【作者】 张勇；

【导师】 刘洪臣；

【作者基本信息】 中国人民解放军军医进修学院 ， 口腔临床医学， 2010， 博士

【摘要】 外伤、肿瘤切除、先天畸形造成的颌骨缺损在临床较常见,目前常采用自体骨移植、异体骨移植、异种骨移植、金属及人工合成材料替代等治疗方法,尚不能完全满足临床的需要。组织工程技术的发展为颌骨缺损的修复提供了新的途径。种子细胞,生长因子和支架材料是组织工程的三大要素。近年来研究发现脂肪组织中存在一些具有自我更新和多向分化潜能的脂肪基质细胞(adipose derived stromal cells, ADSCs),这些细胞在一定条件下可分化为脂肪细胞、成骨细胞、软骨细胞、肌细胞和神经细胞等多种细胞,具有成骨、成脂、成软骨、成肌肉、成神经等多种潜能。而且脂肪组织还具有取材简便、来源丰富等优势,被认为是组织工程中最有前途的种子细胞。其中,ADSCs用于骨缺损的修复倍受关注。生长因子在ADSCs成骨分化中起关键作用。常用的骨生长因子有骨形态发生蛋白2(bone morphogenetic proteins 2, BMP2),骨形态发生蛋白7(bone morphogenetic proteins 7, BMP7),生长分化因子5(Growth differentiation factor 5, GDF-5),转化生长因子-β(transforming growth factor-β, TGF-β),血小板衍生生长因子(platelet-derived growth factor, PDGF),胰岛素样生长因子(insulin-like growth factor, IGF)等。GDF-5是转化生长因子超家族、骨形态发生蛋白亚家族中的一员,在软骨发生和长骨发育中具有重要作用。目前研究主要集中于其促进软骨分化和形成,对于其促进成骨分化和骨形成的作用,国内外的研究较少。因此本实验应用GDF-5诱导hADSCs向成骨细胞分化,并将诱导后的细胞复合纳米羟基磷灰石／胶原／L-聚乳酸(nHAC/PLA)支架植入裸鼠体内,构建组织工程骨三维培养模型,并通过体内体外研究观察成骨情况,为其将来在颌骨组织工程中的应用奠定实验基础。全文共分为三部分：第一部分：hADSCs分离、培养及生物学特性研究目的：研究hADSCs的生长特性、表面抗原特点,横向分化潜能,在此基础上对其进行鉴定。方法：取健康人吸脂术中取出的新鲜脂肪组织,胶原酶消化法分离其中脂肪基质细胞,倒置相差显微镜观察其形态特征；MTT法测hADSCs的生长增殖状况；流式细胞仪分析其表面抗原；波形丝蛋白、角蛋白免疫荧光染色鉴定其来源；在细胞鉴定基础上矿化液诱导其向成骨细胞分化,通过碱性磷酸酶染色、OPN及Ⅰ型胶原免疫荧光染色、钙结节茜素红染色分析其成骨分化潜能；同时,采用成脂诱导液诱导其向脂肪细胞方向分化,油红0染色进行成脂分化鉴定。结果：原代及传代培养的hADSCs形态均匀,呈类成纤维细胞样形态,生长能力旺盛；P3、P5、P10代hADSCs的增殖曲线大致相同,呈近似“S”形；P2代hADSCs表达CD29、CD44、CD105等间充质细胞表面相对特异性标志蛋白,CD14、CD45阴性表达；P4代hADSCs免疫荧光染色波形丝蛋白阳性,角蛋白为阴性。成骨诱导21d后,碱磷酶染色、OPN、Ⅰ型胶原免疫荧光染色阳性,钙结节茜素红染色表现为红色结节。成脂诱导14d后油红0染色有红色着色的脂滴形成。结论：吸脂术来源的细胞具有未分化hADSCs的特征,在体外诱导条件下,可向成骨、成脂方向分化,具有横向分化潜能。第二部分：GDF-5诱导hADSCs成骨分化作用的体外研究目的：研究GDF-5对hADSCs体外成骨分化能力的影响。方法：将GDF-5分成0,1,10,100,500ng／ml 5个浓度,通过MTT法分析不同浓度GDF-5对hADSCs增殖的影响,通过碱性磷酸酶活性、茜素红染色检测GDF-5对其成骨分化能力的影响,确定GDF-5促进成骨的最适浓度。然后取所确定的GDF-5浓度,分成对照组、矿化组、GDF-5三组,通过测定RT-PCR检测成骨诱导后Runx2、OPN、ColI、VEGF基因的表达；Western Blot检测其成骨诱导后Runx2、OPN、ColI、VEGF蛋白的表达,分析该浓度GDF-5对P4代hADSCs的增殖、分化效应。结果：100ng／ml为GDF-5促进hADSCs成骨的最适浓度；三组培养基均能促进hADSCs增殖,但GDF-5组促增殖作用显著低于另两组(P<0.05)；GDF-5组促成骨分化作用最为显著,碱磷酶活性,Runx2、OPN、colI、VEGF基因及蛋白的表达均高于矿化组和细胞组,统计学上有显著性差异(P<0.05)。结论：体外研究表明100ng／mlGDF-5有诱导hADSCs分化为成骨细胞的潜能。第三部分：GDF-5诱导hADSCs复合纳米羟基磷灰石／胶原／聚乳酸体内成骨的研究目的：观察GDF-5诱导的hADSCs复合纳米羟基磷灰石／胶原／L-聚乳酸裸鼠皮下成骨的能力方法：取对数生长期hADSCs,接种于纳米羟基磷灰石／胶原／L-聚乳酸支架材料上,构建细胞／支架材料三维培养模型,扫描电镜(SEM)观察细胞在支架上的生长情况。然后,将其分别置于L-DMEM、矿化液、GDF-5体外培养一周后,移植入裸鼠背部皮下,4w、12w后取材,HE染色、Goldner’s染色观察支架材料复合体在裸鼠皮下成骨情况。结果：hADSCs能粘附在纳米羟基磷灰石／胶原／L-聚乳酸(nanohydroxyapatite/collagen/L-poly lactic acid, nHAC/PLA)支架材料的表面,生长良好；细胞／支架材料复合体经GDF-5诱导后4w、12w后取材均可见类骨质形成,并逐渐取代支架材料,周围未见明显炎症反应。其中GDF-5组成熟的骨及血管形成数量明显多于其他两组。结论：纳米羟基磷灰石／胶原／L-聚乳酸是有利于ADSCs成骨分化的良好的支架材料；GDF-5可促进hADSCs复合纳米羟基磷灰石／胶原／L-聚乳酸异位成骨效应。更多还原

【Abstract】 Bone defects caused by wound, tumour and malformation are common in dental clinic. The main treatment methods, such as autograft, allograft, and bone-grafting materials, couldn’t completely meet the clinical needs. The bone tissue engineering provided novel methods for restoring bone defects. The content of bone tissue engineering includes three elements:seed cells, scaffolds and growth factors. It is hypothesized that human adipose tissue is an abundant reservoir of adult stem cells, which have potent to differentiate into many mature cell lineages varying from adipogenic precursors, osteogenic precursors, chondrogenic precursors to myogenic precursors. ADSCs are easier to obtain, carry relatively lower donor site morbidity, and are available in large numbers of stem cells at harvest. Growth factors play important role in the ADSCs’osteogenic differentiation. The common growth factors were BMP2, BMP7, GDF-5, TGF-βPDGF, IGF and so on. Growth differentiation factor-5 (GDF-5) is a divergent member of the bone morphogenetic proteins(BMPs) subfamily which belongs to the transforming growth factor-β(TGF-β) superfamily. It plays a very important role in the chondrogenesis and long bone development. GDF-5 can promote the osteochondrogenic differentiation of mesenchymal progenitor cells in vitro and in vivo. In order to further elucidate the biological activities of GDF-5 and to expend its applications in bone tissue engineering. Our investigations were presented as followings:Part 1:Experimental study of the isolation, culture and biological characteristics of hADSCs.Objective:To investigate methods for the isolation, culture and biological characteristics of hADSCs in order to provide seed cells for bone tissue engineering. Methods:Human adipose tissues from liposuction were excised and digested with 0.1% collagenase I, and then the cells were cultured in L-DMEM with 20% FBS. The morphology of hADSCs was observed under inserted microscope. The proliferation of the 3rd,5th and 10th passage cells were examined using MTT. The antigens of CD29, CD44, CD 14, CD45, CD 105, the markers of the mesenchymal stem cells, on the 2nd passage cells were examined using flow cytometric methods. The antigen of vimentin was detected by immunofluorescence. ALP, OPN, Col I and calcified nodules of the 4th passage cells were assessed by cytochemical and immunocytochemical staining. Adipogenic differentiation of the 4th passage cells was assessed by Oil Red O staining.Results:The primary hADSCs had long or short spindle-shape, later fibroblast-like. The proliferation curve showed that the 3rd,5th,10th passage cells proliferated rapidly, there were no differences between them. The 4th cells expressed CD29, CD44, CD 105 and vimentin. ALP, OPN and collagen I were positively expressed with histological and immunohistochemic staining. Calcified nodules formed were formed based on the alizarin red stainning. After induction for 14 days, the lipid droplets were presented in the differentiated cells by Oil Red O staining.Conclusions:The hADSCs have the characters of stromal cells and can be used as a source of seed cells for bone tissue engineering.Part II:The effects of GDF-5 on osteogenic differentiation of hADSCs in vitroObjective:To explore the effects of GDF-5 on osteogenic differentiation potential of hADSCs in vitro and make preparations for further research about bone tissue construction in vivo.Methods:Human adipose-derived stromal cells were divided into 5 groups by different concentration of GDF-5 (0,1,10,100,500 ng/ml), MTT, ALP activity and Alizarin red S were used to determine the appropriate concentration. The 4th passage cells were divided into three groups:control group, mineral group and GDF-5 group, their osteogenic differentiation was assessed by MTT, ALP activity respectively. The mRNA expressions of Runx2, OPN, Col I and VEGF were examined by RT-PCR. The protein expressions of Runx2, OPN, Col I and VEGF were examined by Western-blot.Results:The 100ng/ml GDF-5 was the optimal concentration. MTT value in the GDF-5 were significantly lower than the other groups, but ALP activity, mRNA expression and protein expression in the GDF-5 were significantly higher than other groups(P<0.05).Couclusions:The hADSCs could be induced into osteoblast by GDF-5 in vitro.Part III:The effects of GDF-5 on bone formation of hADSCs/nHAC/PLA construct in vivo.Objective:To explore the effects of GDF-5 on bone formation of hADSCs/nHAC/PLA construct in vivo.Methods:The 4th passage hADSCs were seeded onto nHAC/PLA in vitro to construct cells-scaffold composites. They were divided into the control group, mineral group and GDF-5 group respectively. Then the composites were implanted into the back of immunodeficiency disease (SCID) mice.4 weeks and 12 weeks later. The implantation were observed using HE, Goldner’s staining and scanning electron microscope.Results:The hADSCs could adhere and grow on the surfaces of the implants. Bone-like tissues were found to form in the constructs at 4 weeks and 12 weeks in comparison to the control group. The pencentage of newly-formed bone areas in GDF-5 group was signficently higher than those in the other groups.Conclusions:The nHAC/PLA is an ideal scaffold; GDF-5 could enhance ’bone formation of hADSCs/nHAC/PLA in vivo.更多还原