RGD多肽修饰国产多孔钽修复兔桡骨节段性骨缺损的实验研究

发布时间：2018-04-20 10:08

本文选题：多孔钽 + RGD多肽　；参考：《南方医科大学》2016年博士论文

【摘要】：第一章RGD多肽修饰国产多孔钽支架材料的制备目的：利用环RGD多肽表面修饰国产多孔钽支架材料,探讨RGD多肽修饰前、后多孔钽支架材料的形貌特征及亲水性变化。方法：1 RGD多肽修饰多孔钽支架材料制备方法：多孔钽片经消毒后浸泡于浓度为100μmol/L的环RGD多肽磷酸盐缓冲液中,室温下不停振动反应24h,PBS冲洗3次,在层流分流室自然干燥,后经紫外线照射消毒,备用。2多孔钽形貌特征观察：大体及扫描电镜观察经RGD多肽修饰前、后多孔钽。3支架材料亲水性测定：取RGD多肽修饰前、后的各6枚钽片分别称重(W1),于室温下浸泡于去离子水中24h,滤纸除去多余水分,电子天平准确记录其重量(W2)。利用支架材料吸水率计算公式(吸水率=[(W2-W1)/W1]×100%),测定两组样品吸水率,并行统计学分析。结果：1多孔钽经RGD多肽修饰前、后形貌特征变化：大体观察两组支架材料外观颜色均为灰黑色,表面光洁。表面及断面可见蜂窝状孔隙,分布均匀。扫面电镜观察两组支架材料表面可见孔径约400-600μm的微孔结构,孔隙内部有直径为50～200μm的连通小孔。其中RGD多肽修饰后支架材料表面可见斑点状涂层,分布薄厚均匀。2支架材料亲水性测定：测得RGD多肽修饰前、后多孔钽吸水率分别为1.98±0.37%,4.83±0.30%。RGD多肽修饰后多孔钽支架材料吸水率明显高于修饰前支架,经统计学分析两者比较有显著性差异(P0.01)。结论：RGD多肽修饰国产多孔钽支架形貌特征未发生明显变化,而其亲水性明显提高。第二章RGD多肽修饰国产多孔钽支架材料对成骨细胞增殖、黏附影响的体外实验目的：通过体外实验研究探讨RGD多肽修饰多孔钽支架材料对成骨细胞增殖、早期黏附等生物学行为的影响,为国产多孔钽作为骨组织工程支架材料修复骨缺损进行体内实验及临床应用提供实验依据。方法：1成骨细胞分离培养：取新生24 h内新西兰乳兔4只,无菌条件下取颅盖骨,剪成1mm×1mm的小块,置入5ml 0.25%胰蛋白酶离心管中37℃消化30min,加入5ml 0.1％II型胶原酶37℃振荡消化60min,离心后所得沉淀物加入完全培养基,吹打成细胞悬液,使细胞浓度达105/ml,接种于25cm2培养瓶中。待细胞生长至铺满培养瓶底80-90%时,进行传代,取生长状态良好的第2代细胞用于实验。2细胞形态学观察：倒置相差显微镜下观察细胞生长情况并记录下各个时期的细胞形态变化。3成骨细胞鉴定：取生长状态良好的第2代细胞,接种于预置无菌盖玻片的12孔板内,当细胞长满80～90%时,进行ALP染色,鉴定成骨细胞。4实验分组及成骨细胞在多孔钽支架材料培养,形态学及生长情况观察：实验分组A组：环RGD修饰多孔钽组,B组：单纯多孔钽组。空白对照组：将成骨细胞接种于不放支架的24孔板。将第2代生长良好的成骨细胞与多孔钽复合培养,经倒置相差显微镜观察成骨细胞形态变化及生长情况。5 RGD多肽修饰多孔钽对成骨细胞黏附的影响：取生长状态良好的第2代成骨细胞,将其调整成浓度为1×106/ml单细胞悬液,用微量移液器吸取100μ1(1×105个细胞)接种到各组支架表面,于2h、4h分别取出各组支架样本,利用沉淀法检测各组支架成骨细胞黏附率,并行统计学分析。通过扫面电镜观察4h时两组钽支架表面成骨细胞黏附形态、数量情况。6 RGD多肽修饰多孔钽支架材料对成骨细胞增殖的影响：取浓度为2.5×105/ml的单细胞悬液200μl(5×104个细胞)接种到各组支架表面,培养1d、3d、5d、7d,在每个时间点培养基内加入MTT溶液,使用BIO-RAD酶联免疫检测仪(测定波长为490nm,参比波长为650nm)测定各组光吸收值(OD值),绘制各组成骨细胞增殖曲线图,并对不同时间点各组成骨细胞增殖率进行统计学分析。通过扫面电镜观察3d时两组钽支架表面成骨细胞形态及生长增殖情况。结果：1成骨细胞分离培养及鉴定：刚接种的原代细胞悬浮于培养基中,倒置相差显微镜下可见细胞呈透亮的圆球形,且大小一致；接种6h时,细胞有部分贴壁,24h左右细胞完全贴壁铺伸。第3d时,细胞数量增多,体积增大,细胞借伪足样突起相互连接。第6-7d时细胞数量继续增多,相互融合几乎铺满培养瓶底部,成单层或不规则样改变。第2代细胞形态趋于单一化,呈长梭形或多角形。经ALP染色可见细胞浆内可见蓝染颗粒,细胞核为红色,证实为成骨细胞。2倒置相差显微镜下观察成骨细胞与多孔钽支架材料复合培养形态特征及生长情况：两组多孔钽材料边缘黏附的细胞逐渐数量增多,排列密集,形态良好,但A组细胞数量、密集程度均高于B组。3成骨细胞黏附率的测定：在2h、4h两个时间点上,A组黏附率均高于B组及空白对照组,比较差异有统计学意义(P0.05)；而B组与空白对照组黏附率相近,二者无统计学差异(P0.05)。各组细胞黏附率在2h、4h两时间点之间比较均有统计学差异(P0.05)。4成骨细胞增殖率的测定：第1、3、5、7d四个时间点各组间比较,A组OD值均高于B组及空白对照组,并且差异具有统计学意义(P0.05);而B组与空白对照组OD值相近,差别无统计学意义(P0.05)。5扫描电镜下观察成骨细胞在支架上的形态特征及生长情况：在4h时,A组支架材料上成骨细胞伸展变形较好,而B组支架材料上成骨细胞多数为类圆形,伸展变形较慢,同时A组支架材料表面黏附细胞较密集,数量明显高于B组。在第3d时,两组支架材料上成骨细胞生长状态均良好,细胞之间结合紧密,其中A组多孔钽表面的细胞密度及细胞分泌胞外基质的情况均优于B组。结论：1国产多孔钽支架材料具有良好的生物相容性,无细胞毒性,对成骨细胞的黏附、增殖没有影响。2 RGD多肽修饰多孔钽支架材料对成骨细胞的增殖、黏附有促进作用,有望成为骨组织工程的理想支架。第三章RGD多肽修饰国产多孔钽支架修复兔桡骨节段性骨缺损的实验研究目的：建立兔桡骨节段性骨缺损模型,通过影像学、组织学、生物力学等多种检测方法评价RGD多肽修饰多孔钽支架体内生物相容性及骨缺损的修复能力,从而为国产多孔钽临床应用提供体内实验依据。方法：1材料准备及分组：将多孔钽材料制成直径为3.5mm,长15mm的圆柱体,经浓度为100μmol/L的RGD多肽溶液修饰后(操作步骤见第一章),紫外线照射消毒,备用。选取105只6-8月龄新西兰大白兔,随机抽取分为5组,A组：多孔钽+RGD多肽组24只,B组：多孔钽+筋膜包裹组24只,C组：单纯多孔钽组24只,D组：异种骨组24只,E组：空白组9只。2手术步骤：麻醉成功后右前肢切口周围备皮,消毒后取右前臂桡侧中段纵形切口,长约3.5cm,以桡骨弧顶为中心,用卡尺测量出15mm长度,用微型摆锯低速截骨,造成一1.5cm节段性骨缺损,生理盐水冲洗伤口后,以嵌插方式分别植入A、B、C、D组植入物。其中B组筋膜瓣制作：沿桡骨中段切开皮肤,游离并切取约30mmx25mm富含毛细血管网的带蒂筋膜瓣,包裹嵌插至桡骨骨缺损处的植入物。E组为空白组,只做截骨,未加植入物。逐层闭合伤口。术后给予预防感染治疗。3术后实验兔一般情况：观察动物饮食、日常活动及伤口愈合情况。4X线检查：分别于术后当日、4、8、16周行实验兔右桡骨正位X线检查,观察界面骨愈合情况。5大体观察：术后4、8、16周三个时间点,切除各组桡骨标本表面软组织,肉眼观察骨缺损部位的大体修复情况。6组织学检查：术后4、8、16周三个时间点,各组标本经脱钙、石蜡切片HE染色及不脱钙硬组织切片甲苯胺蓝染色观察植入物材料与宿主骨界面及材料内部新生骨生长情况。7植入物与骨界面扫描电镜观察：上述三个时间点观察A、B、C、D组界面、材料表面及孔隙内部类骨样组织生长情况。9生物力学检测：取术后16周A、B、C、D组完整桡骨及对侧正常桡骨标本行三点弯曲试验,检测修复后桡骨生物力学性能。9 Micro-CT扫描评估：取术后16周A、B、C、D组以植入物为中心,截取长度为2.5cm带尺、桡骨标本行Micro-CT扫描、三维重建及新生骨计量学分析。结果：1术后动物一般情况：术后3-7天后动物饮食、精神逐渐恢复正常,切口部位无红肿、渗液及化脓等,均为I期愈合；2周后肢体活动基本恢复正常,跛行消失,体重恢复到术前水平。2 X线观察：术后当日X线可见各组植入物位置良好,无明显位移。术后4、8、16周三个时间点可见随时间延长各组植入物与宿主骨界面结合越来越牢固,骨折线逐渐消失,其中D组骨痂塑形良好,骨髓腔部分再通；在A、B、C三组中,A组界面骨痂生成最多,塑形良好；E组术后16周仍可见骨缺损,两断端骨质逐渐硬化,髓腔封闭。3大体标本观察：术后4、8、16周三个时间点A组材料表面逐渐被骨样组织包裹,界面与宿主骨结合牢固,塑形良好。B、C组界面结合牢固,表面材料孔隙大部分可见骨样组织填充,靠近界面部分表面被骨组织覆盖。D组植入材料逐渐降解,材料降解部分被新生骨样组织替代,有明显的毛细血管懌长入,材料与宿主骨界面融合良好、牢固。术后4周空白组断端可见少量骨痂生长,16周时断端骨质硬化,光滑,髓腔已经封闭,缺损处无骨性连接形成。4组织学检查：术后4、8、16周各组标本经脱钙、石蜡切片HE染色及不脱钙硬组织切片甲苯胺蓝染色,光镜观察可见随着时间推移,A-C三组钽-宿主骨界面新生骨组织数量逐渐增多并沿多孔钽孔隙长入材料内部,骨组织由幼稚到成熟；D组异种骨逐渐降解并被新生骨组织包裹替代；而E组(空白对照组)骨缺损始终存在,洞壁被薄层纤维膜内衬。5植入物与骨界面扫描电镜观察：术后4、8、16周三个时间点观察可见A、B、C三组钽-骨界面、材料表面及孔隙内部骨胶原及成骨细胞逐渐增多,新生骨逐渐成熟并过度为板层骨,钽-骨界面缝隙逐渐结合紧密；D组新生骨组织逐渐增多,并分化为成熟状小梁骨。6生物力学检测：术后16周对A、B、C、D组及对照组正常完整桡骨行三点弯曲试验测定最大载荷力及抗弯曲强度,结果显示A、B、C、D四组无论是最大载荷力还是抗弯曲强度均低于正常桡骨组,并且有统计学差异(P0.01)。A、B、C、D四组间比较,A组力学性能最高,B、C组次之,D组最低,经单因素方差分析各组间差异均有统计学意义(P0.01)。7 Micro-CT扫描观察：术后16周,A、B、C、D四组植入物界面、表面均有大量骨组织覆盖,内部不同程度新生骨组织填充。新生骨体积分数定量分析结果显示D组新生骨组织百分比高于A、B、C三组,其中A组与D组无统计学差异(P0.05),B、C组与D组有统计学差异(P0.01); A, B, C三组间有统计学差异(P0.01)。结论：国产多孔钽材料具有良好的生物相容性,RGD多肽修饰多孔钽支架材料骨传导能力更强,修复兔桡骨节段性骨缺损效果肯定。
[Abstract]:In the first chapter, the preparation of domestic porous tantalum scaffold materials with RGD polypeptide modified homemade porous tantalum scaffold materials: the surface modification of domestic porous tantalum scaffold materials on the surface of RGD polypeptide was used to explore the morphologies and hydrophilicity of the porous tantalum scaffold material before the modification of RGD polypeptide. Method: the preparation method of porous tantalum scaffold materials with 1 RGD polypeptide modified porous tantalum scaffold: porous tantalum slices were dipped after disinfection In the ring RGD polypeptide phosphate buffer solution with a concentration of 100 mol/L, the vibrational reaction of 24h, PBS flush for 3 times at room temperature, then naturally dried in the laminar flow division chamber, and then sterilized by ultraviolet radiation, and observed by the.2 porous tantalum morphology: the hydrophilicity of the porous tantalum.3 scaffold was measured by the general and scanning electron microscope before the RGD polypeptide was modified. Before the modification of RGD polypeptide, the 6 tantalum slices were weighed (W1) respectively. At room temperature, they were soaked in the deionized water 24h, the filter paper removed the excess water, and the weight was recorded accurately by the electronic balance (W2). The water absorption rate of the scaffold was calculated by using the water absorption rate = [(W2-W1) /W1] x 100%), and the water absorption rate of the two groups was measured, and the results were statistically analyzed. The results were 1 porous. The morphology of tantalum was changed after RGD peptide modification. The appearance of two groups of scaffold materials were all gray and black, and the surface and surface were smooth. The surface and cross section showed the honeycomb pore and the distribution was uniform. The surface and the surface of the two groups of scaffolds were observed to have a pore structure of about 400-600 mu m, with a diameter of 50~200 mu m in the pores. On the surface of RGD polypeptide, the surface of the scaffold material can be seen on the surface of the scaffold. The hydrophilicity of the thin and uniform.2 scaffold material is measured. The water absorption rate of the porous tantalum was 1.98 + 0.37% before the RGD polypeptide modification, and the water absorption of the porous tantalum scaffold was significantly higher than that of the pre modified scaffold after the 4.83 + 0.30%.RGD polypeptides modified. Two There was a significant difference (P0.01). Conclusion: the morphology of the domestic porous tantalum scaffold modified by RGD polypeptide did not change obviously, but its hydrophilicity was obviously improved. The effect of the second chapter RGD polypeptide modified domestic porous tantalum scaffold material on the proliferation and adhesion of osteoblasts was in vitro: the modification of RGD polypeptide through the experiment in vitro The effects of porous tantalum scaffold on the biological behavior of osteoblast proliferation and early adhesion are provided for the experiment and clinical application of domestic porous tantalum as scaffold material for bone tissue engineering to repair bone defects. Methods: 1 osteoblasts were isolated and cultured: 4 New Zealand New Zealand milk rabbits were taken from 24 h, and the skull was taken under aseptic conditions. The small pieces of 1mm x 1mm were cut into the 5ml 0.25% trypsin centrifuge tube to digest 30min at 37 degrees C, and 5ml 0.1%II collagenase was added to the 60min. The precipitates were added to the complete medium after centrifugation, and the cells were blown into cell suspension, and the cell concentration was 105/ml and inoculated into the 25cm2 culture bottle. When the cell was grown to the bottom 80-90% of the culture bottle, The second generation cells with good growth state were used to observe the morphological observation of the experimental.2 cells: the cell growth was observed under the inverted phase contrast microscope and the morphological changes of the cells at various stages were recorded and.3 osteoblasts were recorded. The second generation cells with good growth state were inoculated to the 12 foramen of the pre sterile cover glass, and the cell length was long. At the time of 80 to 90%, ALP staining was carried out to identify the.4 experimental group of osteoblasts and the culture of osteoblasts in the porous tantalum scaffold materials, morphology and growth. The experimental group A group: ring RGD modified porous tantalum group, B group: pure porous tantalum group. The blank control group was inoculated to the 24 hole plate without stent. The second generation good growth was good. Good osteoblasts were cultured with porous tantalum, and the morphology and growth of osteoblasts were observed by inverted phase contrast microscope. The effect of.5 RGD polypeptide on the adhesion of porous tantalum on osteoblast: second generation of osteoblasts with good growth status were adjusted to a concentration of 1 x 106/ml single cell suspension, and 100 micron 1 (1 x) was absorbed by a micropipette. 105 cells were inoculated on the surface of each scaffold. The scaffold samples were taken out by 2H and 4H respectively. The adhesion rate of osteoblasts was detected by precipitation method. The adhesion morphology of the two groups of tantalum scaffolds on the surface of tantalum scaffold was observed by scanning electron microscopy. The number of.6 RGD polypeptide modified the porous tantalum scaffold material to osteoblasts. The effect of proliferation: a single cell suspension of 2.5 * 105/ml (5 x 104 cells) was inoculated to the surface of each scaffold, and 1D, 3D, 5D, 7d were cultured. MTT solution was added to each time point culture, and the BIO-RAD enzyme immunodetector (wavelength 490nm, reference wavelength was 650nm) was used to determine the optical absorption values of each group (OD value), and the composition of each component was plotted. The proliferation rate of bone cells in different time points was statistically analyzed. The morphology and growth and proliferation of two groups of tantalum scaffolds on the surface of tantalum scaffold were observed by scanning electron microscopy. Results: 1 osteoblasts were isolated and cultured and identified: primary cell suspension in the culture medium and inverted phase contrast microscope in the two groups At the time of inoculation 6h, the cells were partially adhered to the cells, and the cells were partially adhered to the wall, and the cells were completely plaster and extended. At the time of 3D, the number of cells increased, the volume increased, and the cells were connected by the pseudo foot like protrusions. At the time of 6-7d, the number of cells continued to increase and the phase interfusion almost covered the bottom of the culture bottle, formed monolayer or irregularity. The morphology of the second generation of cells tended to be single, with long spindle shape or polygon. The blue dye particles were visible in the cytoplasm by ALP staining, and the nuclei were red. It was confirmed that the morphological characteristics and growth conditions of osteoblasts and porous tantalum scaffold materials were observed under the inverted phase contrast microscope of.2 osteoblasts: the marginal viscosity of the two groups of porous tantalum materials. The number of cells in the attached cells was increased and the morphology was good, but the number of cells in the A group was higher than that of the B group.3 osteoblast adhesion rate. At the two time points of 2H and 4h, the adhesion rate of group A was higher than that of the B group and the blank control group, and the difference was statistically significant (P0.05), but the adhesion rate of the B group and the blank control group was similar, and the rate of adhesion was similar in the B group and the blank control group. There was no one in the group of B and the blank control group. Statistical difference (P0.05). The cell adhesion rate of each group at 2h, 4H two time points were statistically different (P0.05) the proliferation rate of.4 osteoblast: the 1,3,5,7d four time points were compared in each group, the A group was higher than the B group and the blank control group, and the difference was statistically significant (P0.05), while the B group was similar to the blank control group. There was no statistical significance (P0.05).5 scanning electron microscope to observe the morphological characteristics and growth of osteoblasts on the scaffold: at 4h, the osteoblasts in the A stents were extended and deformed, while the majority of the osteoblasts in the B group were round, and the extensional deformation was slow, and the surface adhesion cells of the A stents were denser and more dense. It was significantly higher than group B. At 3D, the growth state of osteoblasts on the two groups of scaffolds was good and the cells were tightly bonded. The cell density on the surface of the porous tantalum on the A group and the secretion of extracellular matrix were superior to that of the B group. Conclusion: 1 domestic porous tantalum scaffold materials have good biocompatibility, no cytotoxicity, and osteoblasts. Adhesion, proliferation does not affect the proliferation of.2 RGD polypeptide modified porous tantalum scaffold materials for osteoblasts. Adhesion has a promoting effect, and it is expected to be an ideal scaffold for bone tissue engineering. Third experimental research on the repair of rabbit radial segmental bone defect with homemade porous tantalum scaffold modified by RGD polypeptide: to establish a rabbit radial segmental bone defect model, The biocompatibility and repair ability of RGD polypeptides modified porous tantalum scaffold were evaluated by various methods of imaging, histology and biomechanics, so as to provide the experimental basis for the clinical application of domestic porous tantalum. Method: 1 material preparation and grouping: the porous tantalum material was made into a cylinder with a diameter of 3.5mm and a long 15mm. After the modification of the RGD polypeptide solution with a concentration of 100 mol/L (see Chapter 1), UV irradiation and disinfection, the 105 6-8 month old New Zealand white rabbits were selected randomly and divided into 5 groups randomly, A group: 24 porous tantalum +RGD polypeptide group, 24 B group: porous tantalum + fascia package group, C group: pure porous tantalum group 24, D group, 24 bone group, E Group: 9 steps of.2 operation in the blank group: skin preparation around the right forelimb incision after anesthesia was successful. After disinfection, the right forearm radial middle section was taken longitudinally. The length of the radial incision was about 3.5cm. The length of 15mm was measured with a caliper. A 1.5cm segmental bone defect was caused by the micro sawing the low speed osteotomy. After the saline was used, the wound was inserted into the wound. Do not implant the implants of group A, B, C, and D. Among them, group B was made of fascial flap: incision of the skin along the middle of the radius, free and cut off the pedicled fascial flap with the capillary network of 30mmx25mm, and the.E group inserted into the radius bone defect in group.E as the blank group, only osteotomy, no implants and closed wounds. Postoperative.3 surgery was given to prevent infection for.3 surgery. The general condition of the rabbit after the experiment: Observation of animal diet, daily activity and wound healing.4 X - ray examination: on the day after the operation, the right radiography of the right radius of the rabbit was examined on the day of 4,8,16, and the bone healing of the interface was observed by the gross observation of.5: the surface soft tissue of the radius specimens were removed and the naked eye was observed by the naked eye after the operation of the 4,8,16 Wednesday. General repair of the site.6 histological examination: 4,8,16 Wednesday postoperatively, specimens of each group were decalcified, paraffin section HE staining, and DT staining was used to observe the interface between the implant and the host bone and the growth of the internal bone in the material. The.7 implants and the bone interface were observed by scanning electron microscope: the three time A, B, C, D group interface, material surface and internal bone like tissue growth of.9: 16 weeks after the operation: A, B, C, D group complete radius and three point bending test on the contralateral normal radius, and detect the.9 Micro-CT scanning of the radial biomechanics after repair: 16 weeks after the operation. Heart, the length of intercepted length was 2.5cm tape, Micro-CT scan of radial mark, three-dimensional reconstruction and new bone Metrology Analysis. Results: 1 animal general condition after operation: 3-7 days after operation, the animal diet, the spirit gradually resumed normal, the incision site was not red, the seepage and suppuration were all I, after 2 weeks, the extremities were basically restored to normal, limping. .2 X - ray observation of loss, weight recovery to preoperative level: on the day after operation, the X - ray showed that the implants were in good position and no obvious displacement. After 4,8,16 Wednesday, the interface of the implant and the host bone became more and more firm, and the fracture line gradually disappeared, in which the callus of group D was well shaped and the bone marrow cavity was repassed; in A, B, C three groups, A group interface callus formation most, shape good shape, E group 16 weeks after the bone defect still visible, the two broken end bone gradually hardened, the pulp cavity closed.3 gross specimen observation: 4,8,16 Wednesday time point A group material surface gradually wrapped by bone like tissue, the interface and host bone solid, good plastic.B, C group interface firm, C group interface firm, C group.B, C group interface firmly, C Most of the pores in the surface of the surface were filled with bone like tissue, and the.D group was gradually degraded on the surface near the surface of the interface, and the degradation part of the material was replaced by the new bone like tissue. There was a clear capillary vein, and the fusion of the material and the host bone was good and solid. A small amount of callus growth was seen in the 4 weeks after the operation, 1 At 6 weeks, bone sclerosis, smooth, pulp cavity had been closed, and no bone connection was formed in the defect to form a.4 histological examination. After 4,8,16 weeks, the specimens were decalcified, paraffin section HE staining, and decalcified hard tissue section toluidine blue staining. The number of new bone tissue in the tantalum host bone interface of the three groups was observed gradually as time went on, and the number of new bone tissue in the tantalum host bone interface was gradually increased as time went on. The bone tissue was increased from naive to maturity along the porous tantalum pores, and the bone tissue was degraded gradually and replaced by the new bone tissue in D group, while the bone defect existed in group E (blank control group), and the wall was scanned by.5 implants and bone interface by thin layer fibrous membrane, and the observation was observed at the time of 4,8,16 Wednesday after the operation. A, B, C three groups of tantalum bone interface, the material surface and pore bone collagen and osteoblasts increased gradually, the new bone gradually matured and overworked.

【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R318.08;R687

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