Chinese Journal of Tissue Engineering Research ›› 2021, Vol. 25 ›› Issue (13): 2108-2113.doi: 10.3969/j.issn.2095-4344.2187
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Meng Maohua, Li Ying, Chen Xin, Cheng Lu, Dong Qiang
Received:
2020-04-09
Revised:
2020-04-15
Accepted:
2020-05-23
Online:
2021-05-08
Published:
2020-12-29
Contact:
Dong Qiang, MD, Chief physician, Professor, Master’s supervisor, School of Stomatology, Guizhou Medical University/Affiliated Stomatological Hospital, Guizhou Medical University, Guiyang 550004, Guizhou Province, China
About author:
Meng Maohua, Master candidate, School of Stomatology, Guizhou Medical University/Affiliated Stomatological Hospital, Guizhou Medical University, Guiyang 550004, Guizhou Province, China
Supported by:
CLC Number:
Meng Maohua, Li Ying, Chen Xin, Cheng Lu, Dong Qiang. Effects and mechanisms of enamel matrix derivatives on osteogenic differentiation of bone marrow mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2021, 25(13): 2108-2113.
釉基质蛋白衍生物是从幼猪牙胚中提取的釉基质蛋白的衍生物,主要由约95%的釉原蛋白和成釉蛋白、釉成熟蛋白、釉蛋白、硫化釉蛋白、蛋白酶和类生长因子物质等共同构成的混合物,在诱导细胞增殖分化、信号传导和成骨矿化过程中发挥很重要的作用[7-8]。釉基质蛋白衍生物具有良好的生物相容性和生物安全性,在牙根的生长发育过程中起重要作用,具有生物模拟作用。釉基质蛋白衍生物能够促进牙骨质、牙周膜、牙髓等组织再生,提高转化生长因子和骨形态发生蛋白等类生长因子的表达水平,从而给予细胞正性矿化作用,同时促进新生牙周膜纤维的有序排列,诱导牙周组织再生,被广泛应用于口腔牙周疾病的治疗[9]。 2.1 釉基质蛋白衍生物诱导牙周组织再生 釉基质蛋白是牙胚发育过程中由上皮根鞘分泌的一组蛋白质。有研究表明,纯化后的釉基质蛋白与釉基质蛋白衍生物的诱导作用相同,釉基质蛋白是由多个基因编码的多肽的异质性混合物,单个蛋白通过募集间充质祖细胞,诱导牙周组织细胞以正确的空间排列,促进牙周组织再生[10]。釉基质蛋白衍生物可以促进牙周膜细胞增殖,同时抑制上皮细胞生长,牙周膜细胞释放的自分泌生长因子有助于牙周组织的愈合和再生,这一过程模拟了牙根生长发育的自然过程[11]。YUAN 等[12]研究发现釉基质蛋白衍生物组血清中产生了人源性抗pEMD抗体,有助于缓解牙周肿胀和疼痛,有利于牙周炎症的恢复。SHIRAKATA 等[13]建立7只犬牙龈萎缩模型,在牙周手术中使用釉基质蛋白衍生物,结果显示釉基质蛋白衍生物促进牙龈的附着,有牙周再生的作用。在临床中,使用釉基质蛋白衍生物对慢性牙周炎患者进行治疗,术后检查牙周炎症消退和组织再生的效果较好[14-16],而且随访治疗效果维持时间长久[17]。QUEIROZ等[18]研究发现釉基质蛋白衍生物治疗后的龈下微生物群发生改变,减少了病原菌群对牙周组织的破坏能力,促进了牙周组织再生能力。釉基质蛋白衍生物还可以用于治疗正畸牙根吸收[19],在钛表面促进成骨细胞的增殖和分化[20],保护比格犬再植牙的牙根,促进成纤维细胞和胶原纤维的生长[21]。 2.2 釉基质蛋白衍生物诱导牙髓再生 釉基质蛋白衍生物可用于牙髓治疗,促进牙髓细胞的分化和矿化,更好地诱导反应性牙本质的形成[22]。牙髓干细胞被证实有较强的再生能力,可以诱导分化形成牙髓-牙本质复合体,用于牙髓组织修复再生治疗[23]。AJLAN等[24]研究发现在相同的诱导环境下,釉基质蛋白衍生物对人牙髓干细胞的成骨诱导能力强于血小板衍生生长因子和三氧化二矿物团聚体,成骨和矿化基因表达量较高,釉基质蛋白衍生物表现出了更好的诱导牙本质再生能力。釉基质蛋白衍生物可用于直接盖髓、牙髓切断术和根管再治疗等,在牙髓治疗中的应用较广泛,已经成为治疗牙髓疾病常用药物[25]。 2.3 釉基质蛋白衍生物诱导骨髓间充质干细胞 1976年,FRIEDENSTEIN等[26]首次发现了骨髓间充质干细胞。骨髓间充质干细胞来源于中胚层,具有多向分化潜能,可在不同微环境下定向诱导分化成骨细胞、脂肪细胞、软骨细胞和神经细胞等,由多种蛋白和调控因子共同作用,具有良好的治疗应用前景[27]。骨髓间充质干细胞主要来源于骨髓,占人骨髓细胞总量的 1/15 000-1/10 000,可以微创取材,来源组织广泛;自体骨髓间充质干细胞作为细胞治疗的原材料,伦理争议少,使用更方便、更安全。 骨髓间充质干细胞在体外培养技术比较成熟,全骨髓贴壁法和密度梯度离心法最为常用[28]。间充质干细胞的鉴定是根据2006年国际细胞治疗学会间充质和组织干细胞委员会提出的间充质干细胞鉴定标准[29]。骨髓间充质干细胞在体外培养过程中,随着培养时间的延长,细胞趋于衰老并失去其多分化潜能,不能稳定的长期传代培养,体外扩增有一定的限制,骨髓间充质干细胞的规模化生产必须坚持GMP标准化的要求[30];骨髓间充质干细胞的诱导分化潜能与供者年龄有密切关系,供者年龄较小诱导分化功能较佳,随着供者年龄的增长,其分化潜力、数量和寿命都有不同程度的降低,不同的供者之间差异也较大[31]。骨髓间充质干细胞可以定向诱导成骨是该细胞的必备特征之一,诱导成骨早期可以检测碱性磷酸酶活性,成骨中后期可以检测成骨相关基因runx2、骨涎蛋白、骨桥素和骨钙素等,茜素红染色观察矿化结节。随着技术的进步,也研究了很多促进骨髓间充质干细胞成骨的方法,例如药物、基因靶向技术、转化生长因子和细胞共培养技术等,这些新技术的使用,增加了骨髓间充质干细胞在骨组织工程领域的使用范围。 2.3.1 釉基质蛋白衍生物体外诱导骨髓间充质干细胞增殖和成骨分化 釉基质蛋白衍生物含有多种蛋白,可有效诱导多种细胞的增殖分化[32],釉基质蛋白作用于人骨髓间充质干细胞时,促进细胞增殖的作用较明显[33],但对其成骨诱导作用研究报道不一,存在争议。有研究报道使用釉基质蛋白衍生物作用于人骨髓间充质干细胞2周,未检测到成骨相关基因增加,而后加入转化生长因子β培养2周,才检测到成骨相关基因的表达[34]。Katayama等[35]在体外研究中发现使用不同浓度釉基质蛋白衍生物活性成分合成的寡肽(SP)作用于人骨髓间充质干细胞,在质量浓度为10 μg/L时人骨髓间充质干细胞的增殖作用最强。GUIDA 等[36]研究发现釉基质蛋白衍生物以剂量依赖方式刺激人骨髓间充质干细胞的生长,当釉基质蛋白衍生物质量浓度为50 mg/L时,作用24 h的细胞增殖数量为对照组的3.4倍,增殖效果最明显,同时也发现釉基质蛋白衍生物作用后Ⅰ型胶原的合成受到抑制,随着剂量的增加抑制作用越高,体现出抑制成骨分化作用。IZUMIKAWA 等[37]报道了不同的研究结果,在体外分离培养大鼠骨髓间充质干细胞,加入100 μg/L釉原蛋白作用24 h后,通过实时荧光定量RT-PCR检测骨桥蛋白、骨连接蛋白、Ⅰ型胶原和骨钙素的mRNA表达量增高,实验组第10-20天的碱性磷酸酶活性比空白组高,矿化结节更多,实验结果提示釉原蛋白可能促进了大鼠骨髓间充质干细胞的成骨分化和矿化。 吴广升等[38]将大鼠骨髓间充质干细胞接种到壳聚糖温敏水凝胶支架上培养,在质量浓度为100 mg/L釉基质蛋白作用下,从第3天开始碱性磷酸酶活性增加,同时促进了细胞增殖。林智恺等[39]将 200 mg/L釉基质蛋白作用于人骨髓间充质干细胞,成骨相关基因Runx2、碱性磷酸酶、Ⅰ型胶原表达量均增加,结果提示该浓度釉基质蛋白可能促进人骨髓间充质干细胞的成骨和矿化。KEILA等[40]使用25 g/L釉基质蛋白衍生物对大鼠骨髓间充质干细胞进行诱导,细胞增殖数量大约为对照组的3倍,矿化结节数量明显增加,碱性磷酸酶活性增加,成骨诱导能力增强,但是高浓度的釉基质蛋白衍生物诱导时出现抑制细胞矿化的作用,该研究建议在诱导分化时需要反复摸索作用于大鼠骨髓间充质干细胞的最佳浓度,未能给出定论。TANIMOTO等[41]将重组人全长釉原蛋白(Rh174)作用于人骨髓间充质干细胞诱导培养14-26 d,碱性磷酸酶、Ⅰ型胶原、骨桥蛋白和骨涎蛋白的mRNA水平明显高于未处理的对照组。使用100 μg/L重组人全长釉原蛋白处理人骨髓间充质干细胞后,碱性磷酸酶活性和钙离子浓度均增加;釉基质蛋白衍生物组与对照组相比,茜素红染色强度较高,矿化增强,且呈现剂量依赖关系,研究结果提示釉原蛋白在人骨髓间充质干细胞成骨分化过程中具有一定的正性调控作用。有研究使用1 000 μg/L C端釉原蛋白诱导人骨髓间充质干细胞,在第2,4,6天MTS检测提示细胞增殖数量较未干预组显著增加[42]。虽然这些体外实验结果报道不一致,但大部分结果支持釉基质蛋白衍生物在体外能够诱导骨髓间充质干细胞成骨和矿化。 2.3.2 釉基质蛋白衍生物诱导骨髓间充质干细胞体内成骨 釉基质蛋白衍生物可以直接作用于组织细胞或者联合支架材料一起植入体内。在动物实验中,釉基质蛋白衍生物复合不同植入材料的成骨诱导效果报道结果不一,尚无定论。SONG等[43]提取幼猪骨髓间充质干细胞,将釉基质蛋白处理的牙根片表面包裹四氟聚乙烯膜,二者复合后异位植入裸鼠皮下,8周后可见骨髓间充质干细胞分化成骨,沿着根面形成骨样组织,无邻近结缔组织,部分有牙槽骨骨髓样组织生成,电磁脉冲处理的牙根切片上形成细胞牙骨质样组织,与正常猪细胞牙骨质相似。IKAWA等[44]在比格犬双侧下颌骨构建颊侧骨缺损模型,将液态釉基质蛋白衍生物与天然牛骨材料植入到种植体周围骨缺损区域,胶原膜覆盖,3个月后进行组织切片观测,结果显示液态釉基质蛋白衍生物与骨粉相互作用的新骨生成效果最佳,单独使用骨粉的成骨作用较弱,空白对照组有大量结缔组织和小血管生成,无成骨样组织生成。但也有不一致的研究报道,MROZIK等[45]使用釉基质蛋白包裹骨瓷材料,在体外诱导作用72 h后,Ⅰ型胶原、骨桥蛋白和骨膜蛋白mRNA的表达上调,然后植入小鼠皮下2周,可见有血管生成,但碱性磷酸酶活性降低,作者分析其原因可能是釉基质蛋白在体内作用时间过短,未见有成骨样组织生成,异位成骨的效果还有待观察。有关釉基质蛋白衍生物的体内实验相对较少,体内微环境较体外改变较大,还需要更多的体内实验研究来验证。 2.3.3 釉基质蛋白衍生物诱导骨髓间充质干细胞的信号通路 KUNIMATSU等[42]研究发现在C端釉原蛋白的诱导下骨髓间充质干细胞增殖显著,加入抗溶酶体相关膜蛋白1(LAMP1)抗体后,细胞增殖受到抑制;加入丝裂原活化蛋白激酶(MAPK)抑制剂U0126后,ERK1/2磷酸化水平降低,抑制了细胞增殖;C端釉原蛋白通过LAMP1和MAPK-ERK信号通路促进骨髓间充质干细胞增殖。经典Wnt/β-catenin信号通路在釉基质蛋白衍生物诱导骨髓间充质干细胞成骨过程中起着非常重要的作用,釉基质蛋白衍生物通过Wnt/β-catenin信号通路促进骨髓间充质干细胞成骨分化效用较为统一,但关于成脂分化有不同的研究结果。WEN等[46]研究报道富含赖氨酸的釉原蛋白多肽可以通过上调Wnt10b的表达来激活Wnt信号通路,进而促进间充质干细胞的成骨,但同时也促进脂肪生成,而LUO等[47]认为RUNX1通过经典的Wnt/β-catenin途径促进骨髓间充质干细胞成骨,同时抑制脂肪生成,Wnt/β-catenin信号途径可以和非经典途径的Wnt/Ca2+信号途径相互作用[48]。有研究使用ERK1/2抑制剂PD98059处理骨髓间充质干细胞后,细胞增殖和矿化作用被抑制,ERK1/2可能会成为釉基质蛋白衍生物促进骨髓间充质干细胞增殖和成骨分化的信号通路[49]。目前,有关釉基质蛋白衍生物诱导骨髓间充质干细胞成骨分化的机制仍存在争议。 2.4 釉基质蛋白衍生物加强骨髓间充质干细胞膜片 细胞膜片技术是一种组织工程技术,成熟的细胞膜片形成了一个承载更多种子细胞的生物相容性支架,使种子细胞的定向诱导分化能力更强;细胞膜片具有可塑性,可操作性强,使用不同来源的种子细胞膜片组合培养形成复合膜片,共同协同发挥更佳的修复效果,注意细胞膜片折叠和组合后需要保证膜片内部的营养与血供,避免膜片内部液化坏死。细胞膜片联合3D打印技术,使细胞膜片可以更好地进行观察,在需要大面积细胞膜片修复时可以解决膜片内部的营养和血液供应,可以根据需要设计三维空间的形态,使细胞膜片具有可重复性,可以减少生产成本,可操作性更强[50]。利用干细胞诱导形成细胞膜片,进一步显示了干细胞促进组织再生等潜力,减少了干细胞使用的技术局限性,在再生医学中得到了广泛的使用[51]。HU等[52]将人牙髓干细胞膜片移植和单纯细胞直接注射于猪牙周炎骨缺损模型中,3个月后结果显示干细胞膜片组的牙周再生效果优于对照组。ITO等[53]使用骨髓间充质干细胞和骨髓间充质干细胞膜片分别移植到大脑损伤的大鼠同侧新皮质上,4周后细胞膜片组显示了更好的运动功能,细胞膜片上的细胞有更强的成神经分化能力。CHUAH等[54]研究发现在聚二甲基硅氧烷上培养骨髓间充质干细胞形成细胞膜片,增加了诱导软骨形成和骨形成的潜力。 复合细胞膜片较单一细胞膜片植入缺损区域有较多的优势,KOTO等[55]将牙上皮细胞和牙间充质干细胞共培养成复合细胞膜片,在复合膜片中观察到的微环境与牙芽发育期的微环境相似,更有利于细胞再生。作者认为,共培养的复合细胞膜片不仅保留了单细胞膜片的优势,而且促进了各自细胞的功能,在共同作用下组织再生的效果更佳。 PANDUWAWALA等[56]将牙周膜干细胞、人脐静脉内皮细胞或两者共培养的细胞接种到温度敏感型培养皿上,培养成完整的细胞膜片。将细胞膜片以3种不同的组合包裹在处理好的牙根周围,将其植入免疫缺陷的小鼠背部皮下,结果显示共同培养的三维细胞膜片的牙周再生效果优于单独培养的细胞膜片。有研究将血管内皮细胞复合其他细胞共培养植入裸鼠皮下,均观察到功能性血管生成[57]。共培养细胞膜片预血管化的形成更有利于组织修复,例如加速口腔黏膜创面的愈合[58],减少烧伤皮肤缺损创面瘢痕形成[59]。 在釉基质蛋白衍生物联合不同类型细胞膜片技术的有关研究中,WANG等[60]研究发现100 mg/L釉基质蛋白衍生物在体外诱导获取的牙周膜干细胞膜片的细胞层数更多,其细胞外基质分泌更丰富,胶原纤维网连接更致密。在成骨诱导2周时,实时荧光定量PCR检测结果提示,碱性磷酸酶、Ⅰ型胶原、RUNX-2和牙骨质组织特异性基因表达更高,茜素红染色矿化结节更多,釉基质蛋白衍生物增强的细胞膜片比正常诱导的细胞膜片具有更高的成骨潜能。有研究使用釉基质蛋白衍生物加强的颌骨来源骨髓间充质干细胞膜片和牙周膜干细胞膜片包裹表面脱矿的根形牙本质支架,放入中空圆锥形的羟基磷灰石/磷酸三钙支架中,间隙有1 mm模拟牙周结构和微环境为细胞膜片提供空间[61];植入裸鼠体内8周,硬组织切片发现单纯的牙周膜干细胞膜片组有大量有序排列的胶原纤维形成,单纯的颌骨来源骨髓间充质干细胞膜片组有丰富的连续骨样组织形成,复合细胞膜片组含有胶原纤维和骨组织,再生的组织结构类似于牙周复合体的结构组成。釉基质蛋白衍生物可以与新的支架材料共同作用,例如使用RAD16-I肽水凝胶构建临时多层细胞片结构,动态灌注系统培养细胞膜片以稳定三维结构,体外培养48 h后快速获得骨髓间充质干细胞膜片[62],可用于颌骨骨缺损。釉基质蛋白衍生物增强的骨髓间充质干细胞膜片使得干细胞发挥出更大的再生潜能,在口腔牙周软组织和牙槽骨缺损中更加有应用前景。 "
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