多孔钽金属联合骨髓基质干细胞治疗骨坏死的实验研究
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摘要
科研工作来源：骨坏死最常见的原因是由外伤、酗酒、激素等引起,发病率最高的部位是股骨头,股骨头坏死发生后会引起局部疼痛,活动功能障碍,如早期不能得到及时有效的治疗,就会使股骨头塌陷,关节间隙变窄,最后导致骨关节炎,关节失去功能而致残致瘫。股骨头坏死的治疗效果与病情轻重、发现早晚、病程分期有很大关系,病变发现越早,病情越轻,治疗效果就越好,因此股骨头坏死应做到早期诊断和早期治疗,近年来学者越来越重视对该病发病早期治疗的研究。治疗方面,临床上对股骨头坏死做出正确的诊断和分期是决定治疗方案的关键。骨坏死分期方法很多,最早Ficat等根据X线表现将已有临床症状且经组织活检证实的股骨头坏死分为4期。此外还有Ficat6期分法、Marcus6期分法等,但这些方法各有其局限性。1997年世界骨循环研究学会(Association Research Circulation Osseous, ARCO)设立了国际骨坏死标准,简称ARCO分期。该分期较全面而准确地反映了骨坏死面积、部位及与预后的关系,对临床上指导治疗具有重要参考价值。ARCO分期Ⅰ期、Ⅱ期为塌陷前期,具有完好外形的股骨头,此时及时介入治疗有可能终止病程,而Ⅲ期有轻微塌陷,保头治疗的效果较之前差,而到Ⅳ期时,已形成骨性关节炎,只能选择关节置换治疗。年轻患者关节置换后可能要面临几次翻修,假体的植入,骨量的大量丢失,会使翻修越来越困难,因此,早期治疗是避免骨坏死加重,避免致残的关键。
近些年,应用骨髓基质干细胞治疗早期骨坏死成为热点,它具有取材方便、增殖和成骨能力强的优点,在不同理化环境和细胞因子诱导下,可向成骨、成软骨、脂肪和纤维细胞系等多方向分化分离、扩增,并易于被外源基因感染且稳定表达,是基因治疗以及组织工程应用中理想的靶细胞,它被普遍认为是骨组织工程研究中最理想的种子细胞,大量针对早期骨坏死治疗的髓芯减压术式中被应用骨髓基质干细胞,取得了较好的临床效果,但应用的细胞成活于培养基中,注入发病部位时缺乏依附,造成大量的流失,随着组织工程学的快速发展,寻找理想的支架成为人们热衷的研究方向。相继出现脱钙骨基质、聚乳酸(PLA)、聚乙醇酸(PGA)及其共聚物、生物陶瓷类的材料等等,但均有各自的缺点,应用于临床上还为时过早,随着工业技术水平的不断提高,金属类的人工生物材料的研发也有了快速发展,多孔钽金属的制造成功为人体植入物增加一个新的选择。钽金属颜色灰黑,光亮,质地非常坚硬。钽金属的熔点几近3000℃,并且化学性质非常的稳定,将钽金属放入强酸强碱中也不易被腐蚀,钽在生物体内呈惰性,在水及酸碱性环境中均不溶解,常温下不会产生任何化学反应,对机体组织无侵蚀刺激等生物性质,这使得钽具有良好的生物相容性。正是由于钽金属具有熔点高、硬度大、抗磨损等良好的物理性质、耐腐蚀的化学性质以及良好的生物相容性,钽金属成为了临床应用中外科植入体内材料的理想选择。在医疗领域中,钽金属的应用历史已有半个多世纪,主要应用在心脏起搏器、颅骨缺损修补材料、血管夹、股骨柄假体、骨缺损填充材料的制造等方面。有研究表明生物体内吞噬细胞是清除钽的主要物质。实验显示体内吞噬细胞在接触钽尘1小时后,细胞均保持活性并且无细胞变性,仅伴有葡萄糖氧化的明显增加,而相同条件下用矽尘做对比,矽尘会使吞噬细胞出现严重的胞浆变性和死亡,这表明钽金属是无细胞毒性的,对人体没有任何毒害作用。在1997年,美国FDA批准多孔钽金属应用于临床,该材料结构在显微镜下观察如同松质骨,其内孔隙大小400～600u m之间,整体互相连接的孔隙率高达75%～85%,明显高于其他生物固定材料如钻、铬、钛等30%～50%的孔隙率。多孔钽所具有的三维多孔结构更有利于成骨细胞黏附、分化和生长,促进骨长入,从而加强植入体与骨之间的连接,达到生物固定的作用,同时也有利于水分和营养物质在植入体内的传输,多孔钽金属具有良好的弹性模量,能够减少内置物的应力遮挡,多孔设计也使其表面有比其他材料更高的摩擦系数,早期植入后也更有利于局部稳定,多孔结构有利于骨的长入。但是由于骨坏死的发生造成了局部骨细胞活性的降低或死亡,在单独应用多孔钽金属时尽管有了较好的力学支撑,但缺乏足够的种子细胞,因此我们设想骨髓基质干细胞能否粘附于多孔钽金属的微孔内,待骨髓基质干细胞生长增殖到一定数量后,将其联合植入到骨坏死部位,让多孔钽金属起到支撑、提供骨长入空间的作用,又有足够的种子细胞修复坏死部位,在多孔钽金属周围及微孔内重新形成一个健康的骨生长环境,修复骨坏死,防止股骨头的进一步塌陷。
目的及意义：探讨多孔钽金属对骨髓基质干细胞生长增殖的影响,探讨应用多孔钽金属联合骨髓基质干细胞治疗骨坏死的疗效。骨坏死是目前常见骨病之一,该疾病给人们的生活带来了巨大影响,坏死一般都首先发生在股骨头内,坏死过程中股骨头会逐渐塌陷,出现严重的疼痛及功能障碍,造成患者行走困难,劳动力丧失,严重者终身卧床无法行走。骨坏死目前临床常用ARC0分期指导治疗,Ⅰ期和Ⅱ期是塌陷前期,即为早期病例,此时进行积极治疗可能终止病情发展,一般的治疗方法是髓芯减压,髓芯减压联合干细胞治疗,带血管蒂骨瓣转移术等,但均存在不同的缺点。常用的髓芯减压术术后缺乏对软骨下骨的结构支撑,有软骨下骨骨折的风险,联合干细胞治疗时干细胞呈液态,注入骨坏死部位后无法固定位置,带血管蒂骨瓣转移术力学支撑不佳。Ⅲ期病例由于有股骨头的塌陷,即使手术后有成骨表现,也难以阻止病变进一步发展至骨关节炎。治疗上保头治疗及关节置换标准不一,但较统一的观点是保头治疗效果较塌陷前治疗效果差。Ⅳ期时股骨头塌陷及骨性关节炎发生,行关节置换已经成为共识,关节置换后疼痛、关节功能都可以即时得到大幅改善,对于提高人们生活质量有重大意义,但假体关节的磨损、骨质疏松的发生等均会造成关节假体有使用年限的限制,这是关节置换手术一个短时间内无法解决的问题。因此,如何在股骨头坏死塌陷前积极治疗、减少并发症及增加手术成功率是我们此实验的意义所在。
方法：应用全骨髓贴壁分离法提取健康兔骨髓基质干细胞,培养液用Alpha-MEM,内含10%FBS,1%PSN,对其进行培养,通过在特定诱导剂作用下向成脂成骨诱导及细胞集落形成实验来鉴定干细胞性质,用MTT法检测多孔钽金属的细胞毒性,比较多孔钽金属组与钛金属组及对照组的细胞活性,分别在骨髓基质干细胞及多孔钽金属联合培养后的第二天、第五天及第七天用GFP染色及透射电镜观察骨髓基质干细胞在多孔钽金属上的粘附与增殖情况,应用PCR检测多孔钽金属对骨髓基质干细胞的成骨诱导的影响。建立骨坏死模型,根据秦岭教授等造模方法,选取健康成年新西兰大白兔16只,年龄6个月,以10μg/kg剂量肌肉注射大肠杆菌内毒素,注射24小时后,以20mg/kg剂量肌肉注射甲基强地松龙3次,注射间隔时间为24h,所有实验动物无死亡。于第2周造模成功后开始进行体内实验。将其分成2组,每组8只,分别植入单纯多孔钽金属及联合培养后的多孔钽金属和骨髓基质干细胞,通过评估多孔钽金属周围新骨形成情况判断多孔钽金属与骨髓基质干细胞联合培养对骨坏死治疗的疗效。
结果：经过贴壁分离法提取的骨髓基质干细胞,通过成脂诱导后,应用油红染色法证实该细胞可以向脂肪细胞转化,应用茜素红染色法及碱性磷酸酶钴钙染色法证实该细胞可以向成骨细胞方向转化,细胞集落形成实验表明该细胞呈集落式生长,这些均说明提取是细胞为骨髓基质干细胞。应用MTT法检测细胞毒性24小时和48小时两个时间点比较,对照组和实验组的细胞活性比较没有统计学意义(P>0.05),表明多孔钽金属作为植入体内的材料具有良好的生物相容性。骨髓基质干细胞与多孔钽金属联合培养后GFP染色在第二天时可见骨髓基质干细胞均匀分布在钽金属表面,通过显微镜的观察可见清晰度不同的细胞形态,说明这些细胞不在一个平面上,部分散布在微孔内。第五天可见随着时间延长,粘附在多孔钽金属的细胞越来越多,细胞在金属内立体增殖传代,且增殖活跃,可见多条伪足粘附于金属上；第七天骨髓基质干细胞大量增殖,逐渐铺满多孔钽金属的立体结构。电镜下观察在第二天时可见骨髓基质干细胞粘附于多孔钽金属表面及微孔内,数量较少,形态多样；共培养5天时细胞数量逐渐增多,伸出伪足平铺于金属表面及微孔内,在立体结构中增殖良好,数量增多；共培养7天时可见大量骨髓基质干细胞粘附于多孔钽金属表面及微孔内,向各方向伸出伪足,活力良好。PCR基因检测结果显示,BMSCs加入到多孔钽材料上培养两周后,骨钙素(OCN),骨桥蛋白(OPN),转录因子Runx-2mRNA表达水平明显上调；而钽可协同成骨诱导液显著促进OCN表达(A),抑制OPN表达(B),而不影响Runx-2表达(C)；钽材料周围的细胞的基因表达与之类似。因此,可判断钽作为多孔支架促进了BMSCs向成骨细胞方向分化。术后3周可观察到两组植入的钽金属周围均有新生骨生成,但两组均未见有明确的新生骨长入至多孔钽金属的微孔内,单纯植入多孔钽金属组和植入钽金属和干细胞联合培养组的新生骨平均宽度比较无统计学意义(P>0.05)；术后6周可观察到两组植入的钽金属周围新生骨生成明显增多,单纯植入钽金属组和植入钽金属和干细胞联合培养组新生骨宽度的平均值比较有统计学意义(P<0.05),而在骨长入多孔钽金属内部方面,两组硬组织切片荧光显示均有新生骨长入多孔钽金属的微孔内,加入骨髓基质干细胞组的骨长入深度与未加入干细胞组的骨长入深度比较有统计学意义(P<0.05)
结论：多孔钽金属无细胞毒性。多孔钽金属对于骨髓基质干细胞的生长及增殖无消极影响,与对照组比较无统计学意义,在多孔钽金属表面种植的骨髓基质干细胞会随着时间的增加逐渐增殖、扩散至多孔钽金属微孔内,骨髓基质干细胞联合多孔钽金属在治疗骨坏死时可见更多新生骨在多孔钽金属周围,说明骨髓基质干细胞在体内的增殖分化及向成骨细胞转化可以加快新骨的生成,加快机体对坏死骨部位的修复,这样两者在联合治疗骨坏死时,既有多孔钽金属优良的力学支撑及骨长入作用,又有骨髓基质干细胞快速的增殖、分化及向成骨细胞转化的作用,对于加快坏死骨修复及多孔钽金属骨长入方面均有积极意义。
Research Source:The bone necrosis is often caused by trauma, alcohol, hormones and some other causes, the position of femoral head have the highest incidence to become necrosis, and it can cause local pain and activity dysfunction after femoral head necrosis occurring, if the disease cann't get treatment timely and effectively, it will make the femoral head collapse and the joint space narrow, lead to osteoarthritis, loss of function and disability caused by paralysis at last. The treatment result of femoral head necrosis makes a big difference with the severity, discovered sooner or later, and the course of the disease stage, the earlier the lesions detected and the more lighter of the condition, the better the treatment, therefore, osteonecrosis should be done by early diagnosis and early treatment, in recent years, more and more research about the early treatment of the disease are emphasized. For the treatment, the correct diagnosis and stage of the osteonecrosis from clinic is the key to treatment decisions. There are many staging of osteonecrosis, the earliest stage is Ficat, it had divided the osteonecrosis into four stages based on X-ray findings which has clinical symptoms and has been confirmed by biopsy. There are also Ficat four divided stage, Marcus six of points system, but these methods have their limitations.1997World Bone Cycle Research Institute (Association Research Circulation Osseous, ARCO) established the International Standard osteonecrosis, referred to ARCO staging. The stage offers a comprehensive and accurate reflection of osteonecrosis area, location and prognosis,and has an important reference value in guiding clinical treatment. ARCO stages I, II are pre-collapse stage, with the shape of the femoral head intact, then timely intervention may terminate the course, but when there was a slight collapse at phase III, preserveing heads therapeutic effects is worse than before, and at the stage IV, the has formed osteoarthritis, joint replacement therapy can only be choosen. The young patients may have to face several renovations joint replacement prosthesis implantation, a large loss of bone mass, make renovations more difficult, therefore, early treatment is the key to avoiding the aggravation of osteonecrosisand disability.
In recent years, the application of bone marrow stem cells (BMSCs) becomes hot to treat early osteonecrosis, easily obtained, proliferation and osteogenic ability higher are its advantages in different physical and chemical environment and cytokine induction, BMSCs may be directed multi-directionally to osteogenic cell, osteogenic cartilage cell, fat cell and fiber cell and so on, such as isolation, amplification, and is easy to be infected by exogenous gene, then express stablly, so it is ideal target cells to gene therapy and tissue engineering applications, it is generally regarded as the best bone tissue engineering seed cells,lots of the early treatment of osteonecrosis with core decompression surgical procedures applied BMSCs, and achieved good clinical results, it could survival in the medium, but for the reason that the lack of attachment,causing heavy loss.With the rapid development of tissue engineering, looking for the ideal stent become enthusiastic about the research. As their own drawbacks about then appeared demineralized bone matrix, polylactic acid (PLA), polyglycolic acid (PGA) and their copolymers, bio ceramic materials, etc. it is too early to be applied clinically, with increasing levels of industrial technology, research and development of artificial biomaterials metal has also been developing rapidly, manufacture porous tantalum implant becomes success for the body to add a new choice. Tantalum metal's color is grey, light, texture is very hard. The melting point of Tantalum metal is3000℃, and chemical properties of Tantalum metal are very stable, tantalum metal could also be hardly corroded even put in the acid alkaline. Tantalum are inert in organisms, so it is insoluble in water and acid and alkaline environment, and it does not produce any chemical reaction under room temperature, or stimulate tissue with erosion, these make tantalum has good biocompatibility. Tantalum metal became to be the clinical application of surgical choice of implanted materials because of characters of high melting point, high hardness, wear resistance and good physical properties, chemical properties of corrosion resistance and good biocompatibility. In the medical field, the history of the application of tantalum metal is more than half a century, it is mainly used in cardiac pacemaker, skull defect repair materials, vascular clamp, the femoral prosthesis handle, bone defect filler material manufacturing and so on. Studies have shown that phagocytic cells are the main material of removal of tantalum in the body. Experiments showed that phagocytic cells are kept active and no cell degeneration, only accompanied by glucose oxidation increased significantly in the body in contact with tantalum dust after1hour, and under the same conditions with silica dust can cause a serious phagocytes cytoplasm degeneration and death, which suggests that tantalum metal has no cytotoxicity and toxic to the human body. In1997, the American FDA approved of porous tantalum metal applied to clinical, the material looks like cancellous bone structure under the microscope, its pore size is between400-600microns, the whole inter connected porosity is up to75%-85%, significantly higher than the other biological fixation materials, such as cobalt, chromium, titanium which with porosity of30%～50%. Porous tantalum of three-dimensional porous structure has more advantageous to the adhesion, differentiation and growth of osteoblast, promotes the growth of bone, so as to strengthen the connection between the implant and bone, to achieve the effect of biological fixation, as well as to the moisture and nutrients in implanted transmission. Porous tantalum metal has good elastic modulus, it can reduce the stress shelter of implants, porous design also make its surface has a higher coefficient of friction than other materials, early implantation is also more conducive to regional stability, the porous structure is advantageous to the bone ingrowth. Although in a separate application of porous tantalum metal despite better mechanical support, due to the reduce or death of local bone cell activity caused by the occurrence of osteonecrosis, it still lacks of enough seed cells, thus we can conceive of BMSCs adhesion to porous tantalum metal micro hole, after being BMSCs proliferation to a certain number, make them joint into the part of osteonecrosis, let porous tantalum metal have the effect of support, provide bone ingrowth space, and have enough seed cells, repair necrotic areas around the porous tantalum metal and micro hole to form a healthy bone growth environment, repair osteonecrosis, to prevent further collapse of femoral head.
Purpose and meaning:Investigate the porous tantalum metal used on the effects of BMSCs proliferation, discusses the application of porous tantalum metal joint BMSCs curative effect for the treatment of osteonecrosis. Osteonecrosis is currently one of the common bone disease, the disease has brought the huge influence to people's life and necrosis are generally first occurs within the femoral head, femoral head necrosis process will gradually collapse, severe pain and dysfunction make patients with walking difficulties, loss of labor, serious life cannot walk who can only just stay in bed. Osteonecrosis ARCO stage guide the treatment of the clinic disease, commonly in clinical phase Ⅰ and phase Ⅱ is the early stage of the collapse, that is the early cases, active therapy may terminate illness development, the general treatment method is pulp core decompression, pulp core decompression with stem cell therapy, with vascular pedicle bone flap transfer operation, etc., but there are all have some different faults. Commonly pulp core decompression surgery lack the support of the structure of the subchondral bone, that have the risk of subchondral bone fracture, stem cell therapy of stem cells that is the liquid, which inject the parts of the osteonecrosis is not fixed, and the support with vascular pedicle bone flap transfer operation is not well. The period of Ⅲstage with femoral head is collapse, even after surgery osteogenesis performance, also difficult to stop further development to osteoarthritis pathological changes. Protect the head and joint replacement treatment have different standard, but a unified point of view is confirmed head treatment effect is worse than the treatment before the head collapse. In the stage of IV, collapse of the femoral head and osteoarthritis, have joint replacement has become a consensus, joint pain, joint function after replacement are can be significantly improved, that have great significance to improve the quality of people's life, but the wear and tear of prosthetic joint, the occurrence of osteoporosis, etc all can cause joint prosthesis has the limitation of use fixed number of year, this is a joint replacement surgery in a short period of time can't solve the problem. Therefore, how to active treatment in front of the collapse of the femoral head necrosis, decrease complications and increase the success rate of surgery is the meaning of the experiment.
Methods:BMSCs of healthy rabbit were extracted by whole bone marrow adherent method, cultured with Alpha-MEM, containing10%FBS,1%PSN, identify stem cell properties by lipoblast experiment, osteogenesis experiment and colony forming experiment in particular inducer, Detect the cytotoxicity of porous tantalum metal by MTT method, compared the cell activity in more porous tantalum metal group and titanium group and control group respectively, Observe adhesion and proliferation of BMSCs on porous tantalum metal with GFP staining and electron microscope in the second day, five days and seven days after BMSCs jointed porous tantalum metal, detect porous tantalum metal influenced on the distribution of BMSCs induced osteogenic by PCR. Osteonecrosis model is set up, according the methods by professor Qinling,16healthy adult New Zealand white rabbit were selected,6months of age, muscle injection e. coli endotoxin with10g/kg dose, the injection interval time is24h, muscular injection methylprednisolone with20mg/kg dose3times, the injection interval time is24h, all experimental animals were no death. start experiments after models were successful built in2weeks. It is divided into two groups, each group of eight, they were implanted into pure porous tantalum metal and BMSCs jointed porous tantalum metal, judge the therapeutic effect of osteonecrosis with BMSCs jointed porous tantalum metal.by assessing the new bone formation around porous tantalum metal.
Results:we induced BMSCs which extracted by adherent separation by a concomitant, it confirmed that the cells can change to fat cells by the application of oil red staining, it confirmed the cells can transform to the osteoblast by application of alizarin red staining and cobalt calcium alkaline phosphatase staining,cell colony forming experiment indicates that the cells are colony growth, these all show that extracted cells are BMSCs.Detect cytotoxicity by MTT method on24hours and48hours comparing two time points, the cell of activity control group and experimental group were no statistical significance (P>0.05),it showed that porous tantalum metal as a implanted material has good biocompatibility. BMSCs were distributed evenly on surface of tantalum metal in GFP dyeing when BMSCs and porous tantalum metal culturing two days. Different cell morphology under microscope shows that these cells is not on a flat surface, some of them grow into the pores. At the fifth day, more and more cells proliferate and Passage on porous tantalum metal with pseudopodia adhere to the metal. At the seventh day, the three-dimensional structure of porous tantalum metal is full of BMSCs. Under scanning electron microscope, a few cells adhere to porous tantalum metal surface with varied shapes at the second day, at the fifth day cells clearly increased and extended into micro-pore, At the seventh day, pseudopodia of cells stuck out towards all directions and their vigor were nice. After co-culture two weeks, osteocalcin (OCN), osteopontin (OPN) and mRNA transcription factors Runx-2obviously increases determined with PCR, The co-work of tantalum and osteoblast inducing conditional media can enhance the expression of the OCN expression (A), inhibit the expression of OPN (B), and not affect the expression of Runx-2(C); Gene expression of the cell around tantalum material is similar. As a result, we judged that tantalum can promote differentiation of BMSCs to osteoblasts as a porous scaffolds. At3week after surgery, new bone can be observed in two groups around tantalum metal formed, but can not be in microporous. There was no statistical significant differences of average width of new bone in the two group (P>0.05).
At6week after surgery, the new bone increased obviously, the difference were significant in the two group (P<0.05); by Hard tissue section fluorescence technology, both groups have new bone growing into porous tantalum metal, and the depth of group adding BMSCs is greater than the group not adding. The difference was statistically significant (P<0.05).
Conclusion:Porous tantalum metal has no cytotoxicity. It is free of negative effect on the growth and proliferation of BMSCs with no negative impact. There is no significant difference compared with the control group. BMSCs grown on the surface of porous tantalum metal has increased, proliferated and migrated into the microporous. there is more new bone around the porous tantalum metal in the treatment of osteonecrosis using BMSCs and porous tantalum metal, which show the proliferation and differentiation to osteoblasts of BMSCs in the body can speed up the formation of new bone and the repairment of osteonecrosis. The two functions make the repairment of osteonecrosis and the growth of bone into porous tantalum metal even faster.
近些年,应用骨髓基质干细胞治疗早期骨坏死成为热点,它具有取材方便、增殖和成骨能力强的优点,在不同理化环境和细胞因子诱导下,可向成骨、成软骨、脂肪和纤维细胞系等多方向分化分离、扩增,并易于被外源基因感染且稳定表达,是基因治疗以及组织工程应用中理想的靶细胞,它被普遍认为是骨组织工程研究中最理想的种子细胞,大量针对早期骨坏死治疗的髓芯减压术式中被应用骨髓基质干细胞,取得了较好的临床效果,但应用的细胞成活于培养基中,注入发病部位时缺乏依附,造成大量的流失,随着组织工程学的快速发展,寻找理想的支架成为人们热衷的研究方向。相继出现脱钙骨基质、聚乳酸(PLA)、聚乙醇酸(PGA)及其共聚物、生物陶瓷类的材料等等,但均有各自的缺点,应用于临床上还为时过早,随着工业技术水平的不断提高,金属类的人工生物材料的研发也有了快速发展,多孔钽金属的制造成功为人体植入物增加一个新的选择。钽金属颜色灰黑,光亮,质地非常坚硬。钽金属的熔点几近3000℃,并且化学性质非常的稳定,将钽金属放入强酸强碱中也不易被腐蚀,钽在生物体内呈惰性,在水及酸碱性环境中均不溶解,常温下不会产生任何化学反应,对机体组织无侵蚀刺激等生物性质,这使得钽具有良好的生物相容性。正是由于钽金属具有熔点高、硬度大、抗磨损等良好的物理性质、耐腐蚀的化学性质以及良好的生物相容性,钽金属成为了临床应用中外科植入体内材料的理想选择。在医疗领域中,钽金属的应用历史已有半个多世纪,主要应用在心脏起搏器、颅骨缺损修补材料、血管夹、股骨柄假体、骨缺损填充材料的制造等方面。有研究表明生物体内吞噬细胞是清除钽的主要物质。实验显示体内吞噬细胞在接触钽尘1小时后,细胞均保持活性并且无细胞变性,仅伴有葡萄糖氧化的明显增加,而相同条件下用矽尘做对比,矽尘会使吞噬细胞出现严重的胞浆变性和死亡,这表明钽金属是无细胞毒性的,对人体没有任何毒害作用。在1997年,美国FDA批准多孔钽金属应用于临床,该材料结构在显微镜下观察如同松质骨,其内孔隙大小400～600u m之间,整体互相连接的孔隙率高达75%～85%,明显高于其他生物固定材料如钻、铬、钛等30%～50%的孔隙率。多孔钽所具有的三维多孔结构更有利于成骨细胞黏附、分化和生长,促进骨长入,从而加强植入体与骨之间的连接,达到生物固定的作用,同时也有利于水分和营养物质在植入体内的传输,多孔钽金属具有良好的弹性模量,能够减少内置物的应力遮挡,多孔设计也使其表面有比其他材料更高的摩擦系数,早期植入后也更有利于局部稳定,多孔结构有利于骨的长入。但是由于骨坏死的发生造成了局部骨细胞活性的降低或死亡,在单独应用多孔钽金属时尽管有了较好的力学支撑,但缺乏足够的种子细胞,因此我们设想骨髓基质干细胞能否粘附于多孔钽金属的微孔内,待骨髓基质干细胞生长增殖到一定数量后,将其联合植入到骨坏死部位,让多孔钽金属起到支撑、提供骨长入空间的作用,又有足够的种子细胞修复坏死部位,在多孔钽金属周围及微孔内重新形成一个健康的骨生长环境,修复骨坏死,防止股骨头的进一步塌陷。
目的及意义：探讨多孔钽金属对骨髓基质干细胞生长增殖的影响,探讨应用多孔钽金属联合骨髓基质干细胞治疗骨坏死的疗效。骨坏死是目前常见骨病之一,该疾病给人们的生活带来了巨大影响,坏死一般都首先发生在股骨头内,坏死过程中股骨头会逐渐塌陷,出现严重的疼痛及功能障碍,造成患者行走困难,劳动力丧失,严重者终身卧床无法行走。骨坏死目前临床常用ARC0分期指导治疗,Ⅰ期和Ⅱ期是塌陷前期,即为早期病例,此时进行积极治疗可能终止病情发展,一般的治疗方法是髓芯减压,髓芯减压联合干细胞治疗,带血管蒂骨瓣转移术等,但均存在不同的缺点。常用的髓芯减压术术后缺乏对软骨下骨的结构支撑,有软骨下骨骨折的风险,联合干细胞治疗时干细胞呈液态,注入骨坏死部位后无法固定位置,带血管蒂骨瓣转移术力学支撑不佳。Ⅲ期病例由于有股骨头的塌陷,即使手术后有成骨表现,也难以阻止病变进一步发展至骨关节炎。治疗上保头治疗及关节置换标准不一,但较统一的观点是保头治疗效果较塌陷前治疗效果差。Ⅳ期时股骨头塌陷及骨性关节炎发生,行关节置换已经成为共识,关节置换后疼痛、关节功能都可以即时得到大幅改善,对于提高人们生活质量有重大意义,但假体关节的磨损、骨质疏松的发生等均会造成关节假体有使用年限的限制,这是关节置换手术一个短时间内无法解决的问题。因此,如何在股骨头坏死塌陷前积极治疗、减少并发症及增加手术成功率是我们此实验的意义所在。
方法：应用全骨髓贴壁分离法提取健康兔骨髓基质干细胞,培养液用Alpha-MEM,内含10%FBS,1%PSN,对其进行培养,通过在特定诱导剂作用下向成脂成骨诱导及细胞集落形成实验来鉴定干细胞性质,用MTT法检测多孔钽金属的细胞毒性,比较多孔钽金属组与钛金属组及对照组的细胞活性,分别在骨髓基质干细胞及多孔钽金属联合培养后的第二天、第五天及第七天用GFP染色及透射电镜观察骨髓基质干细胞在多孔钽金属上的粘附与增殖情况,应用PCR检测多孔钽金属对骨髓基质干细胞的成骨诱导的影响。建立骨坏死模型,根据秦岭教授等造模方法,选取健康成年新西兰大白兔16只,年龄6个月,以10μg/kg剂量肌肉注射大肠杆菌内毒素,注射24小时后,以20mg/kg剂量肌肉注射甲基强地松龙3次,注射间隔时间为24h,所有实验动物无死亡。于第2周造模成功后开始进行体内实验。将其分成2组,每组8只,分别植入单纯多孔钽金属及联合培养后的多孔钽金属和骨髓基质干细胞,通过评估多孔钽金属周围新骨形成情况判断多孔钽金属与骨髓基质干细胞联合培养对骨坏死治疗的疗效。
结果：经过贴壁分离法提取的骨髓基质干细胞,通过成脂诱导后,应用油红染色法证实该细胞可以向脂肪细胞转化,应用茜素红染色法及碱性磷酸酶钴钙染色法证实该细胞可以向成骨细胞方向转化,细胞集落形成实验表明该细胞呈集落式生长,这些均说明提取是细胞为骨髓基质干细胞。应用MTT法检测细胞毒性24小时和48小时两个时间点比较,对照组和实验组的细胞活性比较没有统计学意义(P>0.05),表明多孔钽金属作为植入体内的材料具有良好的生物相容性。骨髓基质干细胞与多孔钽金属联合培养后GFP染色在第二天时可见骨髓基质干细胞均匀分布在钽金属表面,通过显微镜的观察可见清晰度不同的细胞形态,说明这些细胞不在一个平面上,部分散布在微孔内。第五天可见随着时间延长,粘附在多孔钽金属的细胞越来越多,细胞在金属内立体增殖传代,且增殖活跃,可见多条伪足粘附于金属上；第七天骨髓基质干细胞大量增殖,逐渐铺满多孔钽金属的立体结构。电镜下观察在第二天时可见骨髓基质干细胞粘附于多孔钽金属表面及微孔内,数量较少,形态多样；共培养5天时细胞数量逐渐增多,伸出伪足平铺于金属表面及微孔内,在立体结构中增殖良好,数量增多；共培养7天时可见大量骨髓基质干细胞粘附于多孔钽金属表面及微孔内,向各方向伸出伪足,活力良好。PCR基因检测结果显示,BMSCs加入到多孔钽材料上培养两周后,骨钙素(OCN),骨桥蛋白(OPN),转录因子Runx-2mRNA表达水平明显上调；而钽可协同成骨诱导液显著促进OCN表达(A),抑制OPN表达(B),而不影响Runx-2表达(C)；钽材料周围的细胞的基因表达与之类似。因此,可判断钽作为多孔支架促进了BMSCs向成骨细胞方向分化。术后3周可观察到两组植入的钽金属周围均有新生骨生成,但两组均未见有明确的新生骨长入至多孔钽金属的微孔内,单纯植入多孔钽金属组和植入钽金属和干细胞联合培养组的新生骨平均宽度比较无统计学意义(P>0.05)；术后6周可观察到两组植入的钽金属周围新生骨生成明显增多,单纯植入钽金属组和植入钽金属和干细胞联合培养组新生骨宽度的平均值比较有统计学意义(P<0.05),而在骨长入多孔钽金属内部方面,两组硬组织切片荧光显示均有新生骨长入多孔钽金属的微孔内,加入骨髓基质干细胞组的骨长入深度与未加入干细胞组的骨长入深度比较有统计学意义(P<0.05)
结论：多孔钽金属无细胞毒性。多孔钽金属对于骨髓基质干细胞的生长及增殖无消极影响,与对照组比较无统计学意义,在多孔钽金属表面种植的骨髓基质干细胞会随着时间的增加逐渐增殖、扩散至多孔钽金属微孔内,骨髓基质干细胞联合多孔钽金属在治疗骨坏死时可见更多新生骨在多孔钽金属周围,说明骨髓基质干细胞在体内的增殖分化及向成骨细胞转化可以加快新骨的生成,加快机体对坏死骨部位的修复,这样两者在联合治疗骨坏死时,既有多孔钽金属优良的力学支撑及骨长入作用,又有骨髓基质干细胞快速的增殖、分化及向成骨细胞转化的作用,对于加快坏死骨修复及多孔钽金属骨长入方面均有积极意义。
Research Source:The bone necrosis is often caused by trauma, alcohol, hormones and some other causes, the position of femoral head have the highest incidence to become necrosis, and it can cause local pain and activity dysfunction after femoral head necrosis occurring, if the disease cann't get treatment timely and effectively, it will make the femoral head collapse and the joint space narrow, lead to osteoarthritis, loss of function and disability caused by paralysis at last. The treatment result of femoral head necrosis makes a big difference with the severity, discovered sooner or later, and the course of the disease stage, the earlier the lesions detected and the more lighter of the condition, the better the treatment, therefore, osteonecrosis should be done by early diagnosis and early treatment, in recent years, more and more research about the early treatment of the disease are emphasized. For the treatment, the correct diagnosis and stage of the osteonecrosis from clinic is the key to treatment decisions. There are many staging of osteonecrosis, the earliest stage is Ficat, it had divided the osteonecrosis into four stages based on X-ray findings which has clinical symptoms and has been confirmed by biopsy. There are also Ficat four divided stage, Marcus six of points system, but these methods have their limitations.1997World Bone Cycle Research Institute (Association Research Circulation Osseous, ARCO) established the International Standard osteonecrosis, referred to ARCO staging. The stage offers a comprehensive and accurate reflection of osteonecrosis area, location and prognosis,and has an important reference value in guiding clinical treatment. ARCO stages I, II are pre-collapse stage, with the shape of the femoral head intact, then timely intervention may terminate the course, but when there was a slight collapse at phase III, preserveing heads therapeutic effects is worse than before, and at the stage IV, the has formed osteoarthritis, joint replacement therapy can only be choosen. The young patients may have to face several renovations joint replacement prosthesis implantation, a large loss of bone mass, make renovations more difficult, therefore, early treatment is the key to avoiding the aggravation of osteonecrosisand disability.
In recent years, the application of bone marrow stem cells (BMSCs) becomes hot to treat early osteonecrosis, easily obtained, proliferation and osteogenic ability higher are its advantages in different physical and chemical environment and cytokine induction, BMSCs may be directed multi-directionally to osteogenic cell, osteogenic cartilage cell, fat cell and fiber cell and so on, such as isolation, amplification, and is easy to be infected by exogenous gene, then express stablly, so it is ideal target cells to gene therapy and tissue engineering applications, it is generally regarded as the best bone tissue engineering seed cells,lots of the early treatment of osteonecrosis with core decompression surgical procedures applied BMSCs, and achieved good clinical results, it could survival in the medium, but for the reason that the lack of attachment,causing heavy loss.With the rapid development of tissue engineering, looking for the ideal stent become enthusiastic about the research. As their own drawbacks about then appeared demineralized bone matrix, polylactic acid (PLA), polyglycolic acid (PGA) and their copolymers, bio ceramic materials, etc. it is too early to be applied clinically, with increasing levels of industrial technology, research and development of artificial biomaterials metal has also been developing rapidly, manufacture porous tantalum implant becomes success for the body to add a new choice. Tantalum metal's color is grey, light, texture is very hard. The melting point of Tantalum metal is3000℃, and chemical properties of Tantalum metal are very stable, tantalum metal could also be hardly corroded even put in the acid alkaline. Tantalum are inert in organisms, so it is insoluble in water and acid and alkaline environment, and it does not produce any chemical reaction under room temperature, or stimulate tissue with erosion, these make tantalum has good biocompatibility. Tantalum metal became to be the clinical application of surgical choice of implanted materials because of characters of high melting point, high hardness, wear resistance and good physical properties, chemical properties of corrosion resistance and good biocompatibility. In the medical field, the history of the application of tantalum metal is more than half a century, it is mainly used in cardiac pacemaker, skull defect repair materials, vascular clamp, the femoral prosthesis handle, bone defect filler material manufacturing and so on. Studies have shown that phagocytic cells are the main material of removal of tantalum in the body. Experiments showed that phagocytic cells are kept active and no cell degeneration, only accompanied by glucose oxidation increased significantly in the body in contact with tantalum dust after1hour, and under the same conditions with silica dust can cause a serious phagocytes cytoplasm degeneration and death, which suggests that tantalum metal has no cytotoxicity and toxic to the human body. In1997, the American FDA approved of porous tantalum metal applied to clinical, the material looks like cancellous bone structure under the microscope, its pore size is between400-600microns, the whole inter connected porosity is up to75%-85%, significantly higher than the other biological fixation materials, such as cobalt, chromium, titanium which with porosity of30%～50%. Porous tantalum of three-dimensional porous structure has more advantageous to the adhesion, differentiation and growth of osteoblast, promotes the growth of bone, so as to strengthen the connection between the implant and bone, to achieve the effect of biological fixation, as well as to the moisture and nutrients in implanted transmission. Porous tantalum metal has good elastic modulus, it can reduce the stress shelter of implants, porous design also make its surface has a higher coefficient of friction than other materials, early implantation is also more conducive to regional stability, the porous structure is advantageous to the bone ingrowth. Although in a separate application of porous tantalum metal despite better mechanical support, due to the reduce or death of local bone cell activity caused by the occurrence of osteonecrosis, it still lacks of enough seed cells, thus we can conceive of BMSCs adhesion to porous tantalum metal micro hole, after being BMSCs proliferation to a certain number, make them joint into the part of osteonecrosis, let porous tantalum metal have the effect of support, provide bone ingrowth space, and have enough seed cells, repair necrotic areas around the porous tantalum metal and micro hole to form a healthy bone growth environment, repair osteonecrosis, to prevent further collapse of femoral head.
Purpose and meaning:Investigate the porous tantalum metal used on the effects of BMSCs proliferation, discusses the application of porous tantalum metal joint BMSCs curative effect for the treatment of osteonecrosis. Osteonecrosis is currently one of the common bone disease, the disease has brought the huge influence to people's life and necrosis are generally first occurs within the femoral head, femoral head necrosis process will gradually collapse, severe pain and dysfunction make patients with walking difficulties, loss of labor, serious life cannot walk who can only just stay in bed. Osteonecrosis ARCO stage guide the treatment of the clinic disease, commonly in clinical phase Ⅰ and phase Ⅱ is the early stage of the collapse, that is the early cases, active therapy may terminate illness development, the general treatment method is pulp core decompression, pulp core decompression with stem cell therapy, with vascular pedicle bone flap transfer operation, etc., but there are all have some different faults. Commonly pulp core decompression surgery lack the support of the structure of the subchondral bone, that have the risk of subchondral bone fracture, stem cell therapy of stem cells that is the liquid, which inject the parts of the osteonecrosis is not fixed, and the support with vascular pedicle bone flap transfer operation is not well. The period of Ⅲstage with femoral head is collapse, even after surgery osteogenesis performance, also difficult to stop further development to osteoarthritis pathological changes. Protect the head and joint replacement treatment have different standard, but a unified point of view is confirmed head treatment effect is worse than the treatment before the head collapse. In the stage of IV, collapse of the femoral head and osteoarthritis, have joint replacement has become a consensus, joint pain, joint function after replacement are can be significantly improved, that have great significance to improve the quality of people's life, but the wear and tear of prosthetic joint, the occurrence of osteoporosis, etc all can cause joint prosthesis has the limitation of use fixed number of year, this is a joint replacement surgery in a short period of time can't solve the problem. Therefore, how to active treatment in front of the collapse of the femoral head necrosis, decrease complications and increase the success rate of surgery is the meaning of the experiment.
Methods:BMSCs of healthy rabbit were extracted by whole bone marrow adherent method, cultured with Alpha-MEM, containing10%FBS,1%PSN, identify stem cell properties by lipoblast experiment, osteogenesis experiment and colony forming experiment in particular inducer, Detect the cytotoxicity of porous tantalum metal by MTT method, compared the cell activity in more porous tantalum metal group and titanium group and control group respectively, Observe adhesion and proliferation of BMSCs on porous tantalum metal with GFP staining and electron microscope in the second day, five days and seven days after BMSCs jointed porous tantalum metal, detect porous tantalum metal influenced on the distribution of BMSCs induced osteogenic by PCR. Osteonecrosis model is set up, according the methods by professor Qinling,16healthy adult New Zealand white rabbit were selected,6months of age, muscle injection e. coli endotoxin with10g/kg dose, the injection interval time is24h, muscular injection methylprednisolone with20mg/kg dose3times, the injection interval time is24h, all experimental animals were no death. start experiments after models were successful built in2weeks. It is divided into two groups, each group of eight, they were implanted into pure porous tantalum metal and BMSCs jointed porous tantalum metal, judge the therapeutic effect of osteonecrosis with BMSCs jointed porous tantalum metal.by assessing the new bone formation around porous tantalum metal.
Results:we induced BMSCs which extracted by adherent separation by a concomitant, it confirmed that the cells can change to fat cells by the application of oil red staining, it confirmed the cells can transform to the osteoblast by application of alizarin red staining and cobalt calcium alkaline phosphatase staining,cell colony forming experiment indicates that the cells are colony growth, these all show that extracted cells are BMSCs.Detect cytotoxicity by MTT method on24hours and48hours comparing two time points, the cell of activity control group and experimental group were no statistical significance (P>0.05),it showed that porous tantalum metal as a implanted material has good biocompatibility. BMSCs were distributed evenly on surface of tantalum metal in GFP dyeing when BMSCs and porous tantalum metal culturing two days. Different cell morphology under microscope shows that these cells is not on a flat surface, some of them grow into the pores. At the fifth day, more and more cells proliferate and Passage on porous tantalum metal with pseudopodia adhere to the metal. At the seventh day, the three-dimensional structure of porous tantalum metal is full of BMSCs. Under scanning electron microscope, a few cells adhere to porous tantalum metal surface with varied shapes at the second day, at the fifth day cells clearly increased and extended into micro-pore, At the seventh day, pseudopodia of cells stuck out towards all directions and their vigor were nice. After co-culture two weeks, osteocalcin (OCN), osteopontin (OPN) and mRNA transcription factors Runx-2obviously increases determined with PCR, The co-work of tantalum and osteoblast inducing conditional media can enhance the expression of the OCN expression (A), inhibit the expression of OPN (B), and not affect the expression of Runx-2(C); Gene expression of the cell around tantalum material is similar. As a result, we judged that tantalum can promote differentiation of BMSCs to osteoblasts as a porous scaffolds. At3week after surgery, new bone can be observed in two groups around tantalum metal formed, but can not be in microporous. There was no statistical significant differences of average width of new bone in the two group (P>0.05).
At6week after surgery, the new bone increased obviously, the difference were significant in the two group (P<0.05); by Hard tissue section fluorescence technology, both groups have new bone growing into porous tantalum metal, and the depth of group adding BMSCs is greater than the group not adding. The difference was statistically significant (P<0.05).
Conclusion:Porous tantalum metal has no cytotoxicity. It is free of negative effect on the growth and proliferation of BMSCs with no negative impact. There is no significant difference compared with the control group. BMSCs grown on the surface of porous tantalum metal has increased, proliferated and migrated into the microporous. there is more new bone around the porous tantalum metal in the treatment of osteonecrosis using BMSCs and porous tantalum metal, which show the proliferation and differentiation to osteoblasts of BMSCs in the body can speed up the formation of new bone and the repairment of osteonecrosis. The two functions make the repairment of osteonecrosis and the growth of bone into porous tantalum metal even faster.
引文
[1]Ficat RP. Idiopathic bone necrosis of the femoral head. Early diagnosis and treatment [J] J Bone Joint Surg (Br),1985,3-9.
[2]赵德伟,王卫明,王本杰,等.保留股骨头手术治疗股骨头缺血性坏死1005例临床分析[J].中华外科杂志,2005,43(16)：1054-1057.
[3]张长青,曾炳芳,徐铮宇,等.吻合血管腓骨游离移植在股骨头缺血性坏死中的应用[J].中国修复重建外科杂志,2004,18(5)：367-369.
[4]Vacanti CA, Bonassar J. Clin Orthop,1999; 367(suppl):375～381
[5]Lam K, Esselbrugge H, S chakenraad J, et al. Biodegradable of porous versus non-porous poly (L-1 actic acid) films. JM ater Sci:Mater Med,1994; 5(2):101
[6]Lopez M F, Gutierrez A, Jimenez J A. Surface characterization of new non-toxic titanium alloys for use as biomaterials [J]. Surface Science, 2001,(01):300.
[7]战策,王志伟.钽金属在骨科的临床应用[J].中国矫形外科杂志,2009,17(20)：1547-1549.
[8]Sculco TP. The acetabular component:an elliptical monoblock alternative. J Arthroplasty,2002,17(4 Supply 1):118-120.
[9]Zardiackas LD, Parsell DE, Dillon LD, et al. Structure, metallurgy, and mechanical properties of a porous tantalum foam. J Biomed Mater Res,2001, 58:180-187.
[10]Bobyn JD, Stackpool GJ, Hacking SA, et al. Characteristics of bone ingrowth and interface mechanics of a new porous tantalum biomaterial, J Bone Joint Surg(Br),1999,81:907-914.
[11]Miyaza T, Kim HM, Kokubo T, et al. Mechanism of bonelike apatite formation on bioactive tantalum metal in a simulated body fluid. Biomaterials,2002,23: 827-832.
[12]Matsuno H, Yokoyama A, Wateri F, er al. Biocompatibility and osteogenesis of refeactory metal implants, titanium, hafnium, niobium, tantalum and rhenium. Biomaterials,2001,22:1253-1262
[13]陈凯,蔡郑东.多孔钽金属植入治疗早期股骨头坏死研究进展[J].国际骨科学杂志,2008,29(1)：41-42.
[14]Wiglield C, Robertson J, GilfS, etal. Clinical experience with porous tantalum cervical interbody implants in a prospective randomized controlled trial [J].Br J Neurosurg,2003,17:418-425.
[15]Woodbury D, Schwarz EJ, P rockop DJ, et al. Adult rat and human bone marrow stromal cells differentiate into neurons. J Neuro sci Res,2000,61(4): 3642370.
[16]Johnstone B, Yoo JU. Autologous mesenchymal progenitor cells in cartilage repair [J]. Clin Orthop,1999,367(supple):s156-s162.
[17]Livingston T, kadiyali S, Elkalay M. Repair of canine segmental bone defects using allogeneic mesenchymal stem cells[A].2001.26,49.
[18]Bruder SP, Kurth AA, Shea M, et al. Bone regeneration by implantation of purified culture-expanded human mesenchymal stem cells [J]. J Or thopaedic Res,1998,16(2):155-162.
[19]Heiner AD, Brown TD, Poggie RA. Structural efficacy of a novel porous tantalum implant for osteonecrosis grafting. Trans Orthop Res Soc.2001:26: 480.
[20]Kemp KC, Hows J, Donaldsonc.Bone marrow derived mesenchy mal stem cells (J). Leuk Lymphoma,2005,46(11):1531-1544
[21]秦岭,张戈,盛辉,等.现代生物影像方法评价脂多糖与甲基强的松龙联 合诱导的实验性骨坏死[J].中国修复重建外科杂志,2008,22(3)：258-264.
[22]苏敬阳,韩永台.非创伤性股骨头缺血性坏死股骨头塌陷的早期预测研究进展[J].河北医药,2012,34(8)：1235-1237.
[23]姜文学.2004年国家级股骨头坏死最新治疗进展专题研讨会会议纪要[J].实用骨科杂志,2005,11(2)：190-192
[24]Gangji V, Hauzeur JP. Treatment of osteonecros is of the femoral head with implantation of autologous bone-marrow cells.Surgical technique. J Bone Joint Surg 2005; 87 Supp11(Pt 1):106-112
[25]Jones JP. Epidemiologlcal risk factors for non-traumatic osteonecrosis[J]. Orth opade,2000,29(5):370-379
[26]Hirano K,Tsutsui H,Sugioka Y,et al. Histopathologic alterations of retinacular vessels and osteonecrosis. Clin Orthop,1997,342:192.
[27]Weinstein RS, Jilka RL, Parfitt AM et al. Inhibition of osteoblast ogenesis and promotion of apoptosis of osteoblasts and osteocytes by glucecorticoids. Potential mechanisms of their deleterious effects on bone. J Clin Invest,1998,1 02(2):274-282.
[28]Hofbauer LC, Gorl F Riggs B Let al. Stimulation of osteoprotegerin ligand and inhibition of osteoproteger in production by glucocorticoids in human osteoblastic ineage cells potentia paracrine mechanisms of glucocorticoid-induced osteoporosis. Endocrinologi,1999,140:4382-4389.
[29]W ang G J , Cui Q Balian G The pathogenesis and prevention of steroid-induced osteonecrosis. Clin Orthop,2000,370:295-310
[30]Hernigou P, Beaujean FLambotte JC. Decrease in the mesenchymal stem cell pool in the proximal femur in corticosteroid-induced osteonecrosis. J Bone Joint Surg Bt,1999,81(2):349-355.
[31]Li X, Jin L, Cui Q, et al. Steroid effects on osteogenesis through mesenchymal cel gene expression. Osteoporos Int,2004,15:1.
[32]李月白.激素诱导骨髓基质细胞成脂分化的实验研究.中华骨科杂志,1999,19(11)：687-689.
[33]薛元锁,时述山.激素性股骨头坏死的早期病理生理改变及治疗[J].中国矫形外科杂志,2000,7(2)：187-189.
[34]Ficat RP, Alert J, Hungerford DS. Necrosis of the femoral head, in ischemia and Necrosis of Bone.Baltimore:Williams&Wilkins,1980:53-74.
[35]ARCO(Association Research Circulation Osseous).Committee on Terminology and Classification [J].ARCO News,1992,4:41-46.
[36]Owen M. Marrow stromal stem cells. J Cell Sci(Suppl) 1988:10:63-76.
[37]Johnstone B, Yoo JU. Autologous mesenchymal progenitor cells in articular cartilage repair. Clin Orthop Relat Res 1999; (367 Suppl):S156-162
[38]XU WR, ZHANG XR, QIAN H, et al. Mesenchymal stem cells from adult human bone marrow differentiate into a cardiomyocyte phenotype in vitro [J]. ExpBiol Med,2004,229(7):623-631.
[39]Zhao D, Cui D, Wang B, et al. Treatment of early stage osteonecrosis of the femoral head with autologous implantation of bone marrow-derived and cultured mesenchymal stem cells[J]. Bone,2012,50(1):325-330.
[40]夏万尧,商庆新,崔磊,徐蓉,丁小邦,曹谊林。骨髓基质干细胞向软骨细胞表型定向诱导分化的实验研究。中华整形外科杂志,2002,18：12-14.
[41]Murphy J M, Kavalkovitch K W, Fink D, et al. Regeneration of meniscal tissue and protection of articular cartilage by injection of mesenchymal stem cells[J]. Osteoarthritis cartilage,2000,8(Suppl B):S25.
[42]Cuevas P, Carceller F, Dujovny M, et al. Peripheral nerve regeneration by bone marrow stromal cells[J]. Neurological research,2002,24(7):634-638.
[43]Hutmacher DW. Scaffolds in tissue engineering bone and cartilage. Biomaterials.2000; 21(24):2529-2543.
[44]Chang BS, Lee CK, Hong KS. Osteoconduction at porous hydroxyapatite with various pore configuration. Biomaterials.2000;21(25):1291-1304
[45]战策,王志伟.钽金属在骨科的临床应用[J].中国矫形外科杂志,2009,17(20)：1547-1549.
[46]Schepers S. The biological action of tantalum oxide. Arch Ind Health. 1955;12:121.
[47]夏凤金,刘培生,周茂奇.多孔钽的制备方法[J].科技创新导报,2008,1：83-84.
[48]武垚森,池永龙.小梁金属(多孔钽)在骨科的应用现状[J].中华骨科杂志,2007,27(12)：939-941.
[49]Hacking SA, Bobyn JD, Toh K, et al. Fibrous tissue ingrowth and attachment to porous tantalum. J Biomed Mater Res.2000;52(4):631-638.
[50]Aldegheri G, Taglialavoro A. The tantalum screw for treating femoral head necrosis, rationale and results Strat Traum. Limb Recon.2007;2(2-3):63-68.
[51]Veillette CJ, Mehdian H, Schemitsch EH, et al. Survivorship analysis and radiographic outcome following tantalum rod insertion for osteonecrosis of the femoral head. J Bone Joint Surg Am.2006;88Suppl 3:48-55.
[52]应明.多孔钽及在人工关节中的应用[J].生物骨科材料与临床研究,2006,3(2)：1-3.
[53]Tsao AK, Roberson JR, Christie MJ, et al. Biomechanical and clinical evaluations of a porous tantalum implant for the treatment of early-stage osteonecrosis. J Bone Joint Surg Am.2005;87Suppl 2:22-27.
[54]Zardiackas LD, Parsell DE, Dillon LD, et al. Structure, metallurgy, and mechanical properties of a porous tantalum foam. J Biomed Mater Res. 2001;58(2):180-187.
[55]Johansson CB, Hansson HA, Albrektsson T. Qualitative interfacial study between bone and tantalum, niobium or commercially pure titanium. Biomaterials.1990;11 (4):277-280.
[56]周长忍.生物材料学[M].北京：中国医药科技出版社,2004：111-149.
[57]Levine B, Della Valle CJ, Jacobs JJ. Applications of porous tantalum in total hip arthroplasty. J Am Acad Orthop Surg.2006; 14(12):646-655.
[58]Matsuno H, Yokoyama A, Watari F, et al. Biocompatibility and osteogenesis of refractory metal implants, titanium, hafnium, niobium, tantalum and rhenium. Biomaterials.2001;22(11):1253-1262.
[59]Bobyn JD, Poggie RA, Krygier JJ, et al. Clinical validation of astructural porous tantalum biomaterial for adult reconstruction. J Bone Joint Surg Am. 2004;86-A Suppl 2:123-129.
[60]Balla VK, Bodhak S, Bose S, et al. Porous tantalum structures for bone implants:fabrication, mechanical and in vitro biological properties. Acta Biomater.2010;6(8):3349-3359.
[61]Bobyn JD, Stackpool GJ, Hacking SA, et al. Characteristics of bone ingrowth and interface mechanics of a new porous tantalum biomaterial. J Bone Joint Surg Br.1999;81(5):907-914.
[62]Itala A, Heijink A, Leerapun T, et al. Successful canine patellar tendon reattachment to porous tantalum. Clin Orthop Relat Res.2007;463:202-207.
[63]Macheras GA, Papagelopoulos PJ, Kateros K, etal. Radiological evaluation of the metal-bone interface of a porous tantalum monoblock acetabular component. J Bone Joint Surg Br.2006;88(3):304-309.
[64]Veillette CJ, Mehdian H, Schemitsch EH, et al. Survivorship analysis and radiographic outcome following tantalum rod insertion for osteonecrosis of the femoral head. J Bone Joint Surg Am.2006;88Suppl 3:48-55.
[65]Bobyn JD, Toh KK, Hacking SA, et al. Tissue response to porous tantalum acetabular cups:a canine model. J Arthroplasty.1999;14(3):347-354.
[66]Balla VK, Banerjee S, Bose S, et al. Direct laser processing of a tantalum coating on titanium for bone replacement structures. Acta Biomater. 2010;6(6):2329-2334.
[67]Zhang YM, Bataillon-Linez P, Huang P, et al. Surface analyses of micro-arc oxidized and hydrothermally treated titanium and effect on osteoblast behavior. J Biomed Mater Res A.2004;68(2):383-391.
[68]Schildhauer TA, Robie B, Muhr G, et al. Bacterial adherence to tantalum versus commonly used orthopedic metallic implant materials. J Orthop Trauma. 2006;20(7):476-484.
[69]Zhao D, Zhang Y, Wang W, et al. Tantalum rod implantation and vascularized iliac grafting for osteonecrosis of the femoral head[J]. Orthopedics,2013, 36(6):789-795.
[70]Juan F. Blanco Fermin M. Sa'nchez-Guijo Soraya Carrancio Sandra Muntion Jesus Garl'a-Brin~on Maria-Consuelo del Can" izo Titanium and tantalum as mesenchymal stem cell scaffolds for spinal fusion:and in vitro comparative study
[2]赵德伟,王卫明,王本杰,等.保留股骨头手术治疗股骨头缺血性坏死1005例临床分析[J].中华外科杂志,2005,43(16)：1054-1057.
[3]张长青,曾炳芳,徐铮宇,等.吻合血管腓骨游离移植在股骨头缺血性坏死中的应用[J].中国修复重建外科杂志,2004,18(5)：367-369.
[4]Vacanti CA, Bonassar J. Clin Orthop,1999; 367(suppl):375～381
[5]Lam K, Esselbrugge H, S chakenraad J, et al. Biodegradable of porous versus non-porous poly (L-1 actic acid) films. JM ater Sci:Mater Med,1994; 5(2):101
[6]Lopez M F, Gutierrez A, Jimenez J A. Surface characterization of new non-toxic titanium alloys for use as biomaterials [J]. Surface Science, 2001,(01):300.
[7]战策,王志伟.钽金属在骨科的临床应用[J].中国矫形外科杂志,2009,17(20)：1547-1549.
[8]Sculco TP. The acetabular component:an elliptical monoblock alternative. J Arthroplasty,2002,17(4 Supply 1):118-120.
[9]Zardiackas LD, Parsell DE, Dillon LD, et al. Structure, metallurgy, and mechanical properties of a porous tantalum foam. J Biomed Mater Res,2001, 58:180-187.
[10]Bobyn JD, Stackpool GJ, Hacking SA, et al. Characteristics of bone ingrowth and interface mechanics of a new porous tantalum biomaterial, J Bone Joint Surg(Br),1999,81:907-914.
[11]Miyaza T, Kim HM, Kokubo T, et al. Mechanism of bonelike apatite formation on bioactive tantalum metal in a simulated body fluid. Biomaterials,2002,23: 827-832.
[12]Matsuno H, Yokoyama A, Wateri F, er al. Biocompatibility and osteogenesis of refeactory metal implants, titanium, hafnium, niobium, tantalum and rhenium. Biomaterials,2001,22:1253-1262
[13]陈凯,蔡郑东.多孔钽金属植入治疗早期股骨头坏死研究进展[J].国际骨科学杂志,2008,29(1)：41-42.
[14]Wiglield C, Robertson J, GilfS, etal. Clinical experience with porous tantalum cervical interbody implants in a prospective randomized controlled trial [J].Br J Neurosurg,2003,17:418-425.
[15]Woodbury D, Schwarz EJ, P rockop DJ, et al. Adult rat and human bone marrow stromal cells differentiate into neurons. J Neuro sci Res,2000,61(4): 3642370.
[16]Johnstone B, Yoo JU. Autologous mesenchymal progenitor cells in cartilage repair [J]. Clin Orthop,1999,367(supple):s156-s162.
[17]Livingston T, kadiyali S, Elkalay M. Repair of canine segmental bone defects using allogeneic mesenchymal stem cells[A].2001.26,49.
[18]Bruder SP, Kurth AA, Shea M, et al. Bone regeneration by implantation of purified culture-expanded human mesenchymal stem cells [J]. J Or thopaedic Res,1998,16(2):155-162.
[19]Heiner AD, Brown TD, Poggie RA. Structural efficacy of a novel porous tantalum implant for osteonecrosis grafting. Trans Orthop Res Soc.2001:26: 480.
[20]Kemp KC, Hows J, Donaldsonc.Bone marrow derived mesenchy mal stem cells (J). Leuk Lymphoma,2005,46(11):1531-1544
[21]秦岭,张戈,盛辉,等.现代生物影像方法评价脂多糖与甲基强的松龙联 合诱导的实验性骨坏死[J].中国修复重建外科杂志,2008,22(3)：258-264.
[22]苏敬阳,韩永台.非创伤性股骨头缺血性坏死股骨头塌陷的早期预测研究进展[J].河北医药,2012,34(8)：1235-1237.
[23]姜文学.2004年国家级股骨头坏死最新治疗进展专题研讨会会议纪要[J].实用骨科杂志,2005,11(2)：190-192
[24]Gangji V, Hauzeur JP. Treatment of osteonecros is of the femoral head with implantation of autologous bone-marrow cells.Surgical technique. J Bone Joint Surg 2005; 87 Supp11(Pt 1):106-112
[25]Jones JP. Epidemiologlcal risk factors for non-traumatic osteonecrosis[J]. Orth opade,2000,29(5):370-379
[26]Hirano K,Tsutsui H,Sugioka Y,et al. Histopathologic alterations of retinacular vessels and osteonecrosis. Clin Orthop,1997,342:192.
[27]Weinstein RS, Jilka RL, Parfitt AM et al. Inhibition of osteoblast ogenesis and promotion of apoptosis of osteoblasts and osteocytes by glucecorticoids. Potential mechanisms of their deleterious effects on bone. J Clin Invest,1998,1 02(2):274-282.
[28]Hofbauer LC, Gorl F Riggs B Let al. Stimulation of osteoprotegerin ligand and inhibition of osteoproteger in production by glucocorticoids in human osteoblastic ineage cells potentia paracrine mechanisms of glucocorticoid-induced osteoporosis. Endocrinologi,1999,140:4382-4389.
[29]W ang G J , Cui Q Balian G The pathogenesis and prevention of steroid-induced osteonecrosis. Clin Orthop,2000,370:295-310
[30]Hernigou P, Beaujean FLambotte JC. Decrease in the mesenchymal stem cell pool in the proximal femur in corticosteroid-induced osteonecrosis. J Bone Joint Surg Bt,1999,81(2):349-355.
[31]Li X, Jin L, Cui Q, et al. Steroid effects on osteogenesis through mesenchymal cel gene expression. Osteoporos Int,2004,15:1.
[32]李月白.激素诱导骨髓基质细胞成脂分化的实验研究.中华骨科杂志,1999,19(11)：687-689.
[33]薛元锁,时述山.激素性股骨头坏死的早期病理生理改变及治疗[J].中国矫形外科杂志,2000,7(2)：187-189.
[34]Ficat RP, Alert J, Hungerford DS. Necrosis of the femoral head, in ischemia and Necrosis of Bone.Baltimore:Williams&Wilkins,1980:53-74.
[35]ARCO(Association Research Circulation Osseous).Committee on Terminology and Classification [J].ARCO News,1992,4:41-46.
[36]Owen M. Marrow stromal stem cells. J Cell Sci(Suppl) 1988:10:63-76.
[37]Johnstone B, Yoo JU. Autologous mesenchymal progenitor cells in articular cartilage repair. Clin Orthop Relat Res 1999; (367 Suppl):S156-162
[38]XU WR, ZHANG XR, QIAN H, et al. Mesenchymal stem cells from adult human bone marrow differentiate into a cardiomyocyte phenotype in vitro [J]. ExpBiol Med,2004,229(7):623-631.
[39]Zhao D, Cui D, Wang B, et al. Treatment of early stage osteonecrosis of the femoral head with autologous implantation of bone marrow-derived and cultured mesenchymal stem cells[J]. Bone,2012,50(1):325-330.
[40]夏万尧,商庆新,崔磊,徐蓉,丁小邦,曹谊林。骨髓基质干细胞向软骨细胞表型定向诱导分化的实验研究。中华整形外科杂志,2002,18：12-14.
[41]Murphy J M, Kavalkovitch K W, Fink D, et al. Regeneration of meniscal tissue and protection of articular cartilage by injection of mesenchymal stem cells[J]. Osteoarthritis cartilage,2000,8(Suppl B):S25.
[42]Cuevas P, Carceller F, Dujovny M, et al. Peripheral nerve regeneration by bone marrow stromal cells[J]. Neurological research,2002,24(7):634-638.
[43]Hutmacher DW. Scaffolds in tissue engineering bone and cartilage. Biomaterials.2000; 21(24):2529-2543.
[44]Chang BS, Lee CK, Hong KS. Osteoconduction at porous hydroxyapatite with various pore configuration. Biomaterials.2000;21(25):1291-1304
[45]战策,王志伟.钽金属在骨科的临床应用[J].中国矫形外科杂志,2009,17(20)：1547-1549.
[46]Schepers S. The biological action of tantalum oxide. Arch Ind Health. 1955;12:121.
[47]夏凤金,刘培生,周茂奇.多孔钽的制备方法[J].科技创新导报,2008,1：83-84.
[48]武垚森,池永龙.小梁金属(多孔钽)在骨科的应用现状[J].中华骨科杂志,2007,27(12)：939-941.
[49]Hacking SA, Bobyn JD, Toh K, et al. Fibrous tissue ingrowth and attachment to porous tantalum. J Biomed Mater Res.2000;52(4):631-638.
[50]Aldegheri G, Taglialavoro A. The tantalum screw for treating femoral head necrosis, rationale and results Strat Traum. Limb Recon.2007;2(2-3):63-68.
[51]Veillette CJ, Mehdian H, Schemitsch EH, et al. Survivorship analysis and radiographic outcome following tantalum rod insertion for osteonecrosis of the femoral head. J Bone Joint Surg Am.2006;88Suppl 3:48-55.
[52]应明.多孔钽及在人工关节中的应用[J].生物骨科材料与临床研究,2006,3(2)：1-3.
[53]Tsao AK, Roberson JR, Christie MJ, et al. Biomechanical and clinical evaluations of a porous tantalum implant for the treatment of early-stage osteonecrosis. J Bone Joint Surg Am.2005;87Suppl 2:22-27.
[54]Zardiackas LD, Parsell DE, Dillon LD, et al. Structure, metallurgy, and mechanical properties of a porous tantalum foam. J Biomed Mater Res. 2001;58(2):180-187.
[55]Johansson CB, Hansson HA, Albrektsson T. Qualitative interfacial study between bone and tantalum, niobium or commercially pure titanium. Biomaterials.1990;11 (4):277-280.
[56]周长忍.生物材料学[M].北京：中国医药科技出版社,2004：111-149.
[57]Levine B, Della Valle CJ, Jacobs JJ. Applications of porous tantalum in total hip arthroplasty. J Am Acad Orthop Surg.2006; 14(12):646-655.
[58]Matsuno H, Yokoyama A, Watari F, et al. Biocompatibility and osteogenesis of refractory metal implants, titanium, hafnium, niobium, tantalum and rhenium. Biomaterials.2001;22(11):1253-1262.
[59]Bobyn JD, Poggie RA, Krygier JJ, et al. Clinical validation of astructural porous tantalum biomaterial for adult reconstruction. J Bone Joint Surg Am. 2004;86-A Suppl 2:123-129.
[60]Balla VK, Bodhak S, Bose S, et al. Porous tantalum structures for bone implants:fabrication, mechanical and in vitro biological properties. Acta Biomater.2010;6(8):3349-3359.
[61]Bobyn JD, Stackpool GJ, Hacking SA, et al. Characteristics of bone ingrowth and interface mechanics of a new porous tantalum biomaterial. J Bone Joint Surg Br.1999;81(5):907-914.
[62]Itala A, Heijink A, Leerapun T, et al. Successful canine patellar tendon reattachment to porous tantalum. Clin Orthop Relat Res.2007;463:202-207.
[63]Macheras GA, Papagelopoulos PJ, Kateros K, etal. Radiological evaluation of the metal-bone interface of a porous tantalum monoblock acetabular component. J Bone Joint Surg Br.2006;88(3):304-309.
[64]Veillette CJ, Mehdian H, Schemitsch EH, et al. Survivorship analysis and radiographic outcome following tantalum rod insertion for osteonecrosis of the femoral head. J Bone Joint Surg Am.2006;88Suppl 3:48-55.
[65]Bobyn JD, Toh KK, Hacking SA, et al. Tissue response to porous tantalum acetabular cups:a canine model. J Arthroplasty.1999;14(3):347-354.
[66]Balla VK, Banerjee S, Bose S, et al. Direct laser processing of a tantalum coating on titanium for bone replacement structures. Acta Biomater. 2010;6(6):2329-2334.
[67]Zhang YM, Bataillon-Linez P, Huang P, et al. Surface analyses of micro-arc oxidized and hydrothermally treated titanium and effect on osteoblast behavior. J Biomed Mater Res A.2004;68(2):383-391.
[68]Schildhauer TA, Robie B, Muhr G, et al. Bacterial adherence to tantalum versus commonly used orthopedic metallic implant materials. J Orthop Trauma. 2006;20(7):476-484.
[69]Zhao D, Zhang Y, Wang W, et al. Tantalum rod implantation and vascularized iliac grafting for osteonecrosis of the femoral head[J]. Orthopedics,2013, 36(6):789-795.
[70]Juan F. Blanco Fermin M. Sa'nchez-Guijo Soraya Carrancio Sandra Muntion Jesus Garl'a-Brin~on Maria-Consuelo del Can" izo Titanium and tantalum as mesenchymal stem cell scaffolds for spinal fusion:and in vitro comparative study