VEGF及BMP2基因修饰对血管化组织工程骨的影响及机制研究
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摘要
颌面部骨缺损常见于肿瘤切除、颌骨骨髓炎术后及颌面部外伤等疾患,近年来,我国由上述原因导致的骨缺损患者数量呈逐年上升趋势。揭示骨缺损修复的机制,寻找骨缺损良好修复的方法是骨缺损修复研究领域亟待解决的关键问题。早期的自体骨移植、异体骨移植等传统修复骨缺损的方法对机体损伤较大且效果不佳；之后人工合成的骨替代物因生物相容性差、体内降解慢及机械性能低等不足没有广泛推广；近些年骨组织工程骨的出现为颌骨缺损的修复带来新希望。骨组织工程技术改变了以往创伤性修复的模式,以少量组织细胞修复大面积组织缺损,并可根据需要进行塑形恢复缺损形态,为实现无创修复及良好的生物功能重建提供了理论基础为骨再生修复指明了新的方向。骨组织工程主要包括种子细胞、生长因子及载体支架三个要素。种子细胞是骨组织工程的首要环节和基本要素,其目的是使所构建的组织工程骨兼具骨传导性及骨诱导性,从而更好的修复骨缺损。所以选择最优的种子细胞成为骨组织工程首要解决的问题。骨髓基质干细胞(Bone marrow stromal cells, BMSCs)具有较强的多向分化能力,在一定条件下可分化为骨、软骨、脂肪等细胞,BMSCs也因此成为骨组织工程中应用最广泛的种子细胞。骨形成蛋白2(Bone morphogenetic protein2, BMP2)具有较强的诱导骨形成能力,将因子直接应用于体内由于弥散作用及其半衰期短的原因成骨效果不佳。研究发现采用基因修饰的方式可弥补因子直接应用的不足,所以将BMP2与BMSCs联合应用的方法可为骨组织工程提供新型种子细胞。
以种子细胞复合生物材料的方法修复骨缺损虽然取得了一定的成功,但一些较大面积的骨缺损因植入物缺少充足的血供而发生坏死。因此,有效的血管化已与种子细胞、支架材料及生长因子共同列入骨组织工程的重要因素。预成血管植入是使骨替代物血管化最直接有效的方法,即将血管预先植入骨替代物中,利用显微外科技术将其与宿主体内血管吻合。此种方法可建造独立的血管系统,为植入物提供丰富的血供,可达到良好的血管化效果。但是由于经历多次手术、损伤供血管区及治疗周期长等不足使此方法局限于小面积骨缺损及软组织创伤的修复；诱导血管生成的因子如血管内皮生长因子(Vascular Endothelial Growth Factor, VEGF)、成纤维细胞生长因子(Fibroblast growth factor, FGF)、血管紧张素(Angiopoietin, Ang)的使用可促进血管生成,但因半衰期短局部直接应用效果欠佳。目前将具有血管形成能力的细胞与成骨性细胞的联合应用是血管化组织工程骨最有前景的方法之一,尤为血管内皮前体细胞(Endothelial Progenitor Cells, EPCs)的出现,证明血管发生也存在于发育成熟后的机体组织打破了血管形成的传统理念,为缺血性疾病提供了新思路。EPCs是一种未成熟细胞,可分化为内皮细胞直接参与血管形成,在后肢缺血、心肌缺血及角膜损伤等动物模型的实验研究中取得了一定的成功。但EPCs的细胞数量有限,不足以用于大面积缺血缺损的修复。VEGF基因修饰EPCs可弥补其数量不足的缺点,因为VEGF不仅可以募集、趋化EPCs至缺损处,其本身也可直接诱导血管生成。因此,VEGF与EPCs联合应用应该能够达到事半功倍的效果。基于此,本实验选择具有血管形成能力的细胞与成骨性细胞联合应用的方法促进组织工程骨血管化,观察血管及骨形成情况,探讨VEGF-EPC与BMP2-BMSC联合应用对血管化及成骨作用的影响。
主要实验方法及结果：
1. BMSCs的分离及鉴定：将密度梯度离心法分离的大鼠骨髓单个核细胞通过塑料培养瓶培养、体外扩增传代进行分离纯化获得较高纯度的BMSCs。分别将BMSCs培养于成骨诱导液及成脂诱导液中,茜素红染色及油红O染色证明BMSCs可诱导分化为成骨细胞、成脂肪细胞,通过细胞功能检测鉴定培养的细胞为BMSCs。
2. EPCs的分离及鉴定：采用密度梯度离心法分离大鼠骨髓的单个核细胞,通过特殊培养基诱导,贴壁培养、体外扩增纯化获取细胞,利用细胞形态学、细胞表型检测及细胞免疫双荧光、体外成管实验等细胞功能检测进行鉴定,结果证实此种方法分离诱导纯化的细胞为EPCs。同时将BMSCs及EPCs以不同比例混合培养,MTT法检测细胞的增殖能力,确定BMSCs:EPCs的最佳混合比例为1：1。
3. Ad-BMP2及Ad-VEGF腺病毒载体的构建及有效性鉴定：体外构建Ad-BMP2及Ad-VEGF腺病毒载体,利用AdMax包装系统对其包装、纯化,鉴定重组病毒载体携带目的基因的正确性。
3.1.测定Ad-BMP2的滴度为2.3×1011pfu/ml,Ad-VEGF滴度为3×1011pfu/ml；设置MOI梯度将Ad-BMP2、Ad-VEGF分别转染BMSCs、EPCs,通过对绿色荧光表达的观察确定病毒转染的最佳MOI值为40,并绘制细胞生长曲线证明在MOI=40时细胞的增殖能力没有受到影响；
3.2.提取重组腺病毒转染组、空白病毒转染组及空白细胞组的RNA,实时定量PCR检测显示在RNA水平Ad-BMP2-BMSCs组BMP2及Ad-VEGF-EPCs组VEGF的表达显著高于各自对照组(p<0.05)；分别收集各组细胞上清,Elisa检测BMP2、VEGF蛋白水平的表达,Ad-BMP2-BMSCs组BMP2及Ad-VEGF-EPCs(?)组VEGF的表达显著高于各自对照组(p<0.05)；证明了Ad-BMP2及Ad-VEGF腺病毒载体构建的有效性。
4. VEGF修饰的EPCs对BMP2-BMSC体外成骨分化的影响：将细胞分为BMSC+EPC、Ad-BMP2-BMSC+EPC、BMSC+Ad-VEGF-EPC及Ad-BMP2-BMSC+Ad-VEGF-EPC四组,体外行成骨诱导培养,分别于7d、14d、21d及28d行成骨能力检测。
4.1茜素红染色观察矿化结节：结果显示各组矿化结节数目随时间增加而增多,在第21d及28d,BMP2转染组Ad-BMP2-BMSC+Ad-VEGF-EPC及Ad-BMP2-BMSC+EPC矿化结节数目显著高于其他组(p<0.05)；两组的矿化结节数目在21、28天的变化不明显；
4.2ALP活性检测：随时间增加每组细胞的ALP活性逐渐增强,诱导后第21天各组ALP活性均达最高值,且BMP2转染组Ad-BMP2-BMSC+Ad-VEGF-EPC及Ad-BMP2-BMSC+EPC的ALP活性显著高于其他组(p<0.0J)；但在第28天发现各组间的差异减小,且Ad-BMP2-BMSC+Ad-VEGF-EPC及Ad-BMP2-BMSC+EPC的ALP活性较21d变化微小；
4.3实时定量PCR检测ALP、OC、ColI成骨基因表达：Ad-BMP2-BMSC+EPC、BMSC+Ad-VEGF-EPC及Ad-BMP2-BMSC+Ad-VEGF-EPC组中ALP基因的表达在诱导后第21天达到高峰,其中Ad-BMP2-BMSC+EPC组和Ad-BMP2-BMSC+Ad-VEGF-EPC组ALP的表达显著高于其他各组(P<0.05),每组的OC表达随时间变化增长缓慢,组间没有显著差异；随时间变化各组的Col Ⅰ表达逐渐增多,21天时达到高点,且Ad-BMP2-BMSC+Ad-VEGF-EPC组Col Ⅰ表达水平高于其他各组(P<0.05)。
5. VEGF修饰的EPCs对组织工程骨形成及血管化的影响：将BMSC+EPC、 Ad-BMP2-BMSC+EPC、BMSC+Ad-VEGF-EPC及Ad-BMP2-BMSC+Ad-VEGF-EPC四组细胞分别与藻酸盐凝胶混合注入大鼠股骨外侧肌袋内,每组5只大鼠,分别于4w及6w将动物处死取出植入组织,免疫染色检测骨、Col Ⅰ及血管形成。
5.1石蜡包埋后切片行HE染色检测新生组织中骨生成情况,结果显示混合物注入4周后组织切片HE染色可见藻酸盐凝胶材料数量减少,长圆形、梭形细胞围绕凝胶材料周围；第6周,HE染色仍可观察到材料的残余,能够见到细胞核深染的成骨细胞形成。实验中未见有炎症反应发生。定量分析骨形成面积：在第4周和第6周,Ad-BMP2-BMSC+EPC及Ad-BMP2-BMSC+Ad-VEGF-EPC组新骨形成面积显著高于其他两组(P<0.05)；第6周,Ad-BMP2-BMSC+EPC及Ad-BMP2-BMSC+Ad-VEGF-EPC组组间有显著差异(P<0.05),但Ad-BMP2-BMSC+Ad-VEGF-EPC及Ad-BMP2-BMSC+EPC组新骨形成面积相对第4周增长缓慢；
5.2天狼猩红染色检测Col Ⅰ生成情况：可观察到新胶原组织产生,并间杂有不规则状的未成熟骨样基质。随时间增长,新生胶原组织数量增加。第4及第6周,Ad-BMP2-BMSC+EPC及Ad-BMP2-BMSC+Ad-VEGF-EPC组新胶原形成面积显著高于其他两组(P<0.05)；
5.3血管内皮特殊标记物CD31检测新生物中血管的生成情况,第4及第6周,BMSC+Ad-VEGF-EPC及Ad-BMP2-BMSC+Ad-VEGF-EPC两组血管生成数目显著高于其他两组(P<0.05),且两组间存在显著差异Ad-BMP2-BMSC+Ad-VEGF-EPC组的血管生成数量显著高于BMSC+Ad-VEGF-EPC组(P<0.05)。
6.BMP2及VEGF对Idl表达的影响：分别提取Ad-BMP2-BMSC组、Ad-VEGF-EPC组、空白病毒转染细胞组及空白细胞组的RNA,实时定量PCR检测Ad-BMP2-BMSC组及Ad-VEGF-EPC组Idl基因RNA水平的表达显著高于各自对照组(P<0.05)。
结论：
1.密度梯度离心法分离大鼠骨髓单个核细胞,通过不同的诱导方式对其进行分离、诱导及纯化可分别得到纯度较好的BMSCs及EPCs。
2.通过细胞功能鉴定证明EPCs具有血管形成能力及BMSCs具有成骨分化能力。
3.体外构建Ad-BMP2及Ad-VEGF重组腺病毒表达载体,滴度高转染效率好,可使BMSCs过表达BMP2及EPCs高表达VEGF,可有效促进BMSCs的成骨性能及EPCs的血管形成活性。
4. VEGF修饰的EPCs与BMP2修饰的BMSCs联合应用,可通过细胞间因子交流有效促进血管的形成,VEGF修饰的EPCs为理想的促血管生成种子细胞。
5.BMP2及VEGF诱导Idl的表达升高对成骨细胞终末分化的抑制作用可能会导致骨形成不佳,且Id1可通过募集趋化EPCs促进血管生成；由此推测Idl可能为成骨分化及血管形成的重要分子。Idl在骨形成及血管形成中的作用为骨组织工程的研究提供了新思路。
创新性和意义：
1.本研究的创新点是在国内外首次将BMP修饰的BMSCs与VEGF修饰的EPCs共同作为骨组织工程的种子细胞,利用VEGF-EPC的血管化作用以促进骨缺损的修复,为因血管化不足导致大面积骨组织缺损的修复不良提供解决方法和策略。
2.本研究初步探讨了Idl基因在骨形成及血管形成中的作用,为骨组织工程的研究提供了新思路。
Bone regeneration is required to repair mandible defects arising from trauma, inflammation or tumor. Bone tissue engineering which based on seed cells, grow factors and scaffold material are currently being combined to assess their potential to develop effective concepts for the treatment of extensive loss of osseous tissue. Bone seed cells which share the collective goal of creating osteoconductive and osteoinductive bone graft substitutes is the primary and basic element of bone tissue engineering. Bone marrow stem cells (BMSCs) are pluripotent cells having the potential to differentiate into bone, cartilage, muscle, and ligament have been implicated in the enhancement of bone repair. Although bone morphogenetic protein2(BMP2) can produce orthotopic bone in the hindlimbs of animal model, the short half-life and dispersion limit their effection of directly application. However, the strategy of gene modification can efficiently solve the deficience of directly application and adenovirus vectors have been successfully used for transient gene expression avoiding side effect of gene overexpression. BMP2gene modification of BMSCs provides a new prospect for bone tissue engineering.
Osteogenic stem cells with over-expressed osteogenic factors were less effective in treating large bone defects due to inefficient blood supply in the entire bone graft. The vascularization has been involved in bone tissue engineering with the seed cells, grow factors and scaffold. Implanting the tissue valve embedment with vessel pedicle or blood vessel bundle to reconstruct bone graft blood circulation is an efficient way to vascularization. This method can offer rich blood supply to the construction by an independent vascular system, but multiple surgeries, trauma of vascular area and long treatment cycle limit to a small area and the repair of soft tissue trauma. Administration of angiogenic factors such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) and angiopoietin (Ang) together with biological materials to promote the vascularization. However, the angiogenic factors have short half life in vivo, which cannot reach the effective entration. Angiogenic cells combined with osteogenic cells are a more promising approach to vascularize tissue engineered bone. Endothelial Progenitor Cells (EPCs) with strong ability of promoting the angiogenesis after born updates the traditional concept of angiogenesis, and provides a new thought for ischemic disease. EPCs are a kind of immature cells, which can differentiate into endothelial cells and directly involved in angiogenesis. The treatment of animal model of hindlimb ischemia, myocardial ischemia and injuried cornea by EPCs has achieved some success. But the insufficient number of EPCs limits application in larger bone defect area, VEGF-modified EPCs can compensate for the insufficient number of shortcomings for VEGF can directly induce angiogenesis and induce EPCs recruitment. Thus in this experiment, we adopt the approach of angiogenic cells combined with osteogenic cells to promote vascularize bone graft. In vitro co-culture VEGF-EPC with BMP2-BMSC in osteogenic differentiation medium, then detected ALP activity, expression of osteogenic genes such as ALP, OC and Col I and alizarin red staining observed the number of mineralized nodules. Alginate gel mixed with cells was implanted into lateral femoral muscle, after implantation for4and6weeks immunohistochemistry stainning observed blood vessels and bone formation in order to detect the effction of angiogenesis and osteogenesis after combination of VEGF-EPC and BMP2-BMSC.
The main experimental methods and results are as follows:
1. Mononuclear cells from autogenous animal bone marrow had strong proliferative ability when cultured in plastic culture flask in vitro and would amplified in short period, which finally were induced to BMSCs with high purity and quantity. Under some specific conditions, BMSCs could differentiate into osteoblast and adipocyte. So BMSCs were ideal seed cells of bone tissue engineering.
2. Mononuclear cells were separated and purified from the rat bone marrow by density gradient centrifugation. After amplification and culture under special inductive conditions, high purity EPCs could be obtained in vitro, which were identified by cell morphology, cell phenotype and cell function tests including cellular immune fluorescence and tube formation experiment. Mixed EPCs and BMSCs with different proportions and tested cell proliferation by MTT method, the result shows BMSCs:EPCs best mixing ratio of1:1.
3. BMP2and VEGF gene were constructed into recombinant adenovirus expression vector by AdMax vector system (Ad-BMP2、Ad-VEGF). Purified recombinant adenovirus and determined for virus titer:BMSCs and EPCs were transfected by Ad-BMP2and Ad-VEGF in gradient MOI, according to the expression of green fluorescent observation to determine the best MOI viral transfection is40. Growth curve shown that cells proliferative capacity was not affected after adenovirus transfer at MOI=40. Extract RNA of recombinant adenovirus transfection group and blank virus transfection group and blank cell group, Real-TimePCR detected the BMP2and VEGF level. The expression of BMP2in Ad-BMP2-BMSC group and the expression of VEGF in Ad-VEGF-EPC was significantly higher than their respective control group (p<0.05). Cell supernatant of each groups were collected, Elisa detected the expression of BMP2and VEGF in protein level. The expression of BMP2in Ad-BMP2-BMSCs group and the expression of VEGF in Ad-VEGF-EPC was significantly higher than their respective control group (p<0.05). These results proved the effectiveness of Ad-of BMP2and Ad-VEGF.
4. The co-cultured cells designated as four groups including BMSC+EPC, Ad-BMP2-BMSC+EPC, BMSC+Ad-VEGF-EPC, and Ad-BMP2-BMSC+Ad-VEGF-EPC groups and cultured in osteogenic differentiation medium. Osteoblastic gene expression of cultured cells in vitro was evaluated after transplantation for7,14,21, and28days. ALP gene in Ad-BMP2-BMSC+EPC, BMSC+Ad-VEGF-EPC, and Ad-BMP2-BMSC+Ad-VEGF-EPC groups reached a peak level at the21st day. However, Ad-BMP2-BMSC+EPC and Ad-BMP2-BMSC+Ad-VEGF-EPC groups showed higher ALP expression than other groups (p<0.05). All groups showed slow increase in the expression of OC. Interestingly, the Col I expression of all groups increased time dependently, and the Ad-BMP2-BMSC+Ad-VEGF-EPC group had the highest Col I level at21st day (P<0.05).
5. Four groups including BMSC+EPC, Ad-BMP2-BMSC+EPC, BMSC+Ad-VEGF-EPC and Ad-BMP2-BMSC+Ad-VEGF-EPC groups were seeded on an alginate gel and then implanted in rat intramuscularly to evaluate the effects on angiogenesis and osteogenesis. After implantation for4weeks, decalcified sections stained with H&E and sirius red from all groups exhibited ectopic bone formation and Col I formation. The volume of the alginate gel became smaller and smaller due to the degradation and absorption by host tissues. Long-round or shuttle-like cells were observed along the alginate gel. Many neo-collagen tissues and irregular premature bone-like matrix were also observed on the interface between alginate gel and tissues. Newly formed bone began to cover the surface of scaffolds in all groups after4weeks. At week6, the alginate scaffold still remained in vivo, and more collagen tissues were observed. The quantitative analysis showed that Ad-BMP2-BMSC+EPC and Ad-BMP2-BMSC+Ad-VEGF-EPC groups had higher bone area than others at week4and6, and there was significant difference between these two groups at week6(P<0.05). The amount of Col I formation in Ad-BMP2-BMSC+EPC and Ad-BMP2-BMSC+Ad-VEGF-EPC groups was higher than that in the BMSCs+EPC and BMSC+Ad-VEGF-EPC groups at week4and6, but there was no significant difference between Ad-BMP2-BMSC+EPC and Ad-BMP2-BMSC+Ad-VEGF-EPC groups (P<0.05). The occurrence of angiogenesis at4th and6th week was confirmed by the expression of CD31, a special marker of endothelial cells. The BMSC+Ad-VEGF-EPC and Ad-BMP2-BMSC+Ad-VEGF-EPC groups displayed the higher density of blood vessels than other groups at week4and6, and there was significant difference between these two groups at week6(P<0.05), which was also verified by the quantitative analysis for blood vessel density.
6. The RealTime-PCR revealed that VEGF induced7-fold increases in the expression of Id1gene. The expression of Idl gene was found to be up-regulated more than5-fold in Ad-BMP2-BMSC group.
Conclusions:
1. Bone marrow mononuclear cells were separated by density gradient centrifugation which can be induced to BMSCs and EPCs under certain conditions. Angiogenic ability of EPCs and osteogenic differentiation capabilityof BMSCs can be identified by the cell function test, respectively. This method can obtain the high quality BMSCs and EPCs.
2. BMSCs and EPCs could be infected with Ad-BMP2and Ad-VEGF efficiently in vitro. The BMP2was successfully produced in transfected BMSCs and the VEGF can be efficient secreted in EPCs. Then BMP2can enhance osteogenic differentiation of BMSCs and VEGF induce EPCs into the capillary-like structures in vitro.
3. VEGF-modified EPCs co-cultured with BMP2-modified BMSCs can promote the vascularization of transplant tissues.
4. Id1protein might promote proliferation of early osteoblast progenitor cells and. In this regard, we proposed the overexpression of Id1can be induced by BMP2and VEGF, which down-regulated the terminal differentiation of committed osteoblasts and promote angiogenesis. VEGF-modified EPCs co-cultured with BMP2-modified BMSCs can accelerate osteogenesis and bone formation through promoting the vascularization of transplant tissues. Although this approach did not exhibit perfect bone formation, the relationship between Idl gene and osteogenesis has the potential to provide a new vision for the engineered bone tissues.
Originality
1. We demonstrate, for the first time, that BMP modified BMSCs and VEGF-modified EPCs together as seed cells for bone tissue engineering. The vascularization of VEGF-EPC can promote the repair of bone defects and provide a solution and strategie to a large area of bone defect repair.
2. This study was to explore the role of the Idl gene in osteogenesis and angiogenesis, provides a new idea for the study of bone tissue engineering.
以种子细胞复合生物材料的方法修复骨缺损虽然取得了一定的成功,但一些较大面积的骨缺损因植入物缺少充足的血供而发生坏死。因此,有效的血管化已与种子细胞、支架材料及生长因子共同列入骨组织工程的重要因素。预成血管植入是使骨替代物血管化最直接有效的方法,即将血管预先植入骨替代物中,利用显微外科技术将其与宿主体内血管吻合。此种方法可建造独立的血管系统,为植入物提供丰富的血供,可达到良好的血管化效果。但是由于经历多次手术、损伤供血管区及治疗周期长等不足使此方法局限于小面积骨缺损及软组织创伤的修复；诱导血管生成的因子如血管内皮生长因子(Vascular Endothelial Growth Factor, VEGF)、成纤维细胞生长因子(Fibroblast growth factor, FGF)、血管紧张素(Angiopoietin, Ang)的使用可促进血管生成,但因半衰期短局部直接应用效果欠佳。目前将具有血管形成能力的细胞与成骨性细胞的联合应用是血管化组织工程骨最有前景的方法之一,尤为血管内皮前体细胞(Endothelial Progenitor Cells, EPCs)的出现,证明血管发生也存在于发育成熟后的机体组织打破了血管形成的传统理念,为缺血性疾病提供了新思路。EPCs是一种未成熟细胞,可分化为内皮细胞直接参与血管形成,在后肢缺血、心肌缺血及角膜损伤等动物模型的实验研究中取得了一定的成功。但EPCs的细胞数量有限,不足以用于大面积缺血缺损的修复。VEGF基因修饰EPCs可弥补其数量不足的缺点,因为VEGF不仅可以募集、趋化EPCs至缺损处,其本身也可直接诱导血管生成。因此,VEGF与EPCs联合应用应该能够达到事半功倍的效果。基于此,本实验选择具有血管形成能力的细胞与成骨性细胞联合应用的方法促进组织工程骨血管化,观察血管及骨形成情况,探讨VEGF-EPC与BMP2-BMSC联合应用对血管化及成骨作用的影响。
主要实验方法及结果：
1. BMSCs的分离及鉴定：将密度梯度离心法分离的大鼠骨髓单个核细胞通过塑料培养瓶培养、体外扩增传代进行分离纯化获得较高纯度的BMSCs。分别将BMSCs培养于成骨诱导液及成脂诱导液中,茜素红染色及油红O染色证明BMSCs可诱导分化为成骨细胞、成脂肪细胞,通过细胞功能检测鉴定培养的细胞为BMSCs。
2. EPCs的分离及鉴定：采用密度梯度离心法分离大鼠骨髓的单个核细胞,通过特殊培养基诱导,贴壁培养、体外扩增纯化获取细胞,利用细胞形态学、细胞表型检测及细胞免疫双荧光、体外成管实验等细胞功能检测进行鉴定,结果证实此种方法分离诱导纯化的细胞为EPCs。同时将BMSCs及EPCs以不同比例混合培养,MTT法检测细胞的增殖能力,确定BMSCs:EPCs的最佳混合比例为1：1。
3. Ad-BMP2及Ad-VEGF腺病毒载体的构建及有效性鉴定：体外构建Ad-BMP2及Ad-VEGF腺病毒载体,利用AdMax包装系统对其包装、纯化,鉴定重组病毒载体携带目的基因的正确性。
3.1.测定Ad-BMP2的滴度为2.3×1011pfu/ml,Ad-VEGF滴度为3×1011pfu/ml；设置MOI梯度将Ad-BMP2、Ad-VEGF分别转染BMSCs、EPCs,通过对绿色荧光表达的观察确定病毒转染的最佳MOI值为40,并绘制细胞生长曲线证明在MOI=40时细胞的增殖能力没有受到影响；
3.2.提取重组腺病毒转染组、空白病毒转染组及空白细胞组的RNA,实时定量PCR检测显示在RNA水平Ad-BMP2-BMSCs组BMP2及Ad-VEGF-EPCs组VEGF的表达显著高于各自对照组(p<0.05)；分别收集各组细胞上清,Elisa检测BMP2、VEGF蛋白水平的表达,Ad-BMP2-BMSCs组BMP2及Ad-VEGF-EPCs(?)组VEGF的表达显著高于各自对照组(p<0.05)；证明了Ad-BMP2及Ad-VEGF腺病毒载体构建的有效性。
4. VEGF修饰的EPCs对BMP2-BMSC体外成骨分化的影响：将细胞分为BMSC+EPC、Ad-BMP2-BMSC+EPC、BMSC+Ad-VEGF-EPC及Ad-BMP2-BMSC+Ad-VEGF-EPC四组,体外行成骨诱导培养,分别于7d、14d、21d及28d行成骨能力检测。
4.1茜素红染色观察矿化结节：结果显示各组矿化结节数目随时间增加而增多,在第21d及28d,BMP2转染组Ad-BMP2-BMSC+Ad-VEGF-EPC及Ad-BMP2-BMSC+EPC矿化结节数目显著高于其他组(p<0.05)；两组的矿化结节数目在21、28天的变化不明显；
4.2ALP活性检测：随时间增加每组细胞的ALP活性逐渐增强,诱导后第21天各组ALP活性均达最高值,且BMP2转染组Ad-BMP2-BMSC+Ad-VEGF-EPC及Ad-BMP2-BMSC+EPC的ALP活性显著高于其他组(p<0.0J)；但在第28天发现各组间的差异减小,且Ad-BMP2-BMSC+Ad-VEGF-EPC及Ad-BMP2-BMSC+EPC的ALP活性较21d变化微小；
4.3实时定量PCR检测ALP、OC、ColI成骨基因表达：Ad-BMP2-BMSC+EPC、BMSC+Ad-VEGF-EPC及Ad-BMP2-BMSC+Ad-VEGF-EPC组中ALP基因的表达在诱导后第21天达到高峰,其中Ad-BMP2-BMSC+EPC组和Ad-BMP2-BMSC+Ad-VEGF-EPC组ALP的表达显著高于其他各组(P<0.05),每组的OC表达随时间变化增长缓慢,组间没有显著差异；随时间变化各组的Col Ⅰ表达逐渐增多,21天时达到高点,且Ad-BMP2-BMSC+Ad-VEGF-EPC组Col Ⅰ表达水平高于其他各组(P<0.05)。
5. VEGF修饰的EPCs对组织工程骨形成及血管化的影响：将BMSC+EPC、 Ad-BMP2-BMSC+EPC、BMSC+Ad-VEGF-EPC及Ad-BMP2-BMSC+Ad-VEGF-EPC四组细胞分别与藻酸盐凝胶混合注入大鼠股骨外侧肌袋内,每组5只大鼠,分别于4w及6w将动物处死取出植入组织,免疫染色检测骨、Col Ⅰ及血管形成。
5.1石蜡包埋后切片行HE染色检测新生组织中骨生成情况,结果显示混合物注入4周后组织切片HE染色可见藻酸盐凝胶材料数量减少,长圆形、梭形细胞围绕凝胶材料周围；第6周,HE染色仍可观察到材料的残余,能够见到细胞核深染的成骨细胞形成。实验中未见有炎症反应发生。定量分析骨形成面积：在第4周和第6周,Ad-BMP2-BMSC+EPC及Ad-BMP2-BMSC+Ad-VEGF-EPC组新骨形成面积显著高于其他两组(P<0.05)；第6周,Ad-BMP2-BMSC+EPC及Ad-BMP2-BMSC+Ad-VEGF-EPC组组间有显著差异(P<0.05),但Ad-BMP2-BMSC+Ad-VEGF-EPC及Ad-BMP2-BMSC+EPC组新骨形成面积相对第4周增长缓慢；
5.2天狼猩红染色检测Col Ⅰ生成情况：可观察到新胶原组织产生,并间杂有不规则状的未成熟骨样基质。随时间增长,新生胶原组织数量增加。第4及第6周,Ad-BMP2-BMSC+EPC及Ad-BMP2-BMSC+Ad-VEGF-EPC组新胶原形成面积显著高于其他两组(P<0.05)；
5.3血管内皮特殊标记物CD31检测新生物中血管的生成情况,第4及第6周,BMSC+Ad-VEGF-EPC及Ad-BMP2-BMSC+Ad-VEGF-EPC两组血管生成数目显著高于其他两组(P<0.05),且两组间存在显著差异Ad-BMP2-BMSC+Ad-VEGF-EPC组的血管生成数量显著高于BMSC+Ad-VEGF-EPC组(P<0.05)。
6.BMP2及VEGF对Idl表达的影响：分别提取Ad-BMP2-BMSC组、Ad-VEGF-EPC组、空白病毒转染细胞组及空白细胞组的RNA,实时定量PCR检测Ad-BMP2-BMSC组及Ad-VEGF-EPC组Idl基因RNA水平的表达显著高于各自对照组(P<0.05)。
结论：
1.密度梯度离心法分离大鼠骨髓单个核细胞,通过不同的诱导方式对其进行分离、诱导及纯化可分别得到纯度较好的BMSCs及EPCs。
2.通过细胞功能鉴定证明EPCs具有血管形成能力及BMSCs具有成骨分化能力。
3.体外构建Ad-BMP2及Ad-VEGF重组腺病毒表达载体,滴度高转染效率好,可使BMSCs过表达BMP2及EPCs高表达VEGF,可有效促进BMSCs的成骨性能及EPCs的血管形成活性。
4. VEGF修饰的EPCs与BMP2修饰的BMSCs联合应用,可通过细胞间因子交流有效促进血管的形成,VEGF修饰的EPCs为理想的促血管生成种子细胞。
5.BMP2及VEGF诱导Idl的表达升高对成骨细胞终末分化的抑制作用可能会导致骨形成不佳,且Id1可通过募集趋化EPCs促进血管生成；由此推测Idl可能为成骨分化及血管形成的重要分子。Idl在骨形成及血管形成中的作用为骨组织工程的研究提供了新思路。
创新性和意义：
1.本研究的创新点是在国内外首次将BMP修饰的BMSCs与VEGF修饰的EPCs共同作为骨组织工程的种子细胞,利用VEGF-EPC的血管化作用以促进骨缺损的修复,为因血管化不足导致大面积骨组织缺损的修复不良提供解决方法和策略。
2.本研究初步探讨了Idl基因在骨形成及血管形成中的作用,为骨组织工程的研究提供了新思路。
Bone regeneration is required to repair mandible defects arising from trauma, inflammation or tumor. Bone tissue engineering which based on seed cells, grow factors and scaffold material are currently being combined to assess their potential to develop effective concepts for the treatment of extensive loss of osseous tissue. Bone seed cells which share the collective goal of creating osteoconductive and osteoinductive bone graft substitutes is the primary and basic element of bone tissue engineering. Bone marrow stem cells (BMSCs) are pluripotent cells having the potential to differentiate into bone, cartilage, muscle, and ligament have been implicated in the enhancement of bone repair. Although bone morphogenetic protein2(BMP2) can produce orthotopic bone in the hindlimbs of animal model, the short half-life and dispersion limit their effection of directly application. However, the strategy of gene modification can efficiently solve the deficience of directly application and adenovirus vectors have been successfully used for transient gene expression avoiding side effect of gene overexpression. BMP2gene modification of BMSCs provides a new prospect for bone tissue engineering.
Osteogenic stem cells with over-expressed osteogenic factors were less effective in treating large bone defects due to inefficient blood supply in the entire bone graft. The vascularization has been involved in bone tissue engineering with the seed cells, grow factors and scaffold. Implanting the tissue valve embedment with vessel pedicle or blood vessel bundle to reconstruct bone graft blood circulation is an efficient way to vascularization. This method can offer rich blood supply to the construction by an independent vascular system, but multiple surgeries, trauma of vascular area and long treatment cycle limit to a small area and the repair of soft tissue trauma. Administration of angiogenic factors such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) and angiopoietin (Ang) together with biological materials to promote the vascularization. However, the angiogenic factors have short half life in vivo, which cannot reach the effective entration. Angiogenic cells combined with osteogenic cells are a more promising approach to vascularize tissue engineered bone. Endothelial Progenitor Cells (EPCs) with strong ability of promoting the angiogenesis after born updates the traditional concept of angiogenesis, and provides a new thought for ischemic disease. EPCs are a kind of immature cells, which can differentiate into endothelial cells and directly involved in angiogenesis. The treatment of animal model of hindlimb ischemia, myocardial ischemia and injuried cornea by EPCs has achieved some success. But the insufficient number of EPCs limits application in larger bone defect area, VEGF-modified EPCs can compensate for the insufficient number of shortcomings for VEGF can directly induce angiogenesis and induce EPCs recruitment. Thus in this experiment, we adopt the approach of angiogenic cells combined with osteogenic cells to promote vascularize bone graft. In vitro co-culture VEGF-EPC with BMP2-BMSC in osteogenic differentiation medium, then detected ALP activity, expression of osteogenic genes such as ALP, OC and Col I and alizarin red staining observed the number of mineralized nodules. Alginate gel mixed with cells was implanted into lateral femoral muscle, after implantation for4and6weeks immunohistochemistry stainning observed blood vessels and bone formation in order to detect the effction of angiogenesis and osteogenesis after combination of VEGF-EPC and BMP2-BMSC.
The main experimental methods and results are as follows:
1. Mononuclear cells from autogenous animal bone marrow had strong proliferative ability when cultured in plastic culture flask in vitro and would amplified in short period, which finally were induced to BMSCs with high purity and quantity. Under some specific conditions, BMSCs could differentiate into osteoblast and adipocyte. So BMSCs were ideal seed cells of bone tissue engineering.
2. Mononuclear cells were separated and purified from the rat bone marrow by density gradient centrifugation. After amplification and culture under special inductive conditions, high purity EPCs could be obtained in vitro, which were identified by cell morphology, cell phenotype and cell function tests including cellular immune fluorescence and tube formation experiment. Mixed EPCs and BMSCs with different proportions and tested cell proliferation by MTT method, the result shows BMSCs:EPCs best mixing ratio of1:1.
3. BMP2and VEGF gene were constructed into recombinant adenovirus expression vector by AdMax vector system (Ad-BMP2、Ad-VEGF). Purified recombinant adenovirus and determined for virus titer:BMSCs and EPCs were transfected by Ad-BMP2and Ad-VEGF in gradient MOI, according to the expression of green fluorescent observation to determine the best MOI viral transfection is40. Growth curve shown that cells proliferative capacity was not affected after adenovirus transfer at MOI=40. Extract RNA of recombinant adenovirus transfection group and blank virus transfection group and blank cell group, Real-TimePCR detected the BMP2and VEGF level. The expression of BMP2in Ad-BMP2-BMSC group and the expression of VEGF in Ad-VEGF-EPC was significantly higher than their respective control group (p<0.05). Cell supernatant of each groups were collected, Elisa detected the expression of BMP2and VEGF in protein level. The expression of BMP2in Ad-BMP2-BMSCs group and the expression of VEGF in Ad-VEGF-EPC was significantly higher than their respective control group (p<0.05). These results proved the effectiveness of Ad-of BMP2and Ad-VEGF.
4. The co-cultured cells designated as four groups including BMSC+EPC, Ad-BMP2-BMSC+EPC, BMSC+Ad-VEGF-EPC, and Ad-BMP2-BMSC+Ad-VEGF-EPC groups and cultured in osteogenic differentiation medium. Osteoblastic gene expression of cultured cells in vitro was evaluated after transplantation for7,14,21, and28days. ALP gene in Ad-BMP2-BMSC+EPC, BMSC+Ad-VEGF-EPC, and Ad-BMP2-BMSC+Ad-VEGF-EPC groups reached a peak level at the21st day. However, Ad-BMP2-BMSC+EPC and Ad-BMP2-BMSC+Ad-VEGF-EPC groups showed higher ALP expression than other groups (p<0.05). All groups showed slow increase in the expression of OC. Interestingly, the Col I expression of all groups increased time dependently, and the Ad-BMP2-BMSC+Ad-VEGF-EPC group had the highest Col I level at21st day (P<0.05).
5. Four groups including BMSC+EPC, Ad-BMP2-BMSC+EPC, BMSC+Ad-VEGF-EPC and Ad-BMP2-BMSC+Ad-VEGF-EPC groups were seeded on an alginate gel and then implanted in rat intramuscularly to evaluate the effects on angiogenesis and osteogenesis. After implantation for4weeks, decalcified sections stained with H&E and sirius red from all groups exhibited ectopic bone formation and Col I formation. The volume of the alginate gel became smaller and smaller due to the degradation and absorption by host tissues. Long-round or shuttle-like cells were observed along the alginate gel. Many neo-collagen tissues and irregular premature bone-like matrix were also observed on the interface between alginate gel and tissues. Newly formed bone began to cover the surface of scaffolds in all groups after4weeks. At week6, the alginate scaffold still remained in vivo, and more collagen tissues were observed. The quantitative analysis showed that Ad-BMP2-BMSC+EPC and Ad-BMP2-BMSC+Ad-VEGF-EPC groups had higher bone area than others at week4and6, and there was significant difference between these two groups at week6(P<0.05). The amount of Col I formation in Ad-BMP2-BMSC+EPC and Ad-BMP2-BMSC+Ad-VEGF-EPC groups was higher than that in the BMSCs+EPC and BMSC+Ad-VEGF-EPC groups at week4and6, but there was no significant difference between Ad-BMP2-BMSC+EPC and Ad-BMP2-BMSC+Ad-VEGF-EPC groups (P<0.05). The occurrence of angiogenesis at4th and6th week was confirmed by the expression of CD31, a special marker of endothelial cells. The BMSC+Ad-VEGF-EPC and Ad-BMP2-BMSC+Ad-VEGF-EPC groups displayed the higher density of blood vessels than other groups at week4and6, and there was significant difference between these two groups at week6(P<0.05), which was also verified by the quantitative analysis for blood vessel density.
6. The RealTime-PCR revealed that VEGF induced7-fold increases in the expression of Id1gene. The expression of Idl gene was found to be up-regulated more than5-fold in Ad-BMP2-BMSC group.
Conclusions:
1. Bone marrow mononuclear cells were separated by density gradient centrifugation which can be induced to BMSCs and EPCs under certain conditions. Angiogenic ability of EPCs and osteogenic differentiation capabilityof BMSCs can be identified by the cell function test, respectively. This method can obtain the high quality BMSCs and EPCs.
2. BMSCs and EPCs could be infected with Ad-BMP2and Ad-VEGF efficiently in vitro. The BMP2was successfully produced in transfected BMSCs and the VEGF can be efficient secreted in EPCs. Then BMP2can enhance osteogenic differentiation of BMSCs and VEGF induce EPCs into the capillary-like structures in vitro.
3. VEGF-modified EPCs co-cultured with BMP2-modified BMSCs can promote the vascularization of transplant tissues.
4. Id1protein might promote proliferation of early osteoblast progenitor cells and. In this regard, we proposed the overexpression of Id1can be induced by BMP2and VEGF, which down-regulated the terminal differentiation of committed osteoblasts and promote angiogenesis. VEGF-modified EPCs co-cultured with BMP2-modified BMSCs can accelerate osteogenesis and bone formation through promoting the vascularization of transplant tissues. Although this approach did not exhibit perfect bone formation, the relationship between Idl gene and osteogenesis has the potential to provide a new vision for the engineered bone tissues.
Originality
1. We demonstrate, for the first time, that BMP modified BMSCs and VEGF-modified EPCs together as seed cells for bone tissue engineering. The vascularization of VEGF-EPC can promote the repair of bone defects and provide a solution and strategie to a large area of bone defect repair.
2. This study was to explore the role of the Idl gene in osteogenesis and angiogenesis, provides a new idea for the study of bone tissue engineering.
引文
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