转化生长因子β1基因体外转染兔颞下颌关节滑膜间充质干细胞向纤维软骨转化实验研究
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摘要
第一部分携带TGF-β1基因重组腺相关病毒载体的构建
目的构建携带TGF-β1基因的重组腺相关病毒载体,制备表达TGF-β1基因的重组腺相关病毒rAAV-TGF-β1,检测重组病毒的滴度,为TGF-β1基因的真核表达及临床应用提供实验基础。
方法含TGF-β1 cDNA的质粒pcDNA3-TGF-β1和质粒pAAV-MCS用EcoRⅠ+XbaⅠ进行双酶切后连接,转化大肠杆菌DH5a感受态细胞,获得重组质粒pAAV-MCS-TGF-β1。通过酶切和DNA测序鉴定重组质粒的正确性。采用磷酸钙共沉淀法,以重组质粒pAAV-MCS-TGF-β1和pAAV-RC、pHelper共转染AAV-293细胞,产生具有感染性的病毒颗粒rAAV-TGF-β1并检测重组病毒的滴度。
结果成功构建携带目的基因TGF-β1的重组质粒pAAV-MCS-TGF-β1,并获得携带TGF-β1基因的重组腺相关病毒rAAV-TGF-β1。重组质粒pAAV-MCS—TGF-β1经双酶切和测序证实TGF-β1基因正确插入载体且序列正确。重组腺相关病毒rAAV-TGF-β1经PCR检测,可见1.2kb大小的TGF-β1基因片段。重组病毒的滴度为3×108pfu／ml。
结论成功构建了携带目的基因TGF-β1的重组腺相关病毒载体(rAAV-TGF-β1),为下一步组织工程种子细胞转染目的基因TGF-β1奠定了基础。
第二部分兔颞下颌关节滑膜间充质干细胞的分离与培养
目的探讨兔颞下颌关节滑膜间充质干细胞(SMSCs)的分离纯化及其在体外培养条件下的基本生物学特性,为颞下颌关节组织工程选择合适的种子细胞。
方法在无菌条件下,取3月龄健康新西兰大白兔颞下颌关节腔内面平滑光亮的滑膜组织,采用酶消化法体外培养,有限稀释法进行单细胞克隆,筛选出SMSCs,观察其增殖和生长特性,绘制生长曲线,测定贴壁率。取第3代SMSCs采用不同诱导液进行成脂肪、成骨、成软骨诱导等多向分化,观察细胞的形态变化,在诱导第14d进行油红染色和茜素红染色以鉴定SMSCs成脂肪和成骨分化潜能；在诱导第21d用番红O染色、Ⅱ型胶原免疫组化鉴定SMSCs成软骨分化潜能。
结果SMSCs生物性状稳定,为均一的成纤维样细胞。原代细胞24 h后贴壁,48 h贴壁细胞逐渐增多。第5-6 d细胞数目最多；传代后10-12 h贴壁率高达95%,细胞传至第10代细胞形态无明显变化。不同诱导液诱导后细胞形态发生变化。成骨诱导后可检测到钙化结节形成；成脂肪诱导后可检测到脂滴形成。成软骨诱导番红0染色可见胞浆内有明显的特异性蛋白多糖着色,Ⅱ型胶原免疫组化胞浆内可见棕黄色颗粒。
结论经体外分离纯化的单克隆SMSCs可以表现为成纤维细胞的特性,与其他来源的MSCs具有类似的生物学特性及多向分化潜能,有可能成为颞下颌关节组织工程种子细胞的来源。
第三部分重组腺相关病毒介导TGF-β1基因体外转染SMSCs的实验研究
目的应用重组腺相关病毒rAAV-TGF-β1转染体外培养的SMSCs,检测转染后SMSCs中TGF-β1 mRNA和蛋白的表达。并通过检测Ⅰ型胶原、II型胶原、X型胶原、Sox 9、aggrecan基因及Ⅰ、Ⅱ型胶原蛋白,对TGF-β1诱导SMSCs向纤维软骨细胞分化的表型进行鉴定。
方法将收集好的重组腺相关病毒rAAV-TGF-β1转染SMSCs,按不同处理因素分为3组：转染rAAV-TGF-β1组(实验组),转染空载体组和未转染组(对照组)。于转染后1-7d,应用MTT法检测3组SMSCs的增殖情况。转染后进行SMSCs细胞球团培养,分别于2d、7d、21d收集细胞,应用RT-PCR检测3组细胞TGF-β1 mRNA表达情况,Western blot法检测培养液上清中TGF-β1蛋白含量。并分别于7d、21d采用RT-PCR检测软骨基质Ⅰ、Ⅱ、Ⅹ型胶原、Sox 9、Aggrecan mRNA的表达,同时用免疫组化检测SMSCs及周围基质中Ⅰ、Ⅱ型胶原蛋白的表达。
结果TGF-β1 mRNA及蛋白在基因转染细胞得到正确表达,且随时间的延长,表达量逐渐增加,组间比较有显著性差异(p<0.01)；转染后SMSCs目的基因CollagenⅠ、CollagenⅡ、Sox 9及Aggrecan的表达和软骨特异性基质Ⅰ、Ⅱ型胶原蛋白的分泌增强,且Ⅰ型胶原蛋白的染色明显强于Ⅱ型胶原蛋白。未见X型胶原mRNA的表达。表明SMSCs被诱导向纤维软骨方向分化。
结论重组腺相关病毒介导的外源性基因TGF-β1能够安全有效转染SMSCs,转染后细胞的TGF-β1 mRNA表达和蛋白分泌水平明显增加,高表达的TGF-β1可促进SMSCs向纤维软骨方向分化。
第四部分TGF-B1基因转染滑膜间充质干细胞复合支架构建纤维软骨组织的体外三维培养
目的：研究在体外三维培养条件下,经重组腺相关病毒rAAV-TGF-β1转染的兔滑膜间充质干细胞(SMSCs)复合壳聚糖／Ⅰ型胶原支架培养向纤维软骨分化的生物学特性。
方法：将rAAV-TGF-β1转染的SMSCs以两次沉降法接种到壳聚糖／Ⅰ型胶原支架复合物上,进行三维培养。HE染色观察细胞在复合支架上的生长状况；番红O染色和Ⅱ型胶原免疫组化了解转染后细胞外软骨基质的分泌；扫描电镜观察细胞在材料上的附着和生长情况。MTT检测细胞在支架上的增殖情况。
结果：rAAV-TGF-β1转染的SMSCs在支架中生长黏附良好,有较多的基质形成。番红O染色可见红染的细胞外软骨基质,细胞和基质成分填充于支架材料的孔隙内。免疫组化染色显示胞浆及细胞外均有Ⅱ型胶原蛋白分泌。扫描电镜下可见细胞紧贴支架复合物生长并彼此间有纤维交联,胞体周围有基质样物质分泌。MTT检测细胞具有良好的增殖特性。
结论：壳聚糖／Ⅰ型胶原复合支架材料可为rAAV-TGF-β1转染SMSCs生长分化提供一个良好的环境,建立了较为稳定的SMSCs体外三维培养体系。
PartⅠConstruction of Recombinant Adeno-associated Virus with TGF-β1
Objective To construct and confirm the recombinant adeno-associated virus with transforming growth factorβ1 (TGF-β1).
Methods Both plasmids of pcDNA3-TGF-β1 and pAAV-MCS were linked after being digested by enzyme of EcoR I+Xba I, and the recombinant plasmids of pAVV-MCS-TGF-β1 were collected from the colon bacillus DH5a transformed by the linked plasmids. The recombinant plasmids were confirmed by enzyme digestion and DNA sequence examination. The recombinant virus rAAV-TGF-β1 were collected from the transfected AAV-293 cells which were co-transfected by pAAV-MCS-TGF-β1, pAAV-RC and pHelper through the process of calcium-phosphate coprecipitation. The titers of recombinant virus were detected.
Results The recombinant plasmid pAAV-MCS-TGF-β1 with TGF-β1 were reconstructed, and the recombinant adeno-associated virus with TGF-β1 were also achieved. After being treated by enzyme digestion and sequence analysis, the recombinant pAAV-MCS-TGF-β1 was confirmed that TGF-β1 was inserted correctly into the vector and the sequence of this gene was also corrected. The titers of the recombinant virus (rAAV-TGF-β1) were detected as 3×108pfu/ml and TGF-β1 of rAAV-TGF-β1 was detected by PCR.
Conclusion The recombinant adeno-associated virus vector with TGF-β1 (rAAV-TGF-β1) were reconstructed successfully and might be employed to transduce the synovial mesenchymal stem cells in the tissue engineering of fibrocartilage in the following experiments.
PartⅡIsolation and culture of synovial mesenchymal stem cells of rabbit temporomandibular joint
Objective To explore the isolation and culture of synovial mesenchymal stem cells(SMSCs) for the tissue engineering of temporomandibular joint.
Methods Synovium membrane was harvested from New-Zealand white rabbits (3-month old) under the condition of asepsis. SMSCs were harvested through the method of enzyme digestion and isolated by limited dilution. The proliferation and viability of the isolated SMSCs were detected. For evaluation of the multipotential differentiation of SMSCs, the passage 3 SMSCs were cultured in the defined medium to be differentiated into adipose cells, osteoblasts and chondrocytes. After being cultured in defined medium for 14d, the differentiation of SMSCs into adipose cells and osteoblasts were tested by Oil red O staining or Alizarin red staining. Being induced by TGF-β1 for 21d in vitro, the differentiation of SMSCs into chondrocyte were evaluated by Safranin O staining and collagen typeⅡimmunohistochemical staining.
Results Rabbit SMSCs maintained the fibroblast-like morphology after being cultured 10 passages. Primary SMSCs began attaching on the floor of the flash after 24h, and the attachment ability of SMSCs reached to 80% after 48h. It was found that calcified nodules and lipid drops in the SMSCs after SMSCs being induced and differentiated into osteogenic cells and adipogenic cells. And special extracellular matrix of cartilage could be found in the SMSCs stained by Safranin O and collagen typeⅡstained positive immunohistochemically after the chondrogenic differentiation of SMSCs.
Conclusion It was found that the SMSCs could maintain the fibroblast-like morphology after being cultured several passages, and had multipotential differentiation cultured in the defined medium. Therefore, it suggested that SMSCs might be a choice of seeding cells for temporomandibular joint tissue engineering.
PartⅢStudy on transduction of SMSCs in vitro using recombinant adeno-associated virus with TGF-β1
Objective To detect the expression of TGF-β1 mRNA and protein of the SMSCs transduced by recombinant adeno-associated virus with TGF-β1 (rAAV-TGF-β1) in vitro and investigate the ability of the transduced SMSCs differentiation into fibrocartilage.
Methods Three groups were divided as experimental group of SMSCs transduced by rAAV-TGF-β1, control group of SMSCs transduced by empty vector of adeno-associated virus, and blank control group of SMSCs non-transduced. MTT assay was employed to detect the proliferation of SMSCs after being transduced 1～7d. RT-PCR was applied to explore the expression of TGF-β1, and Western blot was used to evaluate the protein of TGF-β1 of the three groups. Pellet culture of tansduced SMSCs was adopted to detect the expression of mRNA of collagen typeⅠ,ⅡandⅩ, sox9 and aggrecan using RT-PCR, and the collagen typeⅠproteins and collagen typeⅡprotein were investigated by the method of immunocytochemistry staining.
Results Both TGF-β1 mRNA and protein could express in the experimental group of SMSCs transduced by rAAV-TGF-β1; the mRNA expression of collagen typeⅠ,Ⅱ, Sox9 and aggrecan were detected, and the expression of collagen typeⅠprotein is stronger than collagen typeⅡby the method of immunocytochemistry staining. No collagen typeⅩexpressed in transduced SMSCs at any time point during the experiment. In the conrol group, the secretion of cartilage matrixes were not detected. These date suggest that the SMSCs transfected by rAAV-TGF-β1 could differentiate into fibrocartilage.
Conclusion It could be concluded from this study that TGF-β1 could be transduced by adeno-associated virus vector into SMSCs; the transduced cells could express more TGF-β1 mRNA and protein than that of non-transduced cells; and transduced SMSCs could differentiate into fibrocartilage.
PartⅣIn vitro study on fibrocartilage tissue engineering using gene TGF-β1 modified SMSCs and chitosan/collagen typeⅠscaffold
Objective To investigate the potential of the SMSCs transduced by recombinant adeno-associated virus rAAV-TGF-β1 cultured in the fabricated chitosan/collagen typeⅠ(CS/COL-I) scaffold for fibrocartilage tissue engineering.
Methods The SMSCs were transduced by rAAV-TGF-β1 and cultured onto the fabricated chitosan/collagen typeⅠscaffold. After being cultured in defined medium, histological HE staining was applied to examine the cell morphology of SMSCs-CS/COL-I constructs. Safranin O/Fast green staining and immunocyto-chemistry staining were used to detect the the cartilage matrix secreted by transduced SMSCs. The cell morphology and attachment of the SMSCs-CS/COL-I constructs were examined through scanning electronic microscope (SEM). And the proliferation of SMSCs on scaffold was detected by MTT.
Results The SMSCs transduced by rAAV-TGF-β1 attached onto the scaffolds and secreted much extracellular matrix around the cells. The cartilage-like matrix filled in the pores of the scaffolds and was stained red by Safranin O/Fast green. It was found that collagen typeⅡwas immunohistochemically positive in the transduced SMSCs or around the cells. The cells in the constructs communicated each other in the pores of the scaffolds, and matrix surrounded the cells examined through SEM. And the transduced SMSCs cultured in the scaffolds presented good viability and proliferation indicated by MTT assay.
Conclusion The fabricated CS/COL-I coploymer could be a proper three-dimensional scaffold for the SMSCs transduced by rAAV-TGF-β1 to proliferate and differentiate.
目的构建携带TGF-β1基因的重组腺相关病毒载体,制备表达TGF-β1基因的重组腺相关病毒rAAV-TGF-β1,检测重组病毒的滴度,为TGF-β1基因的真核表达及临床应用提供实验基础。
方法含TGF-β1 cDNA的质粒pcDNA3-TGF-β1和质粒pAAV-MCS用EcoRⅠ+XbaⅠ进行双酶切后连接,转化大肠杆菌DH5a感受态细胞,获得重组质粒pAAV-MCS-TGF-β1。通过酶切和DNA测序鉴定重组质粒的正确性。采用磷酸钙共沉淀法,以重组质粒pAAV-MCS-TGF-β1和pAAV-RC、pHelper共转染AAV-293细胞,产生具有感染性的病毒颗粒rAAV-TGF-β1并检测重组病毒的滴度。
结果成功构建携带目的基因TGF-β1的重组质粒pAAV-MCS-TGF-β1,并获得携带TGF-β1基因的重组腺相关病毒rAAV-TGF-β1。重组质粒pAAV-MCS—TGF-β1经双酶切和测序证实TGF-β1基因正确插入载体且序列正确。重组腺相关病毒rAAV-TGF-β1经PCR检测,可见1.2kb大小的TGF-β1基因片段。重组病毒的滴度为3×108pfu／ml。
结论成功构建了携带目的基因TGF-β1的重组腺相关病毒载体(rAAV-TGF-β1),为下一步组织工程种子细胞转染目的基因TGF-β1奠定了基础。
第二部分兔颞下颌关节滑膜间充质干细胞的分离与培养
目的探讨兔颞下颌关节滑膜间充质干细胞(SMSCs)的分离纯化及其在体外培养条件下的基本生物学特性,为颞下颌关节组织工程选择合适的种子细胞。
方法在无菌条件下,取3月龄健康新西兰大白兔颞下颌关节腔内面平滑光亮的滑膜组织,采用酶消化法体外培养,有限稀释法进行单细胞克隆,筛选出SMSCs,观察其增殖和生长特性,绘制生长曲线,测定贴壁率。取第3代SMSCs采用不同诱导液进行成脂肪、成骨、成软骨诱导等多向分化,观察细胞的形态变化,在诱导第14d进行油红染色和茜素红染色以鉴定SMSCs成脂肪和成骨分化潜能；在诱导第21d用番红O染色、Ⅱ型胶原免疫组化鉴定SMSCs成软骨分化潜能。
结果SMSCs生物性状稳定,为均一的成纤维样细胞。原代细胞24 h后贴壁,48 h贴壁细胞逐渐增多。第5-6 d细胞数目最多；传代后10-12 h贴壁率高达95%,细胞传至第10代细胞形态无明显变化。不同诱导液诱导后细胞形态发生变化。成骨诱导后可检测到钙化结节形成；成脂肪诱导后可检测到脂滴形成。成软骨诱导番红0染色可见胞浆内有明显的特异性蛋白多糖着色,Ⅱ型胶原免疫组化胞浆内可见棕黄色颗粒。
结论经体外分离纯化的单克隆SMSCs可以表现为成纤维细胞的特性,与其他来源的MSCs具有类似的生物学特性及多向分化潜能,有可能成为颞下颌关节组织工程种子细胞的来源。
第三部分重组腺相关病毒介导TGF-β1基因体外转染SMSCs的实验研究
目的应用重组腺相关病毒rAAV-TGF-β1转染体外培养的SMSCs,检测转染后SMSCs中TGF-β1 mRNA和蛋白的表达。并通过检测Ⅰ型胶原、II型胶原、X型胶原、Sox 9、aggrecan基因及Ⅰ、Ⅱ型胶原蛋白,对TGF-β1诱导SMSCs向纤维软骨细胞分化的表型进行鉴定。
方法将收集好的重组腺相关病毒rAAV-TGF-β1转染SMSCs,按不同处理因素分为3组：转染rAAV-TGF-β1组(实验组),转染空载体组和未转染组(对照组)。于转染后1-7d,应用MTT法检测3组SMSCs的增殖情况。转染后进行SMSCs细胞球团培养,分别于2d、7d、21d收集细胞,应用RT-PCR检测3组细胞TGF-β1 mRNA表达情况,Western blot法检测培养液上清中TGF-β1蛋白含量。并分别于7d、21d采用RT-PCR检测软骨基质Ⅰ、Ⅱ、Ⅹ型胶原、Sox 9、Aggrecan mRNA的表达,同时用免疫组化检测SMSCs及周围基质中Ⅰ、Ⅱ型胶原蛋白的表达。
结果TGF-β1 mRNA及蛋白在基因转染细胞得到正确表达,且随时间的延长,表达量逐渐增加,组间比较有显著性差异(p<0.01)；转染后SMSCs目的基因CollagenⅠ、CollagenⅡ、Sox 9及Aggrecan的表达和软骨特异性基质Ⅰ、Ⅱ型胶原蛋白的分泌增强,且Ⅰ型胶原蛋白的染色明显强于Ⅱ型胶原蛋白。未见X型胶原mRNA的表达。表明SMSCs被诱导向纤维软骨方向分化。
结论重组腺相关病毒介导的外源性基因TGF-β1能够安全有效转染SMSCs,转染后细胞的TGF-β1 mRNA表达和蛋白分泌水平明显增加,高表达的TGF-β1可促进SMSCs向纤维软骨方向分化。
第四部分TGF-B1基因转染滑膜间充质干细胞复合支架构建纤维软骨组织的体外三维培养
目的：研究在体外三维培养条件下,经重组腺相关病毒rAAV-TGF-β1转染的兔滑膜间充质干细胞(SMSCs)复合壳聚糖／Ⅰ型胶原支架培养向纤维软骨分化的生物学特性。
方法：将rAAV-TGF-β1转染的SMSCs以两次沉降法接种到壳聚糖／Ⅰ型胶原支架复合物上,进行三维培养。HE染色观察细胞在复合支架上的生长状况；番红O染色和Ⅱ型胶原免疫组化了解转染后细胞外软骨基质的分泌；扫描电镜观察细胞在材料上的附着和生长情况。MTT检测细胞在支架上的增殖情况。
结果：rAAV-TGF-β1转染的SMSCs在支架中生长黏附良好,有较多的基质形成。番红O染色可见红染的细胞外软骨基质,细胞和基质成分填充于支架材料的孔隙内。免疫组化染色显示胞浆及细胞外均有Ⅱ型胶原蛋白分泌。扫描电镜下可见细胞紧贴支架复合物生长并彼此间有纤维交联,胞体周围有基质样物质分泌。MTT检测细胞具有良好的增殖特性。
结论：壳聚糖／Ⅰ型胶原复合支架材料可为rAAV-TGF-β1转染SMSCs生长分化提供一个良好的环境,建立了较为稳定的SMSCs体外三维培养体系。
PartⅠConstruction of Recombinant Adeno-associated Virus with TGF-β1
Objective To construct and confirm the recombinant adeno-associated virus with transforming growth factorβ1 (TGF-β1).
Methods Both plasmids of pcDNA3-TGF-β1 and pAAV-MCS were linked after being digested by enzyme of EcoR I+Xba I, and the recombinant plasmids of pAVV-MCS-TGF-β1 were collected from the colon bacillus DH5a transformed by the linked plasmids. The recombinant plasmids were confirmed by enzyme digestion and DNA sequence examination. The recombinant virus rAAV-TGF-β1 were collected from the transfected AAV-293 cells which were co-transfected by pAAV-MCS-TGF-β1, pAAV-RC and pHelper through the process of calcium-phosphate coprecipitation. The titers of recombinant virus were detected.
Results The recombinant plasmid pAAV-MCS-TGF-β1 with TGF-β1 were reconstructed, and the recombinant adeno-associated virus with TGF-β1 were also achieved. After being treated by enzyme digestion and sequence analysis, the recombinant pAAV-MCS-TGF-β1 was confirmed that TGF-β1 was inserted correctly into the vector and the sequence of this gene was also corrected. The titers of the recombinant virus (rAAV-TGF-β1) were detected as 3×108pfu/ml and TGF-β1 of rAAV-TGF-β1 was detected by PCR.
Conclusion The recombinant adeno-associated virus vector with TGF-β1 (rAAV-TGF-β1) were reconstructed successfully and might be employed to transduce the synovial mesenchymal stem cells in the tissue engineering of fibrocartilage in the following experiments.
PartⅡIsolation and culture of synovial mesenchymal stem cells of rabbit temporomandibular joint
Objective To explore the isolation and culture of synovial mesenchymal stem cells(SMSCs) for the tissue engineering of temporomandibular joint.
Methods Synovium membrane was harvested from New-Zealand white rabbits (3-month old) under the condition of asepsis. SMSCs were harvested through the method of enzyme digestion and isolated by limited dilution. The proliferation and viability of the isolated SMSCs were detected. For evaluation of the multipotential differentiation of SMSCs, the passage 3 SMSCs were cultured in the defined medium to be differentiated into adipose cells, osteoblasts and chondrocytes. After being cultured in defined medium for 14d, the differentiation of SMSCs into adipose cells and osteoblasts were tested by Oil red O staining or Alizarin red staining. Being induced by TGF-β1 for 21d in vitro, the differentiation of SMSCs into chondrocyte were evaluated by Safranin O staining and collagen typeⅡimmunohistochemical staining.
Results Rabbit SMSCs maintained the fibroblast-like morphology after being cultured 10 passages. Primary SMSCs began attaching on the floor of the flash after 24h, and the attachment ability of SMSCs reached to 80% after 48h. It was found that calcified nodules and lipid drops in the SMSCs after SMSCs being induced and differentiated into osteogenic cells and adipogenic cells. And special extracellular matrix of cartilage could be found in the SMSCs stained by Safranin O and collagen typeⅡstained positive immunohistochemically after the chondrogenic differentiation of SMSCs.
Conclusion It was found that the SMSCs could maintain the fibroblast-like morphology after being cultured several passages, and had multipotential differentiation cultured in the defined medium. Therefore, it suggested that SMSCs might be a choice of seeding cells for temporomandibular joint tissue engineering.
PartⅢStudy on transduction of SMSCs in vitro using recombinant adeno-associated virus with TGF-β1
Objective To detect the expression of TGF-β1 mRNA and protein of the SMSCs transduced by recombinant adeno-associated virus with TGF-β1 (rAAV-TGF-β1) in vitro and investigate the ability of the transduced SMSCs differentiation into fibrocartilage.
Methods Three groups were divided as experimental group of SMSCs transduced by rAAV-TGF-β1, control group of SMSCs transduced by empty vector of adeno-associated virus, and blank control group of SMSCs non-transduced. MTT assay was employed to detect the proliferation of SMSCs after being transduced 1～7d. RT-PCR was applied to explore the expression of TGF-β1, and Western blot was used to evaluate the protein of TGF-β1 of the three groups. Pellet culture of tansduced SMSCs was adopted to detect the expression of mRNA of collagen typeⅠ,ⅡandⅩ, sox9 and aggrecan using RT-PCR, and the collagen typeⅠproteins and collagen typeⅡprotein were investigated by the method of immunocytochemistry staining.
Results Both TGF-β1 mRNA and protein could express in the experimental group of SMSCs transduced by rAAV-TGF-β1; the mRNA expression of collagen typeⅠ,Ⅱ, Sox9 and aggrecan were detected, and the expression of collagen typeⅠprotein is stronger than collagen typeⅡby the method of immunocytochemistry staining. No collagen typeⅩexpressed in transduced SMSCs at any time point during the experiment. In the conrol group, the secretion of cartilage matrixes were not detected. These date suggest that the SMSCs transfected by rAAV-TGF-β1 could differentiate into fibrocartilage.
Conclusion It could be concluded from this study that TGF-β1 could be transduced by adeno-associated virus vector into SMSCs; the transduced cells could express more TGF-β1 mRNA and protein than that of non-transduced cells; and transduced SMSCs could differentiate into fibrocartilage.
PartⅣIn vitro study on fibrocartilage tissue engineering using gene TGF-β1 modified SMSCs and chitosan/collagen typeⅠscaffold
Objective To investigate the potential of the SMSCs transduced by recombinant adeno-associated virus rAAV-TGF-β1 cultured in the fabricated chitosan/collagen typeⅠ(CS/COL-I) scaffold for fibrocartilage tissue engineering.
Methods The SMSCs were transduced by rAAV-TGF-β1 and cultured onto the fabricated chitosan/collagen typeⅠscaffold. After being cultured in defined medium, histological HE staining was applied to examine the cell morphology of SMSCs-CS/COL-I constructs. Safranin O/Fast green staining and immunocyto-chemistry staining were used to detect the the cartilage matrix secreted by transduced SMSCs. The cell morphology and attachment of the SMSCs-CS/COL-I constructs were examined through scanning electronic microscope (SEM). And the proliferation of SMSCs on scaffold was detected by MTT.
Results The SMSCs transduced by rAAV-TGF-β1 attached onto the scaffolds and secreted much extracellular matrix around the cells. The cartilage-like matrix filled in the pores of the scaffolds and was stained red by Safranin O/Fast green. It was found that collagen typeⅡwas immunohistochemically positive in the transduced SMSCs or around the cells. The cells in the constructs communicated each other in the pores of the scaffolds, and matrix surrounded the cells examined through SEM. And the transduced SMSCs cultured in the scaffolds presented good viability and proliferation indicated by MTT assay.
Conclusion The fabricated CS/COL-I coploymer could be a proper three-dimensional scaffold for the SMSCs transduced by rAAV-TGF-β1 to proliferate and differentiate.
引文
1. Fan J, Varshney RR, Ren L, Cai D, Wang DA. Synovium-derived mesenchymal stem cells:a new cell source for musculoskeletal regeneration. Tissue Eng 2009, Mar;15(1):75-86. Review.
2. Miyamoto C, Matsumoto T, Sakimura K, Shindo H. Osteogenic protein-1 with transforming growth factor-betal:potent inducer of chondrogenesis of synovial mesenchymal stem cells in vitro. J Orthop Sci.2007, Nov;12(6):555-61.
3. Park Y, Sugimoto M, Watrin A, Chiquet M, Hunziker EB. BMP-2 induces the expression of chondrocyte-specific genes in bovine synovium-derived progenitor cells cultured in three-dimensional alginate hydrogel. Osteoarthritis Cartilage. 2005, Jun;13(6):527-36.
4. De Bari C,Dell'Accio F,Luyten FP.Human periosteum-derived cells maintain phenotypic stability and chondrogenic potential throughout expansion regardless of donor age.Arthritis Rheum,2001,44:85-95.
5. Yoo JU,Barthel TS,Nishimura K,et al.The chondrogenic potential of human bone-marrow-derived mesenchymal progenitor cells.J Bone Joint Surg Am,1998, 80:1745-1757.
6. Nishida K, Suzuki T, Kakutani K, Yurube T, Maeno K, Kurosaka M, Doita M. Gene therapy approach for disc degeneration and associated spinal disorders. Eur Spine J.2008, Dec;17 Suppl 4:459-66.
7. Liu X, Li K, Song J, Liang C, Wang X, Chen X. Efficient and stable gene expression in rabbit intervertebral disc cells transduced with a recombinant baculovirus vector. Spine (Phila Pa 1976).2006, Apr 1;31(7):732-5.
8. Lattermann C, Oxner WM, Xiao X, Li J, Gilbertson LG, Robbins PD, Kang JD. Theadeno associated viral vector as a strategy for intradiscal gene transfer in immune competent and pre-exposed rabbits. Spine (Phila Pa 1976).2005, Mar 1;30(5):497-504.
9. Hubert MG, Vadala G, Sowa G, Studer RK, Kang JD. Gene therapy for the
treatment of degenerative disk disease. J Am Acad Orthop Surg.2008, Jun; 16(6):312-9. Review. 10. Nishida K, Doita M, Takada T, Kakutani K, Miyamoto H, Shimomura T, Maeno K,Kurosaka M. Sustained transgene expression in intervertebral disc cells in vivo mediated by microbubble-enhanced ultrasound gene therapy. Spine (Phila Pa 1976).2006, Jun 1;31(13):1415-9.
11. Hildinger M, Baldi L, Stettler M, Wurm FM. High-titer, serum-free production of adeno-associated virus vectors bypolyethyleneimine-mediated plasmidtransfection in mammalian suspension cells. Biotechnol Lett.2007, Nov;29(11):1713-21.
12. Ulrich-Vinther M. Gene therapy methods in bone and joint disorders. Evaluation of the adeno-associated virus vector in experimental models of articular cartilage disorders, periprosthetic osteolysis and bone healing. Acta Orthop Suppl.2007, Apr;78(325):1-64. Review.
13. Timpe J, Bevington J, Casper J, et al. Mechanisms of adeno-associated virus genome encapsidation. Curr Gene Ther.2005,5(3):273-284.
14. Tratschin JD, Miller IL, Carter BJ. Genetic analysis of adeno-associated virus: properties of deletion mutants constructed in vitro and evidence for an adeno-associated virus replication function. J Virol.1984,51(3):611-619.
15. Yan Z, Zak R, Zhang Y, et al. Inverted terminal repeat sequences are important for intermolecular recombination and circularization of adeno-associated virus genomes. J Virol.2005,79(1):364-379.
16. Hauck B, Chen L, Xiao W. Generation and characterization of chimeric recombinant AAV vectors. Mol Ther.2003,7(3):419-425.
17. Gao G, Vandenberghe LH, Alvira MR, et al. Clades of Adeno-associated viruses are widely disseminated in human tissues. J Virol.2004,78(12):6381-6388.
18. Duan D, Sharma P, Yang J, et al. Circular intermediates of recombinant adeno-associated virus have defined structural characteristics responsible for long-term episomal persistence in muscle tissue. J Virol.1998,72(11):8568-8577.
19. Zhang C, Cortez NG, Berns KI. Characterization of a bipartite recombinant adeno-associated viral vector for site-specific integration. Hum Gene Ther.2007, 18(9):787-797.
20. Grieger JC, Samulski RJ. Adeno-associated virus as a gene therapy vector:vector development, production and clinical applications. Adv Biochem Eng Biotechnol. 2005,99:119-145.
21. Duffy AM, O'Doherty AM, O'Brien T, et al. Purification of adenovirus and adeno-associated virus:comparison of novel membrane-based technology to conventional techniques. Gene Ther.2005,12 Suppl 1:S62-72.
22. Wu J, Zhao W, Zhong L, Han Z, Li B, Ma W, Weigel-Kelley KA, Warrington KH,Srivastava A. Self-complementary recombinant adeno-associated viral vectors: packaging capacity and the role of rep proteins in vector purity. Hum Gene Ther. 2007, Feb;18(2):171-82.
23. Wang H, Lieber A. A helper-dependent capsid-modified adenovirus vector expressing adeno-associated virus rep78 mediates site-specific integration of a 27-kilobase transgene cassette. J Virol.2006, Dec;80(23):11699-709.
24. Grieger JC, Samulski RJ. Adeno-associated virus as a gene therapy vector: vector development, production and clinical applications. Adv Biochem Eng Biotechnol.2005,99:119-45. Review.
25. Whiteway A, Deru W, Prentice HG, Anderson R. Construction of adeno-associated virus packaging plasmids and cells that directly select for AAV helper functions.J Virol Methods.2003, Dec;114(1):1-10.
1. Johnstone B, Hering TM, Caplan AI, Goldberg VM, Yoo JU. In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells. Exp Cell Res 1998; 238:265-272.
2. Johnstone B HT, Caplan AI, et al. In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells. Exp Cell Res.1998; 238: 265-272.
3. Park JS, Yang HN, Woo DG, Chung HM, Park KH. In vitro and in vivo chondrogenesis of rabbit bone marrow-derived stromal cells in fibrin matrix mixed with growth factor loaded in nanoparticles. Tissue Eng Part A 2009; 15: 2163-2175.
4. Shirasawa S, Sekiya I, Sakaguchi Y, Yagishita K, Ichinose S, Muneta T. In vitro chondrogenesis of human synovium-derived mesenchymal stem cells: optimal condition and comparison with bone marrow-derived cells. J Cell Biochem 2006; 97:84-97.
5. De Bari C, Dell'Accio F, Tylzanowski P, Luyten FP. Multipotent mesenchymal stem cells from adult human synovial membrane. Arthritis Rheum 2001; 44:1928-1942.
6. Paradossi G, Chiessi E, Venanzi M, Pispisa B, Palleschi A. Branched-chain analogues of linear polysaccharides:a spectroscopic and conformational investigation of chitosan derivatives. Int J Biol Macromol 1992; 14:73-80.
7. Bentley G, Kreutner A, Ferguson AB. Synovial regeneration and articular cartilage changes after synovectomy in normal and steroid-treated rabbits. J Bone Joint Surg Br 1975; 57:454-462.
8. Fowler MR, Nathan CO, Abreo F. Synovial metaplasia, a specialized form of repair. Arch Pathol Lab Med 2002; 126:727-730.
9. Yoshimura H, Muneta T, Nimura A, Yokoyama A, Koga H, Sekiya I. Comparison of rat mesenchymal stem cells derived from bone marrow, synovium, periosteum, adipose tissue, and muscle. Cell Tissue Res 2007; 327: 449-462.
10. Miyazaki M, Zuk PA, Zou J, Yoon SH, Wei F, Morishita Y, et al. Comparison of human mesenchymal stem cells derived from adipose tissue and bone marrow for ex vivo gene therapy in rat spinal fusion model. Spine (Phila Pa 1976) 2008; 33:863-869.
11. Schnabel M, Marlovits S, Eckhoff G, Fichtel I, Gotzen L, Vecsei V, et al. Dedifferentiation-associated changes in morphology and gene expression in primary human articular chondrocytes in cell culture. Osteoarthritis Cartilage 2002; 10:62-70.
12. van Osch GJ, van der Veen SW, Verwoerd-Verhoef HL. In vitro redifferentiation of culture-expanded rabbit and human auricular chondrocytes for cartilage reconstruction. Plast Reconstr Surg 2001; 107:433-440.
13. Jakob M, Demarteau O, Schafer D, Hintermann B, Dick W, Heberer M, et al. Specific growth factors during the expansion and redifferentiation of adult human articular chondrocytes enhance chondrogenesis and cartilaginous tissue formation in vitro. J Cell Biochem 2001; 81:368-377.
1. Hoben GM, Willard VP, Athanasiou KA. Fibrochondrogenesis of hESCs:growth factor combinations and cocultures. Stem Cells Dev.2009,18(2):283-92.
2. Wu MJ, Gu ZY, Sun W. Effects of hydrostatic pressure on cytoskeleton and BMP-2, TGF-beta, SOX-9 production in rat temporomandibular synovial fibroblasts. Osteoarthritis Cartilage.2008,16(1):41-7.
3. Chen S, Emery SE, Pei M. Coculture of synovium-derived stem cells and nucleus pulposus cells in serum-free defined medium with supplementation of transforming growth factor-betal:a potential application of tissue-specific stem cells in disc regeneration. Spine (Phila Pa 1976).2009,34(12):1272-80.
4. Wang L, Lazebnik M, Detamore MS. Hyaline cartilage cells outperform mandibular condylar cartilage cells in a TMJ fibrocartilage tissue engineering application. Osteoarthritis Cartilage.2009,17(3):346-53.
5. Frenkel SR, Saadeh PB, Mehrara BJ, Chin GS, Steinbrech DS, Brent B, Gittes GK, Longaker MT. Transforming growth factor beta superfamily members:role in cartilage modeling. Plast Reconstr Surg.2000,105(3):980-90.
6. Stewart K, Pabbruwe M, Dickinson S, Sims T, Hollander AP, Chaudhuri JB. The effect of growth factor treatment on meniscal chondrocyte proliferation and differentiation on polyglycolic acid scaffolds. Tissue Eng.2007,13 (2):271-80.
7. Steinert AF, Palmer GD, Capito R, Hofstaetter JG, Pilapil C, Ghivizzani SC, Spector M, Evans CH. Genetically enhanced engineering of meniscus tissue using ex vivo delivery of transforming growth factor-beta 1 complementary deoxyribonucleic acid. Tissue Eng.2007,13(9):2227-37.
8. Dai J, Rabie AB. Direct AAV-mediated gene delivery to the temporomandibular joint. Front Biosci.2007,12:2212-20.
9. Hao J, Varshney RR, Wang DA. Engineering osteogenesis and chondrogenesis with gene-enhanced therapeutic cells. Curr Opin Mol Ther,2009,11(4):404-10.
10. Ueblacker P, Wagner B, Vogt S, Salzmann G, Wexel G, Kruger A, Plank C, Brill T, Specht K, Hennig T, Schillinger U, Imhoff AB, Martinek V, Gansbacher B. In vivo analysis of retroviral gene transfer to chondrocytes within collagen caffolds for the treatment of osteochondral defects. Biomaterials.2007,28(30):4480-7.
11. Sakaguchi Y,Sekiya I,Yagishita K,et al.Comparison of human stem cells derived from various mesenchymal tissues:superiority of synovium as a cell source.Arthritis Rheum,2005,52(8):2521-2529.
12. Blunk T,Sieminski AL,et al.Differential effects of growth factors on tissue engineered cartilage.Tissue Eng 2002 Feb;B(1):73-84
13. Murakami S, Kan M, McKeehan WL, et al.Sox9 gene by fibroblast growth factors is Up-regulation of the chondrogenic mediated by the mitogen-activated protein kinase pathway. Proc Natl Acad Sci USA,2000,97(3):1113—1118.14 Attisano L,Wrana JL.Signal transduction by the TGF-beta superfamily. Science. 2002;296(5573):1646-1647.
14. Kawamura K, Chu CR,Sobajima S,et al.Adenoviral-mediated transfer of TGF-betal but not IGF-1 induces chondrogenic differentiation of human mesenchymal stem cells in pellet cultures. Exp Hematol.2005;33(8):865-872.
15. Worster AA,Nixon AJ,Brower-Toland BD,et al.Effect of transforming growth factor betal on chondrogenic differentiation of cultured equine mesenchymal stem cells.Am J Vet Res.2000;61(9):1003-1010.
16. Morales Tl,Joyee ME,et al.Transforming growth factor-beta in calf articular cartilage organ cultures:synthesis and distribution.Arch Biochem Biophys.1991, 288 (2):397-405.
17. Kawamura K,Chu CR,Sobajima S,Robbins PD,Fu FH,Izzo NJ,Niyibizi C. Adenoviral-mediated transfer of TGF-betal but not IGF-I induces chondrogenic differentiation of human mesenchymal stem cells in pellet cultures.Exp Hematol. 2005;33(8):865-72.
18. Kalson NS, Richardson S, Hoyland JA. Strategies for regeneration of the intervertebral disc. Regen Med.2008 Sep;3(5):717-29. Review.
19. Spalazzi JP, Vyner MC, Jacobs MT, Moffat KL, Lu HH. Mechanoactive scaffold induces tendon remodeling and expression of fibrocartilage markers. Clin Orthop Relat Res.2008,466(8):1938-48.
20.郭晓东,杜靖远,郑启新,等.TGF-β1基因修饰的间充质干细胞／仿生基质材料复合移植修复兔关节软骨缺损.中国生物医学工程学报.2002.21：111-117.
21. Lai CM, Lai YK, Rakoczy PE. Adenovirus and adeno-associated virus vectors. DNA Cell Biol,2002,21(12):895-913.
22. Van Vliet KM, Blouin V, Brument N, et al. The role of the adeno-associated virus capsid in gene transfer. Methods Mol Biol,2008,437:51-91.
23. Dai J, Rabie AB. Direct AAV-mediated gene delivery to the temporomandibular joint. Front Biosci.2007 Jan 1; 12:2212-20.
24. Detamore MS, Athanasiou KA. Motivation, characterization, and strategy for tissue engineering thetemporomandibular joint disc[J]. Tissue Eng,2003,9(6): 1065-1087.
25. Shi Y,Massague J.Mechanisms of TGF-beta signaling from cell membrane to the nucleus.Cell,2003,113(6):685-700.
26.Jorgensen C,Noel D,Gross G.Could inflammatory arthritis be trigered by progenitor cells in the joint?Ann Rheum Dis[J],2002,61:629
27. Shi Y,Massague J.Mechanisms of TGF-beta signaling from cell membrane to the nucleus.Cell,2003,113(6):685-700.
28. Wan M,Cao X.BMP signaling in skeletal development.Biochem Biophys Res Commun,2005,328(3):651-657.
29. Bi W,Deng J M,Zhang Z,et al..Nat Gene.[J]1999;22(1):85-89.
30. Bi W,Huang W,Whitworth D J,et al.Proc Natl Acadsci[J].2001;98(12):6698-6708
31. Martin DR,Cox NR,Hachcock TL,et al.Isolation and characterization of multipotential mesenchymal stem cells from feline bone marrow[J].Exp Hematol,2002,30(8):879-886
1. Allen KD, Athanasiou KA. Scaffold and growth factor selection in temporomandibular joint disc engineering. J Dent Res.2008, Feb;87(2):180-5.
2. Schek RM, Taboas JM, Hollister SJ, Krebsbach PH. Tissue engineering osteochondral implants for temporomandibular joint repair. Orthod Craniofac Res. 2005, Nov;8(4):313-9.
3. Lee JE, Kim SE, Kwon IC, Ahn HJ, Cho H, Lee SH, Kim HJ, Seong SC, Lee MC.Effects of a chitosan scaffold containing TGF-betal encapsulated chitosan microspheres on in vitro chondrocyte culture. Artif Organs.2004, Sep;28(9): 829-39.
4. Lumpkins SB, Pierre N, McFetridge PS. A mechanical evaluation of three decellularization methods in the design of a xenogeneic scaffold for tissue engineering the temporomandibular joint disc. Acta Biomater.2008, Jul;4(4):808-16.
5. Ando K, Imai S, Isoya E, Kubo M, Mimura T, Shioji S, Ueyama H, Matsusue Y. Effect of dynamic compressive loading and its combination with a growth factor on the chondrocytic phenotype of 3-dimensional scaffold-embedded chondrocytes. Acta Orthop.2009, Dec;80(6):724-33.
6. Gong Y, He L, Li J, Zhou Q, Ma Z, Gao C, Shen J. Hydrogel-filled polylactide porous scaffolds for cartilage tissue engineering. J Biomed Mater Res B Appl Biomater.2007, Jul;82(1):192-204.
7. Tortelli F, Cancedda R. Three-dimensional cultures of osteogenic and chondrogenic cells:a tissue engineering approach to mimic bone and cartilage in vitro. Eur Cell Mater.2009, Jun 30;17:1-14.
8. Park SH, Park SR, Chung SI, Pai KS, Min BH. Tissue-engineered cartilage using fibrin/hyaluronan composite gel and its in vivo implantation. Artif Organs.2005, 29(10):838-45.
9. Fan H, Hu Y, Li X, Wu H, Lv R, Bai J, Wang J, Qin L. Ectopic cartilage formation induced by mesenchymal stem cells on porous gelatin-chondroitin-hyaluronate scaffold containing microspheres loaded with TGF-betal. Int J Artif Organs.2006, Jun;29(6):602-11.
10. Lee JE, Kim SE, Kwon IC, Ahn HJ, Cho H, Lee SH, Kim HJ, Seong SC, Lee MC. Effects of a chitosan scaffold containing TGF-betal encapsulated chitosan microspheres on in vitro chondrocyte culture. Artif Organs.2004, Sep;28(9): 829-39.
11.Lee JE, Kim KE, Kwon IC, Ahn HJ, Lee SH, Cho H, Kim HJ, Seong SC, Lee MC. Effects of the controlled-released TGF-beta 1 from chitosan microspheres on chondrocytes cultured in a collagen/chitosan/glycosaminoglycan scaffold. Biomaterials.2004, Aug;25(18):4163-73.
12.Lee KY, Ha WS, Park WH. Blood compatibility and biodegradability of partially N-acylated chitosan derivatives. Biomaterials 1995,16:1211-1216.
13.Ragetly GR, Slavik GJ, Cunningham BT, Schaeffer DJ, Griffon DJ. Cartilage tissue engineering on fibrous chitosan scaffolds produced by a replica molding technique. J Biomed Mater Res A.2003,93:46-55.
14.Alves da Silva ML, Crawford A, Mundy JM, Correlo VM, Sol P, Bhattacharya M, et al. Chitosan/polyester-based scaffolds for cartilage tissue engineering: assessment of extracellular matrix formation. Acta Biomater.2006,6:1149-1157.
15.Yang C, Hillas PJ, Baez JA, Nokelainen M, Balan J, Tang J, et al. The application of recombinant human collagen in tissue engineering. BioDrugs.2004,18: 103-119.
16.Kobayashi M, Spector M. In vitro response of the bone marrow-derived mesenchymal stem cells seeded in a type-I collagen-glycosaminoglycan scaffold for skin wound repair under the mechanical loading condition. Mol Cell Biomech. 2009, Dec;6(4):217-27.
17.Fini M, Torricelli P, Giavaresi G, Rotini R, Castagna A, Giardino R. In vitro study comparing two collageneous membranes in view of their clinical application for rotator cuff tendon regeneration. J Orthop Res.2007, Jan;25(1):98-107.
18. Concaro S, Gustavson F, Gatenholm P. Bioreactors for tissue engineering of cartilage. Adv Biochem Eng Biotechnol.2009,112:125-43.
19. Chajra H, Rousseau CF, Cortial D, Ronziere MC, Herbage D, Mallein-Gerin F, Freyria AM. Collagen-based biomaterials and cartilage engineering. Application to osteochondral defects. Biomed Mater Eng.2008,18(1 Suppl):S33-45.
20. Wendt D, Jakob M, Martin I. Bioreactor-based engineering of osteochondral grafts:from model systems to tissue manufacturing. J Biosci Bioeng.2005, Nov;100(5):489-94.
21. Freed LE, Guilak F, Guo XE, Gray ML, Tranquillo R, Holmes JW, Radisic M, Sefton MV, Kaplan D, Vunjak-Novakovic G. Advanced tools for tissue engineering:scaffolds, bioreactors, and signaling. Tissue Eng.2006, Dec;12(12):3285-305.
22. Lin Z, Willers C, Xu J, Zheng MH. The chondrocyte:biology and clinical application. Tissue Eng.2006, Jul;12(7):1971-84.
23. Weng Y, Cao Y, Silva CA, Vacanti MP, Vacanti CA. Tissue-engineered composites of bone and cartilage for mandible condylar reconstruction. J Oral Maxillofac Surg 2001,59:185-190.
24. Galois L,Hutasse S,Cortial D,et al.chondrocyte behaviour in three-dimensional type Ⅰ collagen gel in terms of gel contraction,proliferation and gene expression. Biomaterials.2006,27(1):79-90.
1. Dijkgraaf LC, de Bont LG, Boering G, Liem RS. Structure of the normal synovial membrane of the temporomandibular joint:a review of the literature. J Oral Maxillofac Surg.1996,54(3):332-8.
2. Berkovitz BK, Pacy J. Ultrastructure of the human intra-articular disc of the temporomandibular joint. Eur J Orthod.2002, Apr;24(2):151-8.
3. Berkovitz BK. Collagen crimping in the intra-articular disc and articular surfaces of the human temporomandibular joint. Arch Oral Biol.2000, Sep;45(9):749-56.
4. Detamore MS,Orfanos JG,Almarza AJ,et al.Matrix Biol,2005,24(1):45-57.
5. Zakrzewska JM. Diagnosis and management of non-dental orofacial pain. Dent Update.2007, Apr;34(3):134-6,138-9.
6. Suvinen TI, Reade PC, Kemppainen P, Kononen M, Dworkin SF. Review of aetiological concepts of temporomandibular pain disorders:towards a biopsychosocial model for integration of physical disorder factors with psychological and psychosocial illness impact factors. Eur J Pain.2005, Dec;9(6): 613-33.
7. Fikackova H, Dostalova T, Navratil L, Klaschka J. Effectiveness of low-level laser therapy in temporomandibular joint disorders:a placebo-controlled study.Photomed Laser Surg.2007, Aug;25(4):297-303.
8. Bergstrom I, List T, Magnusson T. A follow-up study of subjective symptoms of temporomandibular disorders in patients who received acupuncture and/or interocclusal appliance therapy 18-20 years earlier. Acta Odontol Scand.2008, Apr;66(2):88-92.
9. Al-Riyami S, Cunningham SJ, Moles DR. Orthognathic treatment and temporomandibular disorders:a systematic review. Am J Orthod Dentofacial Orthop.2009, Nov;136(5):626.e1-16
10. Kashi A, Saha S, Christensen RW. Temporomandibular joint disorders:artificial joint replacements and future research needs. J Long Term Eff Med Implants. 2006,16(6):459-74. Review.
11. Abrahamsson C, Ekberg E, Henrikson T, Bondemark L. Alterations of temporomandibular disorders before and after orthognathic surgery:a systematic review. Angle Orthod.2007, Jul;77(4):729-34. Review.
12. Carter EM, Raggio CL, Genetic and orthopedic aspects of collagen disorders. Curr Opin Pediatr.2009, Feb;21(1):46-54. Review.
13. Nishida K, Suzuki T, Kakutani K, Yurube T, Maeno K, Kurosaka M, Doita M. Genetherapy approach for disc degeneration and associated spinal disorders. Eur Spine J.2008, Dec; 17 Suppl 4:459-66.
14.Ueineken FG and Skalak R.Tissue Engineering: Brief Overview,Journal of Biomechanical Engineering 1991,113:111
15. Tew SR, Murdoch AD, Rauchenberg RP, Hardingham TE. Cellular methods in cartilage research:primary human chondrocytes in culture and chondrogenesis in human bone marrow stem cells. Methods.2008, May;45(1):2-9.
16.Chen FH, Rousche KT, Tuan RS. Technology Insight:adult stem cells in cartilage regeneration and tissue engineering. Nat Clin Pract Rheumatol.2006, Jul;2(7):373-82.
17.Gupta T, Haut Donahue TL. Role of cell location and morphology in the mechanical environment around meniscal cells. Acta Biomater.2006, Sep;2(5): 483-92. Epub 2006 Jul 24.
18.Wim B,Peter M,He8nk M, et al.Growth factors and cartilage repair[J].Clin Orthop Res.2001,391 suppl):244-250.
19. Hanaoka K, Tanaka E, Takata T, Miyauchi M, Aoyama J, Kawai N, Dalla-Bona DA, Yamano E, Tanne K. Platelet-derived growth factor enhances proliferation and matrix synthesis of temporomandibular joint disc-derived cells. Angle Orthod. 2006, May;76(3):486-92.
20. Almarza AJ, Athanasiou KA. Evaluation of three growth factors in combinations of two for temporomandibular joint disc tissue engineering. Arch Oral Biol.2006, Mar;51(3):215-21.
21. Tsutsui TW, Riminucci M, Holmbeck K, Bianco P, Robey PG. Development of craniofacial structures in transgenic mice with constitutively active PTH/PTHrP
receptor. Bone.2008, Feb;42(2):321-31.
22. Shibukawa Y, Young B, Wu C, Yamada S, Long F, Pacifici M, Koyama E. Temporomandibular joint formation and condyle growth require Indian hedgehog signaling. Dev Dyn.2007, Feb;236(2):426-34.
23. Tateishi K,Ando W,Higuchi C,et al.Comparison of human serum with fetal bovine serum for expansion and differentiation of human synovial MSC:potential feasibility for clinical applications.Cell Transplant,2008,17(5):549-557.
24. Yang KG, Saris DB, Geuze RE, Helm YJ, Rijen MH, Verbout AJ, Dhert WJ, Creemers LB. Impact of expansion and redifferentiation conditions on chondrogenic capacity of cultured chondrocytes. Tissue Eng.2006 Sep;12(9): 2435-47.
25. Mercuri LG. Temporomandibular joint reconstruction. Alpha Omegan.2009 Jun;102(2):51-4.15Chladek W, Czerwik I. Mechanical properties of temporomandibular joint disc on the basis of porcine preparation investigations. Acta Bioeng Biomech.2008;10(4):15-20.
26. Naujoks C, Meyer U, Wiesmann HP, Jasche-Meyer J, Hohoff A, Depprich R,Handschel J. Principles of cartilage tissue engineering in TMJ reconstruction. Head Face Med.2008 Feb 25;4:3. Review
27. Takigawa M,Okada M,Takano T,et al.J Dent Res 1984,63 (1):19-22.
28. Tsubai T,Higashi Y,Scott JE.Arch Oral Biol,200045 (6):507-515.
29. Benjamin M,Tyers RN,Ralphs JR.Age-related changes in tendon fibrocartilage. J Anat,1991,179:127-136.
30. Roughley PJ,McNicol D,Santer V,et al.The presence of a cartilage-like roteoglycan in the adult human meniscus.Biochem J,1981,197:77-83.
31. Vanderploeg EJ,Imler SM,Brodkin KR,et al.Oscillatory tension differentially modulates matrix metabolism and cytoskeletal organization in chondrocytes and fibrochondrocytes.J Biomech,2004,37:1941-1952.
32. Darling EM,Athanasiou KA.Rapid phenotypic changes in passaged articular chondrocyte subpopulations[J].J Orthop Res,2005,23:425-432.
33. Weinand C,Peretti GM,Adams SB,et al.An allogenic cell-based implant for meniscal lesions[J].J Sports Med,2006,34:1779-1789.
34. Dozin B,MalpeliM, Camardella L, et al. Response ofyoung, aged and osteoarthritic human articular chondrocytes to inflammatory cytokines:molecular and cellular as-pects[J].Matrix Bio,l 2002,21(5):449-459.
35. Alhadlaq A, Mao JJ. Tissue-engineered neogenesis of human-shaped mandibular condyle from rat mesenchymal stem cells. J Dent Res.2003 Dec;82(12):951-6.
36. Hoben GM,Koay EJ,Athanasiou KA.Fibrochondrogenesis in two embryonic stem cell lines:effects of differentiation timelines[J].Stem Cells,2008,26:422-430.
37.徐青镭,吴海山,周维江,等.兔骨髓干细胞向软骨细胞分化用于半月板组织工程重建[J].中国临床康复,2003,7(6)：912-913.
38. Feinberg SE, Hollister SJ, Halloran JW, Chu TM, Krebsbach PH. Image-based biomimetic approach to reconstruction of the temporomandibular joint. Cells Tissues Organs.2001;169(3):309-21.
39. De Bari C,Dell'Accio F,Tylzanowski P,et al.Multipotent mesenchymal stem cells from adult human synovial membrane[J].Arthritis Rheum,2001,44(8):1928-1942.
40. Djouad F,Bony C,Haupl T,et al.Transcriptional profiles discriminate bone marrow derived and synovium-derived mesenchymal stem cells.Arthritis Res Ther,2005,7 (6):1304-1315.
41. Jo CH, Ahn HJ, Kim HJ, et al. Surface characterization and chondrogenic differentiation of mesenchymal stromal cells derived from synovium. Cytotherapy, 2007,9(4):316-327.
42. Sakaguchi Y,Sekiya I,Yagishita K,et al.Comparison of human stem cells derived from various mesenchymal tissues:superiority of synovium as a cell source. Arthritis Rheum,2005,52(8):2521-2529.
43. Shintani,Kurth T,Hunziker EB.Expression of cartilage-related genes in bovine synovial tissue.J Orthop Res,2007,25(6):813-819.
44. Yoshimura H,Muneta T,Nimura A,et al.Comparison of rat mesenchymal stem cells derived from bone marrow,synovium,periosteum, adipose tissue,and muscle.Cell Tissue Res,2007,327(3):449-462.
45.李健龙星柯金等。颞下颌关节滑膜间充质干细胞的分离与培养。中华口腔医学杂志,2005,40(5)：362-364.
46. Yoo JU,Barthel TS,Nishimura K,et al.The chondrogenic potential of human bone-marrow-derived mesenchymal progenitor cells J Bone Joint Surg Am,1998, 80:1745-1757.
47. Worster AA,Brower-Toland BD,Fortier LA,et al.Chondrocytic differentiation of mesenchymal stem cells sequentially exposed to transforming growth factor-betal in monolayer and insulin-like growth factor-I in a three-dimensional matrix.J Orthop Res,2001,19:738-749.
48. Majumdar MK,Banks V,Peluso DP,et al.Isolation,characterization,and chondrogenic potential of human bone marrow-derived multipotential stromal cells.J Cell Physiol,2000,185:98-106.
49. Wang L,Detamore MS.Effects of growth factors and glucosamine on porcine mandibular condylar cartilage cells and hyaline cartilage cells for tissue engineering applications [J].Arch Oral Biol,2008,54(1):1-5
50. Gotz W,Lehmann TS,Appel TR.Distribution of insulin-like growth factors in condylar hyperplasia[J].Ann Anat,2007,189(4):347-349
51. Visnapuu V,Peltomki T,Rnning O.Growth hormone and insulin-like growth factor I receptors in the temporomandibular joint of the rat.J Dent Res,2001,80(10): 1903-1907
52.王健,陈新明,汪说之.TGF-β在不同发育阶段胎儿髁突软骨中的表达.口腔医学纵横,2001,17(4)：277
53.Madry H,Emkey G,Zurakowski D,et al. Over expression of human fibroblast growth factor 2 stimulates cell proliferation in an ex vivo model of articular chondrocyte transplantation. J Gene Med,2004,6(2):238-245
54.Tanaka E,Aoyama J,Miyauchi M.Vascular endothelial growth factor plays an important autocrine/paracrine role in the progression of osteoarthritis[J].Histochem Cell Biol,2005,123(3):275-281
55. Staudenmaier R, Froelich K, Birner M, Kindermann J, The Hoang N, Pueschel RC, Mandlik V. Optimization of platelet isolation and extraction of autogenous TGF-beta in cartilage tissue engineering. Artif Cells Blood Substit Immobil Biotechnol.2009;37 (6):265-72.
56. Emin N, Koc A, Durkut S, Elgin AE, Elgin YM. Engineering of rat articular cartilage on porous sponges:effects of tgf-beta 1 and microgravity bioreactor culture. Artif Cells Blood Substit Immobil Biotechnol.2008;36(2):123-37.
57. Wrotniak M, Bielecki T, Gazdzik TS. Current opinion about using the platelet-rich gel in orthopaedics and trauma surgery. Ortop Traumatol Rehabil. 2007 May-Jun;9(3):227-38. Review.
58. Miura Y, Parvizi J, Fitzsimmons JS, O'Driscoll SW. Brief exposure to high-dose transforming growth factor-β1 enhances periosteal chondrogenesis in vitro:a preliminary report. J Bone Joint Surg Am,2002,84-A(5):793-799.
59. Park H,Temenoff JS,Tabata Y,et al.Injectable biodegradable hydrogel composites for rabbit marrow mesenchymal stem cell and growth factor delivery for cartilage tissue engineering. Biomaterials.2007,28(21):3217-3227.
60.Guo X, Zheng Q, Yang S, Shao Z, Yuan Q, Pan Z, Tang S, Liu K, Quan D. Repair of full-thickness articular cartilage defects by cultured mesenchymal stem cells transfected with the transforming growth factor betal gene. Biomed Mater.2006 Dec;1(4):206-15.
61. Yasushi Miura,Javad Jarvizi,er al.Brief exposure to high-dose transfouming growth factor-β1 enhances periosteal chondrogenesis in vitro.The Journal of Bone&Joint Surgery,2002,5:793-799
62. Stevens MM, Marini RP, Martin I, Langer R, Prasad Shastri V. FGF-2 enhances TGF-betal-induced periosteal chondrogenesis. J Orthop Res.2004 Sep;22(5): 1114-9.
63. Ma Z, Gao C, Gong Y, Shen J. Cartilage tissue engineering PLLA scaffold with surface immobilized collagen and basic fibroblast growth factor. Biomaterials. 2005 Apr;26(11):1253-9.
64. Johns DE, Athanasiou KA. Growth factor effects on costal chondrocytes for tissue engineering fibrocartilage. Cell Tissue Res.2008 Sep;333(3):439-47. Epub 2008 Jul 3. PubMed PMID:18597118.
65. Arevalo-Silva CA,Cao YL,Weng YL,et al.The effect of fibroblast growth factor and transforming growth factor-Bon porcine chondrocytes and tissue-engineered autologous elastic cartilage.Tissue Eng 2001,7(1):81-8
66. Martin I,Vunjak-Novakovic G,Yang J,et al.Mammalian chondrocytes expanded in the presence of fibroblast growth factor 2 mainatin the ability to diffrenetiate and regenerate three-dimensional cartilaginous tissue.Exp Cell Res 1999,253(2): 681-8
67.焦岩涛,王大章,韩文利,等.bFGF、IGF-I及TGF-β对人髁突软骨细胞增殖的影响.中华口腔医学杂志2000,35(5)：346-349.
68. Takafuji H, Suzuki T, Okubo Y.Regeneration of articular cartilage defect in the temporomandibular joint of rabbits by fibroblast growth factor-2:a pilot study.Int J Oral Maxillofac Surg 2007,36(10):934-937.
69. Wang L, Detamore MS.Effects of growth factors and glucosamine on porcine mandibular condylar cartilage cells and hyaline cartilage cells for tissue engineering applications.Arch Oral Biol,2009,54(1):1-5.
70. Delatte M, Von den Hoff JW, Maltha JC, et al.Growth stimulation of mandibular condyles and femoral heads of newborn rats by IGF-I.Arch Oral Biol,2004, 49(3):165-175.
71.赖文莉,赵美英,罗颂椒.功能矫形大鼠下颌前伸后髁突胰岛素分布规律的研究.中华口腔医学杂志,1996,31(2)：91-93.
72. Kramer J,Hegert C,Guan K,et al.Embryonic stem cell-derived chondrogenic differentiation in vitro:activition by BMP-2 and BMP-4.Mech Dev.2000,2(2): 193-205
73. Blunk T,Sieminski AL,Appel B,et al.Bone morphogenetic protein 9:a potent modulator of cartilage development in vitro.Growth Factors.2003,21(2):71-7
74. Loeser RF,Pacione CA,Chubinskaya S.The combination of insulin-like growth factor 1 and osteogenic protein 1 promotes increased survival of and matrix synthesis by normal and osteoarthritic human articular chondrocytes.Arthritis Rheum.2003,48(8):2188-96
75. Ferrara N,Gerber HP,LeCouter J.The biology of VEGF and its receptors. Nat Med,2003,9(6):669-676.
76. Simpson AH,Mills L,Noble B.The role of growth factors and related agents in accelerating fracture healing J Bone Joint SurgBr,2006,88(6):701-705.
77.Sato J,Segami N,Kaneyama K,et al.Vascular endothelial growth factor concentrations in synovial fluids of patients with symptomatic internal derangement of the temporomandibular joint. J Oral Pathol Med.2005,34(3): 170-177.
78. Leonardi R, Lo Muzio L, Bernasconi G, Caltabiano C, Piacentini C, Caltabiano M. Expression of vascular endothelial growth factor in human dysfunctional temporomandibular joint discs. Arch Oral Biol.2003, Mar;48(3):185-92.
79. Yee G, Yu Y, Walsh WR, et al.The immunolocalisation of VEGF in the articular cartilage of sheep mandibular condyles.J Craniomaxillofac Surg.2003,31(4): 244-251.
80. Li Y, Tew SR, Russell AM, Gonzalez KR, Hardingham TE, Hawkins RE. Transduction of passaged human articular chondrocytes with adenoviral, retroviral, and lentiviral vectors and the effects of enhanced expression of SOX9. Tissue Eng.2004, Mar-Apr;10(3-4):575-84.
81. Partridge KA, Oreffo RO. Gene delivery in bone tissue engineering:progress and prospects using viral and nonviral strategies. Tissue Eng.2004, Jan-Feb; 10(1-2):295-307.
82. Ueblacker P, Wagner B, Vogt S, Salzmann G, Wexel G, Kruger A, Plank C, Brill T, Specht K, Hennig T, Schillinger U, Imhoff AB, Martinek V, Gansbacher B. In vivo analysis of retroviral gene transfer to chondrocytes within collagen scaffolds for the treatment of osteochondral defects. Biomaterials.2007, Oct;28(30):4480-7.
83. Tew SR, Li Y, Pothacharoen P, Tweats LM, Hawkins RE, Hardingham TE. Retroviral transduction with SOX9 enhances re-expression of the chondrocyte phenotype in passaged osteoarthritic human articular chondrocytes. Osteoarthritis Cartilage.2005, Jan;13(1):80-9.
84. Feng G, Wan Y, Balian G, Laurencin CT, Li X. Adenovirus-mediated expression of growth and differentiation factor-5 promotes chondrogenesis of adipose stem cells. Growth Factors.2008, Jun;26(3):132-42.
85. Schek RM, Taboas JM, Hollister SJ, Krebsbach PH. Tissue engineering osteochondral implants for temporomandibular joint repair. Orthod Craniofac Res. 2005, Nov;8(4):313-9.
86. Jin X, Sun Y, Zhang K, Wang J, Shi T, Ju X, Lou S. Ectopic neocartilage formation from predifferentiated human adipose derived stem cells induced by adenoviral-mediated transfer of hTGF beta2. Biomaterials.2007, Jul;28(19): 2994-3003.
87. Dai J, Rabie AB. The use of recombinant adeno-associated virus for skeletal gene therapy. Orthod Craniofac Res.2007, Feb;10(1):1-14. Review.
88. Izal I, Acosta CA, Ripalda P, Zaratiegui M, Ruiz J, Forriol F. IGF-1 gene therapy to protect articular cartilage in a rat model of joint damage. Arch Orthop Trauma Surg.2008, Feb;128(2):239-47.
89. Pagnotto MR, Wang Z, Karpie JC, Ferretti M, Xiao X, Chu CR. Adeno-associated viral gene transfer of transforming growth factor-betal to human mesenchymal stem cells improves cartilage repair. Gene Ther.2007, May;14(10):804-13.
90. Li QF, Rabie AB. A new approach to control condylar growth by regulating angiogenesis. Arch Oral Biol.2007, Nov;52(11):1009-17. Epub 2007 Jul 20.
91. Chen T, Hudnall SD. Anatomical mapping of human herpesvirus reservoirs of infection. Mod Pathol.2006, May;19(5):726-37.
92. Stove J, Fiedler J, Huch K, Gunther KP, Puhl W, Brenner R. Lipofection of rabbit chondrocytes and long lasting expression of a lacZ reporter system in alginate beads. Osteoarthritis Cartilage.2002, Mar;10(3):212-7.
2. Miyamoto C, Matsumoto T, Sakimura K, Shindo H. Osteogenic protein-1 with transforming growth factor-betal:potent inducer of chondrogenesis of synovial mesenchymal stem cells in vitro. J Orthop Sci.2007, Nov;12(6):555-61.
3. Park Y, Sugimoto M, Watrin A, Chiquet M, Hunziker EB. BMP-2 induces the expression of chondrocyte-specific genes in bovine synovium-derived progenitor cells cultured in three-dimensional alginate hydrogel. Osteoarthritis Cartilage. 2005, Jun;13(6):527-36.
4. De Bari C,Dell'Accio F,Luyten FP.Human periosteum-derived cells maintain phenotypic stability and chondrogenic potential throughout expansion regardless of donor age.Arthritis Rheum,2001,44:85-95.
5. Yoo JU,Barthel TS,Nishimura K,et al.The chondrogenic potential of human bone-marrow-derived mesenchymal progenitor cells.J Bone Joint Surg Am,1998, 80:1745-1757.
6. Nishida K, Suzuki T, Kakutani K, Yurube T, Maeno K, Kurosaka M, Doita M. Gene therapy approach for disc degeneration and associated spinal disorders. Eur Spine J.2008, Dec;17 Suppl 4:459-66.
7. Liu X, Li K, Song J, Liang C, Wang X, Chen X. Efficient and stable gene expression in rabbit intervertebral disc cells transduced with a recombinant baculovirus vector. Spine (Phila Pa 1976).2006, Apr 1;31(7):732-5.
8. Lattermann C, Oxner WM, Xiao X, Li J, Gilbertson LG, Robbins PD, Kang JD. Theadeno associated viral vector as a strategy for intradiscal gene transfer in immune competent and pre-exposed rabbits. Spine (Phila Pa 1976).2005, Mar 1;30(5):497-504.
9. Hubert MG, Vadala G, Sowa G, Studer RK, Kang JD. Gene therapy for the
treatment of degenerative disk disease. J Am Acad Orthop Surg.2008, Jun; 16(6):312-9. Review. 10. Nishida K, Doita M, Takada T, Kakutani K, Miyamoto H, Shimomura T, Maeno K,Kurosaka M. Sustained transgene expression in intervertebral disc cells in vivo mediated by microbubble-enhanced ultrasound gene therapy. Spine (Phila Pa 1976).2006, Jun 1;31(13):1415-9.
11. Hildinger M, Baldi L, Stettler M, Wurm FM. High-titer, serum-free production of adeno-associated virus vectors bypolyethyleneimine-mediated plasmidtransfection in mammalian suspension cells. Biotechnol Lett.2007, Nov;29(11):1713-21.
12. Ulrich-Vinther M. Gene therapy methods in bone and joint disorders. Evaluation of the adeno-associated virus vector in experimental models of articular cartilage disorders, periprosthetic osteolysis and bone healing. Acta Orthop Suppl.2007, Apr;78(325):1-64. Review.
13. Timpe J, Bevington J, Casper J, et al. Mechanisms of adeno-associated virus genome encapsidation. Curr Gene Ther.2005,5(3):273-284.
14. Tratschin JD, Miller IL, Carter BJ. Genetic analysis of adeno-associated virus: properties of deletion mutants constructed in vitro and evidence for an adeno-associated virus replication function. J Virol.1984,51(3):611-619.
15. Yan Z, Zak R, Zhang Y, et al. Inverted terminal repeat sequences are important for intermolecular recombination and circularization of adeno-associated virus genomes. J Virol.2005,79(1):364-379.
16. Hauck B, Chen L, Xiao W. Generation and characterization of chimeric recombinant AAV vectors. Mol Ther.2003,7(3):419-425.
17. Gao G, Vandenberghe LH, Alvira MR, et al. Clades of Adeno-associated viruses are widely disseminated in human tissues. J Virol.2004,78(12):6381-6388.
18. Duan D, Sharma P, Yang J, et al. Circular intermediates of recombinant adeno-associated virus have defined structural characteristics responsible for long-term episomal persistence in muscle tissue. J Virol.1998,72(11):8568-8577.
19. Zhang C, Cortez NG, Berns KI. Characterization of a bipartite recombinant adeno-associated viral vector for site-specific integration. Hum Gene Ther.2007, 18(9):787-797.
20. Grieger JC, Samulski RJ. Adeno-associated virus as a gene therapy vector:vector development, production and clinical applications. Adv Biochem Eng Biotechnol. 2005,99:119-145.
21. Duffy AM, O'Doherty AM, O'Brien T, et al. Purification of adenovirus and adeno-associated virus:comparison of novel membrane-based technology to conventional techniques. Gene Ther.2005,12 Suppl 1:S62-72.
22. Wu J, Zhao W, Zhong L, Han Z, Li B, Ma W, Weigel-Kelley KA, Warrington KH,Srivastava A. Self-complementary recombinant adeno-associated viral vectors: packaging capacity and the role of rep proteins in vector purity. Hum Gene Ther. 2007, Feb;18(2):171-82.
23. Wang H, Lieber A. A helper-dependent capsid-modified adenovirus vector expressing adeno-associated virus rep78 mediates site-specific integration of a 27-kilobase transgene cassette. J Virol.2006, Dec;80(23):11699-709.
24. Grieger JC, Samulski RJ. Adeno-associated virus as a gene therapy vector: vector development, production and clinical applications. Adv Biochem Eng Biotechnol.2005,99:119-45. Review.
25. Whiteway A, Deru W, Prentice HG, Anderson R. Construction of adeno-associated virus packaging plasmids and cells that directly select for AAV helper functions.J Virol Methods.2003, Dec;114(1):1-10.
1. Johnstone B, Hering TM, Caplan AI, Goldberg VM, Yoo JU. In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells. Exp Cell Res 1998; 238:265-272.
2. Johnstone B HT, Caplan AI, et al. In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells. Exp Cell Res.1998; 238: 265-272.
3. Park JS, Yang HN, Woo DG, Chung HM, Park KH. In vitro and in vivo chondrogenesis of rabbit bone marrow-derived stromal cells in fibrin matrix mixed with growth factor loaded in nanoparticles. Tissue Eng Part A 2009; 15: 2163-2175.
4. Shirasawa S, Sekiya I, Sakaguchi Y, Yagishita K, Ichinose S, Muneta T. In vitro chondrogenesis of human synovium-derived mesenchymal stem cells: optimal condition and comparison with bone marrow-derived cells. J Cell Biochem 2006; 97:84-97.
5. De Bari C, Dell'Accio F, Tylzanowski P, Luyten FP. Multipotent mesenchymal stem cells from adult human synovial membrane. Arthritis Rheum 2001; 44:1928-1942.
6. Paradossi G, Chiessi E, Venanzi M, Pispisa B, Palleschi A. Branched-chain analogues of linear polysaccharides:a spectroscopic and conformational investigation of chitosan derivatives. Int J Biol Macromol 1992; 14:73-80.
7. Bentley G, Kreutner A, Ferguson AB. Synovial regeneration and articular cartilage changes after synovectomy in normal and steroid-treated rabbits. J Bone Joint Surg Br 1975; 57:454-462.
8. Fowler MR, Nathan CO, Abreo F. Synovial metaplasia, a specialized form of repair. Arch Pathol Lab Med 2002; 126:727-730.
9. Yoshimura H, Muneta T, Nimura A, Yokoyama A, Koga H, Sekiya I. Comparison of rat mesenchymal stem cells derived from bone marrow, synovium, periosteum, adipose tissue, and muscle. Cell Tissue Res 2007; 327: 449-462.
10. Miyazaki M, Zuk PA, Zou J, Yoon SH, Wei F, Morishita Y, et al. Comparison of human mesenchymal stem cells derived from adipose tissue and bone marrow for ex vivo gene therapy in rat spinal fusion model. Spine (Phila Pa 1976) 2008; 33:863-869.
11. Schnabel M, Marlovits S, Eckhoff G, Fichtel I, Gotzen L, Vecsei V, et al. Dedifferentiation-associated changes in morphology and gene expression in primary human articular chondrocytes in cell culture. Osteoarthritis Cartilage 2002; 10:62-70.
12. van Osch GJ, van der Veen SW, Verwoerd-Verhoef HL. In vitro redifferentiation of culture-expanded rabbit and human auricular chondrocytes for cartilage reconstruction. Plast Reconstr Surg 2001; 107:433-440.
13. Jakob M, Demarteau O, Schafer D, Hintermann B, Dick W, Heberer M, et al. Specific growth factors during the expansion and redifferentiation of adult human articular chondrocytes enhance chondrogenesis and cartilaginous tissue formation in vitro. J Cell Biochem 2001; 81:368-377.
1. Hoben GM, Willard VP, Athanasiou KA. Fibrochondrogenesis of hESCs:growth factor combinations and cocultures. Stem Cells Dev.2009,18(2):283-92.
2. Wu MJ, Gu ZY, Sun W. Effects of hydrostatic pressure on cytoskeleton and BMP-2, TGF-beta, SOX-9 production in rat temporomandibular synovial fibroblasts. Osteoarthritis Cartilage.2008,16(1):41-7.
3. Chen S, Emery SE, Pei M. Coculture of synovium-derived stem cells and nucleus pulposus cells in serum-free defined medium with supplementation of transforming growth factor-betal:a potential application of tissue-specific stem cells in disc regeneration. Spine (Phila Pa 1976).2009,34(12):1272-80.
4. Wang L, Lazebnik M, Detamore MS. Hyaline cartilage cells outperform mandibular condylar cartilage cells in a TMJ fibrocartilage tissue engineering application. Osteoarthritis Cartilage.2009,17(3):346-53.
5. Frenkel SR, Saadeh PB, Mehrara BJ, Chin GS, Steinbrech DS, Brent B, Gittes GK, Longaker MT. Transforming growth factor beta superfamily members:role in cartilage modeling. Plast Reconstr Surg.2000,105(3):980-90.
6. Stewart K, Pabbruwe M, Dickinson S, Sims T, Hollander AP, Chaudhuri JB. The effect of growth factor treatment on meniscal chondrocyte proliferation and differentiation on polyglycolic acid scaffolds. Tissue Eng.2007,13 (2):271-80.
7. Steinert AF, Palmer GD, Capito R, Hofstaetter JG, Pilapil C, Ghivizzani SC, Spector M, Evans CH. Genetically enhanced engineering of meniscus tissue using ex vivo delivery of transforming growth factor-beta 1 complementary deoxyribonucleic acid. Tissue Eng.2007,13(9):2227-37.
8. Dai J, Rabie AB. Direct AAV-mediated gene delivery to the temporomandibular joint. Front Biosci.2007,12:2212-20.
9. Hao J, Varshney RR, Wang DA. Engineering osteogenesis and chondrogenesis with gene-enhanced therapeutic cells. Curr Opin Mol Ther,2009,11(4):404-10.
10. Ueblacker P, Wagner B, Vogt S, Salzmann G, Wexel G, Kruger A, Plank C, Brill T, Specht K, Hennig T, Schillinger U, Imhoff AB, Martinek V, Gansbacher B. In vivo analysis of retroviral gene transfer to chondrocytes within collagen caffolds for the treatment of osteochondral defects. Biomaterials.2007,28(30):4480-7.
11. Sakaguchi Y,Sekiya I,Yagishita K,et al.Comparison of human stem cells derived from various mesenchymal tissues:superiority of synovium as a cell source.Arthritis Rheum,2005,52(8):2521-2529.
12. Blunk T,Sieminski AL,et al.Differential effects of growth factors on tissue engineered cartilage.Tissue Eng 2002 Feb;B(1):73-84
13. Murakami S, Kan M, McKeehan WL, et al.Sox9 gene by fibroblast growth factors is Up-regulation of the chondrogenic mediated by the mitogen-activated protein kinase pathway. Proc Natl Acad Sci USA,2000,97(3):1113—1118.14 Attisano L,Wrana JL.Signal transduction by the TGF-beta superfamily. Science. 2002;296(5573):1646-1647.
14. Kawamura K, Chu CR,Sobajima S,et al.Adenoviral-mediated transfer of TGF-betal but not IGF-1 induces chondrogenic differentiation of human mesenchymal stem cells in pellet cultures. Exp Hematol.2005;33(8):865-872.
15. Worster AA,Nixon AJ,Brower-Toland BD,et al.Effect of transforming growth factor betal on chondrogenic differentiation of cultured equine mesenchymal stem cells.Am J Vet Res.2000;61(9):1003-1010.
16. Morales Tl,Joyee ME,et al.Transforming growth factor-beta in calf articular cartilage organ cultures:synthesis and distribution.Arch Biochem Biophys.1991, 288 (2):397-405.
17. Kawamura K,Chu CR,Sobajima S,Robbins PD,Fu FH,Izzo NJ,Niyibizi C. Adenoviral-mediated transfer of TGF-betal but not IGF-I induces chondrogenic differentiation of human mesenchymal stem cells in pellet cultures.Exp Hematol. 2005;33(8):865-72.
18. Kalson NS, Richardson S, Hoyland JA. Strategies for regeneration of the intervertebral disc. Regen Med.2008 Sep;3(5):717-29. Review.
19. Spalazzi JP, Vyner MC, Jacobs MT, Moffat KL, Lu HH. Mechanoactive scaffold induces tendon remodeling and expression of fibrocartilage markers. Clin Orthop Relat Res.2008,466(8):1938-48.
20.郭晓东,杜靖远,郑启新,等.TGF-β1基因修饰的间充质干细胞／仿生基质材料复合移植修复兔关节软骨缺损.中国生物医学工程学报.2002.21：111-117.
21. Lai CM, Lai YK, Rakoczy PE. Adenovirus and adeno-associated virus vectors. DNA Cell Biol,2002,21(12):895-913.
22. Van Vliet KM, Blouin V, Brument N, et al. The role of the adeno-associated virus capsid in gene transfer. Methods Mol Biol,2008,437:51-91.
23. Dai J, Rabie AB. Direct AAV-mediated gene delivery to the temporomandibular joint. Front Biosci.2007 Jan 1; 12:2212-20.
24. Detamore MS, Athanasiou KA. Motivation, characterization, and strategy for tissue engineering thetemporomandibular joint disc[J]. Tissue Eng,2003,9(6): 1065-1087.
25. Shi Y,Massague J.Mechanisms of TGF-beta signaling from cell membrane to the nucleus.Cell,2003,113(6):685-700.
26.Jorgensen C,Noel D,Gross G.Could inflammatory arthritis be trigered by progenitor cells in the joint?Ann Rheum Dis[J],2002,61:629
27. Shi Y,Massague J.Mechanisms of TGF-beta signaling from cell membrane to the nucleus.Cell,2003,113(6):685-700.
28. Wan M,Cao X.BMP signaling in skeletal development.Biochem Biophys Res Commun,2005,328(3):651-657.
29. Bi W,Deng J M,Zhang Z,et al..Nat Gene.[J]1999;22(1):85-89.
30. Bi W,Huang W,Whitworth D J,et al.Proc Natl Acadsci[J].2001;98(12):6698-6708
31. Martin DR,Cox NR,Hachcock TL,et al.Isolation and characterization of multipotential mesenchymal stem cells from feline bone marrow[J].Exp Hematol,2002,30(8):879-886
1. Allen KD, Athanasiou KA. Scaffold and growth factor selection in temporomandibular joint disc engineering. J Dent Res.2008, Feb;87(2):180-5.
2. Schek RM, Taboas JM, Hollister SJ, Krebsbach PH. Tissue engineering osteochondral implants for temporomandibular joint repair. Orthod Craniofac Res. 2005, Nov;8(4):313-9.
3. Lee JE, Kim SE, Kwon IC, Ahn HJ, Cho H, Lee SH, Kim HJ, Seong SC, Lee MC.Effects of a chitosan scaffold containing TGF-betal encapsulated chitosan microspheres on in vitro chondrocyte culture. Artif Organs.2004, Sep;28(9): 829-39.
4. Lumpkins SB, Pierre N, McFetridge PS. A mechanical evaluation of three decellularization methods in the design of a xenogeneic scaffold for tissue engineering the temporomandibular joint disc. Acta Biomater.2008, Jul;4(4):808-16.
5. Ando K, Imai S, Isoya E, Kubo M, Mimura T, Shioji S, Ueyama H, Matsusue Y. Effect of dynamic compressive loading and its combination with a growth factor on the chondrocytic phenotype of 3-dimensional scaffold-embedded chondrocytes. Acta Orthop.2009, Dec;80(6):724-33.
6. Gong Y, He L, Li J, Zhou Q, Ma Z, Gao C, Shen J. Hydrogel-filled polylactide porous scaffolds for cartilage tissue engineering. J Biomed Mater Res B Appl Biomater.2007, Jul;82(1):192-204.
7. Tortelli F, Cancedda R. Three-dimensional cultures of osteogenic and chondrogenic cells:a tissue engineering approach to mimic bone and cartilage in vitro. Eur Cell Mater.2009, Jun 30;17:1-14.
8. Park SH, Park SR, Chung SI, Pai KS, Min BH. Tissue-engineered cartilage using fibrin/hyaluronan composite gel and its in vivo implantation. Artif Organs.2005, 29(10):838-45.
9. Fan H, Hu Y, Li X, Wu H, Lv R, Bai J, Wang J, Qin L. Ectopic cartilage formation induced by mesenchymal stem cells on porous gelatin-chondroitin-hyaluronate scaffold containing microspheres loaded with TGF-betal. Int J Artif Organs.2006, Jun;29(6):602-11.
10. Lee JE, Kim SE, Kwon IC, Ahn HJ, Cho H, Lee SH, Kim HJ, Seong SC, Lee MC. Effects of a chitosan scaffold containing TGF-betal encapsulated chitosan microspheres on in vitro chondrocyte culture. Artif Organs.2004, Sep;28(9): 829-39.
11.Lee JE, Kim KE, Kwon IC, Ahn HJ, Lee SH, Cho H, Kim HJ, Seong SC, Lee MC. Effects of the controlled-released TGF-beta 1 from chitosan microspheres on chondrocytes cultured in a collagen/chitosan/glycosaminoglycan scaffold. Biomaterials.2004, Aug;25(18):4163-73.
12.Lee KY, Ha WS, Park WH. Blood compatibility and biodegradability of partially N-acylated chitosan derivatives. Biomaterials 1995,16:1211-1216.
13.Ragetly GR, Slavik GJ, Cunningham BT, Schaeffer DJ, Griffon DJ. Cartilage tissue engineering on fibrous chitosan scaffolds produced by a replica molding technique. J Biomed Mater Res A.2003,93:46-55.
14.Alves da Silva ML, Crawford A, Mundy JM, Correlo VM, Sol P, Bhattacharya M, et al. Chitosan/polyester-based scaffolds for cartilage tissue engineering: assessment of extracellular matrix formation. Acta Biomater.2006,6:1149-1157.
15.Yang C, Hillas PJ, Baez JA, Nokelainen M, Balan J, Tang J, et al. The application of recombinant human collagen in tissue engineering. BioDrugs.2004,18: 103-119.
16.Kobayashi M, Spector M. In vitro response of the bone marrow-derived mesenchymal stem cells seeded in a type-I collagen-glycosaminoglycan scaffold for skin wound repair under the mechanical loading condition. Mol Cell Biomech. 2009, Dec;6(4):217-27.
17.Fini M, Torricelli P, Giavaresi G, Rotini R, Castagna A, Giardino R. In vitro study comparing two collageneous membranes in view of their clinical application for rotator cuff tendon regeneration. J Orthop Res.2007, Jan;25(1):98-107.
18. Concaro S, Gustavson F, Gatenholm P. Bioreactors for tissue engineering of cartilage. Adv Biochem Eng Biotechnol.2009,112:125-43.
19. Chajra H, Rousseau CF, Cortial D, Ronziere MC, Herbage D, Mallein-Gerin F, Freyria AM. Collagen-based biomaterials and cartilage engineering. Application to osteochondral defects. Biomed Mater Eng.2008,18(1 Suppl):S33-45.
20. Wendt D, Jakob M, Martin I. Bioreactor-based engineering of osteochondral grafts:from model systems to tissue manufacturing. J Biosci Bioeng.2005, Nov;100(5):489-94.
21. Freed LE, Guilak F, Guo XE, Gray ML, Tranquillo R, Holmes JW, Radisic M, Sefton MV, Kaplan D, Vunjak-Novakovic G. Advanced tools for tissue engineering:scaffolds, bioreactors, and signaling. Tissue Eng.2006, Dec;12(12):3285-305.
22. Lin Z, Willers C, Xu J, Zheng MH. The chondrocyte:biology and clinical application. Tissue Eng.2006, Jul;12(7):1971-84.
23. Weng Y, Cao Y, Silva CA, Vacanti MP, Vacanti CA. Tissue-engineered composites of bone and cartilage for mandible condylar reconstruction. J Oral Maxillofac Surg 2001,59:185-190.
24. Galois L,Hutasse S,Cortial D,et al.chondrocyte behaviour in three-dimensional type Ⅰ collagen gel in terms of gel contraction,proliferation and gene expression. Biomaterials.2006,27(1):79-90.
1. Dijkgraaf LC, de Bont LG, Boering G, Liem RS. Structure of the normal synovial membrane of the temporomandibular joint:a review of the literature. J Oral Maxillofac Surg.1996,54(3):332-8.
2. Berkovitz BK, Pacy J. Ultrastructure of the human intra-articular disc of the temporomandibular joint. Eur J Orthod.2002, Apr;24(2):151-8.
3. Berkovitz BK. Collagen crimping in the intra-articular disc and articular surfaces of the human temporomandibular joint. Arch Oral Biol.2000, Sep;45(9):749-56.
4. Detamore MS,Orfanos JG,Almarza AJ,et al.Matrix Biol,2005,24(1):45-57.
5. Zakrzewska JM. Diagnosis and management of non-dental orofacial pain. Dent Update.2007, Apr;34(3):134-6,138-9.
6. Suvinen TI, Reade PC, Kemppainen P, Kononen M, Dworkin SF. Review of aetiological concepts of temporomandibular pain disorders:towards a biopsychosocial model for integration of physical disorder factors with psychological and psychosocial illness impact factors. Eur J Pain.2005, Dec;9(6): 613-33.
7. Fikackova H, Dostalova T, Navratil L, Klaschka J. Effectiveness of low-level laser therapy in temporomandibular joint disorders:a placebo-controlled study.Photomed Laser Surg.2007, Aug;25(4):297-303.
8. Bergstrom I, List T, Magnusson T. A follow-up study of subjective symptoms of temporomandibular disorders in patients who received acupuncture and/or interocclusal appliance therapy 18-20 years earlier. Acta Odontol Scand.2008, Apr;66(2):88-92.
9. Al-Riyami S, Cunningham SJ, Moles DR. Orthognathic treatment and temporomandibular disorders:a systematic review. Am J Orthod Dentofacial Orthop.2009, Nov;136(5):626.e1-16
10. Kashi A, Saha S, Christensen RW. Temporomandibular joint disorders:artificial joint replacements and future research needs. J Long Term Eff Med Implants. 2006,16(6):459-74. Review.
11. Abrahamsson C, Ekberg E, Henrikson T, Bondemark L. Alterations of temporomandibular disorders before and after orthognathic surgery:a systematic review. Angle Orthod.2007, Jul;77(4):729-34. Review.
12. Carter EM, Raggio CL, Genetic and orthopedic aspects of collagen disorders. Curr Opin Pediatr.2009, Feb;21(1):46-54. Review.
13. Nishida K, Suzuki T, Kakutani K, Yurube T, Maeno K, Kurosaka M, Doita M. Genetherapy approach for disc degeneration and associated spinal disorders. Eur Spine J.2008, Dec; 17 Suppl 4:459-66.
14.Ueineken FG and Skalak R.Tissue Engineering: Brief Overview,Journal of Biomechanical Engineering 1991,113:111
15. Tew SR, Murdoch AD, Rauchenberg RP, Hardingham TE. Cellular methods in cartilage research:primary human chondrocytes in culture and chondrogenesis in human bone marrow stem cells. Methods.2008, May;45(1):2-9.
16.Chen FH, Rousche KT, Tuan RS. Technology Insight:adult stem cells in cartilage regeneration and tissue engineering. Nat Clin Pract Rheumatol.2006, Jul;2(7):373-82.
17.Gupta T, Haut Donahue TL. Role of cell location and morphology in the mechanical environment around meniscal cells. Acta Biomater.2006, Sep;2(5): 483-92. Epub 2006 Jul 24.
18.Wim B,Peter M,He8nk M, et al.Growth factors and cartilage repair[J].Clin Orthop Res.2001,391 suppl):244-250.
19. Hanaoka K, Tanaka E, Takata T, Miyauchi M, Aoyama J, Kawai N, Dalla-Bona DA, Yamano E, Tanne K. Platelet-derived growth factor enhances proliferation and matrix synthesis of temporomandibular joint disc-derived cells. Angle Orthod. 2006, May;76(3):486-92.
20. Almarza AJ, Athanasiou KA. Evaluation of three growth factors in combinations of two for temporomandibular joint disc tissue engineering. Arch Oral Biol.2006, Mar;51(3):215-21.
21. Tsutsui TW, Riminucci M, Holmbeck K, Bianco P, Robey PG. Development of craniofacial structures in transgenic mice with constitutively active PTH/PTHrP
receptor. Bone.2008, Feb;42(2):321-31.
22. Shibukawa Y, Young B, Wu C, Yamada S, Long F, Pacifici M, Koyama E. Temporomandibular joint formation and condyle growth require Indian hedgehog signaling. Dev Dyn.2007, Feb;236(2):426-34.
23. Tateishi K,Ando W,Higuchi C,et al.Comparison of human serum with fetal bovine serum for expansion and differentiation of human synovial MSC:potential feasibility for clinical applications.Cell Transplant,2008,17(5):549-557.
24. Yang KG, Saris DB, Geuze RE, Helm YJ, Rijen MH, Verbout AJ, Dhert WJ, Creemers LB. Impact of expansion and redifferentiation conditions on chondrogenic capacity of cultured chondrocytes. Tissue Eng.2006 Sep;12(9): 2435-47.
25. Mercuri LG. Temporomandibular joint reconstruction. Alpha Omegan.2009 Jun;102(2):51-4.15Chladek W, Czerwik I. Mechanical properties of temporomandibular joint disc on the basis of porcine preparation investigations. Acta Bioeng Biomech.2008;10(4):15-20.
26. Naujoks C, Meyer U, Wiesmann HP, Jasche-Meyer J, Hohoff A, Depprich R,Handschel J. Principles of cartilage tissue engineering in TMJ reconstruction. Head Face Med.2008 Feb 25;4:3. Review
27. Takigawa M,Okada M,Takano T,et al.J Dent Res 1984,63 (1):19-22.
28. Tsubai T,Higashi Y,Scott JE.Arch Oral Biol,200045 (6):507-515.
29. Benjamin M,Tyers RN,Ralphs JR.Age-related changes in tendon fibrocartilage. J Anat,1991,179:127-136.
30. Roughley PJ,McNicol D,Santer V,et al.The presence of a cartilage-like roteoglycan in the adult human meniscus.Biochem J,1981,197:77-83.
31. Vanderploeg EJ,Imler SM,Brodkin KR,et al.Oscillatory tension differentially modulates matrix metabolism and cytoskeletal organization in chondrocytes and fibrochondrocytes.J Biomech,2004,37:1941-1952.
32. Darling EM,Athanasiou KA.Rapid phenotypic changes in passaged articular chondrocyte subpopulations[J].J Orthop Res,2005,23:425-432.
33. Weinand C,Peretti GM,Adams SB,et al.An allogenic cell-based implant for meniscal lesions[J].J Sports Med,2006,34:1779-1789.
34. Dozin B,MalpeliM, Camardella L, et al. Response ofyoung, aged and osteoarthritic human articular chondrocytes to inflammatory cytokines:molecular and cellular as-pects[J].Matrix Bio,l 2002,21(5):449-459.
35. Alhadlaq A, Mao JJ. Tissue-engineered neogenesis of human-shaped mandibular condyle from rat mesenchymal stem cells. J Dent Res.2003 Dec;82(12):951-6.
36. Hoben GM,Koay EJ,Athanasiou KA.Fibrochondrogenesis in two embryonic stem cell lines:effects of differentiation timelines[J].Stem Cells,2008,26:422-430.
37.徐青镭,吴海山,周维江,等.兔骨髓干细胞向软骨细胞分化用于半月板组织工程重建[J].中国临床康复,2003,7(6)：912-913.
38. Feinberg SE, Hollister SJ, Halloran JW, Chu TM, Krebsbach PH. Image-based biomimetic approach to reconstruction of the temporomandibular joint. Cells Tissues Organs.2001;169(3):309-21.
39. De Bari C,Dell'Accio F,Tylzanowski P,et al.Multipotent mesenchymal stem cells from adult human synovial membrane[J].Arthritis Rheum,2001,44(8):1928-1942.
40. Djouad F,Bony C,Haupl T,et al.Transcriptional profiles discriminate bone marrow derived and synovium-derived mesenchymal stem cells.Arthritis Res Ther,2005,7 (6):1304-1315.
41. Jo CH, Ahn HJ, Kim HJ, et al. Surface characterization and chondrogenic differentiation of mesenchymal stromal cells derived from synovium. Cytotherapy, 2007,9(4):316-327.
42. Sakaguchi Y,Sekiya I,Yagishita K,et al.Comparison of human stem cells derived from various mesenchymal tissues:superiority of synovium as a cell source. Arthritis Rheum,2005,52(8):2521-2529.
43. Shintani,Kurth T,Hunziker EB.Expression of cartilage-related genes in bovine synovial tissue.J Orthop Res,2007,25(6):813-819.
44. Yoshimura H,Muneta T,Nimura A,et al.Comparison of rat mesenchymal stem cells derived from bone marrow,synovium,periosteum, adipose tissue,and muscle.Cell Tissue Res,2007,327(3):449-462.
45.李健龙星柯金等。颞下颌关节滑膜间充质干细胞的分离与培养。中华口腔医学杂志,2005,40(5)：362-364.
46. Yoo JU,Barthel TS,Nishimura K,et al.The chondrogenic potential of human bone-marrow-derived mesenchymal progenitor cells J Bone Joint Surg Am,1998, 80:1745-1757.
47. Worster AA,Brower-Toland BD,Fortier LA,et al.Chondrocytic differentiation of mesenchymal stem cells sequentially exposed to transforming growth factor-betal in monolayer and insulin-like growth factor-I in a three-dimensional matrix.J Orthop Res,2001,19:738-749.
48. Majumdar MK,Banks V,Peluso DP,et al.Isolation,characterization,and chondrogenic potential of human bone marrow-derived multipotential stromal cells.J Cell Physiol,2000,185:98-106.
49. Wang L,Detamore MS.Effects of growth factors and glucosamine on porcine mandibular condylar cartilage cells and hyaline cartilage cells for tissue engineering applications [J].Arch Oral Biol,2008,54(1):1-5
50. Gotz W,Lehmann TS,Appel TR.Distribution of insulin-like growth factors in condylar hyperplasia[J].Ann Anat,2007,189(4):347-349
51. Visnapuu V,Peltomki T,Rnning O.Growth hormone and insulin-like growth factor I receptors in the temporomandibular joint of the rat.J Dent Res,2001,80(10): 1903-1907
52.王健,陈新明,汪说之.TGF-β在不同发育阶段胎儿髁突软骨中的表达.口腔医学纵横,2001,17(4)：277
53.Madry H,Emkey G,Zurakowski D,et al. Over expression of human fibroblast growth factor 2 stimulates cell proliferation in an ex vivo model of articular chondrocyte transplantation. J Gene Med,2004,6(2):238-245
54.Tanaka E,Aoyama J,Miyauchi M.Vascular endothelial growth factor plays an important autocrine/paracrine role in the progression of osteoarthritis[J].Histochem Cell Biol,2005,123(3):275-281
55. Staudenmaier R, Froelich K, Birner M, Kindermann J, The Hoang N, Pueschel RC, Mandlik V. Optimization of platelet isolation and extraction of autogenous TGF-beta in cartilage tissue engineering. Artif Cells Blood Substit Immobil Biotechnol.2009;37 (6):265-72.
56. Emin N, Koc A, Durkut S, Elgin AE, Elgin YM. Engineering of rat articular cartilage on porous sponges:effects of tgf-beta 1 and microgravity bioreactor culture. Artif Cells Blood Substit Immobil Biotechnol.2008;36(2):123-37.
57. Wrotniak M, Bielecki T, Gazdzik TS. Current opinion about using the platelet-rich gel in orthopaedics and trauma surgery. Ortop Traumatol Rehabil. 2007 May-Jun;9(3):227-38. Review.
58. Miura Y, Parvizi J, Fitzsimmons JS, O'Driscoll SW. Brief exposure to high-dose transforming growth factor-β1 enhances periosteal chondrogenesis in vitro:a preliminary report. J Bone Joint Surg Am,2002,84-A(5):793-799.
59. Park H,Temenoff JS,Tabata Y,et al.Injectable biodegradable hydrogel composites for rabbit marrow mesenchymal stem cell and growth factor delivery for cartilage tissue engineering. Biomaterials.2007,28(21):3217-3227.
60.Guo X, Zheng Q, Yang S, Shao Z, Yuan Q, Pan Z, Tang S, Liu K, Quan D. Repair of full-thickness articular cartilage defects by cultured mesenchymal stem cells transfected with the transforming growth factor betal gene. Biomed Mater.2006 Dec;1(4):206-15.
61. Yasushi Miura,Javad Jarvizi,er al.Brief exposure to high-dose transfouming growth factor-β1 enhances periosteal chondrogenesis in vitro.The Journal of Bone&Joint Surgery,2002,5:793-799
62. Stevens MM, Marini RP, Martin I, Langer R, Prasad Shastri V. FGF-2 enhances TGF-betal-induced periosteal chondrogenesis. J Orthop Res.2004 Sep;22(5): 1114-9.
63. Ma Z, Gao C, Gong Y, Shen J. Cartilage tissue engineering PLLA scaffold with surface immobilized collagen and basic fibroblast growth factor. Biomaterials. 2005 Apr;26(11):1253-9.
64. Johns DE, Athanasiou KA. Growth factor effects on costal chondrocytes for tissue engineering fibrocartilage. Cell Tissue Res.2008 Sep;333(3):439-47. Epub 2008 Jul 3. PubMed PMID:18597118.
65. Arevalo-Silva CA,Cao YL,Weng YL,et al.The effect of fibroblast growth factor and transforming growth factor-Bon porcine chondrocytes and tissue-engineered autologous elastic cartilage.Tissue Eng 2001,7(1):81-8
66. Martin I,Vunjak-Novakovic G,Yang J,et al.Mammalian chondrocytes expanded in the presence of fibroblast growth factor 2 mainatin the ability to diffrenetiate and regenerate three-dimensional cartilaginous tissue.Exp Cell Res 1999,253(2): 681-8
67.焦岩涛,王大章,韩文利,等.bFGF、IGF-I及TGF-β对人髁突软骨细胞增殖的影响.中华口腔医学杂志2000,35(5)：346-349.
68. Takafuji H, Suzuki T, Okubo Y.Regeneration of articular cartilage defect in the temporomandibular joint of rabbits by fibroblast growth factor-2:a pilot study.Int J Oral Maxillofac Surg 2007,36(10):934-937.
69. Wang L, Detamore MS.Effects of growth factors and glucosamine on porcine mandibular condylar cartilage cells and hyaline cartilage cells for tissue engineering applications.Arch Oral Biol,2009,54(1):1-5.
70. Delatte M, Von den Hoff JW, Maltha JC, et al.Growth stimulation of mandibular condyles and femoral heads of newborn rats by IGF-I.Arch Oral Biol,2004, 49(3):165-175.
71.赖文莉,赵美英,罗颂椒.功能矫形大鼠下颌前伸后髁突胰岛素分布规律的研究.中华口腔医学杂志,1996,31(2)：91-93.
72. Kramer J,Hegert C,Guan K,et al.Embryonic stem cell-derived chondrogenic differentiation in vitro:activition by BMP-2 and BMP-4.Mech Dev.2000,2(2): 193-205
73. Blunk T,Sieminski AL,Appel B,et al.Bone morphogenetic protein 9:a potent modulator of cartilage development in vitro.Growth Factors.2003,21(2):71-7
74. Loeser RF,Pacione CA,Chubinskaya S.The combination of insulin-like growth factor 1 and osteogenic protein 1 promotes increased survival of and matrix synthesis by normal and osteoarthritic human articular chondrocytes.Arthritis Rheum.2003,48(8):2188-96
75. Ferrara N,Gerber HP,LeCouter J.The biology of VEGF and its receptors. Nat Med,2003,9(6):669-676.
76. Simpson AH,Mills L,Noble B.The role of growth factors and related agents in accelerating fracture healing J Bone Joint SurgBr,2006,88(6):701-705.
77.Sato J,Segami N,Kaneyama K,et al.Vascular endothelial growth factor concentrations in synovial fluids of patients with symptomatic internal derangement of the temporomandibular joint. J Oral Pathol Med.2005,34(3): 170-177.
78. Leonardi R, Lo Muzio L, Bernasconi G, Caltabiano C, Piacentini C, Caltabiano M. Expression of vascular endothelial growth factor in human dysfunctional temporomandibular joint discs. Arch Oral Biol.2003, Mar;48(3):185-92.
79. Yee G, Yu Y, Walsh WR, et al.The immunolocalisation of VEGF in the articular cartilage of sheep mandibular condyles.J Craniomaxillofac Surg.2003,31(4): 244-251.
80. Li Y, Tew SR, Russell AM, Gonzalez KR, Hardingham TE, Hawkins RE. Transduction of passaged human articular chondrocytes with adenoviral, retroviral, and lentiviral vectors and the effects of enhanced expression of SOX9. Tissue Eng.2004, Mar-Apr;10(3-4):575-84.
81. Partridge KA, Oreffo RO. Gene delivery in bone tissue engineering:progress and prospects using viral and nonviral strategies. Tissue Eng.2004, Jan-Feb; 10(1-2):295-307.
82. Ueblacker P, Wagner B, Vogt S, Salzmann G, Wexel G, Kruger A, Plank C, Brill T, Specht K, Hennig T, Schillinger U, Imhoff AB, Martinek V, Gansbacher B. In vivo analysis of retroviral gene transfer to chondrocytes within collagen scaffolds for the treatment of osteochondral defects. Biomaterials.2007, Oct;28(30):4480-7.
83. Tew SR, Li Y, Pothacharoen P, Tweats LM, Hawkins RE, Hardingham TE. Retroviral transduction with SOX9 enhances re-expression of the chondrocyte phenotype in passaged osteoarthritic human articular chondrocytes. Osteoarthritis Cartilage.2005, Jan;13(1):80-9.
84. Feng G, Wan Y, Balian G, Laurencin CT, Li X. Adenovirus-mediated expression of growth and differentiation factor-5 promotes chondrogenesis of adipose stem cells. Growth Factors.2008, Jun;26(3):132-42.
85. Schek RM, Taboas JM, Hollister SJ, Krebsbach PH. Tissue engineering osteochondral implants for temporomandibular joint repair. Orthod Craniofac Res. 2005, Nov;8(4):313-9.
86. Jin X, Sun Y, Zhang K, Wang J, Shi T, Ju X, Lou S. Ectopic neocartilage formation from predifferentiated human adipose derived stem cells induced by adenoviral-mediated transfer of hTGF beta2. Biomaterials.2007, Jul;28(19): 2994-3003.
87. Dai J, Rabie AB. The use of recombinant adeno-associated virus for skeletal gene therapy. Orthod Craniofac Res.2007, Feb;10(1):1-14. Review.
88. Izal I, Acosta CA, Ripalda P, Zaratiegui M, Ruiz J, Forriol F. IGF-1 gene therapy to protect articular cartilage in a rat model of joint damage. Arch Orthop Trauma Surg.2008, Feb;128(2):239-47.
89. Pagnotto MR, Wang Z, Karpie JC, Ferretti M, Xiao X, Chu CR. Adeno-associated viral gene transfer of transforming growth factor-betal to human mesenchymal stem cells improves cartilage repair. Gene Ther.2007, May;14(10):804-13.
90. Li QF, Rabie AB. A new approach to control condylar growth by regulating angiogenesis. Arch Oral Biol.2007, Nov;52(11):1009-17. Epub 2007 Jul 20.
91. Chen T, Hudnall SD. Anatomical mapping of human herpesvirus reservoirs of infection. Mod Pathol.2006, May;19(5):726-37.
92. Stove J, Fiedler J, Huch K, Gunther KP, Puhl W, Brenner R. Lipofection of rabbit chondrocytes and long lasting expression of a lacZ reporter system in alginate beads. Osteoarthritis Cartilage.2002, Mar;10(3):212-7.