DBM/rhBMP-2/CPC复合材料修复大段长骨缺损的实验研究
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摘要
目的骨缺损修复是当前骨科临床上常见的难题,长期以来,人们寻求一种良好的人工替代材料用来修复骨缺损,但结果不理想。本实验制备脱钙骨基质颗粒(DBM)/磷酸钙骨水泥(CPC)/重组人骨形成蛋白-2(rhBMP-2)的复合材料,探讨其力学性能、结构特征和生物相容性,找出该复合材料的最佳配方。进一步观察复合材料植入骨缺损内的超微结构、X线及组织学特征,评价该材料修复大段长骨缺损的能力和降解性能,为临床应用和批量化生产提供依据。
方法:(1)预制兔DBM,用吸附法将rhBMP-2与DBM复合后,按DBM :复合材料的质量比为分别是0.1,0.2,0.3,0.4,0.5,0.6,0.7的配方与CPC复合,制备的复合材料含rhBMP-2的量约1.2×106 mg/ m3。(2)将DBM质量比为分别是0.1,0.2,0.3,0.4复合材料行体外抗压强度测定和扫描电镜(SEM)观察,探讨其力学性能、结构特征,找出该复合材料的最佳配方。(3)通过毒性实验、凝血实验、溶血实验、热原实验和肌肉植入实验评价材料的生物相容性。(4)将DBM质量比为0.2的柱状复合材料植入兔股骨髁部0.5cm缺损,术后6、12、24周处死取材进行组织学和扫描电镜观察,评价其成骨性能。(5)将质量比为0.2和0.3的柱状复合材料植入兔1.5cm长桡骨缺损和股骨髁部缺损,术后6、12、24、36周观察X线征象、组织学形态及超微结构,评价其修复大段长骨缺损的能力和降解性能。
结果:(1)DBM的质量比在0.2～0.4的范围内,复合材料中存在较多100μm以上的不规则裂隙。DBM的质量比≤0.1时,材料内部的大部分间隙<100μm。DBM的质量比≥0.5时,DBM和CPC丧失结合及塑型能力。随DBM质量比的增加,材料抗压极限强度递减,质量比为0.1,0.2,0.3,0.4的抗压极限强度分别为(8.12±0.79)MPa,(5.46±1.13)MPa,(5.13±1.18)MPa,(1.49±0.61)MPa。各组数据经方差分析,有统计学差异(P<0.05),其中质量比为0.2,0.3两组比较无统计学差异(P>0.05),而(1.49±0.61)MPa小于人体松质骨的平均抗压强度。(2)复合材料具有良好的生物相容性。(3)组织学和扫描电镜观察见:复合材料植入股骨髁部缺损后第6周复合材料-骨界面模糊,宿骨发出纤维连续通过界面,编织骨样结构开始向材料内部长入。第12周血管和成骨细胞在复合材料内部形成。第24周骨缺损已修复,形成骨性连接,材料大部分被新骨替代。(4)将复合材料植入兔1.5cm长桡骨缺损,X线示:第6周复合材料-骨界面模糊,周围有骨痂再生。第12周骨痂生长丰富,材料-骨界面消失,材料被降解失去原有外形。第24周材料被降解更明显,髓腔开始再通。第36周材料被新骨替代,髓腔再通。组织学检查也支持此结果。复合材料植入兔股骨髁部缺损,X线示:第6周～24周,材料逐渐被降解,密度逐渐降低。扫描电镜观察结果也得出同样的结论。
结论:随着DBM质量比的增加,孔隙越丰富而材料力学强度逐渐降低。DBM质量比为0.2～0.3的复合材料可用于修复低承重部位松质骨的骨缺损。复合材料具有良好的生物相容性,植入骨缺损内可以促进细胞、血管、新骨的长入、易降解和被自体骨替代,能够用于修复大段骨缺损。
OBJECTIVE: It is common to be met in orthopedic clinic that the bone defects haven’t ideal methods to repair at present.Scientist seek a kind of perfect and artificial bone substitute for repair of bone defects in long times, but the effect is not perfect. The aim of the present experiment is to prepare the composite material of decalcified bone matrix(DBM) impregnated calcium phosphate cement(CPC) with recombinant human bone morphogenetic protein-2(rhBMP-2), to analyze the mechanical, microstructure and biocompatibility properties of composite material, and to investigate the best component proportion of this compositions. More of the present experiment is to study the ultramicrostructure, radiographic and histological properties of composition implanted in bone defect, to evaluate the efficacy of the composite materials in healing of the bone segmental defect of long bone and to analyze the biodegradable properties for the use in clinic and the batch of production.
METHODS:(1) The rabbit DBM were prepared beforehand. The rhBMP-2 and DBM were mixed with adsorption. The mixture was added into CPC material at the proportion of DBM weight being 0.1, 0.2, 0.3, 0.4, 0.5, 0.6and 0.7, with the proportion of rhBMP-2 being 1.2×106 mg/ m3. (2) With the proportion of DBM being 0.1, 0.2, 0.3 and 0.4, the composition were solidified and examined by biomechanics and scanning electron microscope(SEM).The mechanical and microstructure properties of composite material were analyzed for investigating the best component proportion of the compositions. (3) By study of systemic reaction of animals’body for implanting the composition, the biocompatibility was evaluated. By exvivio test of haemocytolysis, pyrogen reaction and disturbance of blood coagulation was caused by implants, the biocompatibility was analyzed. The biocompatibility was verified by view of adverse cellular reactions in the interface region between implants and muscle. (4) Rabbits underwent femur condyles osteotomies with creation of a 0.5 cm cylindrical defect and the defect was filled with implants of composite material with the proportion of DBM being 0.2.They were killed at 6,12 or 24 weeks, regenerated bone was studied by scanning electron microscope and histology. (5) Rabbits underwent osteotomies with creation of a 1.5 cm radial defect and a 0.5 cm femur condyles defect. The defect was filled with cylindrical implants of composite material with the proportion of DBM being 0.2 or 0.3. The efficacy of the composition in healing of the bone segmental defect of long bone was valuated by radiographic evaluation and histology. The biodegradable property of the composition was studied by radiographic evaluation and ultramicrostructure properties.
RESULTS: (1) There were many irregular gaps of more than 100μm in the composite materials, when the proportion of DBM was from 0.2 to 0.4. Most of gaps’diameters were less than 100μm when the proportion of DBM was no more than 0.1. The DBM and CPC couldn’t be consolidated when the proportion of DBM was no less than 0.5. The compressive strength of the compositions was progressively decreased, in accordance with the increase of proportions of DBM. With the proportion of DBM being 0.1, 0.2, 0.3 and 0.4, the ultimate compressive strength was (8.12±0.79), (5.46±1.13), (5.13±1.18), (1.49±0.61). A significant difference (P<0.05) was found in the statistical comparison of the different groups with regard to the composition’s ultimate compressive strength, except between 0.2 and 0.3. But the mean value of human cancellous bone’s compressive strength was strikingly higher than 1.493 MPa. (2) The composition has a perfect biodegradable property. (3) At 6 weeks after femur condyles osteotomies, a significant difference in the interface region between implants and bone was not viewed by histology and scanning electron microscope. It was viewed by histology and scanning electron microscope that the interface region between implants and bone was filled with osteoid substance with colonization inside the materials by new bone formed. At 12 weeks, Histology showed formation of blood vessel and osteoblasts inside the materials. At 24 weeks, the defect in femur condyles were almost completely repaired by bone tissues, most of implants was resorbed and replaced by new bone. (4) At 6 weeks after radial osteotomies, a significant difference in the interface region between implants and bone was not observed on radiographs. Radiographs displayed new bone formation around the implants. At 12 weeks, radiographs showed an increase of new bone amount around implants. The interface region was not seen, on radiographs, and the profile of implants was changed with absorption of materials particle. At 24 weeks, radiographs showed the resorption amount of implants are increase and the uniformly radiopaque segment filled with implants became partly radioparent. At 36 weeks, radiographs showed that most of implants were replaced by the bone tissues and the radiopaque segment almostly became radioparent. The study by histology supported a coincident conclusion. From 6 to 24weeks, in femur condyles osteotomies group, radiographs showed that with absorption of materials particle, the significant decrease in the size and density of implants. The same things were observed by scanning electron microscope.
Conclusions: Along with the increase of proportions of DBM, the porosity of compositions is more plenty, contrarily the compressive strength of compositions is descended accordingly. Compositions with DBM proportion of 0.2 ~ 0.3 could be used in repairing cancellous bone’s gap where do need to bear lower load. The composition has a perfect biodegradable property. The composite materials implanted in bone segment defect can stimulate the growth of osteoblast, blood vessel and new bones. The materials can be replaced by autogenous bone for its biodegradable property.
方法:(1)预制兔DBM,用吸附法将rhBMP-2与DBM复合后,按DBM :复合材料的质量比为分别是0.1,0.2,0.3,0.4,0.5,0.6,0.7的配方与CPC复合,制备的复合材料含rhBMP-2的量约1.2×106 mg/ m3。(2)将DBM质量比为分别是0.1,0.2,0.3,0.4复合材料行体外抗压强度测定和扫描电镜(SEM)观察,探讨其力学性能、结构特征,找出该复合材料的最佳配方。(3)通过毒性实验、凝血实验、溶血实验、热原实验和肌肉植入实验评价材料的生物相容性。(4)将DBM质量比为0.2的柱状复合材料植入兔股骨髁部0.5cm缺损,术后6、12、24周处死取材进行组织学和扫描电镜观察,评价其成骨性能。(5)将质量比为0.2和0.3的柱状复合材料植入兔1.5cm长桡骨缺损和股骨髁部缺损,术后6、12、24、36周观察X线征象、组织学形态及超微结构,评价其修复大段长骨缺损的能力和降解性能。
结果:(1)DBM的质量比在0.2～0.4的范围内,复合材料中存在较多100μm以上的不规则裂隙。DBM的质量比≤0.1时,材料内部的大部分间隙<100μm。DBM的质量比≥0.5时,DBM和CPC丧失结合及塑型能力。随DBM质量比的增加,材料抗压极限强度递减,质量比为0.1,0.2,0.3,0.4的抗压极限强度分别为(8.12±0.79)MPa,(5.46±1.13)MPa,(5.13±1.18)MPa,(1.49±0.61)MPa。各组数据经方差分析,有统计学差异(P<0.05),其中质量比为0.2,0.3两组比较无统计学差异(P>0.05),而(1.49±0.61)MPa小于人体松质骨的平均抗压强度。(2)复合材料具有良好的生物相容性。(3)组织学和扫描电镜观察见:复合材料植入股骨髁部缺损后第6周复合材料-骨界面模糊,宿骨发出纤维连续通过界面,编织骨样结构开始向材料内部长入。第12周血管和成骨细胞在复合材料内部形成。第24周骨缺损已修复,形成骨性连接,材料大部分被新骨替代。(4)将复合材料植入兔1.5cm长桡骨缺损,X线示:第6周复合材料-骨界面模糊,周围有骨痂再生。第12周骨痂生长丰富,材料-骨界面消失,材料被降解失去原有外形。第24周材料被降解更明显,髓腔开始再通。第36周材料被新骨替代,髓腔再通。组织学检查也支持此结果。复合材料植入兔股骨髁部缺损,X线示:第6周～24周,材料逐渐被降解,密度逐渐降低。扫描电镜观察结果也得出同样的结论。
结论:随着DBM质量比的增加,孔隙越丰富而材料力学强度逐渐降低。DBM质量比为0.2～0.3的复合材料可用于修复低承重部位松质骨的骨缺损。复合材料具有良好的生物相容性,植入骨缺损内可以促进细胞、血管、新骨的长入、易降解和被自体骨替代,能够用于修复大段骨缺损。
OBJECTIVE: It is common to be met in orthopedic clinic that the bone defects haven’t ideal methods to repair at present.Scientist seek a kind of perfect and artificial bone substitute for repair of bone defects in long times, but the effect is not perfect. The aim of the present experiment is to prepare the composite material of decalcified bone matrix(DBM) impregnated calcium phosphate cement(CPC) with recombinant human bone morphogenetic protein-2(rhBMP-2), to analyze the mechanical, microstructure and biocompatibility properties of composite material, and to investigate the best component proportion of this compositions. More of the present experiment is to study the ultramicrostructure, radiographic and histological properties of composition implanted in bone defect, to evaluate the efficacy of the composite materials in healing of the bone segmental defect of long bone and to analyze the biodegradable properties for the use in clinic and the batch of production.
METHODS:(1) The rabbit DBM were prepared beforehand. The rhBMP-2 and DBM were mixed with adsorption. The mixture was added into CPC material at the proportion of DBM weight being 0.1, 0.2, 0.3, 0.4, 0.5, 0.6and 0.7, with the proportion of rhBMP-2 being 1.2×106 mg/ m3. (2) With the proportion of DBM being 0.1, 0.2, 0.3 and 0.4, the composition were solidified and examined by biomechanics and scanning electron microscope(SEM).The mechanical and microstructure properties of composite material were analyzed for investigating the best component proportion of the compositions. (3) By study of systemic reaction of animals’body for implanting the composition, the biocompatibility was evaluated. By exvivio test of haemocytolysis, pyrogen reaction and disturbance of blood coagulation was caused by implants, the biocompatibility was analyzed. The biocompatibility was verified by view of adverse cellular reactions in the interface region between implants and muscle. (4) Rabbits underwent femur condyles osteotomies with creation of a 0.5 cm cylindrical defect and the defect was filled with implants of composite material with the proportion of DBM being 0.2.They were killed at 6,12 or 24 weeks, regenerated bone was studied by scanning electron microscope and histology. (5) Rabbits underwent osteotomies with creation of a 1.5 cm radial defect and a 0.5 cm femur condyles defect. The defect was filled with cylindrical implants of composite material with the proportion of DBM being 0.2 or 0.3. The efficacy of the composition in healing of the bone segmental defect of long bone was valuated by radiographic evaluation and histology. The biodegradable property of the composition was studied by radiographic evaluation and ultramicrostructure properties.
RESULTS: (1) There were many irregular gaps of more than 100μm in the composite materials, when the proportion of DBM was from 0.2 to 0.4. Most of gaps’diameters were less than 100μm when the proportion of DBM was no more than 0.1. The DBM and CPC couldn’t be consolidated when the proportion of DBM was no less than 0.5. The compressive strength of the compositions was progressively decreased, in accordance with the increase of proportions of DBM. With the proportion of DBM being 0.1, 0.2, 0.3 and 0.4, the ultimate compressive strength was (8.12±0.79), (5.46±1.13), (5.13±1.18), (1.49±0.61). A significant difference (P<0.05) was found in the statistical comparison of the different groups with regard to the composition’s ultimate compressive strength, except between 0.2 and 0.3. But the mean value of human cancellous bone’s compressive strength was strikingly higher than 1.493 MPa. (2) The composition has a perfect biodegradable property. (3) At 6 weeks after femur condyles osteotomies, a significant difference in the interface region between implants and bone was not viewed by histology and scanning electron microscope. It was viewed by histology and scanning electron microscope that the interface region between implants and bone was filled with osteoid substance with colonization inside the materials by new bone formed. At 12 weeks, Histology showed formation of blood vessel and osteoblasts inside the materials. At 24 weeks, the defect in femur condyles were almost completely repaired by bone tissues, most of implants was resorbed and replaced by new bone. (4) At 6 weeks after radial osteotomies, a significant difference in the interface region between implants and bone was not observed on radiographs. Radiographs displayed new bone formation around the implants. At 12 weeks, radiographs showed an increase of new bone amount around implants. The interface region was not seen, on radiographs, and the profile of implants was changed with absorption of materials particle. At 24 weeks, radiographs showed the resorption amount of implants are increase and the uniformly radiopaque segment filled with implants became partly radioparent. At 36 weeks, radiographs showed that most of implants were replaced by the bone tissues and the radiopaque segment almostly became radioparent. The study by histology supported a coincident conclusion. From 6 to 24weeks, in femur condyles osteotomies group, radiographs showed that with absorption of materials particle, the significant decrease in the size and density of implants. The same things were observed by scanning electron microscope.
Conclusions: Along with the increase of proportions of DBM, the porosity of compositions is more plenty, contrarily the compressive strength of compositions is descended accordingly. Compositions with DBM proportion of 0.2 ~ 0.3 could be used in repairing cancellous bone’s gap where do need to bear lower load. The composition has a perfect biodegradable property. The composite materials implanted in bone segment defect can stimulate the growth of osteoblast, blood vessel and new bones. The materials can be replaced by autogenous bone for its biodegradable property.
引文
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