张 迟 李 梅,2 赵基源*
1(宁波大学医学院,浙江省病理生理学技术研究重点实验室,浙江 宁波 315211)2(宁波市医学科学研究所, 浙江 宁波 315211)
细胞外基质材料在骨组织工程中的研究进展
张 迟1李 梅1,2赵基源1*
1(宁波大学医学院,浙江省病理生理学技术研究重点实验室,浙江 宁波 315211)2(宁波市医学科学研究所, 浙江 宁波 315211)
由疾病、外部创伤等原因引起的大骨骼缺损的治疗需要通过骨移植手术,寻找安全易得的替代骨已经成为临床上的重要课题,近年来快速发展的组织工程骨为解决这一难题提供了一种新的途径。支架材料作为组织工程的核心要素,其表面性状、结构,机械性能和生物学性能均能调控细胞的各种生命活动和体内组织的修复再生。细胞外基质由于其天然性、低免疫排斥性和优异的生物相容性等特点,已被广泛用作再生医学的支架材料。通过回顾近些年来细胞外基质材料在骨组织工程中的应用,阐述多种细胞外基质材料的构建修饰方法及其体外、体内的生物学效应,并对其在骨再生领域的应用前景进行展望。
骨移植材料;细胞外基质材料;胶原;生物相容性;骨组织工程
创伤导致的骨缺损的治疗,特别是大段骨缺损的治疗是临床上的难点之一。中国每年约有350万人因不同原因出现骨缺损,骨移植手术约为150万例。自体骨一直被认为是骨移植材料的金标准[1],但来源有限、易造成供骨部位的二次创伤,使其在临床上的应用受到限制。异体骨虽然具有与自体骨类似的机械性能及生物学活性[2],但存在免疫排斥反应,有时甚至导致移植的失败。随着组织工程技术的不断发展,人工骨可以实现大批量生产,是自体骨、异体骨所无法比拟的;同时,新一代的人工骨由于其优异的生物相容性、成骨传导性与成骨诱导性等优点,其前景被广泛看好。支架材料在人工骨的构建中起着至关重要的作用,理想的支架材料应该具有和天然骨类似的组成成分,提供类似的机械强度和生物微环境,利于细胞在材料上的募集、生长、增殖、迁移和分化。细胞外基质(extracellular matrix,ECM),是由细胞分泌到细胞外空间、由蛋白和多糖构成的精密有序的网络结构,三维微观结构能提供最接近于体内细胞生长的微环境,富含的各种活性分子为各种细胞活动提供了基础,被认为是理想的组织工程材料[3]。骨的基质包括有机成分和无机成分,两大成分的紧密结合使骨组织坚硬而有韧性。骨基质主要ECM组分功能见表1。
表1 骨的主要细胞外基质组分
骨基质组分作为支架材料,具有生物相容性好、仿生性优异等特点,已被广泛应用于组织工程和再生医学领域的研究。通过不同骨基质组分的复合、骨基质组分与活性分子、细胞的复合等方法可以改性材料,增强其生物相容性、生物仿生性和生物学活性,满足不同需求。
1.1 不同骨基质组分的复合
单一的骨基质组分难以模拟复杂的成骨微环境,两种或多种材料的复合不仅可将两种或多种材料的优点结合起来,往往还会具有协同效应。因此,构建不同的骨基质组分复合支架材料成为发展趋势。胶原与羟基磷灰石(HAp)是天然骨组织的重要组成成分,这两者材料在骨组织修复与再生中应用广泛。将胶原和HAp通过物理、化学方法组合,制备成多种形态的复合材料模拟骨基质微环境,植入体内后也表现出优良的生物相容性和成骨传导性[37]。He等以HAp为钙磷源,与胶原复合后作为实验组(HAp-胶原),HAp和多聚物PLGA-PEG-PLGA 复合作为对照(HAp- PLGA-PEG-PLGA),在这两种基质上种鼠骨髓间充质干细胞(MSC),RT-PCR结果显示胶原的加入能上调Runx2、Alp、Opn、Ocn等成骨相关基因的表达,表明HAp-Col复合材料能促进MSCs成骨分化[38]。然后将两组材料分别植入体内,4周后的组织学分析显示,HAp-Col复合材料能促进体内骨骼的再生。Xu等在体外用CaCl2和H3PO4处理得到矿化的胶原(mineralizedcollagen,MC),分别在MC和HAp上种人骨髓间充质干细胞(hMSC),结果显示细胞在MC上黏附更好、增殖更快,碱性磷酸酶(ALP)活性更强,说明MC具有更强的促hMSC成骨分化的能力[39]。基因芯片结果还发现,MC能同时上调hMSC的BMP-2、COL1A1、CTSK等成骨相关基因,验证了MC的成骨诱导特性。
由于新的制备工艺出现,复合材料的制备方式出现了新的趋势。Hatakeyama等分别通过纳米技术、传统技术制备得到两种不同粒径的HAp(n-HAp、m-HAp),与胶原复合得到两组复合材料n-HAp-Col和m-HAp-Col[40]。实验对比发现,n-HAp-Col组较m-HAp-Col组成骨能力有显著提高,表明支架材料的许多特征与粒径大小密切相关,纳米尺寸的支架材料不仅能够满足生物相容性、生物活性、力学性能等要求,其高孔隙率的三维立体结构更适合种子细胞的增殖与分化;在诱导成骨缺损修复方面,具有很好的应用前景。Inzana等用3D打印技术制备得到磷酸钙支架(3DP-CPS)和磷酸钙-胶原复合支架(CPS-Col),两组支架上分别培养C3H10T1/2细胞,发现胶原作为黏合剂能增加细胞在支架上的存活率[41]。然后,将两组支架分别植入小鼠股骨2mm临界缺损处,9周后 micro-CT 结果显示,CPS-Col组缺损处新生骨的体积显著增加,并且缺损处新骨的生长速率与支架的降解速率保持一致。冷冻干燥技术可用于制备多孔聚合物,提高支架表面积,从而改善生物学活性。胶原与多孔β-磷酸三钙经冻干、热交联(dehydrothermal,DHT)处理后,得到具有高孔隙率、高机械强度、易降解的复合材料,在经过严格的灭菌消毒后,该支架不仅能促进细胞增殖,同时也能增强对药物释放的可控性。毒理测试还发现,细胞在该支架上存活率高,生物相容性优异[42]。
1.2 骨基质组分支架材料的修饰
促骨再生的生物活性分子对体内骨骼的修复有着非常重要的作用。它能够通过上调骨修复相关基因的表达,促进体外成骨分化和体内骨再生。骨基质组分支架材料作为一个良好的载体,用活性分子修饰,往往能加速骨修复。Kim等将KLD12-SP肽与PLA/β-TCP多孔支架复合,植入大鼠头盖骨缺损处,与仅植入PLA/β-TCP的对照组比较发现,PLA/β-TCP/ KLD12-SP组能募集大量细胞,同时也能促进细胞的黏附、增殖和分化[43]。术后24周组织学结果显示缺损处的愈合情况,PLA/β-TCP组的新骨生成率仅有20%,而PLA/β-TCP/ KLD12-SP组的新骨生成率与对照组相比上升至42%,且缺损处有丰富的血管出现。Yamada等发现,含小分子半胱氨酸(NAC)培养基能上调成骨细胞碱性磷酸酶(ALP)的表达,以Col海绵作为载体,载药NAC植入大鼠骨缺损处,观察3、6周的愈合情况[44]。空白组、Col海绵组、Col海绵+NAC组3组比较,Col海绵+NAC组的愈合速度最快,表明经NAC修饰的胶原支架能加速体内新骨的生成。 Kim等以胶原/PLGA为载体载上成骨小分子药物地塞米松,得到复合材料(胶原/PLGA-DEX),胶原作为对照组,两组材料植入大鼠8 mm直径颅骨缺损处[45]。8周后,micro-CT结果显示,胶原/PLGA-DEX组新生骨体积与缺损组织总体积比值(BV/TV)高达约50%,对照组BV/TV仅为34%,表明构建的活性分子复合体系能很好地促进成骨。金属镁(Mg)作为新一代的生物可降解材料,在体内能直接与细胞外基质中的生物大分子、细胞相互作用,Zhao等发现,Mg与细胞外基质中I型胶原的组装有关[46],胶原分别与3种不同粒径的Mg混合,得到3种具有不同表面粗糙度的胶原支架。与其余两组比较,胶原与最小粒径的Mg混合后具有最大的比表面积,不仅能促进胶原支架在体内的吸收与重建,而且能增强成骨细胞的黏附、增殖,具有很好的生物学活性。
1.3 细胞与骨基质组分支架材料的复合
体内骨修复是细胞与周围基质的协同作用过程,骨基质组分支架材料的三维微观结构能为宿主细胞提供良好黏附、增殖和迁移的微环境。将不同的细胞与骨基质组分支架材料在体外复合后植入体内进行治疗,往往能有效减少炎症反应和瘢痕生成,促进血管新生和骨骼的再生等。目前用于骨基质组分支架材料复合治疗的细胞主要是间充质干细胞(MSC),具有向成骨方向分化的潜能。Cooper等以胶原支架作为对照组,将MSC与胶原复合制备复合支架(MSC-胶原),两组分别植入SD大鼠颅骨临界缺损处,术后28 d观察治疗情况,发现MSC-胶原组与对照组比较新骨生成率从19.07%提高到44.21%,说明复合MSC后的支架材料治疗效果显著[47]。Gao等从小鼠骨髓分离得到MSC,种在胶原凝胶上制备MSC/胶原复合支架,将该复合支架与仅含有胶原的支架分别植入小鼠的股骨缺损处,术后4 d micro-CT结果显示, MSC/胶原组的新骨体积和缺损组织总体积的比值(BV/TV)为11%,而对照组BV/TV值仅为4%,说明MSC和胶原的相互作用能显著促进新骨的生成[48]。 Lu等制备多聚物PDLLA/磷酸钙复合支架,该支架对脂肪间充质干细胞(ADSC)的黏附、成骨分化均具有促进作用,该支架复合ADSC植入体内骨缺损处,8周后组织学染色及免疫组化结果显示,与对照组PDLLA/磷酸钙组比较,PDLLA/磷酸钙/ADSC组COL-I、OCN等成骨相关蛋白表达量、ECM含量显著增加、矿物化水平也有明显提升[49]。
上述骨基质组分支架材料的构建修饰方法虽然对骨缺损有显著的治疗效果,但难以精确模拟人体骨组织中有机与无机成分的组成与配比,实现骨组织工程材料形态结构和生物力学的仿生。而天然组织经脱细胞处理后得到去细胞基质支架材料较传统的人工合成支架蕴含重要的生物信息,形成的三维空间结构与真正的骨组织类似,具有很好的仿生性及生物活性,故受到越来越多的关注。去细胞后的同种异体骨或异种骨支架虽然被广泛地用于组织工程及再生医学的研究,但存在免疫排斥及制备工艺复杂等限制,使此类去细胞化的细胞外基质支架材料具有一定的局限性。近几年来,采用不同的诱导方法诱导细胞产生特定的细胞外基质来改良支架表面的思路逐渐盛行,将细胞培养在支架表面,培养过程中形成大量细胞外基质来模拟体内组织微环境。该支架不仅能为体内细胞提供正确的模板支架,而且具有很强的生物仿生性。与传统的构建磷酸盐/胶原支架模型比较,具有更好的仿生结构、可塑性、无免疫原性等特点。
2.1 细胞合成的ECM支架的制备及成骨诱导作用
不同细胞所产生的细胞外基质均不相同,因此具有的生物学功能也不尽相同。体内成骨修复过程与成纤维细胞、成骨细胞、间充质干细胞密切相关,这三种细胞相互协调、合作形成新生骨组织。Bae等将成纤维细胞、成骨细胞、成软骨细胞分别培养在盖玻片上,6 d后脱细胞处理得到3种细胞外基质:成纤维细胞衍生的细胞外基质(FDM)、成软骨细胞衍生的细胞外基质(CHDM)、成骨细胞衍生的细胞外基质(PDM)[50]。SEM观察发现,不同细胞分泌的胞外基质有独特的表面纹理及纤维排列方向。然后,分别将前成骨细胞、鼠MSCs作为种子细胞种植在3种不同基质上,FDM与PDM的成骨诱导能力相当,CHDM诱导成骨的能力最差,且FDM在初期对细胞的增殖有明显的促进作用。
成纤维细胞具有旺盛的分裂增殖能力,短时间内能大量增殖并分泌丰富的ECM蛋白基质, 形成大量结缔组织,刺激新骨形成[51]。Xing等通过分析FDM组分,发现FDM中富含胶原蛋白、弹性蛋白、糖胺聚糖等活性蛋白及成纤维细胞生长因子(bFGF)、血管内皮生(VEGF)等生物活性因子[52]。在FDM上培养hMSCs发现,FDM能显著增强细胞的增殖、黏附,说明这种FDM具有很好的生物相容性。
成骨细胞是骨形成的主要功能细胞,骨缺损处骨的发育、重建与该位点招募成骨细胞能力密切相关[53]。PDM具有完整的骨组织具有的细胞外成分,各组分的组成和排列均类似于自然骨。Rutledge等利用微球烧结技术制备得到PLGA支架,随后将人成骨细胞种植在微球表面经过14天的培养去细胞得到PDM-PLGA[54]。成骨细胞在PDM-PLGA上培养,矿物化沉积和碱性磷酸酶含量显著增加,用人胚胎干细胞(hESCs)检测该支架的成骨诱导性,hESCs分别在PDM-PLGA、PLGA支架上培养,发现与对照组相比,hESCs在PDM-PLGA支架上的矿物化沉积、骨钙蛋白、RUNX2等成骨相关蛋白含量显著增加。Tour等将成骨细胞种植在HAp支架上,抗坏血酸诱导处理21 d后去细胞,得到复合支架HAp-ECM;HAp-ECM与HAp两组支架分别植入到大鼠颅骨8 mm直径缺损处,术后12周观察发现,成骨细胞合成的ECM能加强HAp的成骨能力[55]。
间充质干细胞具有很强的增殖能力和多向分化的潜能,其合成的细胞外基质对骨缺损处组织的重建起着非常重要的作用。Ravindran等将人间充质干细胞种植在胶原与壳聚糖(1∶1)复合基质上,成骨诱导培养基(含100 μg/mL抗坏血酸、10 mMβ-甘油磷酸盐、10 mM地塞米松)处理4周后,脱细胞后得到骨基质,随后用核磁共振技术检测证实了此基质的成骨性[56]。Lyu等把小鼠骨髓来源的MSC 种植到静电纺丝的PLGA网状支架上,成功创建出了MSC衍生的矿化细胞外基质[57]。与PLGA支架对比,细胞种植在ECM修饰的PLGA支架上,与细胞发育和成骨相关的OSX、RUNX2等基因表达量显著提高,细胞分泌的ECM呈现高度有序排列。以上两项研究均说明,间充质干细胞经诱导合成的ECM具有很好的成骨特性。Deutsch等发现,体外诱导MSC向成骨分化,去细胞得到的ECM不仅能使细胞分泌的矿物化沉积、碱性磷酸酶含量增加,而且ECM和胶原复合治疗大鼠股骨缺损,与对照组胶原比较,MSC合成的ECM能显著促进体内骨骼的修复[58]。
细胞外的微环境对组织的形成、重塑、修复起着非常重要的作用。由于纳米技术、3D打印技术等材料修饰技术的快速发展,不仅使人工组装的胶原和羟基磷灰石/磷酸钙复合材料宏观上实现了模拟天然骨的化学组成,也使此类复合材料在微观结构上排列更有序、更精确。但是,体内环境复杂,不同组织、同一组织的不同部位所含细胞外基质的量差异很大,细胞外微环境的精密结构让人工合成天然生物材料具有一定的局限性。随着组织工程骨的不断深入研究,用细胞制备细胞外基质模拟与自体骨类似的微环境逐渐受到组织工程研究人员的关注。细胞分泌的细胞外基质较合成材料,能更好地模拟骨组织中各种基质成分的组成、分布和生物学功效。但是,由于ECM的复杂性和动态性,目前尚无对细胞合成细胞外基质体系的系统研究报道,利用体外三维培养系统构建可控基质组分的细胞外基质复合材料,将可能成为未来的热点研究方向。
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Research Progress on Extracellular Matrix Materials in Bone Tissue Engineering
Zhang Chi1Li Mei1, 2Zhao Jiyuan1*
1(ZhejiangKeyLaboratoryofPathophysiology,NingboUniversitySchoolofMedicine,Ningbo315211,Zhejiang,China)2(NingboInstituteofMedicalSciences,Ningbo315211,Zhejiang,China)
The limited ability of the body to fully repair large bone defects beyond critical sizes often necessitates the implantation of replacement material to promote healing.While the current clinical strategies to address such bone defects generally carry associated limitations, bone-tissue engineering approaches seek to minimize any adverse effects and facilitate complete regeneration of the lost tissue. Extracellular matrix, due to its excellent biocompatibility, unique biomechanical properties and biological activities,has been widely investigated as a scaffold in regenerative medicine. This review focused on hybrid constructs and modification on ECM materials, as well as its biological effectsinvitroandinvivo. Application prospects in bone regeneration were also discussed.
bone transplantation materials;extracellular matrix materials;collagen;biocompatibility;bone tissue engineering
10.3969/j.issn.0258-8021. 2017. 01.013
2016-07-07, 录用日期:2016-08-26
国家自然科学基金青年基金(31300800);宁波市自然科学基金(2014A610238);宁波市自然科学基金(2014A610220)
R318
A
0258-8021(2017) 01-0103-06
*通信作者(Corresponding author), E-mail: zhaojiyuan@nbu.edu.cn