitis patients, the formation of TRACP⁺ MNC was similar for all three culture conditions; thus supplementation of the cytokines was not needed to induce MNC formation. In control cultures, however, M-CSF or M-CSF/RANKL resulted in higher numbers compared with cultures without cytokines. Upregulations of osteoclast marker mRNA cathepsin K and carbonic anhydrase II confirmed the osteoclastic character. Bone resorption was only observed when PBMCs were cultured in the presence of M-CSF and RANKL.
Conclusion: Our data indicate that PBMCs from periodontitis patients do not need priming by M-CSF to become osteoclast-like cells, suggesting that PBMCs from periodontitis patients are present in the circulation in a different state of activity.     

不同浓度破骨细胞分化因子和巨噬细胞集落刺激因子体外诱导大鼠破骨样细胞形成的研究

目的探讨破骨细胞分化因子和巨噬细胞集落刺激因子在大鼠破骨样细胞形成、分化中的作用,为进一步研究正畸牙齿移动的生物力学机制奠定基础。方法应用不同浓度的破骨细胞分化因子和巨噬细胞集落刺激因子对提取的SD大鼠骨髓单个核细胞进行诱导培养,分别于培养1、3、5、7 d利用TRAP染色、骨吸收陷窝检测等对单核及多核破骨样细胞的生成进行鉴定,对TRAP(+)的细胞进行计数和统计学分析。结果巨噬细胞集落刺激因子和破骨细胞分化因子对TRAP(+)的单核及多核破骨样细胞的诱导分化作用与时间有关:第3 d时逐渐增多,以第5 d和第7 d为最多;缺乏巨噬细胞集落刺激因子时,骨髓单个核细胞不能有效地分化为TRAP(+)的细胞;在破骨细胞分化因子浓度一定的条件下(50 ng/ml),TRAP(+)的细胞数与巨噬细胞集落刺激因子的浓度有关,当其浓度超过30 ng/ml时,TRAP(+)细胞数不再显著增加,曲线趋于平缓;当巨噬细胞集落刺激因子浓度一定的条件(50 ng/ml)下,TRAP(+)的细胞数与破骨细胞分化因子的浓度无关。结论以巨噬细胞集落刺激因子和破骨细胞分化因子作为诱导因子,对大鼠骨髓单个核细胞进行诱导培养,可获得TRAP(+)吸收陷窝(+)的破骨样细胞,且巨噬细胞集落刺激因子30 ng/ml、破骨细胞分化因子50 ng/ml时具有最强的生物学效应。     

Human osteoclast formation and activity on an equine spongy bone substitute

Objectives: The aim of the present study was to evaluate the in vitro formation and activity of human osteoclasts (OCLs) generated on a new type of xenograft for bone substitution, an equine spongy bone.
Material and methods: Peripheral blood mononuclear cells from healthy volunteers were used to generate OCLs in vitro in the presence of macrophage colony stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL) on bovine bone slices (positive control) and equine spongy bone. Morphological and biochemical methods were used to assess OCLs formation and activity.
Results: Cells generated after 21 days of culture on equine spongy bone showed similar morphology to those on the positive control and displayed typical OCL markers and features, indicating that this material supported OCL formation. Moreover, these cells were functionally active on equine spongy bone with statistically significant differences compared with the control in the release of tartrate-resistant acid phosphatase (TRAcP5b) at days 14 and 21 of culture. With regard to the resorption, on equine bone, OCLs formed smaller discontinuous island-like lacunae rather than the typical lobulated, tracking resorption lacunae observed on the control.
Conclusions: This study enables clinicians to tailor the usage of equine spongy bone and presents a model, which can be applied to the preclinical assessment of bone substitute material's resorbability and resorption rates.     

Cholera toxin and forskolin stimulate formation of osteoclast-like cells in mouse marrow cultures and cultured mouse calvarial bones

Osteoclasts are hematopoietic in origin and formed by proliferation, differentiation and fusion of osteoclast progenitor cells. However, the signal transducing mechanisms involved in generation of osteoclasts are not clear. We have used two well-known adenylate cyclase stimulators to examine the effect of cyclic AMP (cAMP) on the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells in cultured mouse calvarial bones and in mouse bone marrow cultures. The effects of forskolin and cholera toxin were compared with those of parathyroid hormone (PTH) and 1,25(OH)2vitaminD3 (1,25(OH)2D3). PTH, as well as forskolin and cholera toxin, increased the number of osteoclast profiles/mm bone in 24-h and 120-h cultures of mouse calvarial bones. In mouse bone marrow cultures, 1,25(OH)2D3 or PTH stimulated formation of TRAP-positive multinucleated cells. Moreover, forskolin or cholera toxin produced dose-dependent stimulation of these cells at a range of concentrations correlating with their effect on cAMP production. The osteoclastic phenotype of the TRAP-positive cells was demonstrated by autoradiography of 125I-labelled calcitonin binding and by the bone-resorbing activity of the cells. The sustained presence (0-9 d) of forskolin or PTH was required to obtain maximal formation of osteoclasts. However, the presence of 1,25(OH)2D3 was required only for the last 3 d of culture for maximal osteoclast formation. We conclude that PTH may stimulate osteoclast generation using the adenylate cyclase cAMP system as a signal transduction mechanism.     

The gingipains from Porphyromonas gingivalis do not directly induce osteoclast differentiation in primary mouse bone marrow cultures

Background and Objective:  Porphyromonas gingivalis is a major aetiological agent in the development of periodontitis, the major clinical hallmark of which is bone resorption. The cysteine proteases (gingipains) produced by P. gingivalis have a critical role in the pathogenesis of the disease, and previous studies on whole bacteria have implicated these enzymes in osteoclastogenesis, a process which serves to upregulate bone resorption. The effects of the gingipains from P. gingivalis on osteoclast differentiation were investigated here to determine whether the enzymes directly contribute to osteoclastogenesis and thus to bone resorption.
Material and Methods:  The effects of the gingipains on osteoclast differentiation were investigated in primary mouse bone marrow cultures. The cultures harvested from C57BL6/J mice were incubated in the presence of parathyroid hormone, a known osteoclastogenic factor, or active/inactivated forms of three gingipains. Osteoclast differentiation was quantified by counting the number of multinucleated cells positive for tartrate-resistant acid phosphatase, an enzyme marker for these cells.
Results:  After 10 days of culture, the gingipains, either active or inactive, failed to stimulate osteoclast differentiation in comparison to the parathyroid hormone.
Conclusion:  The data presented here demonstrate that the gingipains do not induce osteoclast differentiation in this system, indicating that the bacterium uses other mechanisms to induce bone loss.     

破骨细胞分化因子诱导小鼠骨髓细胞形成破骨细胞的实验研究

目的:探讨破骨细胞分化因子(ODF)和巨噬细胞集落刺激因子(M-CSF)联合应用进行体外诱导小鼠骨髓细胞形成破骨细胞(OC)的能力。方法:收集5～6周小鼠骨髓细胞,在含M-CSF(10 ng/ml)的α-MEM全培养液中培养24h,然后将悬浮生长的细胞接种到24孔培养板,并加入不同浓度的ODF和M-CSF。,通过观察抗酒石酸盐酸性磷酸酶(TRAP)染色的阳性细胞和能否在骨磨片上形成吸收陷窝来鉴定OC形成情况。结果:在只加入ODF或M-CSF一种细胞因子时,未见有TRAP阳性或CTR阳性细胞形成,同时加入ODF和M-CSF,可形成典型的OC。TRAP阳性多核细胞的数目和培养液中钙离子浓度的增加与ODF的浓度呈正相关。结论:小鼠骨髓来源的单核细胞在ODF和M-CSF共同作用下可形成具有骨吸收功能的OC,为体外研究OC的分化发育和功能调节提供了一种新的方法。     

Effects of whole cell sonicates of Treponema lecithinolyticum on osteoclast differentiation

BACKGROUND: Alveolar bone destruction is a characteristic feature of periodontal diseases and multinucleated osteoclast cells derived from hemopoietic cells are responsible for bone resorption. Treponema lecithinolyticum is a novel oral spirochete isolated from the periodontal lesions. METHODS: The effect of whole cell sonicates on the osteoclast differentiation was examined in a co-culture system of hemopoietic mouse bone marrow cells and calvaria derived-osteoblastic cells to clarify the role of T. lecithinolyticum in the alveolar bone destruction associated with periodontal diseases. The differentiated osteoclasts were confirmed by tartrate-resistant acid phosphatase (TRAP) staining. RESULTS: Sonicates of this bacterium stimulated the osteoclast formation in the co-culture system in a dose-dependent manner. The sonicates-induced osteoclast formation was partially inhibited by the heat treatment of sonicates. Indomethacin, which is a prostaglandin inhibitor, decreased the osteoclast formation induced by the bacterial sonicates. CONCLUSIONS: These findings suggest that T. lecithinolyticum induces osteoclast differentiation by a prostaglandin E2-dependent mechanism and that heat-labile components may be involved in this process.     

Dental follicle cell-conditioned medium enhances the formation of osteoclast-like multinucleated cells

An influx of mononuclear cells and the subsequent increase of osteoclasts around tooth germs suggests that the dental follicle (DF) regulates or influences bone resorption required for tooth eruption. In order to study the effects of DF cell products on osteoclast formation during tooth eruption, a conditioned medium (CM) was created in which DF cells were added to mouse bone marrow cultures. Tartrate-resistant acid phosphatase (TRAP)-positive osteoclast-like multinucleated cells were formed in the presence of 10 nM 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. The CM, dose-dependently, stimulated the formation of TRAP-positive cells in the presence of 1,25(OH)2D3 for 14 days culture. The number of these cells decreased due to degradation in the control culture. A semi-solid methylcellulose assay in the presence of CM showed little expression of colony-stimulating activity. These results suggest that the DF cells of a developing tooth produce factor(s) that enhance osteoclast formation and bone resorption necessary for tooth eruption.