Osteogenic differentiation of adipose-derived stem cells on a composite scaffold in the repair of osteoporotic bone defects

doi:10.3969/j.issn.2095-4344.014.41.024

Abstract

Abstract:

BACKGROUND: The traditional treatment methods for osteoporosis accompanied by bone defects, such as autogenous bone graft, allograft, biomaterial implants, have significant limitations. The regenerative medicine approach using adipose-derived stem cells as seed cells offers a new way for the repair of bone defects following osteoporosis.

OBJECTIVE: To review the pathogenesis of osteoporosis and its impacts on the repair of bone defects, the signal pathway regulation of osteogenic differentiation of adipose-derived stem cells, and the feasibility of adipose-derived stem cells for repairing osteoporotic bone defects.

METHODS: A computer-based online search of CNKI database and PubMed database was performed to retrieve the relevant articles published from January 1998 to September 2014 with the key words of “adipose-derived stem cells, osteoporosis, bone defect, osteogenic differentiation, bone regeneration” in Chinese and English, respectively. Finally 77 articles were included for review after deleting unrelated and repetitive ones.

RESULTS AND CONCLUSION: In recent years, adipose-derived stem cells have been widely used in regenerative medicine research. With the development of relevant disciplines such as regenerative medicine, tissue engineering, molecular biology, and material science, in-depth studies on regulatory mechanisms of osteogenic differentiation of adipose-derived stem cells have been developed. Adipose-derived stem cells combined with biological scaffolds for construction of tissue-engineered bones provides a new way to repair osteoporotic bone defects.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

全文链接：

Key words: stem cells, adipose tissue, osteoporosis, bone regeneration

CLC Number:

R318

Huang Cheng-long, Xiao Jin-gang. Osteogenic differentiation of adipose-derived stem cells on a composite scaffold in the repair of osteoporotic bone defects[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(41): 6696-6702.

Figures/Tables 1

2.1 骨质疏松症的发病机制及对骨缺损修复的影响 2.1.1 骨质疏松症的发病机制成骨细胞和破骨细胞活动的平衡维护骨稳态。此过程受到多种激素及生长因子的调节，如雌激素、骨保护素、甲状旁腺激素和生长因子等。目前公认的骨质疏松症发病机制是成骨细胞和破骨细胞在骨重塑过程中失衡，成骨细胞数量和活性降低[2]，破骨细胞数量增加、功能活跃[3]，导致骨吸收大于骨形成，造成骨质疏松。新成骨细胞和破骨细胞凋亡时间比率的平衡失调也是导致骨质疏松的重要原因[4]，而其中的关键因素是成骨细胞的不足[5]。成骨细胞在骨重建过程中起到核心作用，其不仅调节骨的矿化，还间接调节破骨细胞的骨吸收功能，它的数量和功能变化直接影响代谢性骨病的发生、发展和预后[6]。 2.1.2 骨质疏松症对骨缺损修复的影响当机体骨质疏松时，由于破骨细胞活性增强，成骨细胞的不足，骨的吸收大于骨的形成，故新骨生成能力下降，导致骨质疏松骨缺损的愈合时间明显延迟，而新生骨痂减少，愈合后的骨生物力学强度差，容易骨折，同时也常伴有松质骨压缩及骨缺损。与常规骨缺损的治疗相比，治疗骨质疏松骨缺损的花费要更大且传统方法疗效欠佳。 2.2 信号通路对脂肪干细胞成骨分化的调控脂肪干细胞成骨分化是修复骨质疏松症骨缺损的关键环节，其成骨分化受到多种信号通路的调控[7]。已经证实骨形成蛋白、成纤维细胞生长因子、Notch、Wnt/β-Catenin等信号通路参与调控脂肪干细胞的成骨分化[8]。骨形成蛋白是转化生长因子β家族的成员，在骨再生过程中，骨形成蛋白具有刺激脂肪干细胞成骨分化的作用。骨形成蛋白通过与细胞表面骨形成蛋白受体结合，磷酸化下游信号分子R-Smad(Smad 1、5和8)，磷酸化的R-Smad与Smad 4结合形成复合体，进而与核内转录因子如Runx2/Cbfa1和Osterix等结合，调控成骨相关靶基因骨桥蛋白、骨钙蛋白和Ⅰ型胶原蛋白等的转录[9]。有学者用骨形成蛋白2转染脂肪干细胞，修复雌性去卵巢大鼠骨缺损，发现脂肪干细胞对去卵巢大鼠骨质疏松症骨缺损有很好的修复作用[10]。国外大量研究也证实骨形成蛋白2在脂肪干细胞中过表达具有促进脂肪干细胞介导的骨修复作用[11-13]。通过激活骨形成蛋白信号通路，促进脂肪干细胞的成骨分化，抑制成脂分化。基于骨形成蛋白和脂肪干细胞构建的组织工程骨修复骨质疏松症骨缺损具有巨大前景[14]。成纤维细胞生长因子信号在软骨内和膜内成骨中扮演复杂角色，且对脂肪干细胞的体内和体外成骨分化也具有重要影响[15-16]。成纤维细胞生长因子2作为骨细胞的自分泌和旁分泌因子，既能调节成骨又能调节成软骨，是成纤维细胞生长因子家族中调控骨形成的一个典型因子[15]。虽然成纤维细胞生长因子2促进脂肪干细胞介导的骨修复已被报道过，证明其能够通过拮抗视黄酸介导的上调BMPR-1B通路抑制晚期成骨分化[16-18]。这个明显的矛盾该如何解释？一方面可认为成纤维细胞生长因子2促进了骨祖细胞的增殖，维持和扩大骨祖细胞库，为随后的及时分化作准备。另一方面可认为成纤维细胞生长因子2能激活MAP/ERK通路、PKC通路和PI3K通路，促进增殖和分化[15,19]。在成骨过程中由于成纤维细胞生长因子信号的复杂作用，以及其他信号通路的参与，若能调节一个特定的信号通路，基于脂肪干细胞的骨组织再生疗法将具有巨大优势。Kwan等[17]学者研究证实通过化学方法控制成纤维细胞生长因子2的分泌，能促进脂肪干细胞修复颅骨极量缺损。 Artavanis-Tsakona等[20]最初在果蝇神经系统发育的研究中，发现一条在多种细胞的特化中起关键作用的信号途径，称为Notch信号通路。Notch信号传递过程中与相邻细胞表面的配体结合后，Notch的胞内区被水解切割，产生Notch胞内域(Notch Intracellular Domain， NICD)，NICD从细胞膜上脱离被转运进入细胞核，与转录抑制因子RBPJK结合发生经典信号转导，产生激活效应诱导靶基因的表达。体外实验研究表明，Notch信号通路是一条影响细胞命运，保守而重要的信号转导通路，可以调节细胞增殖、分化和凋亡，在多种干细胞或前体细胞中都起作用，影响甚至决定这些细胞的命运[21-22]。Notch信号通路对于脂肪干细胞成骨分化具有正反两种调控作用[23]。阻断Notch信号通路导致骨量增加，细胞成骨向分化减少，并维持细胞的干性[24]。Notch1及其下游Hey1和Hes1抑制Runx2转录活性从而间接抑制成骨向分化[25]。一些学者认为Notch信号促进细胞成骨向分化[26]。相反，也有学者认为Notch信号抑制成骨细胞形成和成骨向分化[27-28]。鉴于这种情况，Notch信号通路调控脂肪干细胞成骨分化中，何种作用起主导作用值得深入研究。对间充质干细胞骨向分化的研究发现，Wnt 信号通路在其骨向分化中具有重要调控作用，可以调控间充质干细胞的细胞增殖和凋亡，决定细胞命运[29]。国内外近期研究亦表明，Wnt/β-catenin是调控干细胞骨向分化的关键信号通路，对维持骨平衡和调节间充质祖细胞骨向分化具有重要作用[30-31]。近年来的报道称经典Wnt信号通路是生理成骨必需的，无论Wnt信号分子的表达增强还是Wnt抑制因子的缺失都将导致骨形成的增强。低密度脂蛋白受体相关蛋白(LPR5)获得性功能突变会导致高骨量表型，而缺失性功能突变将引起骨质疏松[32]。通过小鼠遗传学研究发现，经典Wnt信号通路的激活增加了骨量和骨强度，而其抑制则导致骨形成减少、骨强度降低[30]。小鼠中增加的Wnt 信号水平促使祖细胞直接分化为成骨细胞系，增加了骨形成，促进了骨折修复，并抑制了成骨细胞凋亡[33]。经典 Wnt 信号中的Wnt10b 基因在间充质祖细胞中过表达可以促进成骨细胞的发生，在体内会使骨密度增加，减少了雌激素缺乏导致的骨丢失；Wnt10b基因敲除(Wnt10b-/-)小鼠中间充质祖细胞数量减少、成骨相关基因减少，骨小梁和血清骨钙蛋白亦减少，进而骨量减少[34]。LRP5的激活导致骨量增高、阻止了年老性骨形成减少和脂肪形成增加，功能失活突变则引起骨质疏松症[35]。对成体干细胞的研究表明，Wnt/β-catenin信号路径的激活促进了端突干细胞的增殖和骨向分化[36]。生理状态下具有多向分化潜能的祖细胞中成骨和成脂转录因子维持在生理水平，Wnt/β-catenin信号通路激活后，通过抑制成脂转录因子C/EBPα和 PPARγ的表达，增加成骨转录因子 Runx2、Dlx5和osterix的表达，从而促使间充质干细胞向成骨细胞分化，同时抑制了成脂形成[37]。而锶能够在体内外激发β-catenin表达，激活Wnt/β-catenin信号路径，在体外增强人间充质干细胞的骨向分化、上调细胞外基质相关基因的表达，体内实验证实促进了骨形成[38]。不同细胞信号通路之间不仅能发生协同作用，也可能产生拮抗作用，这就是信号通路之间的“串话”[39-40]。间充质细胞在Wnt/β-catenin激活的情况下直接分化为成骨前体细胞；相反则分化为脂肪细胞或软骨细胞[41]。Wnt/β-catenin和骨形成蛋白对成骨前体细胞有着相反的作用，Wnt/β-catenin通路能维持这些细胞的前体状态和促进细胞的增殖，骨形成蛋白信号则能刺激这些细胞分化为成熟的成骨细胞[42-43]。当成骨前体细胞分化为成骨细胞时，Wnt/β-catenin和骨形成蛋白则发挥协同作用，促进细胞进一步分化和合成碱性磷酸酶[44]。Wnt/β-catenin与骨形成蛋白在骨生成的不同阶段发挥不同的作用(拮抗或协同)[41]。 Notch信号通路在调节成骨过程中与Wnt信号通路存在相互拮抗的关系。在ST2骨髓基质细胞中，过表达NICD抑制骨形成蛋白2和Wnt3a的作用，降低碱性磷酸酶活性。同时，通过Hes1抑制Wnt依赖受体活性和β-catenin水平，最终抑制成骨发生[45]。 Notch和骨形成蛋白在成骨过程中有协同增强的作用。过表达NICD促进骨形成蛋白所引起的成骨向分化，碱性磷酸酶表达增高、钙化结节形成增多，证明NICD促进骨形成蛋白介导的成骨过程[46]。当不与骨形成蛋白2共同作用时，过表达的Dll1和Jag1激活内源性NICD对细胞分化没有作用。此外，在加入Noth1抑制剂后可观察到骨形成蛋白目的基因启动子活性下降和碱性磷酸酶活性下降[47]。这些证据表明Notch和骨形成蛋白协同促进成骨细胞分化。成纤维细胞生长因子和骨形成蛋白对骨形成也具有相互作用。成纤维细胞生长因子2和成纤维细胞生长因子9能增加其他成骨因子的表达，如骨形成蛋白2和转化生长因子β1，以及内源性FGF/FGFR信号在上游调节骨形成蛋白2介导的成骨细胞分化[48]。成纤维细胞生长因子2和骨形成蛋白2对骨折愈合具有协同效应：在早期阶段成纤维细胞生长因子2的一个关键功能是为稍后阶段骨形成蛋白2促进矿化作准备[49]。成纤维细胞生长因子2基因敲除小鼠Runx2的聚集已受损，阻碍骨形成蛋白2诱导的骨形成和碱性磷酸酶活性[50]。在颅骨发育中，Runx2是介导骨形成蛋白2表达响应成纤维细胞生长因子刺激的关键环节[51]。成纤维细胞生长因子和骨形成蛋白在骨形成中的协同效应可以通过Rnnx2的激活来调控。 2.3 脂肪干细胞修复骨质疏松症骨缺损的可行性根据骨质疏松症的发病机制，理论上通过增加成骨细胞的量可以达到治愈骨质疏松症且修复骨缺损的目的。成骨细胞起源于多能的骨髓基质的间质细胞——间质干细胞。目前虽可从多种组织中分离到间质干细胞，但包括骨髓在内的组织中蕴藏的间充质干细胞比例都非常低[52]。脂肪干细胞相对容易获得而且远比其他组织来源间充质干细胞产量高，更易培养、生长快且不易衰老[53-54]。此外，脂肪干细胞在体内和体外均显示出相似于骨髓间充质干细胞的成骨分化能力[55]，一些研究人员甚至认为脂肪干细胞在某些方面优于骨髓间充质干细胞[56]，成为近年来的研究热点。脂肪组织供体的差异可能影响分离的脂肪干细胞的活性。一般认为脂肪干细胞的增殖和分化能力随供体年龄增加而降低。Zhu等[57-58]研究指出脂肪干细胞的成脂能力与年龄关系不大，但成骨潜能明显与年龄相关。脂肪干细胞的增殖和成骨分化能力比骨髓间充质干细胞少受年龄和传代的影响，这表明以脂肪干细胞为基础的治疗，尤其是治疗老年患者骨质疏松更有潜力。不同性别的脂肪分布有重要的异质性，女性脂肪干细胞更具持久的细胞表面分子表达和增殖[59]。不同部位的皮下脂肪组织有不同的血液供应、细胞因子信号和基因表达谱，导致成骨能力的不同，研究发现内脏脂肪比皮下脂肪具有更大的成骨能力[60]。还有研究表明，不同个体的脂肪干细胞活性各异。脂肪干细胞的需求量大，常需冻存。进行细胞冻存和在这种条件下的长期储存不可避免的会改变细胞的过程和特性[61]。对解冻细胞的体外细胞参数包括增殖、成骨和成脂分化能力的评估，证明冷冻保存会降低增殖和分化能力[62-63]。James等[64]研究发现即使冻存细胞只有2周时间，对细胞的成骨分化能力还是有不良影响。因此，虽然冻存的细胞能够进行成骨分化，但是直接使用刚获得的脂肪干细胞可能更有利于成骨重建。临床上已经有使用自体脂肪干细胞与β-磷酸三钙和骨形成蛋白2组合修复10 cm长的下颌骨缺损的报道[65]。最近，有学者通过对比同种异体与自体脂肪干细胞复合天然珊瑚支架修复比格犬的颅骨极量缺损，发现同种异体脂肪干细胞是可以修复颅面骨缺损，并且无需使用免疫抑制剂治疗，脂肪干细胞具有一定的免疫调节功能，能够抑制T淋巴细胞的增殖与活化[66-67]。还有实验证明未分化的与已分化的脂肪干细胞都能促进免疫抑制作用，异体脂肪干细胞可以代替自体脂肪干细胞和间充质干细胞作为骨组织工程的种子细胞来源[68]。然而，是否适用于临床，还有待进一步研究。 Cao等[69]将自体浓缩的骨髓间充质干细胞与多孔β-磷酸三钙支架相结合，植入骨质疏松山羊的股骨内侧髁极量骨缺损处，移植16周后用X射线，显微CT，组织切片染色发现植入骨髓间充质干细胞复合β-磷酸三钙支架组的骨缺损得到修复，成骨能力显著提高。赵铭等[10]发现人骨形态生发蛋白2基因修饰的脂肪干细胞对去卵巢大鼠骨质疏松性骨缺损有很好的修复作用，为绝经后所致的骨质疏松性骨缺损提示了新的治疗思路。Tao等[70]将体外分离培养至第6代的脂肪干细胞经尾静脉注射到骨质疏松大鼠模型，4周后发现，腰椎及股骨的骨密度均明显增加，破骨细胞数目减少，骨吸收下降，骨小梁的形成增多，骨小梁变厚变粗，骨小梁间的连接增加，陷窝减少。说明脂肪干细胞可提高骨密度，降低骨吸收，促进骨形成，有效改善骨组织微结构。有研究表明，系统移植沉默Zfp467的脂肪干细胞到去势小鼠体内，可同时激活成骨和破骨，且骨形成大于骨吸收，恢复骨质疏松引起的骨质流失[71]。也有学者研究发现异体脂肪源干细胞移植对糖皮质激素致骨质疏松大鼠有一定的治疗作用，能够促进骨形成，增加骨密度，改善骨生物力学性能，降低骨折发生率。进一步提示在骨质疏松的研究中应该更加关注提高机体干细胞的数量和促进干细胞向成骨细胞分化及骨组织的形成方面，为临床上治疗骨质疏松提供了一种新的研究思路[72]。李冬松[73]将血管内皮生长因子基因修饰的脂肪干细胞凝胶海绵材料植入糖尿病大鼠骨质疏松性骨缺损区，术后2周发现植入区即有大量细小血管呈网状生成，并在第4周保持较多生长，使孔隙临近骨质密度明显提高，自体骨与材料之间产生明显的骨化融合带。汪玉海等[74]构建大鼠骨质疏松症骨折模型，右侧股骨骨折植入脂肪干细胞复合聚乳酸-羟基乙酸共聚物，左侧植入单纯脂肪干细胞，术后4、8周测定最大载荷值和最大应力值发现，脂肪干细胞复合聚乳酸-羟基乙酸共聚物组明显高于单纯细胞植入组，说明脂肪干细胞复合聚乳酸-羟基乙酸共聚物可以提高骨质疏松骨折后骨质愈合的质量，为临床骨质疏松骨折的治疗提供实验依据。Ye等[75]首先构建兔的骨质疏松模型，将经成骨诱导后的自体脂肪干细胞复合藻酸钙凝胶植入一侧股骨远端内侧髁网状空隙处，对侧植入空白材料。12周时显微CT和组织学评估显示，细胞复合材料治疗的股骨比空白材料组有更多的新骨形成。Liu等[76]利用流式细胞术鉴定年轻和老年雌性SAMP8小鼠脂肪干细胞，然后将分离获取的脂肪干细胞植入骨质疏松小鼠(OVX-SAMP8)骨髓，评估其骨形成能力。术后4个月发现，年轻小鼠脂肪干细胞比老年小鼠脂肪干细胞骨密度平均提高24.3%，老年的脂肪干细胞可能通过减少成骨信号使脂肪干细胞改善骨质疏松作用降低，进一步说明脂肪干细胞作为种子细胞治疗骨质疏松症有很好的潜力。Lee等[77]研究发现人脂肪干细胞可通过激活 Smad/ERK(extracellular signal-regulated kinase)/JNK (c-jun NH(2) -terminal kinase)刺激成骨细胞的增殖和分化，可通过激活ERK/JNK/p38刺激破骨细胞的分化。在骨质疏松症骨缺损的修复中，如何利用好信号通路之间的相互作用来促进骨再生是以后的研究重点。"

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