【作者】 韩大庆；

【导师】 张其清；

【作者基本信息】 中国协和医科大学 ， 生物医学工程， 2006， 博士

【摘要】 近来，越来越多的证据明确表明了破骨细胞的调节作用对于正常的骨形成是至关重要的。破骨细胞缺乏动物所表现出来的骨形成异常在目前的组织工程化骨中也都有所反映。因此，破骨细胞参与到骨体外重建策略中来不仅能洞察移植后骨重塑以及成骨细胞和破骨细胞相互作用，而且也能为完善组织工程化骨提供必要的解决办法。本研究主要探索既符合生理过程又实际有效的破骨细胞引入途径。目的：1)获取破骨细胞前体（融合前破骨细胞，pOCs）并描述其生物学特征；2)实验分析成骨细胞的分化程度与pOCs成熟的关系；3)建立创新的破骨细胞在组织工程化骨生成方法；4)观察破骨细胞的生成，功能表达，以及对支架类骨结构的影响。方法：2个月昆明鼠全骨髓细胞与新生鼠原代颅骨成骨细胞共同培养在含有1，25（OH）₂ D₃的培养液中6天。通过轻轻吹打新加的介质，打下黏附不紧密的细胞作为pOCs使用。当这种细胞悬液接种于培养板，半小时后经抗酒石酸酸性磷酸酶（TRAP）染色和碱性磷酸酶（ALP）染色，确定pOCs的存在和数目以及有无多核细胞和成骨细胞的混杂。TRAP荧光染色进一步对结果进行确认。通过融合试验来研究原代成骨细胞对pOCs融合的影响。为了研究成骨细胞分化程度与pOCs成熟的关系，成骨细胞系MC3T3-El细胞被接种到培养板，使用成骨介质培养到细胞汇聚（4d）、矿化开始（16d）和高度矿化（28d），矿化是通过茜素红染色来判定。这三种培养物分别代表骨形成过程中细胞增殖、基质沉积和成熟以及基质矿化。pOCs分别接种到三种培养物，在生长介质中共培养3d。然后进行TRAP染色确定类破骨细胞的形成；纤维肌动蛋白（F-actin）荧光染色判断其吸收活性。在建立破骨细胞符合生理过程引入实验中，MC3T3-El细胞首先接种于支架上，在成骨介质中培养5w和12w，接着pOCs分别接种到低矿化（5w）和高矿化（12w）支架结构上，在生长介质中培养3d和8d。支架的矿化程度通过支架上磷含量检测来判定。然后进行无任何处理的环境扫描电镜（ESEM）和一般电镜检测（SEM），观察类破骨细胞的形成和功能表达；支架切片F-actin荧光染色判断其吸收活性。结果：在pOCs制备物中，没发现多核细胞和ALP阳性的成骨细胞，TRAP阳性细胞约占接种细胞总数的60％。融合试验表明成骨细胞的存在对于pOCs融合是必不可少的。在分析成骨细胞分化与pOCs成熟关系实验中，在高度矿化培养物中，可见到大小不等的矿化结节。结节的矿化程度可用透光度来表示。这些结节，依其透光度，可分为带有黑色纹理的不充分矿化结节、除边缘以外全黑的相对充分矿化结节和全黑的充分矿化结节。TRAP染色后，在增殖期和刚矿化共培养物中，TRAP阳性细胞分散存在，没发现大于两个核的TRAP阳性细胞。在高度矿化共培养物中，可见到TRAP阳性细胞聚集在为数很少的大的不充分和相对充分矿化结节周围。约95％大于两个核TRAP阳性细胞在这样的细胞中存在。这表明pOCs的成熟不仅与基质的矿化有关也与矿化量有关。聚集在相对充分矿化结节周围的TRAP阳性细胞有向其集中的趋势。大的充分矿化结节周围无明显细胞聚集现象；但一些原本不透光的结节却有不同程度的TRAP染色。这种TRAP染色结节说明矿物有吸收；间接反映了所形成的类破骨细胞有骨吸收活性。细胞在大矿化结节周围分布的差异反映了在大小类似的情况下，结节矿化程度越高，对周围的TRAP阳性细胞的趋化能力越强。F-actin荧光染色显示了在大矿化节结上F-actin环的出现，这是破骨细胞具有吸收活性的特征标志，直接证明了所形成的类破骨细胞具有骨吸收活性。在研究破骨细胞生理引入实验中，通过ESEM和SEM观察到各种形态的类破骨细胞生成。在高度矿化支架结构上（12周），类破骨细胞更容易发现并且尺寸更大；这可能是类破骨细胞的形成与骨基质功能状态相适应的反映。而且使用ESEM观察到正在吸收的类破骨细胞，这个细胞展示了面向骨面的细胞膜特化结构—皱褶缘和封闭区及其足体，同时吸收坑也清晰可见。这无疑表明使用这一策略产生的类破骨细胞能够吸收由成骨细胞在体外产生的类骨基质。支架切片F-actin荧光染色显示了F-actin环的出现，进一步确认类破骨细胞的吸收活性。在原本总是被成骨细胞异常生长所形成的膜状或条索状结构占据的支架孔壁上，出现巨大的细胞融合体。在类破骨细胞出现的地方，观察不到明显的成骨细胞异常生长结构。这些现象表明类破骨细胞能以某种方式去除异常细胞结构。结论：1)通过机械分离从破骨细胞分化培养物中获取pOCs是一种简单、有效的获取破骨细胞来源细胞的方法。2)在模拟骨组织发育和形成过程中，pOCs的成熟需要骨基质的形成和累积。3)由pOCs在细胞矿化支架结构上生成破骨细胞是生理、有效、可行的破骨细胞引入方法。4)破骨细胞的引入可以起到改善组织工程化骨结构的作用。更多还原

【Abstract】 There is ever-increasing evidence that regulatory effects of osteoclasts areessential for normal bone formation. The abnormalities of bone formation present inosteoclast-deficient animals are largely reflected in current tissue-engineered bones.Therefore, the involvement of osteoclasts in bone-like tissue reconstruction in vitronot only allows insight into remodeling post-implantation and interactions betweenosteoclasts and osteoblasts, but also provides a necessary means to improvetissue-engineered bone. This present study attempts to present a physiological feasiblestrategy for osteoclast introduction. Objective: 1) to harvest and biologicallycharacterize preosteoclasts （pOCs）, the cells just before fusion to form multinucleatedcells （MNCs）; 2) to experimentally analyze the relationship of pOC maturation todifferentiation levels of the osteoblast; 3) to establish a novel method forosteoclastogenesis on tissue-engineered bone; and 4) to observe the appearance andfunctional expression of osteoclast-like cells as well as their potential effect onbone-like structure. Methods: pOCs were obtained by gentle pipetting of 6-daycocultures of mouse bone marrow cells and primary mouse osteoblastic cells in thepresence of la, 25（OH）₂ D₃. After the plating of pOC preparations, the presence andpercentage of pOCs were determined by tartrate-resistant acid phosphatase （TRAP）histochemical or fluorescent staining and the contamination of osteoblasts was judgedby alkaline phosphatase staining. Effect of the presence or absence of primaryosteoblastic cells on pOC fusion was evaluated using fusion assay system. Toinvestigate roles of osteoblastic differentiation levels in pOC maturation, MC3T3-Elcells （El cells） were first cultured to confluence （4d）, initial （16d） and heavy （28d）mineralization （judging by alizarin red S staining）; respectively representingosteoblastic proliferation, deposition and maturation, and mineralization of matrixduring bone formation, pOCs were then added to these three cultures and coculturedin growth medium without any osteotropic hormon for 3 days. The formation ofmultinucleated osteoclast-like cells and their activities were determined by TRAPstaining and by F-actin fluorescent staining, respectively. To set up a physiologically relevant method for osteoclast introduction, pOCs were seeded into lowly （5w） andhighly （12w） mineralized scaffold constructs of El cells （judging by phosphate contentassay） and cocultured in growth medium for 3 or 8 days. The formation and functionalexpression of multinucleated osteoclast-like cells were demonstrated byEnvironmental scanning electron microscopy （ESEM） and Scanning electronmicroscopy （SEM） and their activities were confirmed by F-actin fluorescent stainingof cross-sections. Results: The population of pOCs was about 60% of the cellsenriched with pOCs isolated from the osteoclast differentiation cultures. When theisolated pOCs were seeded on a plate, neither TRAP（+） MNCs nor osteoblastic cellswere detected. Fusion assay showed the presence of osteoblasts is essential for pOCfusion. In the heavily mineralized cultures of El cells, mineralized modules greatlyvaried in size and were classified according to their degree of mineralizationrepresented by their ability to transmit light rays, that is, as not completed black butwith texture, relatively completely black but without sharp outlines, and completelyblack with sharp outlines. In the coculture of pOCs and differently differentiatedcultures of El cells, TRAP（+）MNCs （＞2 nuclei） were only seen in highly mineralizedcultures. These MNCs （95%） were mostly found among TRAP（+） cells concentratedaround and on large not completely and relatively completely mineralized nodulesonly; even in the nodules covered with cells, nodule size affected cell concentration.This phenomenon suggests that pOC maturation is the formation and accumulation ofbone matrix. The relatively completely mineralized nodules had the tendency to focusthe surrounding cells on themselves. Some of the large black completely mineralizednodules locally became transparent and stained red for TRAP. This can provideindirect evidence that these osteoclast-like cells had bone-resorbing ability, as TRAPstaining of the black nodules—black because of their not transmitting lightrays—would not be visible under the inverted microscope unless the nodules wereresorbed by infiltrating TRAP（+） cells. F-actin fluorescent staining showedappearance of F-actin rings as characteristic of osteoclastic bone-resorbing activity onlarge mineralized nodules and directly confirmed resorbing activities of these cells.The difference in the surrounding cell distribution between large not completely and completely mineralized nodules might reflect their abilities to chemoattract. In thecoculture of pOCs and mineralized scaffold constructs of Elcells, variousmorphologies of osteoclast-like cells were visualized by MSEM and SEM.osteoclast-like cells were usually much large on the highly mineralized constructs （12weeks） in comparison with the low mineralized constructs （5 weeks）, suggesting thatthe formation of osteoclast-like cells adapted to functional state of bone-like matrix.ESEM showed on one highly mineralized construct that partial displacement of oneosteoclast exposed specialized structures of the cellular membrane facing the bonesurface, namely, the ruffled border and podosomes of the sealing zone, as well as theresorption pit under the ruffled border, unequivocally indicating that osteoclast-likecells formed have the ability to resorb bone-like matrix produced by osteoblasts invitro. F-actin fluorescent staining of cross-sections showed appearance of F-actinrings and confirmed resorbing activities of these cells. Before the seeding of pOCs, itwas invariably observed by ESEM or SEM that El cells grew across the spacebetween pore-walls to form cords or film-shaped structures. After 8 days of coculture,ESEM showed that a great body of cells aggregated and fused to form hugecell-fusion bodies on the pore-walls of the highly mineralized constructs that wereotherwise occupied by abnormal cell growth structures. These abnormal structuresfailed to be visualized by ESEM or SEM in the places where osteoclast-like cellsappeared. These phenomena suggest that osteoclast-like cells can somehow eliminatethe abnormal cell structures. Conchusions: 1) mechanical isolation of pOCs from theosteoclast differentiation cultures is a simple effective method for harvest ofosteoclast-sourcing cells. 2) pOC maturation requires formation and accumulation ofbone matrix in the process of the mimicking of the development and formation ofnatural bone tissue. 3) osteoclastogenesis from preosteoclasts on tissue-engineeredbone is a physiological feasible method for osteoclast introduction. 4) osteoclastintroduction serves to improve the bone-like tissue structure by removing abnormalcell growth-related structures.更多还原