复合PRP联合bBMP组织工程骨修复兔桡骨缺损的实验研究
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摘要
背景及目的
骨缺损的修复和再生一直是骨科学研究领域中的重要课题,目前研究热点是利用生长因子来促进、增强骨的爬行替代和新骨形成的能力,对骨缺损的修复及愈合起重要作用。富血小板血浆(platelet-rich plasma, PRP)是自体全血经过离心得到的血小板浓缩物,其血小板浓度至少为全血中血小板浓度的4倍以上血小板释放的生物活性物质包括ADP、ATP、血管生成素-2(Angiopoietin-2, Ang-2)、结缔组织激活肽-3(Connective Tissue-Activating PeptideⅢ, CATPⅢ)、表皮生长因子(Epidermal Growth Factor, EGF)、碱性成纤维细胞生长因子(basic Fibroblast Growth Factor, bFGF)、胰岛素样生长因子(Insulin-like Growth Factor-I, IGF-Ⅰ)、骨钙素(Osteocalcin P-Selectin, GMP-140)、血小板源性内皮细胞生长因子(Platelet-derived endothelial cell growth factor, PDECGF)、血小板衍生生长因子(Platelet-derived Growth Factor, PDGF)、转化生长因子(Transforming Growth Factor-b1, TGF-b1)、血管内皮生长因子(Vascular Endothelial Growth Factor, VEGF),以上成分均具有促进组织愈合和组织再生的功能。富血小板血浆因其制备简单,价格低廉,自体取血既无毒性又无免疫原性,其释放的多种生长因子非常的符合人体生长因子的配比,因此被视为较为理想的符合临床治疗骨缺损需要的生长因子来源。骨形态发生蛋白(Bone morphogenetic protein,BMP)能诱导未分化的间充质细胞向骨系细胞转化,促进骨细胞的生长增殖。并进而合成胶原,形成钙化的骨组织能力,它可以促进原位或异位骨的形成。骨形态发生蛋白和PRP激活后所释放的多种生长因子联合作用具有明显的促进骨组织缺损的愈合和重塑。
本实验通过组织学观察及计算机图像分析等方法,观察自体富含血小板血浆及牛骨形态发生蛋白对兔桡骨缺损区骨组织再生与修复能力的影响,为临床骨缺损修复的应用提供理论依据。
材料及方法
1动物分组及模型制备
选择健康新西兰大白兔48只随机分为A、B、C、D三组,每组12只。A组桡骨缺损处植入复合型组织工程骨(PRP+FG+bBMP); B、C两组分别植入复合材料(PRP+FG)和(bBMP+FG); D组为空白组,只植入FG。根据骨缺损大小植入材料体积约2.0ml,上述材料均为胶状。逐层缝合切口,术后3d肌注青霉素40万u/d。
2 PRP的制作
整个制作过程严格无菌,0.03g/L戊巴比妥钠耳缘静脉注射麻醉,从兔耳中央动脉抽取5ml自体血,装在预先放有0.5ml复方枸橼酸钠抗凝剂的10ml离心管中,参照Landeaserg法进行PRP提取。
3组织工程骨的构建
取48mg bBMP放入蒸馏水中浸泡,4°C冰箱过夜。取出bBMP,研磨成碎末状后与PRP和FG根据骨缺损创面大小注入容器中,然后取凝血剂(由1ml：10%的氯化钙与1000u凝血酶混合而成)按照1ml：200mg将PRP/bBMP混合物制成凝胶,然后将PRP/bBMP混合物凝胶贴于桡骨骨缺损处。凝胶制作及应用应在40分钟内完成。
4大体观察
动物饮食、精神状态,切口愈合情况,观察植入物的体积变化,与周围组织结合的紧密程度,植入物表面的变化,有无炎症反应。
5组织学观察
取术后4、8、12周各组骨缺损处中央组织,10%甲醛固定,复合脱钙液脱钙,石蜡包埋,,切片HE染色观察其成骨情况。
6 X线片检查
术后4、8和12周拍摄双前肢正侧位X线片,观察骨缺损修复情况。
7计算机图像分析
每个标本选取2张HE切片,每个切片取4个视野,应用QWIN图像分析系统采集图像并分析,统计四组新生骨在骨缺损区所占的面积百分比,并进行比较。
8统计学方法
数据以均数±标准差(x)表示,采用SPSS 16.0统计软件进行单因素方差分析,同一时间点各组间的差异LSD法比较。检验水准取a=0.05。
结果
1一般情况术后所有动物饮食、行动、精神、毛发色泽、大小便正常。手术切口无红肿、渗血及脓性分泌物,切口均一期愈合。
2组织学观察术后4周,A组骨缺损处见新生血管丰富,可见较多的纤维性骨痂形成。局部见较多成骨细胞及软骨细胞增生活跃,可见与骨干平行的骨样组织。B、C组可见纤维组织结构,其间散在数量不等的成骨细胞和软骨细胞,成骨作用较A组差。D组见纤维组织生长,材料降解为网状,周围有炎性细胞和少量骨母细胞。术后8周,A组骨缺损区可见大量的新生骨样组织,骨基质丰富,可见钙盐沉积,软骨组织数量减少。B、C组新生骨组织呈交织状,可见骨基质和少量钙盐沉积,数量小于A组。D组可见未被完全机化的纤维组织和少量炎性细胞。12周,A组为成熟骨组织结构,主要为板层骨,散在少量的编织骨,可见少量破骨细胞,骨髓腔完全再通。B组编织骨较多,可见成骨细胞,破骨细胞及少量骨髓组织。C组骨组织成熟度稍差,亦可见髓腔内有血细胞。D组仍为纤维组织填充,较少的骨基质和新生骨组织。
3 X线片观察A组术后4周骨缺损处呈斑片状模糊阴影,断端较清晰,两断端部密度稍高。术后8周时两断端模糊,骨缺损处为连续性高密度阴影,桡骨缺损处可见连续性骨痂形成,两断端间形成桥接。12周时骨缺损处呈均匀一致高密度影,骨皮质连续,髓腔贯通。B组术后4周,骨缺损处为连续性絮状模糊阴影,断端清晰。术后8周骨缺损区模糊,为连续性高密度阴影,骨痂量较A组为少。12周时骨缺损基本修复,髓腔大部分再通。C组术后4周时,截骨端及缺损区有密度较低的骨痂形成,缺损区部分为骨痂填充。术后8和12周时,骨缺损处部分由新生骨所填充,可见髓腔部分再通。D组术后4周时,骨缺损区未见明显成骨,断端清晰,缺损区域未见修复。术后8和12周时,髓腔部分闭合,骨缺损区未见明显修复,断端髓腔闭合。
4.计算机图像分析随着时间的推移,新骨形成逐渐增多,材料逐渐被降解。术后4、8、12周,A组桡骨缺损处修复性新骨形成均多于B、C和D组(P<0.01)；术后4周,B组新骨形成略多于C组,但两组间无明显差异(P>0.05)；术后8周、12周,B组新骨形成多于C组(P<0.05)
结论
1在兔桡骨缺损的修复和愈合方面单独应用PRP和单独应用BMP均可促进骨缺损的修复,但前者较后者更有利于促进骨愈合；
2 PRP联合BMP组织工程骨在促进兔桡骨缺损的修复和愈合等方面明显优于单独应用PRP和BMP；
3以PRP+FG+BMP构建的组织工程骨材料因其操作,制备较为简单,无免疫原性,成骨效果明确,有较高的临床应用价值。
Background & Objectives
How to promote the healing of bone defect has been an important issue in the research field of bone repair. At present, the focus on the healing of bone defect is how to promote and strengthen the creeping substitution in bone and formation of new bone by using a variety of growth factors. Platelet-rich plasma (PRP) is platelet concentrates got by concentrating autologous whole blood. The platelet concentration in PRP is at least 4 times more than that in the whole blood. PRP contains high concentrations of various growth factors, including ADP, ATP, Angiopoietin-2, Tissue-Activating Peptide-Ⅲ, Platelet-derived growth factor (PDGF), Transforming growth factor-P (TGF-(3), Vascular endo-thelial growth factor (VEGF), Insulinlike growth factor (IGF), Epidermal growth factor (EGF) and etc. These factors have the functions of accelerating the healing and regeneration of injury tissue. The preparation of PRP is cheap and simple. Autologous blood has neither toxicity nor immunogenicity. The growth factors released by PRP are in accordance with the ratio of factors from autologous blood. Therefore it is regarded as an ideal source of growth factors for clinical treatment of bone defect. Bone morphogenetic protein (BMP) can induce undifferentiated mesenchymal cells differentiating into Osteoblast and enhance the growth and proliferation of bone cell. It can also enhance the synthesis and calcification of collagen so as to form new bone and promote ectopic bone formation or bone formation in situ. The joint effect of several growth factors released by activated PRP can obviously promote the healing and remodeling on bone defect. We make use of histological observation and computer image analysis to observe the influence of PRP and BMP on bone defect, providing a theroy basis for clinical applications in bone defect and repair.
Materials and Methods
1 Animal grouping and model preparation 48 healthy New Zealand white rabbits were randomly divided into A, B, C and D group. Every group contained 12 New Zealand white rabbits. The radial bone defects of rabbits in group A were implanted with the compound tissue engineering bone (PRP+FG+bBMP). B and C group was respectively implanted with compound materials (PRP+FG) and (bBMP+FG). According to the size of bone defects. we implanted about 2.0 ml these compound materials. After that, we sutured the incision and injected penicillin 400000u/d for 3 days.
2 The preparation of PRP The whole process of PRP preparation was strictly sterile.0.03g/L sodium pentobarbital was injected into the ear vein of rabbits for anesthesia. Then we extracted 5ml autologous blood from the rabbit central ear artery and put it into 10ml centrifuge tubes according to the Landeaserg PRP extraction method.
3 Construction of tissue engineered bone 48mg bBMP was immersed into distilled water and then put it into 4℃refrigerator overnight. Then bBMP was ground into powder and was mixed with PRP and FG. We put coagulant (1ml mixture of 10% calcium chloride and 1000u thrombin) into container which contained this complex. According to 1ml:200u PRP/bBMP, we made the mixture of PRP, bBMP and FG into gel, which was implanted into the radial defects in rabbits. The production and application of the PRP compound gel should be finished within 40 minutes.
4 General observation Animal diet, mental state, wound healing, changes of implants volume and the extent in which the implants had the tightness with the surrounding tissue and changes in implant surface and whether it had inflammatory response, etc.
5 Histological observation We took the central tissue of bone defects after 4,8 and 12 weeks and then observed its bone formation through HE slice.
6 X-ray observation After 4,8 and 12 weeks, the two fore limbs of rabbits were X-rayed to observe the repair on radius'defect.
7 Computer image analysis Two HE sections were selected from each specimen. Four visions were taken from each section. Then we used QWIN image analysis systems to compare area percentage of new bone formation among the three groups.
8 Statistical Methods The statistical software SSPS16.0 for windows was exploited to analyis data. All the data were expressed by (x±s). Deviation and difference were compared using the one way ANOVA and LSD test between each group in every time point. The significant difference was judged by P<0.05.
Results
1 General situation The rabbit's consciousness, diet, action, hair color, urine and stool was normal. There was no swelling, bleeding and purulent secretion on the incision of rabbits. The wound healed within six days.
2 Histological observation 4 weeks later, In group A, we could see there was affluent neovascularization on bone defects and more fibrous callus formation. Osteoblasts and chondrocytes were active in proliferation. In group B and C, we could see fibrous tissue structure on bone defects in which there was various amount of osteoblasts and chondrocytes. In group D, we could see fibrous tissue structure on bone defects surrounded by small amount of inflammatory cells and osteoblasts. The osteogenesis in group A was better than that in group B and C.8 weeks later, In group A, abundant bone matrix and new bone formation on radius'defects and the number of cartilage cells decreased. The new-formed bone interweaved on radius'defects of rabbits in group B and C. The Bone matrix and calcium deposition in group B and C was not as good as that in group A.12 weeks later, in group A, mature bone structure was mainly lamellar bone and small amount of scattered woven bone. The bone marrow cavity is completely re-passed. In group B, There was more woven bone, osteoblasts, osteoclasts and a small amount of myeloid tissue could be seen. In group C, Bone maturation was not good and we could see blood cells in the marrow cavity. In group D, we could see that the radius'defect was still filled with fibrous tissue and small amount of bone matrix and new bone.
3 X-ray observation Group A:4 weeks after operation, we could see that blurred shadow was patchy in distribution on the radius'defects. The two ends of fracture was clear and had highter density.8 weeks after operation, we could see continuous high-density shadow and callus formation on the radius'defects, which formed a bridge between the two ends.12 weeks after operation, we could see a uniform shadow of high density on the radius'defects. The bone marrow cavity was re-passed, with the cortex bone shell of long bone continued. Group B:4 weeks later, we could see continuous flocculent blurred shadow on the radius'defects.8 weeks later, we could see continuous high-density shadow on the radius'defects and the callus formation in group B was less than that in group A.12 weeks later, the radius'defects in rabbits was nearly repaired and the bone marrow cavity was mostly re-passed. Group C:4 weeks later, we could see that there was low-density callus formation on the radius'defects and the two ends of fracture.8 and 12 weeks after operation, we could see that the radius'defects were filled partly with new bone and the bone marrow cavity was partly re-passed. Group D:4 weeks after surgery, there was no obvious new bone formation and the two ends of bone defects were very clear.8 and 12 weeks later, Marrow cavity was partly closed. The bone defects didn't be repaired.
4 Computer image analysis Over time, new bone was gradually on the increase and the compound bio-material was gradually decomposed.4,8 and 12 weeks after surgery, area ratio of newly-formed bone on the surface area of radius defects in group A was significantly higher than that in group B, C and D. Area ratio of newly-formed bone on the radius in group B was significantly higher than that on the radius in group C from 8 to 12 weeks after surgery.
Conclusions
1 Both PRP and BMP have an effect on improving the generation and repair of radius'defects in rabbits, but the former is better than the latter in the respects of forming new bone.
2 The tissue engineering material containing PRP and bBMP have a better effect on the bone generation and maturity of radius'defects in rabbits than tissue engineering material only containing PRP and bBMP.
3 The tissue engineering material containing PRP, FG and bBMP has a high value of clinical application owing to its simple preparation, obvious osteogenic effects and non-immunogenicity,.
骨缺损的修复和再生一直是骨科学研究领域中的重要课题,目前研究热点是利用生长因子来促进、增强骨的爬行替代和新骨形成的能力,对骨缺损的修复及愈合起重要作用。富血小板血浆(platelet-rich plasma, PRP)是自体全血经过离心得到的血小板浓缩物,其血小板浓度至少为全血中血小板浓度的4倍以上血小板释放的生物活性物质包括ADP、ATP、血管生成素-2(Angiopoietin-2, Ang-2)、结缔组织激活肽-3(Connective Tissue-Activating PeptideⅢ, CATPⅢ)、表皮生长因子(Epidermal Growth Factor, EGF)、碱性成纤维细胞生长因子(basic Fibroblast Growth Factor, bFGF)、胰岛素样生长因子(Insulin-like Growth Factor-I, IGF-Ⅰ)、骨钙素(Osteocalcin P-Selectin, GMP-140)、血小板源性内皮细胞生长因子(Platelet-derived endothelial cell growth factor, PDECGF)、血小板衍生生长因子(Platelet-derived Growth Factor, PDGF)、转化生长因子(Transforming Growth Factor-b1, TGF-b1)、血管内皮生长因子(Vascular Endothelial Growth Factor, VEGF),以上成分均具有促进组织愈合和组织再生的功能。富血小板血浆因其制备简单,价格低廉,自体取血既无毒性又无免疫原性,其释放的多种生长因子非常的符合人体生长因子的配比,因此被视为较为理想的符合临床治疗骨缺损需要的生长因子来源。骨形态发生蛋白(Bone morphogenetic protein,BMP)能诱导未分化的间充质细胞向骨系细胞转化,促进骨细胞的生长增殖。并进而合成胶原,形成钙化的骨组织能力,它可以促进原位或异位骨的形成。骨形态发生蛋白和PRP激活后所释放的多种生长因子联合作用具有明显的促进骨组织缺损的愈合和重塑。
本实验通过组织学观察及计算机图像分析等方法,观察自体富含血小板血浆及牛骨形态发生蛋白对兔桡骨缺损区骨组织再生与修复能力的影响,为临床骨缺损修复的应用提供理论依据。
材料及方法
1动物分组及模型制备
选择健康新西兰大白兔48只随机分为A、B、C、D三组,每组12只。A组桡骨缺损处植入复合型组织工程骨(PRP+FG+bBMP); B、C两组分别植入复合材料(PRP+FG)和(bBMP+FG); D组为空白组,只植入FG。根据骨缺损大小植入材料体积约2.0ml,上述材料均为胶状。逐层缝合切口,术后3d肌注青霉素40万u/d。
2 PRP的制作
整个制作过程严格无菌,0.03g/L戊巴比妥钠耳缘静脉注射麻醉,从兔耳中央动脉抽取5ml自体血,装在预先放有0.5ml复方枸橼酸钠抗凝剂的10ml离心管中,参照Landeaserg法进行PRP提取。
3组织工程骨的构建
取48mg bBMP放入蒸馏水中浸泡,4°C冰箱过夜。取出bBMP,研磨成碎末状后与PRP和FG根据骨缺损创面大小注入容器中,然后取凝血剂(由1ml：10%的氯化钙与1000u凝血酶混合而成)按照1ml：200mg将PRP/bBMP混合物制成凝胶,然后将PRP/bBMP混合物凝胶贴于桡骨骨缺损处。凝胶制作及应用应在40分钟内完成。
4大体观察
动物饮食、精神状态,切口愈合情况,观察植入物的体积变化,与周围组织结合的紧密程度,植入物表面的变化,有无炎症反应。
5组织学观察
取术后4、8、12周各组骨缺损处中央组织,10%甲醛固定,复合脱钙液脱钙,石蜡包埋,,切片HE染色观察其成骨情况。
6 X线片检查
术后4、8和12周拍摄双前肢正侧位X线片,观察骨缺损修复情况。
7计算机图像分析
每个标本选取2张HE切片,每个切片取4个视野,应用QWIN图像分析系统采集图像并分析,统计四组新生骨在骨缺损区所占的面积百分比,并进行比较。
8统计学方法
数据以均数±标准差(x)表示,采用SPSS 16.0统计软件进行单因素方差分析,同一时间点各组间的差异LSD法比较。检验水准取a=0.05。
结果
1一般情况术后所有动物饮食、行动、精神、毛发色泽、大小便正常。手术切口无红肿、渗血及脓性分泌物,切口均一期愈合。
2组织学观察术后4周,A组骨缺损处见新生血管丰富,可见较多的纤维性骨痂形成。局部见较多成骨细胞及软骨细胞增生活跃,可见与骨干平行的骨样组织。B、C组可见纤维组织结构,其间散在数量不等的成骨细胞和软骨细胞,成骨作用较A组差。D组见纤维组织生长,材料降解为网状,周围有炎性细胞和少量骨母细胞。术后8周,A组骨缺损区可见大量的新生骨样组织,骨基质丰富,可见钙盐沉积,软骨组织数量减少。B、C组新生骨组织呈交织状,可见骨基质和少量钙盐沉积,数量小于A组。D组可见未被完全机化的纤维组织和少量炎性细胞。12周,A组为成熟骨组织结构,主要为板层骨,散在少量的编织骨,可见少量破骨细胞,骨髓腔完全再通。B组编织骨较多,可见成骨细胞,破骨细胞及少量骨髓组织。C组骨组织成熟度稍差,亦可见髓腔内有血细胞。D组仍为纤维组织填充,较少的骨基质和新生骨组织。
3 X线片观察A组术后4周骨缺损处呈斑片状模糊阴影,断端较清晰,两断端部密度稍高。术后8周时两断端模糊,骨缺损处为连续性高密度阴影,桡骨缺损处可见连续性骨痂形成,两断端间形成桥接。12周时骨缺损处呈均匀一致高密度影,骨皮质连续,髓腔贯通。B组术后4周,骨缺损处为连续性絮状模糊阴影,断端清晰。术后8周骨缺损区模糊,为连续性高密度阴影,骨痂量较A组为少。12周时骨缺损基本修复,髓腔大部分再通。C组术后4周时,截骨端及缺损区有密度较低的骨痂形成,缺损区部分为骨痂填充。术后8和12周时,骨缺损处部分由新生骨所填充,可见髓腔部分再通。D组术后4周时,骨缺损区未见明显成骨,断端清晰,缺损区域未见修复。术后8和12周时,髓腔部分闭合,骨缺损区未见明显修复,断端髓腔闭合。
4.计算机图像分析随着时间的推移,新骨形成逐渐增多,材料逐渐被降解。术后4、8、12周,A组桡骨缺损处修复性新骨形成均多于B、C和D组(P<0.01)；术后4周,B组新骨形成略多于C组,但两组间无明显差异(P>0.05)；术后8周、12周,B组新骨形成多于C组(P<0.05)
结论
1在兔桡骨缺损的修复和愈合方面单独应用PRP和单独应用BMP均可促进骨缺损的修复,但前者较后者更有利于促进骨愈合；
2 PRP联合BMP组织工程骨在促进兔桡骨缺损的修复和愈合等方面明显优于单独应用PRP和BMP；
3以PRP+FG+BMP构建的组织工程骨材料因其操作,制备较为简单,无免疫原性,成骨效果明确,有较高的临床应用价值。
Background & Objectives
How to promote the healing of bone defect has been an important issue in the research field of bone repair. At present, the focus on the healing of bone defect is how to promote and strengthen the creeping substitution in bone and formation of new bone by using a variety of growth factors. Platelet-rich plasma (PRP) is platelet concentrates got by concentrating autologous whole blood. The platelet concentration in PRP is at least 4 times more than that in the whole blood. PRP contains high concentrations of various growth factors, including ADP, ATP, Angiopoietin-2, Tissue-Activating Peptide-Ⅲ, Platelet-derived growth factor (PDGF), Transforming growth factor-P (TGF-(3), Vascular endo-thelial growth factor (VEGF), Insulinlike growth factor (IGF), Epidermal growth factor (EGF) and etc. These factors have the functions of accelerating the healing and regeneration of injury tissue. The preparation of PRP is cheap and simple. Autologous blood has neither toxicity nor immunogenicity. The growth factors released by PRP are in accordance with the ratio of factors from autologous blood. Therefore it is regarded as an ideal source of growth factors for clinical treatment of bone defect. Bone morphogenetic protein (BMP) can induce undifferentiated mesenchymal cells differentiating into Osteoblast and enhance the growth and proliferation of bone cell. It can also enhance the synthesis and calcification of collagen so as to form new bone and promote ectopic bone formation or bone formation in situ. The joint effect of several growth factors released by activated PRP can obviously promote the healing and remodeling on bone defect. We make use of histological observation and computer image analysis to observe the influence of PRP and BMP on bone defect, providing a theroy basis for clinical applications in bone defect and repair.
Materials and Methods
1 Animal grouping and model preparation 48 healthy New Zealand white rabbits were randomly divided into A, B, C and D group. Every group contained 12 New Zealand white rabbits. The radial bone defects of rabbits in group A were implanted with the compound tissue engineering bone (PRP+FG+bBMP). B and C group was respectively implanted with compound materials (PRP+FG) and (bBMP+FG). According to the size of bone defects. we implanted about 2.0 ml these compound materials. After that, we sutured the incision and injected penicillin 400000u/d for 3 days.
2 The preparation of PRP The whole process of PRP preparation was strictly sterile.0.03g/L sodium pentobarbital was injected into the ear vein of rabbits for anesthesia. Then we extracted 5ml autologous blood from the rabbit central ear artery and put it into 10ml centrifuge tubes according to the Landeaserg PRP extraction method.
3 Construction of tissue engineered bone 48mg bBMP was immersed into distilled water and then put it into 4℃refrigerator overnight. Then bBMP was ground into powder and was mixed with PRP and FG. We put coagulant (1ml mixture of 10% calcium chloride and 1000u thrombin) into container which contained this complex. According to 1ml:200u PRP/bBMP, we made the mixture of PRP, bBMP and FG into gel, which was implanted into the radial defects in rabbits. The production and application of the PRP compound gel should be finished within 40 minutes.
4 General observation Animal diet, mental state, wound healing, changes of implants volume and the extent in which the implants had the tightness with the surrounding tissue and changes in implant surface and whether it had inflammatory response, etc.
5 Histological observation We took the central tissue of bone defects after 4,8 and 12 weeks and then observed its bone formation through HE slice.
6 X-ray observation After 4,8 and 12 weeks, the two fore limbs of rabbits were X-rayed to observe the repair on radius'defect.
7 Computer image analysis Two HE sections were selected from each specimen. Four visions were taken from each section. Then we used QWIN image analysis systems to compare area percentage of new bone formation among the three groups.
8 Statistical Methods The statistical software SSPS16.0 for windows was exploited to analyis data. All the data were expressed by (x±s). Deviation and difference were compared using the one way ANOVA and LSD test between each group in every time point. The significant difference was judged by P<0.05.
Results
1 General situation The rabbit's consciousness, diet, action, hair color, urine and stool was normal. There was no swelling, bleeding and purulent secretion on the incision of rabbits. The wound healed within six days.
2 Histological observation 4 weeks later, In group A, we could see there was affluent neovascularization on bone defects and more fibrous callus formation. Osteoblasts and chondrocytes were active in proliferation. In group B and C, we could see fibrous tissue structure on bone defects in which there was various amount of osteoblasts and chondrocytes. In group D, we could see fibrous tissue structure on bone defects surrounded by small amount of inflammatory cells and osteoblasts. The osteogenesis in group A was better than that in group B and C.8 weeks later, In group A, abundant bone matrix and new bone formation on radius'defects and the number of cartilage cells decreased. The new-formed bone interweaved on radius'defects of rabbits in group B and C. The Bone matrix and calcium deposition in group B and C was not as good as that in group A.12 weeks later, in group A, mature bone structure was mainly lamellar bone and small amount of scattered woven bone. The bone marrow cavity is completely re-passed. In group B, There was more woven bone, osteoblasts, osteoclasts and a small amount of myeloid tissue could be seen. In group C, Bone maturation was not good and we could see blood cells in the marrow cavity. In group D, we could see that the radius'defect was still filled with fibrous tissue and small amount of bone matrix and new bone.
3 X-ray observation Group A:4 weeks after operation, we could see that blurred shadow was patchy in distribution on the radius'defects. The two ends of fracture was clear and had highter density.8 weeks after operation, we could see continuous high-density shadow and callus formation on the radius'defects, which formed a bridge between the two ends.12 weeks after operation, we could see a uniform shadow of high density on the radius'defects. The bone marrow cavity was re-passed, with the cortex bone shell of long bone continued. Group B:4 weeks later, we could see continuous flocculent blurred shadow on the radius'defects.8 weeks later, we could see continuous high-density shadow on the radius'defects and the callus formation in group B was less than that in group A.12 weeks later, the radius'defects in rabbits was nearly repaired and the bone marrow cavity was mostly re-passed. Group C:4 weeks later, we could see that there was low-density callus formation on the radius'defects and the two ends of fracture.8 and 12 weeks after operation, we could see that the radius'defects were filled partly with new bone and the bone marrow cavity was partly re-passed. Group D:4 weeks after surgery, there was no obvious new bone formation and the two ends of bone defects were very clear.8 and 12 weeks later, Marrow cavity was partly closed. The bone defects didn't be repaired.
4 Computer image analysis Over time, new bone was gradually on the increase and the compound bio-material was gradually decomposed.4,8 and 12 weeks after surgery, area ratio of newly-formed bone on the surface area of radius defects in group A was significantly higher than that in group B, C and D. Area ratio of newly-formed bone on the radius in group B was significantly higher than that on the radius in group C from 8 to 12 weeks after surgery.
Conclusions
1 Both PRP and BMP have an effect on improving the generation and repair of radius'defects in rabbits, but the former is better than the latter in the respects of forming new bone.
2 The tissue engineering material containing PRP and bBMP have a better effect on the bone generation and maturity of radius'defects in rabbits than tissue engineering material only containing PRP and bBMP.
3 The tissue engineering material containing PRP, FG and bBMP has a high value of clinical application owing to its simple preparation, obvious osteogenic effects and non-immunogenicity,.
引文
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