Dental pulp stem cells in regenerative medicine: application and development

doi:10.3969/j.issn.2095-4344.2016.19.020

Abstract

Abstract:

BACKGROUND: Dental pulp stem cells are characterized by multi-lineage differentiation and proliferation abilities and are easy to obtain, so they are becoming an issue of concern in regenerative medicine.
OBJECTIVE: To provide clues and direction for further study by analyzing progress of domestic and overseas research on dental pulp stem cells, and summarizing their application in regenerative medicine.
METHODS: The “dental pulp stem cell, regenerative medicine, tissue engineering” in Chinese and English served as the search terms to search articles related to dental pulp stem cells and regenerative medicine, published from 2000 to 2015 in Medline, PubMed, CNKI, Wanfang and Cqvip databases. Totally 46 articles were selected for overview.
RESULTS AND CONCLUSION: Dental pulp stem cells, which hold the capacity of self-renewal and multi-lineage differentiation, are relatively easy to obtain, and exhibit a great potential in regenerative medicine. The research of dental pulp stem cells in repairing bone defects has entered the clinical trial phase, but the research of cell differentiation into other tissues is still in basic trial phase and needs further development.

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

Key words: Stem Cells, Dental Pulp, Regenerative Medicine, Tissue Engineering

CLC Number:

R394.2

Ma Zi-yang, Guo Xiao-xia. Dental pulp stem cells in regenerative medicine: application and development[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(19): 2872-2878.

Figures/Tables 1

2.1 牙髓干细胞的生物学性状牙髓干细胞来源于神经嵴，位于牙髓，属于成体干细胞，具有一定的自我更新和定向分化能力[2]。间充质干细胞的一些特异性表面标记物可以在研究中被用于识别牙源性干细胞，如常被用于识别硬组织的间充质干细胞基质细胞抗原1(stromal cell antigen 1, STRO-1)已被广泛应用于牙髓干细胞的识别，显示为阳性；间充质干细胞主要表面标记物CD29、CD44、CD73、CD90、CD105、CD117、CD146、CD166和CD271在牙髓干细胞中呈阳性表达，其中CD105和CD117在牙髓干细胞上阳性表达，提示它有良好的增殖分化能力[4]。造血干细胞主要表面标记物CD45等呈阴性表达，CD34在不同的研究中阳、阴性表达有争议[4-5]。国际细胞治疗协会把CD105+/CD73+/CD34-/CD45-作为间充质干细胞的特征[6]。Yasui等[7]根据细胞表达2种表面标记物LNGFR(CD271)和THY-1(CD90)确定牙髓组织特异的细胞群，纯化人牙髓干细胞，这些细胞同时表达已知的间充质细胞标记物。分离的牙髓来源的 LNGFRLow+ THY-1High+细胞代表高纯度的克隆形成细胞群，这些细胞表现出长期增殖、多向分化潜能，能促进新骨形成。因此建议，LNGFRLow+ THY-1High+牙髓来源的细胞可为骨再生提供一种很好的细胞来源。牙髓干细胞具有多向分化潜能，可以在特定条件下分化为骨、软骨、神经细胞、肌细胞、脂肪细胞和角膜上皮等多种组织细胞，可用于牙本质再生、骨组织再生、神经修复等研究领域[8-9]。在临床治疗中已有成功被用于骨组织再生的报道[10]。 2.2 牙髓干细胞的分离与储存相比其他成体干细胞，牙髓干细胞可以较容易的从正畸拔除的牙齿、脱落的乳牙、拔除的阻生齿的牙髓中获取。但是，到目前为止，大多数分离、扩增方法还不能完全令人满意，因为这些方法有可能改变细胞的生物学特性和分化细胞的质量[11]。Hilkens等[12]研究发现，目前广泛应用的牙髓组织酶消化法(the enzymatic digestion of the pulp tissue，DPSC-EZ)和外植体法(the explant method，DPSC-OG)分离的牙髓干细胞，均可较好的应用于骨组织再生。Eubanks等[13]研究发现，用以上两种分离方法从1个第三磨牙分离的细胞，经过2周培养均能回收大约106个细胞。不断有研究认为，酶消化法有可能影响细胞的表型和性能，故不适于治疗用细胞分离[14-16]。相比而言，外植体法更容易、快捷、安全、经济，更符合GMP指南获得临床级数量的间充质干细胞，而且获得的牙髓干细胞显示相似或更高的分化能力[17]。Murakami等[18]用粒细胞集落刺激因子(granulocyte-colony stimulating factor，GCSF)分离牙髓干细胞，并诱导其定向分化，然后移植入免疫缺陷大鼠，结果显示其高效安全。目前用于分离牙髓干细胞的粒细胞集落刺激因子已有商品化试剂NEUTROGIN®。牙髓可以储存在4 ℃冷藏环境中过夜，在-85 ℃到-196 ℃冷冻环境中可以储存1周，从冷藏或冷冻的牙髓中分离牙髓干细胞除成功分离数量有所下降外，其表面抗原仍有活性，免疫功能和分化能力上并无损失[19]。Munévar等将分离出的牙髓干细胞(CD105+)分别用Kamath法[20]：10%二甲基亚砜+体积分数为70%胎牛血清+20% NH(non-hematopoietic)干细胞培养液；Papaccio法[21]：10%二甲基亚砜+体积分数为90%胎牛血清冻存。通过比较两种方法冻存30 d的细胞，发现Papaccio法(59.5%)比Kamath法(56.2%)有更高的细胞生存率，而对于冻存1 d(65.5%)和7 d (56%)的细胞Kamath法更优。冻存后的人牙髓干细胞表达间充质干细胞标记物，但冻存时间可能通过改变细胞膜蛋白空间构型或者在某个分化水平抑制细胞，从而影响表面标记物的表达[22]。因此，Ducret等[23]推荐在分离和培养细胞时用人胎盘Ⅰ型和Ⅲ型胶原等量混合预涂培养皿，因为这两种胶原是牙髓细胞外基质中含量最丰富的胶原。用无异种分离试剂和确定的培养基(如SPE-IV®，含临床级人白蛋白，α-MEM，rhIGF-1，rhFGF-2)取代通用产品培养和传代细胞。经过上述方法分离培养的牙髓细胞在无血清培养基中冻存后，不会影响细胞的倍增时间和收集细胞数量，细胞活力与新鲜细胞相比有所下降，但与文献报道相似[24]。一些发达国家如日本、美国和挪威先后建立牙齿银行，为牙髓干细胞的基础和临床应用研究提供便利，但牙齿银行用于临床储存维持费用较高，较难为发展中国家接受，而最新的全牙储存方法可以在保证牙髓干细胞免疫功能及分化能力不受影响的同时降低储存成本[25]。 2.3 牙髓干细胞在再生医学中的应用牙髓干细胞具有多向分化、获取简便的特性，被广泛应用于各种退行性疾病的再生医学治疗研究[4]。目前，牙髓干细胞用于骨组织修复已经进入临床应用研究阶段，在神经、心肌、肝细胞、血管修复等方面尚处于动物实验研究阶段。牙髓干细胞在再生医学方面已经显示出巨大潜能，有可能用于治疗各种人类疾病。 2.3.1 应用牙髓干细胞修复牙髓和牙本质成人牙髓干细胞亚组分CD31-/CD146- SP细胞和CD105+细胞，具有较高的血管和神经分化潜力，可以作为潜在的临床牙髓修复材料[26]。EI-Backly等[27]将新西兰白兔牙髓干细胞接种在聚乳酸乙醇酸支架上，然后移植入兔皮下12 d，观察到类似于牙本质小管的结构。Suzuki等[28]将人类牙髓干细胞接种在胶原支架上，然后在体外加入骨形态发生蛋白7，观察到牙髓干细胞的钙化。在牙髓干细胞胶原支架复合体中添加碱性成纤维细胞生长因子，移植入大鼠背部，观察到牙髓样细胞及血管的再生。研究发现在龋齿根管治疗后，将牙髓干细胞用于牙髓再生的效果更好。但目前尚无法将牙髓干细胞培养为完整的牙髓-牙本质复合体。 2.3.2 应用牙髓干细胞修复骨组织 cDNA微阵列分析表明，牙髓干细胞参与颅面结构，包括颅面部骨骼与软骨的形成[29]。牙髓干细胞向骨组织分化的能力在较多实验中得到验证。在体外，牙髓干细胞表达成骨标记物包括骨涎蛋白(bone sialoprotein，BSP)、碱性磷酸酶(alkaline phosphatase，ALP)、牙本质涎蛋白(entin sialoprotein，DSP)，可以分化为成骨细胞[9]。Graziano等[30]应用磁激活细胞分选法分选人牙髓干细胞，被选出的CD34+干细胞能分化为前成骨细胞，不需通过扩增，将其黏附于聚乳酸支架，移植入免疫缺陷大鼠，结果发现回收的骨结节类似于原始支架尺寸，在免疫荧光和X射线下呈现骨组织的特征，且新生成的血管具有人类血管特征。de Mendoca Costa等[31]将人牙髓干细胞与胶原膜黏合，移植入全层颅骨缺损的非免疫缺陷大鼠，观察到颅骨缺损的修复。聚合酶链反应扩增发现新生成的骨细胞含有人类DNA，提示颅骨的修复源于牙髓干细胞。Tabatabaei等[32]通过正畸治疗中的机械力刺激人牙髓干细胞，与对照组相比，成骨标记物碱性磷酸酶和骨桥蛋白含量显著提高，结果提示不使用试剂，仅通过机械力可以诱导牙髓干细胞向成骨方向分化。d’Aquino等[10]分离提取人第三磨牙内牙髓干细胞，扩增后黏附于胶原蛋白海绵支架，填充于由于骨吸收造成的自体下颌骨的骨缺损处，3个月后临床、X射线及组织学检测表明骨组织完全再生，1年后再生骨组织功能达到最佳，提示人牙髓干细胞可以完全恢复人类下颌骨缺损。 2.3.3 应用牙髓干细胞修复神经组织 Sakai等[8]发现，牙髓干细胞能诱发脊髓损伤后的神经再生。将牙髓干细胞移植到大鼠的脊髓横断处，可分化为成熟的少突胶质细胞，通过促进轴突再生，抑制轴突生长抑制因子，从而抑制神经元、星形胶质细胞、少突胶质细胞的凋亡达到再生神经的目的。de Almeida等[33]采用小鼠挤压神经损伤模型也支持上述结论。Apel等[34]在阿尔茨海默病和帕金森病体外模型中将大鼠牙髓干细胞加入至原代神经元培养，2 d后加入神经毒素，发现牙髓干细胞可以减弱毒素的毒性，保护原代神经元并释放骨形态发生蛋白2和神经营养因子(包括神经生长因子、胶质细胞源性神经营养因子、脑源性神经营养因子)。Chang等[35]发现，在电刺激下，含有牙髓干细胞的培养基中含有更多的βⅢ微管蛋白、胶质纤维酸性蛋白和少突胶质细胞，提示牙髓干细胞可以向成熟的多巴胺能神经元分化。Matsubara等[36]将人牙髓干细胞鞘内注射入脊髓急性损伤大鼠，牙髓干细胞通过释放单核细胞趋化蛋白1和唾液酸结合性免疫球蛋白样凝集素9，加速损伤修复，使大鼠恢复行走能力。 2.3.4 应用牙髓干细胞改善循环系统功能 Gandia等[37]采用心肌内注射法将牙髓干细胞注入心肌梗死裸大鼠模型内，牙髓干细胞通过分泌多种促血管生成与抗凋亡因子，包括血管内皮生长因子、胰岛素样生长因子1和2、干细胞因子和粒细胞集落刺激因子，促进生成新的血管和心肌细胞。实验组大鼠心前壁增厚，梗死面积减小，心脏功能改善并有新血管生成。Iohara等[38]将牙髓干细胞移植入后肢缺血小鼠模型，通过表达促血管生成因子如血管内皮生长因子，促进毛细血管的生成。 2.3.5 牙髓干细胞在其他组织修复中的应用研究发现牙髓干细胞还具有分化为肌肉、角膜、肝细胞、胰岛素生成细胞、皮肤等多种细胞与组织的能力。Yang等[39]将人牙髓干细胞注射入骨骼肌缺损小鼠模型中，发现有肌肉再生的现象。牙髓干细胞通过产生抗肌萎缩蛋白(dystrophin)，改善肌营养不良症状。Gomes等[40]使用兔眼角膜烧伤模型，将组织工程牙髓干细胞片移植入切除烧伤部位的角膜床中，3个月后角膜透明度有所改善，有少量新血管生成。Ishkitieve等[41]在体外培养人牙髓干细胞，使用肝生长因子刺激，培养出高纯度的肝细胞样细胞。此后，Ishkitieve等[42]使用人牙髓干细胞治疗胆汁淤积型肝硬化，使肝功能完全恢复，且与骨髓干细胞相比出现恶性肿瘤的风险有大幅度降低。石建峰等[43]通过构建重组转录因子hFOXA2和hPDX1慢病毒载体，转染人牙髓干细胞成功诱导其形成胰岛素生成样细胞，提示牙髓干细胞可用于1型糖尿病的治疗。Nishino等[44]使用裸鼠全层皮肤缺损模型，将人牙髓干细胞移植入伤口组织，结果皮肤组织加速愈合，缺损区域有人类Ⅰ型胶原的形成。"

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