Chinese Journal of Tissue Engineering Research ›› 2013, Vol. 17 ›› Issue (6): 1111-1115.doi: 10.3969/j.issn.2095-4344.2013.06.027
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Jiang Quan-chun, Zhao Xiu-lan
Received:
2012-11-08
Revised:
2012-11-20
Online:
2013-02-05
Published:
2013-02-05
About author:
Jiang Quan-chun, Attending physician, Department of Stomatology, Dalian Eco-tech Development Zone Hospital, Dalian 116600, Liaoning Province, China springjqc_42@sina.com
CLC Number:
Jiang Quan-chun, Zhao Xiu-lan. Reconstruction of tissue-engineered bone using adipose-derived stem cells and Bio-oss scaffold for the treatment of peri-implantitis[J]. Chinese Journal of Tissue Engineering Research, 2013, 17(6): 1111-1115.
2.1 脂肪干细胞 研究表明,组织工程化骨的构建主要涉及3个重要因素,即种子细胞、生物支架、构建方法;其中种子细胞和支架材料是当前研究的重点。理想的骨组织工程种子细胞应具有以下几个特点:取材容易,对机体的损伤小;体外扩增能力强;易定向分化为成骨细胞;植入体内后能很好地适应受区生理、病理、应力等环境并保持成骨活性。 脂肪干细胞是存在于脂肪组织基质中[10],在干细胞领域展开广泛研究的一类成体干细胞。脂肪组织在人体分布广泛,来源充足,易于获取,抽吸脂肪组织对患者无额外创伤,患者易于接受,无心理上顾虑。自1994年Sah 等[11]从患者皮下脂肪组织中发现异位成骨现象,从而受到启发,提出脂肪前体细胞可以向成骨细胞分化后,至2001年Zuk等[12-13]报道脂肪组织含有的细胞不仅与间充质干细胞有相同干细胞表面标志,也具有向骨、软骨、脂肪、肌肉和神经等细胞分化的能力,明确了脂肪基质中存在具有干细胞特性的细胞,脂肪干细胞的概念由此提出。继之有学者应用脂肪干细胞在免疫功能缺陷裸鼠皮下形成骨组织[14]。 崔磊等[15]通过对脂肪干细胞表面HLA分子表达的研究证明,其检测的脂肪干细胞均表达HLA Ⅰ类分子,但未检测到HLA Ⅱ类分子阳性表达。人干扰素γ刺激48 h后,HLA Ⅰ表达未见明显增高,HLA Ⅱ类分子表达明显增高。培养第2代的脂肪干细胞CD44、CD45、CD29、CDI05、CDI66表达率维持较高水平,表明细胞均一性较强,而D34、CD14、CD45、CD31等基本未见表达,排除了造血干细胞与内皮细胞污染的可能,提示脂肪干细胞可能具有调节淋巴细胞反应的免疫调节功能。通过进一步观察与检测脂肪干细胞对淋巴细胞反应调节作用,研究发现,单纯的脂肪干细胞未能刺激异体淋巴细胞产生增殖反应,说明脂肪干细胞具有较低的抗原反应性。在干扰素γ作用后,虽然脂肪干细胞表达了HLA Ⅱ类分子,但淋巴细胞未见明显增殖,结果表明脂肪干细胞具有较低免疫原性,此外,其体外实验表明脂肪干细胞能够抑制同种异体抗原引起的淋巴细胞增殖,也能抑制PHA等多克隆激活剂引起的淋巴细胞增殖。进一步实验表明脂肪干细胞向成骨细胞分化后仍具有上述免疫学特性[16]。有学者还发现脂肪基质干细胞贴壁能力强,体外易于培养,营养需求低,在基础培养基中生长旺盛,体外倍增时间16-48 h可传代。此外许多学者还证明成体干细胞脂肪干细胞具有既不表达MHCH分子,也不表达B7等与淋巴细胞激活有关的分子免疫学特点[15]。体外实验表明脂肪干细胞能够抑制同种异体抗原引起的淋巴细胞增殖,也能抑制PHA等多克隆激活剂引起的淋巴细胞增殖。进一步实验表明脂肪干细胞向成骨细胞分化后仍具有上述免疫学特性[16]。同时实验过程中还发现脂肪基质干细胞贴壁能力强,体外易于培养,营养需求低,在基础培养基中生长旺盛,体外倍增时间16-48 h可传代[17]。脂肪干细胞体外连续传代15代,细胞仍具有稳定的增殖能力和较低的老化水平[18]。与骨髓基质干细胞相比,脂肪基质干细胞可能更易于在体外培养中大量扩增,从而获得足够的细胞量以满足实验及临床应用的需要。另外,脂肪干细胞分化能力强,易于接受基因修饰,特别是血管内皮生长因子、骨形态发生蛋白2等,骨形态发生蛋白2可以调控脂肪干细胞的信号通路。 目前脂肪干细胞在口腔标记牙齿再生、软骨再生方面均有一定的研究报道,特别是在成骨及骨再生方面较系统地研究了PONX2,OSX,骨形态发生蛋白2等信号通路在间充质干细胞成骨分化中的调控作用,采用基因治疗的方法正式转入外源性骨形态发生蛋白2基因可以进一步增强脂肪干细胞的成骨能力,加速新骨形成,提高新骨形成质量[19]。另外,发现脂肪干细胞均来源于外周细胞,这些细胞静息状态下维持血管形态和压力,当组织损伤时,可以迁移至病损区域,一部分维持外周细胞的原有状态,吸引和帮助内皮细胞形成新生微血管,为病损区域提供营养支持,另一部分外周细胞分化成为不同的功能细胞,如成骨细胞、脂肪细胞、肌细胞、神经细胞等来参与病损组织修复。 2.2 外源性生长因子——骨形态发生蛋白2 骨和软骨组织中含有多种参与调解骨髓发育及生长的多肽类生长因子,在诸多因子中,骨形态发生蛋白是惟一能够单独诱导骨组织形成的局部生长因子,其中骨形态发生蛋白2是诱导成骨活性最强的骨形态发生蛋白之一,属于转化生长因子超家族成员。 骨形态发生蛋白2主要从两方面促进骨形成,一是直接促进成骨细胞的分化:骨基质中的骨形态发生蛋白2可募集并诱导干细胞分化为成骨细胞及软骨细胞,再通过钙盐沉寂形成新骨;骨形态发生蛋白2还可通过环磷酸腺苷(cyclic adenosine monophosphate,cAMP)诱导间充质细胞不可逆地分化为骨细胞;骨形态发生蛋白2在骨的再生和修复过程中也促进成骨细胞的分化并抑制其凋亡[20]。二是促进其他成骨因子的表达;骨形态发生蛋白2能够促进成骨细胞标志基因骨诱导蛋白(osteogenid protein,OP)核心结合因子α-1(core,binding factor alpha.1 chfa1)碱性磷酸酶,脂肪酸偶联蛋白4等的表达,这些基因的相应蛋白在成骨细胞分化中也起非常关键的作用[21]。成骨细胞内各种调控因素的调节,其中骨形态发生蛋白2是主要的调控因素之一。当成骨细胞内骨形态发生蛋白2表达增高时,细胞自分泌与旁分泌骨形态发生蛋白2增多。成骨细胞功能减退,导致新骨形成量减少,对骨吸收陷窝的修补能力减弱,造成骨小梁变细、薄弱、穿孔,皮质骨出现多孔性改变,从而引起骨质疏松的发生[22]。有文献报道,骨形态发生蛋白2主要对未分化间充质细胞和骨系细胞起到募集和分化作用[23]。在骨形成早期,骨形态发生蛋白2使未分化间充质细胞向骨形成中心募集,并分化为骨系细胞,并使成纤维细胞、成肌细胞及骨髓的基细胞逆转分化为骨系细胞。其中主要过程是:增加或抑制这些细胞内的某些特异性蛋白的分泌,使成纤维细胞分化为成骨细胞,成肌细胞快速分化为肥大的软骨细胞,并促进基质钙化。对于成骨细胞骨形态发生蛋白2可使之维持其特有细胞表型,并诱导成骨细胞标志物的增高,促进细胞外基质钙化。在骨形成后期,骨形态发生蛋白2还作为一种破骨细胞分化因子与其他支持破骨细胞分化因子直接或间接刺激破骨细胞分化,参与骨的重建[22]。 2.3 支架材料 骨组织工程技术能否取得成功,支架材料的选择至关重要。支架材料目前被广泛的研究,各种骨移植材料都在被试验。理想的组织细胞外基质材料必须具有如下特征,即:①良好的生物相容性。②良好的生物降解性。③具有三维立体多孔结构。④良好的可塑性和一定的机械强度。⑤良好的材料细胞界面。⑥材料须易于消毒。众多学者不断地尝试新的载体材料,迄今为止,应用磷酸盐支架,纤维蛋白胶,自体骨移植物等作为组织工程的支架材料己经取得初步成功[24]。但这些支架材料存在着生物相容性差,机械强度低,不能提供有效支持空间,组织创伤大,感染机率高等缺点,未能广泛应用。如何促进干细胞与理想材料相容生长并成骨一直是骨组织工程研究的热点[25-26]。 Bio-Oss是一种天然的具有骨引导作用的多孔骨移植材料,作为理想的骨代品,其理化性质与人体骨组织结构非常相似。经过特殊工艺加工,去除松质骨中所有有机成分,从而使精细的骨小梁结构和内部空隙被保存下来,为成骨细胞的长入提供了支架,利于细胞吸附,增殖及发挥成骨效能[27]。作为骨形成的引导支架,Bio-oss的多孔无机结构可为干细胞提供巨大的接触面积,能与骨组织很好地结合,对骨细胞的生长起导向作用,有利于新骨形成。Bio-oss的内部结构中有大小不一的空隙,能促进血管再生长入和血凝块的稳定,为组织再生重建提供最早期的营养供应。Bio-oss的小晶体尺寸,还可被破骨细胞缓慢吸收降解,为血管的再生和骨组织的重建提供足够的时间和支持空间,同时防止了新生骨组织过早吸收[28-29]。Bio-oss在骨缺损区占据特定的解剖位置,并在该区域诱导骨的形成,与此同时,Bio-oss逐渐降解吸收,当完全吸收时,该部位的新骨全部形成,并取代了Bio-oss原先占据的位置,这些新生成的骨具有完整功能。 脂肪干细胞作为理想的种子细胞,骨形态发生蛋白2作为理想的细胞因子与Bio-oss生物材料混合,构成三维环境,Bio-oss晶体内广泛交织的空隙结构,有利于脂肪干细胞的迁移。骨形态发生蛋白2与脂肪干细胞复合,促进控制其分化向骨形成中心募集,并分化为骨系细胞,通过钙盐沉积形成新骨,达到治疗种植体周围骨缺损的目的。"
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