鹿茸多肽纳米复合材料对成骨细胞及骨愈合的影响
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摘要
临床上由于创伤、肿瘤、感染所造成的骨缺损很常见,目前缺乏较为理想的植骨材料：自体骨移植常受到自身供体有限性的限制以及术后供区损伤、感染等并发症,异体骨主要存在移植后的免疫排斥反应及传播疾病等问题。组织工程学的问世为人体组织器官缺损的再造和修复开辟了新的途径。模仿天然骨的成分和结构特征制造的骨替代材料,可为细胞提供与天然骨相类似的微环境。β-磷酸三钙(Beta-tricalcium phosphate, P-TCP)因其具有良好的生物相容性和成骨活性,在水溶液和体液中的溶解度是羟基磷灰石(HAP)的10-15倍,能够克服HAP在体内的降解问题而成为骨组织修复领域的主要研究对象。但因脆性大和不易成型的缺点,严重制约了其在临床上的应用。将p-TCP与具有良好韧性的有机物复合,形成无机/有机复合生物材料,既可以解决p-TCP的脆性问题,也可以利用有机物来提高材料的生物学活性。纳米β-磷酸三钙是利用纳米技术在纳米结构单元或纳米数量级(1～100nm)下生产的新型材料,具有小尺寸效应、表面效应,可以在三维空间上提高材料的强度。胶原是人体骨组织中的主要有机成分,在体内可以明显增进细胞间的相互作用,如细胞趋化、细胞增殖等。明胶是胶原蛋白的水解产物,具有与胶原相同的氨基酸,良好的生物相容性,同时还具有较好的黏附作用和骨诱导性,可被人体降解吸收以及较低的免疫原性。鹿茸多肽为鹿茸的主要药理活性成分,具有促进骨髓间质细胞分化,软骨细胞、表皮细胞及成纤维细胞增殖等作用,可明显加速骨组织再生、周围神经组织再生及创面愈合。将鹿茸多肽复合到纳米磷酸三钙明胶微球中不仅可以发挥纳米磷酸三钙材料的骨传导性又可以解决多肽类药物不易通过生物屏障,在伤口及创面易被水解酶破坏等问题,可以持续、稳定、高效地控制鹿茸多肽药物的释放速度,延长半衰期,实现药物的靶向释放,大幅度提高药物的生物利用度,从而达到修复骨缺损与药物治疗的双重效果。基于以上认识,本实验研制了鹿茸多肽纳米复合材料。
实验分五个部分进行观察研究：
1.鹿茸多肽纳米复合材料的制备及性能研究
利用反相微乳液法制备了鹿茸多肽纳米复合材料(nano TCP/gel/VAP),扫描电镜观察纳米复合微球形态,SELDI-TOF-MS质谱仪检测复合材料的组成成分。
2.鹿茸多肽纳米复合材料的生物相容性评价研究
根据国际标准化组织(IS010993)我国(GB/T 16886)对于生物材料相容性的测试要求,通过急性毒性实验、溶血实验、细胞增殖实验和细胞毒性实验研究复合材料的生物安全性。
3.鹿茸多肽纳米复合材料对成骨细胞生物学特性的影响
采用MTT比色法、茜素红染色方法检测nano TCP/gel/VAP对体外培养人成骨细胞的增殖、细胞分泌细胞外钙基质的影响；采用全自动生化分析仪检测成骨细胞碱性磷酸酶活性,通过电镜形态学观察复合材料对受损成骨细胞的影响。
4.鹿茸多肽纳米复合材料对兔下颌骨缺损修复影响的实验研究
建立兔下颌骨缺损模型,通过鹿茸多肽纳米复合材料和单纯的纳米复合材料置兔下颌骨缺损处,术后4、8、12周时通过表面观察、Micro-CT、组织病理学和扫描电镜观察评价鹿茸多肽纳米复合材料的骨修复效果。
5.鹿茸多肽纳米复合材料促成骨细胞早期分化机制的初步研究
通过p38MAPK信号转导阻断剂SB203580对成骨细胞增殖的影响与鹿茸多肽纳米复合材料对SB203580作用下成骨细胞分泌ALP、ColⅠmRNA表达的影响,观察鹿茸多肽纳米复合材料在成骨细胞早期分化过程中的作用。
实验获得以下主要结果和结论：
1.本研究研制了鹿茸多肽纳米复合材料(nano TCP/gel/VAP),扫描电镜观察纳米复合微球形貌良好,直径约20～40μm,具有高度的分散性和均一性,微球表面有纳米级孔洞；通过质谱分析,检测到明胶分子与鹿茸多肽的存在,鹿茸多肽通过静电作用包覆于明胶微球内部。微球形貌规则、尺寸可控,通过调节微球的尺寸可以满足不同骨修复的需要。
2.根据国际标准化组织(ISO10993)和我国(GB/T 16886)对于生物材料相容性的测试要求,证明鹿茸多肽纳米复合材料无细胞毒性,无急性毒性,不引起溶血反应,符合相应的标准要求,具有良好的生物安全性。
3.鹿茸多肽纳米复合材料可以促进人成骨细胞的黏附、增殖,并促进成骨细胞分泌细胞外基质和形成钙结节,促进成骨细胞的分化成熟,对受损的成骨细胞有保护和修复的作用。
4.鹿茸多肽纳米复合材料修复兔下颌骨缺损术后12周,nano TCP/gel/VAP组的骨缺损由骨组织修复,空白对照组的则由纤维组织覆盖,nano TCP/gel组介于上两组之间,边缘是骨组织覆盖,而中心区是纤维组织。Micro-CT检查显示,复合材料组优于空白对照组,nano TCP/gel/VAP组优于nano TCP/gel组。组织学观察nano TCP/gel/VAP组见致密的板层骨,可见哈佛管系统,髓腔再通,板层骨与原骨组织紧密结合；nano TCP/gel组为编织骨修复,缺损中心仍为纤维组织；空白对照组在缺损周边部位可见极少骨岛生成,中心部位仍由纤维组织填充。扫描电镜观察nano TCP/gel/VAP组,为致密的骨样组织,nano TCP/gel组新生骨组织与边界融合不紧密,孔隙较多,结构疏松。空白对照组未见骨组织生成,仍为纤维组织。表明鹿茸多肽纳米复合材料因鹿茸多肽成分的引入在磷酸三钙的传导性基础上增加了骨诱导性能,提高了兔下颌骨缺损的修复效果。
5. p38MAPK阻断剂SB203580使人成骨细胞增殖能力下降,20μM为阻断剂SB203580半数抑制浓度；ALP活性检测结果显示,阻断剂组ALP活性最低,复合材料组ALP活性最高,复合材料加阻断剂组ALP活性低于复合材料组；实时荧光定量PCR结果显示,阻断剂组ColⅠmRNA表达均低于对照组、复合材料组和复合材料加阻断剂组；与复合材料组比较,复合材料加阻断剂组ColⅠmRNA表达在干预初期二者相近,干预时间延长则复合材料加阻断剂组明显低于复合材料组,表明p38MAPK通路在人成骨细胞的分化成熟中起着重要的调控作用,鹿茸多肽纳米复合材料能够逆转阻断剂的作用,进一步促进细胞的ALP活性增高,ColⅠmRNA表达量增加。阻断p38MAPK通路并不能完全抑制复合材料促成骨细胞早期分化作用,提示鹿茸多肽纳米复合材料还可能通过其他信号通路调节成骨细胞的早期分化。
本研究的创新之处,首次将天然活性多肽—鹿茸多肽复合到纳米磷酸三钙、明胶体系中,制备了具有生物活性的鹿茸多肽纳米复合材料。该项研究填补了鹿茸多肽在骨修复新型材料应用中的空白。首次证明了该新型复合材料对下颌骨缺损具有明显的修复作用。该项研究为临床骨缺损修复的新型材料的研究提供了理论和实验依据。
Bone defect caused by injury, tumor and infection is quite common in clinical practice. However, there is still a deficiency of ideal bone graft material. Autogenous bone grafts are often limited by their own limitations and by the injury, infection and other complications of the donor site after operation, while allograft bone has problems in immunologic rejection after transplantation and dissemination of diseases. The advent of tissue engineering provides a innovative approach for the reconstruction and repair of human tissue and organ defects. The bone replacement materials manufactured by modeling the composition and structure of natural bone can provide the cells with microenvironment similar to natural bones. With good biocompatibility and osteogenic activity, the solubility ofβ-TCP in aqueous solution and in body fluids is 10-15 times of hydroxyapatite (HAP) which makes it the main subject in the field of bone tissue repair as it can solve the problem of HAP degradation in the body. On the other hand, the shortcomings of brittle and difficult to shape severely restrict its clinical applications. Compounding the (3-TCP and the organic compound with good toughness to form inorganic/organic composite biomaterials can solve the brittleness of (3-TCP and improve the biological activity by use of organic materials. Nanoβ-TCP is the new materials under the use of nano technology in nano-structure unit or nanometer scale (1～100nm). With a small size effect and surface effect, it can improve the strength of the material three-dimensionally. Collagen is the major organic components of human bones which can significantly enhance the interaction between cells in the body, such as cell chemotaxis and cell proliferation. Gelatin is a collagen hydrolyzate. with the same amino acid as collagen and good biocompatibility, better adhesion as well as bone induction, which can be degraded and absorbed by the body with lower immunogenicity. VAP is the major pharmacologically active component of antler which can promote differentiation of bone marrow stromal cells, proliferation of chondrocytes, epidermal cells and fibroblasts and can significantly accelerate bone tissue regeneration, peripheral nerve tissue regeneration as well as wound healing. The compounding of VAP to the nano-TCP microspheres in the gelatin can not only make full use of bone conduction of nano-TCP materials but also solve the problem that peptide drugs cannot easily go through biological barriers. As to the problem that wound and wound surface can be easily damaged by hydrolase. it can conduct sustainable, stable and efficient control of drug release rate of VAP. extend half-life period, achieve targeted release of drugs, and greatly improve the bioavailability of drugs, thus can repair bone defect and drug treatment. Based on the above understandings, nano TCP/gel/VAP was developed in this study.
Five parts of experimental observation are explained as follows:
1. Preparation and Introduction of nano TCP/gel/VAP Properties
By use of reverse microemulsion. nano TCP/gel/VAP was developed. The specification of nano-composite microspheres was also observed through SEM. Examining components of composite materials by SELDI-TOF-MS mass spectrometer was also part of the experiment.
2. Evaluation of biocompatibility of nano TCP/gel/VAP
According to ISO10993 and China biological material compatibility testing requirements (GB/T 16886). the biological safety of composite materials was studied through the acute toxicity test, hemolysis test, cell proliferation test and cell toxicity test.
3. The effects of nano TCP/gel/VAP on Biological characteristics of osteoblasts
MTT colorimetry and Alizarin red staining were used to detect the effects of nano TCP/gel/VAP on culturing human osteoblasts proliferation, secretion of extracellular calcium matrix by cells in vitro. ALP activity was detected by automatic biochemical analyzer.
4. Experimental study of nano TCP/gel/VAP on the repair of rabbit mandibular defects
By making a rabbit mandible defect model and seting nano TCP/gel/VAP and simple nanocomposites respectively at the defect site,, the effects of nano TCP/gel/ VAP on bone repair effects by gross observation, Micro-CT, histopathology and SEM was evaluated and examined respectively 4 weeks,8 weeks and 12 weeks after surgery.
5. The experimental study of nano TCP/gel/VAP in promoting early differentiation of osteogenic cells
Through the effect of p38MAPK signal transduction inhibitor SB203580 on the proliferation of osteoblasts, the effect of SB203580 and nano TCP/gel/VAP on ALP, ColⅠmRNA secreting from osteogenic cells, the effects of nano TCP/gel/VAP on the process of early differentiation of osteogenic cells were observed.
Main findings and conclusions of the experiments are as followings:
1. This research developes nano TCP/gel/VAP. With the diameter of about 20-40μm, specification of nanocomposite microspheres looks good through SEM, with high degree of dispersion and uniformity. Meanwhile, there are nano-holes on microsphere surface. Through mass spectrometry analysis, the existence of gelatin molecules and VAP are detected. VAP coats on the inside of gelatin microspheres by electrostatic interaction. With a regular shape and controllable size, microspheres can meet the different needs of bone repair by adjusting the size.
2. According to ISO10993 and China biological material compatibility testing requirements(GB/T 16886), it's proved that nano TCP/gel/VAP has no cytotoxicity, no acute toxicity, does not cause hemolysis reaction in line with corresponding standards, and ensures biological safety.
3. Nano TCP/gel/VAP can promote the proliferation of osteoblasts from human embryo, secretion of extracellular matrix from osteoblasts and formation of calcium nodules which promote differentiation and maturation of osteoblasts.
4.Within 12 weeks after the surgery of rabbit mandibular defects repair by the effects of nano TCP/gel/VAP. bone defects in nano TCP/gel/VAP group are repaired by bone tissues, while the ones in blank control group are covered by the fibrous tissues. Nano TCP/gel group is between the two groups in which the edges are covered by bone tissues while the central areas are covered by fibrous tissues. The examination of Micro-CT shows that the composite material group is superior to the blank control group; nano TCP/gel/VAP group is superior to nano TCP/gel group. Dense lamellar bone can be seen by histological nano TCP/gel/VAP group. Harvard tube system, medullary cavity recanalization and integration between lamellar bones and the original bone tissues are also visible. Nano TCP/gel group can repair for bones, while the defect center is still fibrous tissue. In blank control group, it is rare to see bone island generation around the defects and the center is still filled with fibrous tissues. It is dense bone-like tissue under the examination of SEM in nano TCP/gel/VAP group. New bone tissues of nano TCP/gel group are not closely integrated with the boundary, with small holes and loose structure. The blank control group is still fibrous tissue without bone tissue formation. This indicates that nano TCP/gel/VAP increase bone induction on the basis of conductivity of TCP by the introduction of VAP and improves the repair effects of rabbit mandibular defects.
5. p38MAPK inhibitor SB203580 decreases ability of osteoblast proliferation. 20μM is the half density for inhibitor SB203580 to restrain the proliferation of osteoblasts. The result of ALP activity test shows that ALP activity of inhibitor group
实验分五个部分进行观察研究：
1.鹿茸多肽纳米复合材料的制备及性能研究
利用反相微乳液法制备了鹿茸多肽纳米复合材料(nano TCP/gel/VAP),扫描电镜观察纳米复合微球形态,SELDI-TOF-MS质谱仪检测复合材料的组成成分。
2.鹿茸多肽纳米复合材料的生物相容性评价研究
根据国际标准化组织(IS010993)我国(GB/T 16886)对于生物材料相容性的测试要求,通过急性毒性实验、溶血实验、细胞增殖实验和细胞毒性实验研究复合材料的生物安全性。
3.鹿茸多肽纳米复合材料对成骨细胞生物学特性的影响
采用MTT比色法、茜素红染色方法检测nano TCP/gel/VAP对体外培养人成骨细胞的增殖、细胞分泌细胞外钙基质的影响；采用全自动生化分析仪检测成骨细胞碱性磷酸酶活性,通过电镜形态学观察复合材料对受损成骨细胞的影响。
4.鹿茸多肽纳米复合材料对兔下颌骨缺损修复影响的实验研究
建立兔下颌骨缺损模型,通过鹿茸多肽纳米复合材料和单纯的纳米复合材料置兔下颌骨缺损处,术后4、8、12周时通过表面观察、Micro-CT、组织病理学和扫描电镜观察评价鹿茸多肽纳米复合材料的骨修复效果。
5.鹿茸多肽纳米复合材料促成骨细胞早期分化机制的初步研究
通过p38MAPK信号转导阻断剂SB203580对成骨细胞增殖的影响与鹿茸多肽纳米复合材料对SB203580作用下成骨细胞分泌ALP、ColⅠmRNA表达的影响,观察鹿茸多肽纳米复合材料在成骨细胞早期分化过程中的作用。
实验获得以下主要结果和结论：
1.本研究研制了鹿茸多肽纳米复合材料(nano TCP/gel/VAP),扫描电镜观察纳米复合微球形貌良好,直径约20～40μm,具有高度的分散性和均一性,微球表面有纳米级孔洞；通过质谱分析,检测到明胶分子与鹿茸多肽的存在,鹿茸多肽通过静电作用包覆于明胶微球内部。微球形貌规则、尺寸可控,通过调节微球的尺寸可以满足不同骨修复的需要。
2.根据国际标准化组织(ISO10993)和我国(GB/T 16886)对于生物材料相容性的测试要求,证明鹿茸多肽纳米复合材料无细胞毒性,无急性毒性,不引起溶血反应,符合相应的标准要求,具有良好的生物安全性。
3.鹿茸多肽纳米复合材料可以促进人成骨细胞的黏附、增殖,并促进成骨细胞分泌细胞外基质和形成钙结节,促进成骨细胞的分化成熟,对受损的成骨细胞有保护和修复的作用。
4.鹿茸多肽纳米复合材料修复兔下颌骨缺损术后12周,nano TCP/gel/VAP组的骨缺损由骨组织修复,空白对照组的则由纤维组织覆盖,nano TCP/gel组介于上两组之间,边缘是骨组织覆盖,而中心区是纤维组织。Micro-CT检查显示,复合材料组优于空白对照组,nano TCP/gel/VAP组优于nano TCP/gel组。组织学观察nano TCP/gel/VAP组见致密的板层骨,可见哈佛管系统,髓腔再通,板层骨与原骨组织紧密结合；nano TCP/gel组为编织骨修复,缺损中心仍为纤维组织；空白对照组在缺损周边部位可见极少骨岛生成,中心部位仍由纤维组织填充。扫描电镜观察nano TCP/gel/VAP组,为致密的骨样组织,nano TCP/gel组新生骨组织与边界融合不紧密,孔隙较多,结构疏松。空白对照组未见骨组织生成,仍为纤维组织。表明鹿茸多肽纳米复合材料因鹿茸多肽成分的引入在磷酸三钙的传导性基础上增加了骨诱导性能,提高了兔下颌骨缺损的修复效果。
5. p38MAPK阻断剂SB203580使人成骨细胞增殖能力下降,20μM为阻断剂SB203580半数抑制浓度；ALP活性检测结果显示,阻断剂组ALP活性最低,复合材料组ALP活性最高,复合材料加阻断剂组ALP活性低于复合材料组；实时荧光定量PCR结果显示,阻断剂组ColⅠmRNA表达均低于对照组、复合材料组和复合材料加阻断剂组；与复合材料组比较,复合材料加阻断剂组ColⅠmRNA表达在干预初期二者相近,干预时间延长则复合材料加阻断剂组明显低于复合材料组,表明p38MAPK通路在人成骨细胞的分化成熟中起着重要的调控作用,鹿茸多肽纳米复合材料能够逆转阻断剂的作用,进一步促进细胞的ALP活性增高,ColⅠmRNA表达量增加。阻断p38MAPK通路并不能完全抑制复合材料促成骨细胞早期分化作用,提示鹿茸多肽纳米复合材料还可能通过其他信号通路调节成骨细胞的早期分化。
本研究的创新之处,首次将天然活性多肽—鹿茸多肽复合到纳米磷酸三钙、明胶体系中,制备了具有生物活性的鹿茸多肽纳米复合材料。该项研究填补了鹿茸多肽在骨修复新型材料应用中的空白。首次证明了该新型复合材料对下颌骨缺损具有明显的修复作用。该项研究为临床骨缺损修复的新型材料的研究提供了理论和实验依据。
Bone defect caused by injury, tumor and infection is quite common in clinical practice. However, there is still a deficiency of ideal bone graft material. Autogenous bone grafts are often limited by their own limitations and by the injury, infection and other complications of the donor site after operation, while allograft bone has problems in immunologic rejection after transplantation and dissemination of diseases. The advent of tissue engineering provides a innovative approach for the reconstruction and repair of human tissue and organ defects. The bone replacement materials manufactured by modeling the composition and structure of natural bone can provide the cells with microenvironment similar to natural bones. With good biocompatibility and osteogenic activity, the solubility ofβ-TCP in aqueous solution and in body fluids is 10-15 times of hydroxyapatite (HAP) which makes it the main subject in the field of bone tissue repair as it can solve the problem of HAP degradation in the body. On the other hand, the shortcomings of brittle and difficult to shape severely restrict its clinical applications. Compounding the (3-TCP and the organic compound with good toughness to form inorganic/organic composite biomaterials can solve the brittleness of (3-TCP and improve the biological activity by use of organic materials. Nanoβ-TCP is the new materials under the use of nano technology in nano-structure unit or nanometer scale (1～100nm). With a small size effect and surface effect, it can improve the strength of the material three-dimensionally. Collagen is the major organic components of human bones which can significantly enhance the interaction between cells in the body, such as cell chemotaxis and cell proliferation. Gelatin is a collagen hydrolyzate. with the same amino acid as collagen and good biocompatibility, better adhesion as well as bone induction, which can be degraded and absorbed by the body with lower immunogenicity. VAP is the major pharmacologically active component of antler which can promote differentiation of bone marrow stromal cells, proliferation of chondrocytes, epidermal cells and fibroblasts and can significantly accelerate bone tissue regeneration, peripheral nerve tissue regeneration as well as wound healing. The compounding of VAP to the nano-TCP microspheres in the gelatin can not only make full use of bone conduction of nano-TCP materials but also solve the problem that peptide drugs cannot easily go through biological barriers. As to the problem that wound and wound surface can be easily damaged by hydrolase. it can conduct sustainable, stable and efficient control of drug release rate of VAP. extend half-life period, achieve targeted release of drugs, and greatly improve the bioavailability of drugs, thus can repair bone defect and drug treatment. Based on the above understandings, nano TCP/gel/VAP was developed in this study.
Five parts of experimental observation are explained as follows:
1. Preparation and Introduction of nano TCP/gel/VAP Properties
By use of reverse microemulsion. nano TCP/gel/VAP was developed. The specification of nano-composite microspheres was also observed through SEM. Examining components of composite materials by SELDI-TOF-MS mass spectrometer was also part of the experiment.
2. Evaluation of biocompatibility of nano TCP/gel/VAP
According to ISO10993 and China biological material compatibility testing requirements (GB/T 16886). the biological safety of composite materials was studied through the acute toxicity test, hemolysis test, cell proliferation test and cell toxicity test.
3. The effects of nano TCP/gel/VAP on Biological characteristics of osteoblasts
MTT colorimetry and Alizarin red staining were used to detect the effects of nano TCP/gel/VAP on culturing human osteoblasts proliferation, secretion of extracellular calcium matrix by cells in vitro. ALP activity was detected by automatic biochemical analyzer.
4. Experimental study of nano TCP/gel/VAP on the repair of rabbit mandibular defects
By making a rabbit mandible defect model and seting nano TCP/gel/VAP and simple nanocomposites respectively at the defect site,, the effects of nano TCP/gel/ VAP on bone repair effects by gross observation, Micro-CT, histopathology and SEM was evaluated and examined respectively 4 weeks,8 weeks and 12 weeks after surgery.
5. The experimental study of nano TCP/gel/VAP in promoting early differentiation of osteogenic cells
Through the effect of p38MAPK signal transduction inhibitor SB203580 on the proliferation of osteoblasts, the effect of SB203580 and nano TCP/gel/VAP on ALP, ColⅠmRNA secreting from osteogenic cells, the effects of nano TCP/gel/VAP on the process of early differentiation of osteogenic cells were observed.
Main findings and conclusions of the experiments are as followings:
1. This research developes nano TCP/gel/VAP. With the diameter of about 20-40μm, specification of nanocomposite microspheres looks good through SEM, with high degree of dispersion and uniformity. Meanwhile, there are nano-holes on microsphere surface. Through mass spectrometry analysis, the existence of gelatin molecules and VAP are detected. VAP coats on the inside of gelatin microspheres by electrostatic interaction. With a regular shape and controllable size, microspheres can meet the different needs of bone repair by adjusting the size.
2. According to ISO10993 and China biological material compatibility testing requirements(GB/T 16886), it's proved that nano TCP/gel/VAP has no cytotoxicity, no acute toxicity, does not cause hemolysis reaction in line with corresponding standards, and ensures biological safety.
3. Nano TCP/gel/VAP can promote the proliferation of osteoblasts from human embryo, secretion of extracellular matrix from osteoblasts and formation of calcium nodules which promote differentiation and maturation of osteoblasts.
4.Within 12 weeks after the surgery of rabbit mandibular defects repair by the effects of nano TCP/gel/VAP. bone defects in nano TCP/gel/VAP group are repaired by bone tissues, while the ones in blank control group are covered by the fibrous tissues. Nano TCP/gel group is between the two groups in which the edges are covered by bone tissues while the central areas are covered by fibrous tissues. The examination of Micro-CT shows that the composite material group is superior to the blank control group; nano TCP/gel/VAP group is superior to nano TCP/gel group. Dense lamellar bone can be seen by histological nano TCP/gel/VAP group. Harvard tube system, medullary cavity recanalization and integration between lamellar bones and the original bone tissues are also visible. Nano TCP/gel group can repair for bones, while the defect center is still fibrous tissue. In blank control group, it is rare to see bone island generation around the defects and the center is still filled with fibrous tissues. It is dense bone-like tissue under the examination of SEM in nano TCP/gel/VAP group. New bone tissues of nano TCP/gel group are not closely integrated with the boundary, with small holes and loose structure. The blank control group is still fibrous tissue without bone tissue formation. This indicates that nano TCP/gel/VAP increase bone induction on the basis of conductivity of TCP by the introduction of VAP and improves the repair effects of rabbit mandibular defects.
5. p38MAPK inhibitor SB203580 decreases ability of osteoblast proliferation. 20μM is the half density for inhibitor SB203580 to restrain the proliferation of osteoblasts. The result of ALP activity test shows that ALP activity of inhibitor group
引文
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