hBMP-2基因修饰BMSCs细胞膜片的异体冻干骨输送盘构建及其成骨机制的研究
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摘要
目的
一些先天性发育因素以及后天的外伤、肿瘤术后等因素引起的颌面部巨大骨缺损严重影响了人们的生存与生活质量。大段的颌面骨缺损的修复一直是临床上困扰颌面外科医生的一大难题。传统所采用的骨缺损修复方法(包括自体或异体骨移植、人工合成材料等)尽管能达到相当的治疗效果,但仍存在着许多不可避免的缺点。牵张成骨(distraction osgeogenesis,DO)是利用骨创伤后骨痂愈合机理产生新骨的一项内源性骨组织工程新技术,近年来该技术在颅颌面整形、肿瘤术后重建、牙槽种植等方面展现了广阔的应用前景。输送盘牵张成骨(Transport distraction osteogenesis, TDO)是牵张成骨(Distraction osteogenesis, DO)技术的一种模式,该技术为颅颌面骨大段骨缺损的修复提供了一种新的治疗途径。但TDO需要在骨缺损一端制作合适的骨输送盘(即尽量保持其骨膜和软组织附着)并装上牵张器后才可进行牵张成骨治疗。对于那些严重的骨缺损或者严重的骨畸形而无法在骨缺损区制作合适输送盘的患者来说,传统的TDO技术就难以奏效。如何解决这一难题,让牵张成骨技术拥有更广阔的应用前景,正成为众多学者研究的热点。导师周诺教授应用游离自体骨输送盘(即完全剥离骨膜、无软组织附着)进行牵张成骨,成功构建了“下颌骨非血管化骨游离输送盘牵张成骨”的动物模型,并在国际上率先提出了“非血管化游离输送盘牵张成骨”的新模式,这个新模式的提出为无法利用骨缺损区自身骨段制作合适输送盘的巨大骨缺损修复提供了新的思路。在此基础上,我们设想能否研制一种安全而又有效的异体骨输送盘来替代自体骨输送盘,既可解决前述的问题,又可避免供区二度创伤。然而由于牵张成骨周期较长,异体骨输送盘的应用可能会给导致时间进一步延长,给患者带来诸多不适。
因此,我们在成功构建的“非血管化游离输送盘牵张成骨”动物模型基础上,本研究将种子细胞工程、基因工程和骨组织工程两者的技术结合起来,运用细胞膜片技术,将稳定表达骨形态发生蛋白-2(hBMP-2)的骨髓间充质干细胞(BMSCs)制作成细胞膜片,再与冻干骨复合构建成游离的组织工程化生物输送盘,最终利用该生物输送盘进行下颌骨的骨缺损区输送盘牵张成骨术修复,通过构建一种新的生物输送盘,进一步探索一种新的牵张成骨模式的可行性,并探讨其成骨机理。同时模拟胚胎骨形成自然发生过程,通过细胞膜片可控地在需要的时段持续表达BMP-2,诱导输送盘上或牵张成骨自身骨断端区的多潜能干细胞以及可能的成纤维细胞向成骨或成软骨细胞分化,使成骨级联再次启动,重续定向成骨分化,最终达到促进骨缺损区新骨形成和改建的目的,从而开辟另一种牵张骨新模式,为进一步在临床上广泛应用牵张成骨技术治疗各种严重骨缺损或者骨畸形提供理论依据。
方法
1.犬骨髓间充质干细胞(BMSCs)的体外分离培养及鉴定的实验研究：取1-2月龄健康杂种幼犬,穿刺抽取胫骨骨髓3m1,采用全骨髓贴壁培养法进行分离培养犬骨髓间充质干细胞；通过倒置相差显微镜观察细胞形态；流式细胞术检测细胞表面标志物；通过茜素红染色检测钙结节形成和碱性磷酸酶水平的检测鉴定犬BMSCs的成骨分化；通过HE染色和甲苯胺蓝染色鉴定犬BMSCs的成软骨细胞分化。
2.慢病毒介导hBMP-2转染犬BMSCs的实验研究：取犬第2代BMSCs,用慢病毒作为载体介导hBMP-2基因转染入犬BMSCs中；荧光显微镜和流式细胞仪检测其稳定转染后的转染率；MTT法检测慢病毒转染后对细胞的增殖和生长能力的影响；通过逆转录-聚合酶链反应(RT-PCR)检测hBMP-2mRNA的表达情况。慢病毒转染后再用G418筛选2周,筛选出稳定的阳性转染细胞,收集备用。经过筛选后的犬BMSCs培养4周后,通过免疫印迹(WB)和免疫细胞化学检测hBMP-2蛋白的表达情况。
3. BMSCs的细胞膜片制备及其生物学特性的实验研究：取生长旺盛的第2代犬BMSCs,按1×106密度接种于直径6cm的细胞培养皿中,加入含维生素C的诱导液培养7-10天后形成细胞膜片,通过倒差显微镜、HE染色、免疫组织化学、透射电镜进行观察该细胞膜片的生物学特性；通过实时荧光定量-聚合酶链反应(qPCR)比较犬BMSCs的细胞膜片和酶消化下来的犬BMSCs细胞外基质中I型胶原纤维(COLL Ⅰ)、纤维连接蛋白(Fibronectin)表达水平。通过茜素红染色鉴定BMSCs细胞膜片成骨分化。
4.犬同种异体冻干骨的制备及其生物相容的实验研究：取新鲜的同种异体犬下颌骨,行骨块制备、深冻、脱脂、冷冻干燥、灭菌制成冻干骨。取犬BMSCs制成细胞膜片,并与冻干骨复合培养。HE染色观察冻干骨的组织结构。MTT比色法检测冻干骨对犬BMSCs的活性影响。扫描电镜观察细胞在冻干骨表面的生长情况。
5. hBMP-2修饰BMSCs细胞膜片的异体冻干骨作为生物输送盘在犬下颌骨牵张成骨的实验研究：取健康杂种犬18只随机分为3组,每组6只。实验组(hBMP-2基因修饰BMSCs细胞膜片的异体冻干骨输送盘)、对照组(BMSCs细胞膜片的异体冻干骨输送盘)、空白组(异体冻干骨输送盘)。建立犬下颌骨输送盘牵张动物模型,各动物分组按设计要求将前期实验制备的输送盘分别植入犬下颌骨缺损区,并安装牵张器。于牵张固定期2周、4周、8周后,各实验组分别处死动物各2只,通过大体病理、组织学、摄X线片观察新骨形成和改建情况,通过实时荧光定量聚合酶链反应(qPCR)、免疫组织化学等手段检测BMP-2基因的表达情况。
结果
1.慢病毒介导的hBMP-2转染犬BMSCs的实验结果：原代培养BMSCs呈纤维状贴壁集落式生长,第7-10天后细胞集落扩大并相互融合铺满瓶底,即可传代。传代后细胞保持BMSCs的典型形态和生长方式。细胞表面标志物CD90、CD29、CD44分子阳性,CD34和HLA-DR分子阴性。经成骨诱导后钙结节形成并茜素红染色阳性,诱导期间碱性磷酸酶活性持续增高。成软骨细胞诱导后,肉眼可见白色团块状物形成,行HE染色见软骨组织,甲苯胺蓝染色见细胞外基质异染成紫红色。
2.慢病毒介导hBMP-2转染犬BMSCs的实验结果：通过慢病毒介导的hBMP-2基因转染犬BMSCs,转染效率约为70%。转染48小时细胞可看见绿色荧光,96h后荧光强度最强。MTT法显示转染后的犬BMSCs生长增殖能力无变化。RT-PCR检测到hBMP-2mRNA的表达。用G418成功筛选出稳定转染的细胞,该细胞培养4周后用免疫细胞化学、免疫印迹(WB)检测到1iBMP-2蛋白稳定表达。
3.犬BMSCs的细胞膜片制备及其生物学特性的实验结果：倒差显微镜下可见细胞呈复层生长,细胞呈长梭形,完全融合,细胞界限不清。HE染色显示：骨髓间充质干细胞膜片数层细胞构成,细胞之间连接紧密。透射电镜：可见细胞之间紧密连接,细胞生长分化良好。细胞周围可见基质和胶原纤维qPCR结果显示：犬BMSCs的细胞膜片与酶消化下来的犬BMSCs比,犬BMSCs的细胞膜片的细胞外基质中I型胶原蛋白、纤维连接蛋白都显著增高(P<0.05)。免疫组织化学：膜片的I型胶原蛋白、纤维连接蛋白表达均为阳性。犬BMSCs的细胞膜片成骨分化诱导后可形成钙结节,茜素红染色阳性。
4.犬同种异体冻干骨的制备及其生物相容的实验结果：实验成功制备同种异体犬冻干骨,MTT法显示该冻干骨对BMSCs的生长增殖活性无影响(P>0.05)。扫描电镜下观察可见细胞紧密粘附冻干骨生长,细胞形态呈多角形。
5. hBMP-2基因修饰BMSCs细胞膜片的异体冻干骨作为生物输送盘在犬下颌骨牵张成骨的实验结果：所有实验组动物均成功完成牵张成骨手术,手术区与口腔内无穿通,伤口无感染,且可以顺利牵张；实验动物均存活至预定时间,生长状况良好。通过大体病理、HE染色组织切片、X线和扫描电镜观察,牵张区新骨均愈合良好,牵张区新骨形成质量由高到低：A组>B组>C组。通过免疫组织化学观察并经过光密度值统计软件分析,在固定期2周、4周、8周,A组牵张间隙骨组织质量明显高于B和C组(p<0.05),B组明显高于C组(P<0.05)。
结论
1.成功构建hBMP-2基因修饰BMSCs细胞膜片的异体冻干骨作为新的游离生物输送盘。
2.验证了"hBMP-2基因修饰BMSCs细胞膜片的异体冻干骨”作为游离输送盘牵张成骨模式的可行性。
3. hBMP-2基因修饰的BMSCs细胞膜片可促进牵张区的新骨形成。
Objective
Congenital aplasia, maxillofacial trauma, tumor, surgery that lead to the large bone defects seriously affect the people's survival and quality of life. Large block of maxillofacial bone defect repair has been a difficulty of maxillofacial surgeon clinically. The use of traditional bone defect repair method (including autologous or allogeneic bone graft, synthetic materials, etc.) to achieve a considerable therapeutic effect, but there are still many shortcomings unavoidable. Distraction osteogenesis(DO) is one of an endogenous bone tissue engineering new technologies to generate new bone tissue of which mechanism is the utilization of the bone callus after trauma healing,and the technology in craniofacial surgery, cancer reconstruction surgery, dental slot planting areas show the broad application prospects. However, TDO that must make a suitable bone transport disc near the bone defect area can be carried out with a treatment, and it will be ineffective for the patients with severe bone defects or severe bone deformity and bone defects can't be fitted with a suitable bone transport disc. How to solve this problem, and let distraction osteogenesis have a broader application prospects, which has been a hot research for many scholars. Zhou Nuo(professor and instructor) using free autogenous bone transport disk (ie, fully periosteal stripping, no soft tissue attachment) for distraction osteogenesis successfully build the animal model of "non-vascularized transport disc distraction osteogenesis of mandible", and internationally firstly proposed the new model of "non-vascularized transport disc distraction osteogenesis", which provides a new way of thinking for large bone defects repair that can't treated through producing proper bone transport disc with its own bone in bone deficit area. However, autologous free bone transport disc still need donor bone in donor site, and if a new bone transport disc instead of autologous bone as free transport disc, avoiding secondary trauma of the donor site and reducing the pain of patients caused by surgery.
Therefore, on the basis of animal model of "non-vascularized transport disc distraction osteogenesis", this study will use cell sheet technology, with combination of seed cell engineering, genetic engineering and bone tissue engineering, to make bone marrow mesenchymal stem cells (BMSCs) which stablely express bone morphogenetic protein-2(hBMP-2) into cell sheets, and then will be constructed with freeze-dried bone to produce free tissue engineering biological transport disc, which will be finally used to undergo transporting disc distraction osteogenesis repair in mandibular bone defect area. Generally, this study further explore a new mode of distraction osteogenesis feasibility through building a new kind of biological transport disc, and to explore the mechanism of bone formation. Simultaneously, naturally occurring process of embryonic bone formation is simulated, through the pathway that cell sheets controllably sustained expression of BMP-2at the desired time, and pluripotent stem cells and fibroblasts in transport disc or ends zone of distraction osteogenesis own bone will be induced into bone or cartilage cell differentiation, so that osteogenic cascade will start again and renew directed osteogenic differentiation, and ultimately to promote new bone formation and remodeling in bone defect area, thus open another new model of bone distraction, and provide a theoretical basis for further widely used in the clinical treatment of distraction osteogenesis for a variety of severe bone defects or bone deformities.
Methods
1The experimental study of canine bone marrow mesenchymal stem cells (BMSCs) separation, cultivation and identification in vitro:BMSCs were aspirated from baby dog tibia's bone marrow and isolated by the whole bone marrow adherence method, and identified by cell morphology and cell surface antigen. Then they were induced their differentiation into osteoblasts and chondroblast which were identified by histochemistry staining and Trace enzyme standard method.
2The experimental study of integrating hBMP-2into BMSCs by lentivirus gene transfection:Take passage2BMSCs, with lentivirus as gene avector for integrating BMP-2gene into BMSCs, and transfection rate was by detected by Flow cytometry instrument, and the cell proliferation and growth of the ability after transfection were tested, and the expression of hBMP-2mRNA was detected by RT-PCR. G418was used to sift the stable expression cells for two weeks after transfection. And then the expression of BMP-2protien was tested by western blot and immunochemistry.
3BMSCs cell sheet's preparation and its biological characteristics research: Taken passage2BMSCs, the cells were cultured in a6cm culture dish with the density of1×106till7～10days after the formation of cell sheet. The biological characteristics of cell sheet was observed through the inverted microscope, HE staining, immunochemistry, TEM ect. Dog BMSCs cell sheet and enzyme digestion of dog BMSCs were compared with the expression level of collagen I and fibronectin in the extracellular matrix by real-time fluorescent quantitative polymerase chain reaction (qPCR).
4Preparation of freeaze-dried boneand evaluation of the cellular compatibility: Canine freeze-dried bone was made through block preparation, deep frozen degreasing, freeze drying and sterilization. BMSCs were cultured on FDB in vitro. The tissue structure of FDB was observed by HE staining. The influence of FDB on the proliferation of BMCSs was observed by MTT method. The adhesion and growth of BMSCs were observed by scanning electron microscope (SEM).
5hBMP-2modified BMSCs cell sheet composite freeze-dried bone as a biological transport disc in canine mandibular TDO experiment research: Eighteen healthy mongel dogs were randomly divided into3groups, and6dogs of each group. A-group is experimental group for hBMP-2modified BMSCs cell sheet composite freeze-dried bone as a biological transport disc. B-group is for BMSCs cell sheet composite freeze-dried bone as a biological transport disc. And C-group is for freeze-dried bone as a biological transport disc. A animal model of TDO for canine mandible was constructed. Each experimental group will fix the biological transport disc that had prepared before the operation of TDO. On the time point of two weeks, four weeks and eight weeks after distracting in TDO, the formation of new bone and reconstruction of bone was observed by general pathology, morphology, and X-ray test, and the expression of BMP-2protein was detected by immunochemistry.
Results
1The results of the experimental study of canine bone marrow mesenchymal stem cells (BMSCs) separation, cultivation and identification in vitro:The primary and passages of BMSCs spindle in shape had adhesive character, and grew in colonies. These cells were positive for essential MSC surface molecules (CD90, CD44, CD29) and negative for most haematopoietic markers (CD34, HLA-DR). The result of alizarin red staining was positive after differentiation to osteogenesis. In addition, the result of HE staining show cartilage tissue after differentiation along chondrogenic pathways. And the result of toluidine blue staining showed that cartilage extracellular matrix was dyed indigo blue after differentiation to chondrogenesis.
2The results of the experimental study of integrating hBMP-2into BMSCs by lentivirus gene transfection:Through use lentivirus as gene avector for integrating BMP-2gene into BMSCs, the transfection rate is about70percent of cells, and the green fluorescence was seen after48h transfection, and the intensity of the green fluorescence was strongest after96h transfection. The results of MTT showed the growth proliferation capacity of the canine BMSCs after transfection was no change. The results of RT-PCR show the expression of hBMP-2mRNA in the canine BMSCs after transfection. G418has successfully shift the stable expression the canine BMSCs. The expression of BMP-2of these cells could be detected after4weeks culture by immunochemistry and western blot.
3The results of BMSCs cell sheet's preparation and its biological characteristics research:Under the invert microscope we can see that the cell of BMSCs cell sheet group as multilayer, the cell are long fusiform and full fusion. The cell boundaries were not clear. By HE staining we can look the BMSCs cell sheet made of multilayer cells, and compact connection between cells. By SEM we can observe compact connection between cells, the cell group and differentiation are very good. We can see the extracellular matrix and collagen fiber around the cells. The results of qPCR show canine BMSCs cell sheet are more expression of COLL I and fibronectin(p＜0.05). The cell sheet is detected the expression of COLL I and fibronectin by immunochemistry.
4The results of preparation of freeaze-dried bone(FDB) and to evaluate the cellular compatibility:The freeze-dried bone has been successfully prepared. The results of MTT showed that canine FDB had no influence on the proliferation of BMSCs. Under the SEM, the BMSCs adhered on the surface of the canine FDB tightly, and they grew well and showed polygon shape.
5The results of hBMP-2modified BMSCs cell sheet composite freeze-dried bone as a biological transport disc in canine mandibular TDO experiment research:All experimental animals were successfully completed with a surgery of transport distraction osteogenesis. Operation area and the mouth without a passageway. The operation incision had no infection. The animal model can successfully be distraction. The results of general pathology, tissue section HE staining and X-ray examination show that the quantity of regeneration of new bone ranked as follows:group A> group B> group C. The gray value of the results of IHC showed that the expression of BMP-2of group A was obviously higher than that of group B and group C (p<0.05), and group B was higher than group C (p<0.05).
Conclusions
1hBMP-2modified BMSCs cell sheet of freeze-dried bone allograft has been successfully built as a new biological transport disc.
2The feasibility of this TDO model that used "hBMP-2modified BMSCs cell sheet of freeze-dried bone allograft as a new biological transport disc" has been Verified.
3hBMP-2modified BMSCs cell sheet can promote the formation of new bone.
一些先天性发育因素以及后天的外伤、肿瘤术后等因素引起的颌面部巨大骨缺损严重影响了人们的生存与生活质量。大段的颌面骨缺损的修复一直是临床上困扰颌面外科医生的一大难题。传统所采用的骨缺损修复方法(包括自体或异体骨移植、人工合成材料等)尽管能达到相当的治疗效果,但仍存在着许多不可避免的缺点。牵张成骨(distraction osgeogenesis,DO)是利用骨创伤后骨痂愈合机理产生新骨的一项内源性骨组织工程新技术,近年来该技术在颅颌面整形、肿瘤术后重建、牙槽种植等方面展现了广阔的应用前景。输送盘牵张成骨(Transport distraction osteogenesis, TDO)是牵张成骨(Distraction osteogenesis, DO)技术的一种模式,该技术为颅颌面骨大段骨缺损的修复提供了一种新的治疗途径。但TDO需要在骨缺损一端制作合适的骨输送盘(即尽量保持其骨膜和软组织附着)并装上牵张器后才可进行牵张成骨治疗。对于那些严重的骨缺损或者严重的骨畸形而无法在骨缺损区制作合适输送盘的患者来说,传统的TDO技术就难以奏效。如何解决这一难题,让牵张成骨技术拥有更广阔的应用前景,正成为众多学者研究的热点。导师周诺教授应用游离自体骨输送盘(即完全剥离骨膜、无软组织附着)进行牵张成骨,成功构建了“下颌骨非血管化骨游离输送盘牵张成骨”的动物模型,并在国际上率先提出了“非血管化游离输送盘牵张成骨”的新模式,这个新模式的提出为无法利用骨缺损区自身骨段制作合适输送盘的巨大骨缺损修复提供了新的思路。在此基础上,我们设想能否研制一种安全而又有效的异体骨输送盘来替代自体骨输送盘,既可解决前述的问题,又可避免供区二度创伤。然而由于牵张成骨周期较长,异体骨输送盘的应用可能会给导致时间进一步延长,给患者带来诸多不适。
因此,我们在成功构建的“非血管化游离输送盘牵张成骨”动物模型基础上,本研究将种子细胞工程、基因工程和骨组织工程两者的技术结合起来,运用细胞膜片技术,将稳定表达骨形态发生蛋白-2(hBMP-2)的骨髓间充质干细胞(BMSCs)制作成细胞膜片,再与冻干骨复合构建成游离的组织工程化生物输送盘,最终利用该生物输送盘进行下颌骨的骨缺损区输送盘牵张成骨术修复,通过构建一种新的生物输送盘,进一步探索一种新的牵张成骨模式的可行性,并探讨其成骨机理。同时模拟胚胎骨形成自然发生过程,通过细胞膜片可控地在需要的时段持续表达BMP-2,诱导输送盘上或牵张成骨自身骨断端区的多潜能干细胞以及可能的成纤维细胞向成骨或成软骨细胞分化,使成骨级联再次启动,重续定向成骨分化,最终达到促进骨缺损区新骨形成和改建的目的,从而开辟另一种牵张骨新模式,为进一步在临床上广泛应用牵张成骨技术治疗各种严重骨缺损或者骨畸形提供理论依据。
方法
1.犬骨髓间充质干细胞(BMSCs)的体外分离培养及鉴定的实验研究：取1-2月龄健康杂种幼犬,穿刺抽取胫骨骨髓3m1,采用全骨髓贴壁培养法进行分离培养犬骨髓间充质干细胞；通过倒置相差显微镜观察细胞形态；流式细胞术检测细胞表面标志物；通过茜素红染色检测钙结节形成和碱性磷酸酶水平的检测鉴定犬BMSCs的成骨分化；通过HE染色和甲苯胺蓝染色鉴定犬BMSCs的成软骨细胞分化。
2.慢病毒介导hBMP-2转染犬BMSCs的实验研究：取犬第2代BMSCs,用慢病毒作为载体介导hBMP-2基因转染入犬BMSCs中；荧光显微镜和流式细胞仪检测其稳定转染后的转染率；MTT法检测慢病毒转染后对细胞的增殖和生长能力的影响；通过逆转录-聚合酶链反应(RT-PCR)检测hBMP-2mRNA的表达情况。慢病毒转染后再用G418筛选2周,筛选出稳定的阳性转染细胞,收集备用。经过筛选后的犬BMSCs培养4周后,通过免疫印迹(WB)和免疫细胞化学检测hBMP-2蛋白的表达情况。
3. BMSCs的细胞膜片制备及其生物学特性的实验研究：取生长旺盛的第2代犬BMSCs,按1×106密度接种于直径6cm的细胞培养皿中,加入含维生素C的诱导液培养7-10天后形成细胞膜片,通过倒差显微镜、HE染色、免疫组织化学、透射电镜进行观察该细胞膜片的生物学特性；通过实时荧光定量-聚合酶链反应(qPCR)比较犬BMSCs的细胞膜片和酶消化下来的犬BMSCs细胞外基质中I型胶原纤维(COLL Ⅰ)、纤维连接蛋白(Fibronectin)表达水平。通过茜素红染色鉴定BMSCs细胞膜片成骨分化。
4.犬同种异体冻干骨的制备及其生物相容的实验研究：取新鲜的同种异体犬下颌骨,行骨块制备、深冻、脱脂、冷冻干燥、灭菌制成冻干骨。取犬BMSCs制成细胞膜片,并与冻干骨复合培养。HE染色观察冻干骨的组织结构。MTT比色法检测冻干骨对犬BMSCs的活性影响。扫描电镜观察细胞在冻干骨表面的生长情况。
5. hBMP-2修饰BMSCs细胞膜片的异体冻干骨作为生物输送盘在犬下颌骨牵张成骨的实验研究：取健康杂种犬18只随机分为3组,每组6只。实验组(hBMP-2基因修饰BMSCs细胞膜片的异体冻干骨输送盘)、对照组(BMSCs细胞膜片的异体冻干骨输送盘)、空白组(异体冻干骨输送盘)。建立犬下颌骨输送盘牵张动物模型,各动物分组按设计要求将前期实验制备的输送盘分别植入犬下颌骨缺损区,并安装牵张器。于牵张固定期2周、4周、8周后,各实验组分别处死动物各2只,通过大体病理、组织学、摄X线片观察新骨形成和改建情况,通过实时荧光定量聚合酶链反应(qPCR)、免疫组织化学等手段检测BMP-2基因的表达情况。
结果
1.慢病毒介导的hBMP-2转染犬BMSCs的实验结果：原代培养BMSCs呈纤维状贴壁集落式生长,第7-10天后细胞集落扩大并相互融合铺满瓶底,即可传代。传代后细胞保持BMSCs的典型形态和生长方式。细胞表面标志物CD90、CD29、CD44分子阳性,CD34和HLA-DR分子阴性。经成骨诱导后钙结节形成并茜素红染色阳性,诱导期间碱性磷酸酶活性持续增高。成软骨细胞诱导后,肉眼可见白色团块状物形成,行HE染色见软骨组织,甲苯胺蓝染色见细胞外基质异染成紫红色。
2.慢病毒介导hBMP-2转染犬BMSCs的实验结果：通过慢病毒介导的hBMP-2基因转染犬BMSCs,转染效率约为70%。转染48小时细胞可看见绿色荧光,96h后荧光强度最强。MTT法显示转染后的犬BMSCs生长增殖能力无变化。RT-PCR检测到hBMP-2mRNA的表达。用G418成功筛选出稳定转染的细胞,该细胞培养4周后用免疫细胞化学、免疫印迹(WB)检测到1iBMP-2蛋白稳定表达。
3.犬BMSCs的细胞膜片制备及其生物学特性的实验结果：倒差显微镜下可见细胞呈复层生长,细胞呈长梭形,完全融合,细胞界限不清。HE染色显示：骨髓间充质干细胞膜片数层细胞构成,细胞之间连接紧密。透射电镜：可见细胞之间紧密连接,细胞生长分化良好。细胞周围可见基质和胶原纤维qPCR结果显示：犬BMSCs的细胞膜片与酶消化下来的犬BMSCs比,犬BMSCs的细胞膜片的细胞外基质中I型胶原蛋白、纤维连接蛋白都显著增高(P<0.05)。免疫组织化学：膜片的I型胶原蛋白、纤维连接蛋白表达均为阳性。犬BMSCs的细胞膜片成骨分化诱导后可形成钙结节,茜素红染色阳性。
4.犬同种异体冻干骨的制备及其生物相容的实验结果：实验成功制备同种异体犬冻干骨,MTT法显示该冻干骨对BMSCs的生长增殖活性无影响(P>0.05)。扫描电镜下观察可见细胞紧密粘附冻干骨生长,细胞形态呈多角形。
5. hBMP-2基因修饰BMSCs细胞膜片的异体冻干骨作为生物输送盘在犬下颌骨牵张成骨的实验结果：所有实验组动物均成功完成牵张成骨手术,手术区与口腔内无穿通,伤口无感染,且可以顺利牵张；实验动物均存活至预定时间,生长状况良好。通过大体病理、HE染色组织切片、X线和扫描电镜观察,牵张区新骨均愈合良好,牵张区新骨形成质量由高到低：A组>B组>C组。通过免疫组织化学观察并经过光密度值统计软件分析,在固定期2周、4周、8周,A组牵张间隙骨组织质量明显高于B和C组(p<0.05),B组明显高于C组(P<0.05)。
结论
1.成功构建hBMP-2基因修饰BMSCs细胞膜片的异体冻干骨作为新的游离生物输送盘。
2.验证了"hBMP-2基因修饰BMSCs细胞膜片的异体冻干骨”作为游离输送盘牵张成骨模式的可行性。
3. hBMP-2基因修饰的BMSCs细胞膜片可促进牵张区的新骨形成。
Objective
Congenital aplasia, maxillofacial trauma, tumor, surgery that lead to the large bone defects seriously affect the people's survival and quality of life. Large block of maxillofacial bone defect repair has been a difficulty of maxillofacial surgeon clinically. The use of traditional bone defect repair method (including autologous or allogeneic bone graft, synthetic materials, etc.) to achieve a considerable therapeutic effect, but there are still many shortcomings unavoidable. Distraction osteogenesis(DO) is one of an endogenous bone tissue engineering new technologies to generate new bone tissue of which mechanism is the utilization of the bone callus after trauma healing,and the technology in craniofacial surgery, cancer reconstruction surgery, dental slot planting areas show the broad application prospects. However, TDO that must make a suitable bone transport disc near the bone defect area can be carried out with a treatment, and it will be ineffective for the patients with severe bone defects or severe bone deformity and bone defects can't be fitted with a suitable bone transport disc. How to solve this problem, and let distraction osteogenesis have a broader application prospects, which has been a hot research for many scholars. Zhou Nuo(professor and instructor) using free autogenous bone transport disk (ie, fully periosteal stripping, no soft tissue attachment) for distraction osteogenesis successfully build the animal model of "non-vascularized transport disc distraction osteogenesis of mandible", and internationally firstly proposed the new model of "non-vascularized transport disc distraction osteogenesis", which provides a new way of thinking for large bone defects repair that can't treated through producing proper bone transport disc with its own bone in bone deficit area. However, autologous free bone transport disc still need donor bone in donor site, and if a new bone transport disc instead of autologous bone as free transport disc, avoiding secondary trauma of the donor site and reducing the pain of patients caused by surgery.
Therefore, on the basis of animal model of "non-vascularized transport disc distraction osteogenesis", this study will use cell sheet technology, with combination of seed cell engineering, genetic engineering and bone tissue engineering, to make bone marrow mesenchymal stem cells (BMSCs) which stablely express bone morphogenetic protein-2(hBMP-2) into cell sheets, and then will be constructed with freeze-dried bone to produce free tissue engineering biological transport disc, which will be finally used to undergo transporting disc distraction osteogenesis repair in mandibular bone defect area. Generally, this study further explore a new mode of distraction osteogenesis feasibility through building a new kind of biological transport disc, and to explore the mechanism of bone formation. Simultaneously, naturally occurring process of embryonic bone formation is simulated, through the pathway that cell sheets controllably sustained expression of BMP-2at the desired time, and pluripotent stem cells and fibroblasts in transport disc or ends zone of distraction osteogenesis own bone will be induced into bone or cartilage cell differentiation, so that osteogenic cascade will start again and renew directed osteogenic differentiation, and ultimately to promote new bone formation and remodeling in bone defect area, thus open another new model of bone distraction, and provide a theoretical basis for further widely used in the clinical treatment of distraction osteogenesis for a variety of severe bone defects or bone deformities.
Methods
1The experimental study of canine bone marrow mesenchymal stem cells (BMSCs) separation, cultivation and identification in vitro:BMSCs were aspirated from baby dog tibia's bone marrow and isolated by the whole bone marrow adherence method, and identified by cell morphology and cell surface antigen. Then they were induced their differentiation into osteoblasts and chondroblast which were identified by histochemistry staining and Trace enzyme standard method.
2The experimental study of integrating hBMP-2into BMSCs by lentivirus gene transfection:Take passage2BMSCs, with lentivirus as gene avector for integrating BMP-2gene into BMSCs, and transfection rate was by detected by Flow cytometry instrument, and the cell proliferation and growth of the ability after transfection were tested, and the expression of hBMP-2mRNA was detected by RT-PCR. G418was used to sift the stable expression cells for two weeks after transfection. And then the expression of BMP-2protien was tested by western blot and immunochemistry.
3BMSCs cell sheet's preparation and its biological characteristics research: Taken passage2BMSCs, the cells were cultured in a6cm culture dish with the density of1×106till7～10days after the formation of cell sheet. The biological characteristics of cell sheet was observed through the inverted microscope, HE staining, immunochemistry, TEM ect. Dog BMSCs cell sheet and enzyme digestion of dog BMSCs were compared with the expression level of collagen I and fibronectin in the extracellular matrix by real-time fluorescent quantitative polymerase chain reaction (qPCR).
4Preparation of freeaze-dried boneand evaluation of the cellular compatibility: Canine freeze-dried bone was made through block preparation, deep frozen degreasing, freeze drying and sterilization. BMSCs were cultured on FDB in vitro. The tissue structure of FDB was observed by HE staining. The influence of FDB on the proliferation of BMCSs was observed by MTT method. The adhesion and growth of BMSCs were observed by scanning electron microscope (SEM).
5hBMP-2modified BMSCs cell sheet composite freeze-dried bone as a biological transport disc in canine mandibular TDO experiment research: Eighteen healthy mongel dogs were randomly divided into3groups, and6dogs of each group. A-group is experimental group for hBMP-2modified BMSCs cell sheet composite freeze-dried bone as a biological transport disc. B-group is for BMSCs cell sheet composite freeze-dried bone as a biological transport disc. And C-group is for freeze-dried bone as a biological transport disc. A animal model of TDO for canine mandible was constructed. Each experimental group will fix the biological transport disc that had prepared before the operation of TDO. On the time point of two weeks, four weeks and eight weeks after distracting in TDO, the formation of new bone and reconstruction of bone was observed by general pathology, morphology, and X-ray test, and the expression of BMP-2protein was detected by immunochemistry.
Results
1The results of the experimental study of canine bone marrow mesenchymal stem cells (BMSCs) separation, cultivation and identification in vitro:The primary and passages of BMSCs spindle in shape had adhesive character, and grew in colonies. These cells were positive for essential MSC surface molecules (CD90, CD44, CD29) and negative for most haematopoietic markers (CD34, HLA-DR). The result of alizarin red staining was positive after differentiation to osteogenesis. In addition, the result of HE staining show cartilage tissue after differentiation along chondrogenic pathways. And the result of toluidine blue staining showed that cartilage extracellular matrix was dyed indigo blue after differentiation to chondrogenesis.
2The results of the experimental study of integrating hBMP-2into BMSCs by lentivirus gene transfection:Through use lentivirus as gene avector for integrating BMP-2gene into BMSCs, the transfection rate is about70percent of cells, and the green fluorescence was seen after48h transfection, and the intensity of the green fluorescence was strongest after96h transfection. The results of MTT showed the growth proliferation capacity of the canine BMSCs after transfection was no change. The results of RT-PCR show the expression of hBMP-2mRNA in the canine BMSCs after transfection. G418has successfully shift the stable expression the canine BMSCs. The expression of BMP-2of these cells could be detected after4weeks culture by immunochemistry and western blot.
3The results of BMSCs cell sheet's preparation and its biological characteristics research:Under the invert microscope we can see that the cell of BMSCs cell sheet group as multilayer, the cell are long fusiform and full fusion. The cell boundaries were not clear. By HE staining we can look the BMSCs cell sheet made of multilayer cells, and compact connection between cells. By SEM we can observe compact connection between cells, the cell group and differentiation are very good. We can see the extracellular matrix and collagen fiber around the cells. The results of qPCR show canine BMSCs cell sheet are more expression of COLL I and fibronectin(p＜0.05). The cell sheet is detected the expression of COLL I and fibronectin by immunochemistry.
4The results of preparation of freeaze-dried bone(FDB) and to evaluate the cellular compatibility:The freeze-dried bone has been successfully prepared. The results of MTT showed that canine FDB had no influence on the proliferation of BMSCs. Under the SEM, the BMSCs adhered on the surface of the canine FDB tightly, and they grew well and showed polygon shape.
5The results of hBMP-2modified BMSCs cell sheet composite freeze-dried bone as a biological transport disc in canine mandibular TDO experiment research:All experimental animals were successfully completed with a surgery of transport distraction osteogenesis. Operation area and the mouth without a passageway. The operation incision had no infection. The animal model can successfully be distraction. The results of general pathology, tissue section HE staining and X-ray examination show that the quantity of regeneration of new bone ranked as follows:group A> group B> group C. The gray value of the results of IHC showed that the expression of BMP-2of group A was obviously higher than that of group B and group C (p<0.05), and group B was higher than group C (p<0.05).
Conclusions
1hBMP-2modified BMSCs cell sheet of freeze-dried bone allograft has been successfully built as a new biological transport disc.
2The feasibility of this TDO model that used "hBMP-2modified BMSCs cell sheet of freeze-dried bone allograft as a new biological transport disc" has been Verified.
3hBMP-2modified BMSCs cell sheet can promote the formation of new bone.
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