充填型及大段型活性人工植骨材料的研制和相关研究
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摘要
骨缺损的治疗是临床骨科研究的重要课题之一。由于传统的治疗方法一自体或异体骨移植都存在一定的问题，因此人们一直在进行人工植骨材料的研究。本研究以基因重组人骨形态发生蛋白-2(rhBMP-2)为诱导成骨的活性生长因子，进行了充填型活性人工植骨材料和大段活性人工骨的研制，并通过动物实验对两种活性人工植骨材料的成骨活性和修复骨缺损的能力进行了检验，为今后临床应用提供理论依据。
一、充填型活性人工植骨材料的研制及相关研究
1．rhBMP-2异位诱导成骨活性的实验
目的：检验本研究所使用的利用原核生物表达的rhBMP-2的成骨活性，了解其复合载体以后的成骨能力。方法：实验设三组，单纯rhBMP-2组、rhBMP-2·明胶复合组及单纯明胶组，将材料植入小鼠股部肌袋，术后3天、1、2、3周通过大体观察及影像学和组织学检查，评价异位成骨效果；计算术后3周两个实验组中单位剂量rhBMP-2的诱导成骨量，比较成骨活性。结果：对照组仅出现炎症反应，无新骨形成。rhBMP-2组和rhBMP-2·明胶复合体组于术后2～3周形成小梁骨；术后3周复合体组单位剂量rhBMP-2的诱导成骨量是rhBMP-2组的8.8倍。结论：rhBMP-2有良好的异位诱导成骨活性；明胶作为载体可使rhBMP-2的异位诱导成果活性增大8.8倍。
2．三种充填型活性植骨材料的制备及修复兔长骨骨缺损的实验研究
第四军医大学博士学位论文
一
目的：将hBMP－二分别与明胶·羟基磷灰石、卵磷脂、甲壳素复合形成三
种充填型活性植骨材料，通过兔挠骨1．scm骨缺损模型观察三种植骨材料修复
骨缺损的能力，比较不同载体材料对n］．－－MP－2成骨作用的影响。方法：将含等
量f］．－MP－2的三种充填型活性植骨材料植入兔挠骨缺损中，并设空白对照组，
通过影像学、组织学、骨密度检查，评价骨缺损的修复效果。结果：术后12周
时，明胶·羟基磷灰石组和卵磷脂组的骨缺损全部得到修复，两组骨痴密度分
别为正常值的H＊％和陀．1％：甲壳素组形成被纤维组织包裹的包块，无新骨形
成，缺损未修复；空白组无骨俪形成，缺损未修复；载体材料的降解情况是明
胶·羟基磷灰石2周内降解，卵磷脂1周内降解，甲壳素术后12周仍有材料残
留。结论：ti］．－－MP－2在兔体内有肯定的诱导成骨作用。明胶·羟基磷灰石和卵
磷脂都是hBMP－2的良好载体，形成植骨材料后对骨缺损都有良好的修复效果，
相比较而言，明胶·羟基磷灰石的缓释时间更长，复合hBMP－2后对骨缺损的
修复效果更好。本实验所用的甲壳素在兔体内可以引起异物反应，不适合作为
rbBMPZ的载体c
3．充填型活性植骨材料修复犬长骨骨缺损的实验研究
目的：将hBto－2与明胶、羟基磷灰石、卵磷脂复合形成充填型活性植骨
材料，并通过犬挠骨缺损修复实验观察该充填型活性人工植骨材料对犬长骨骨
缺损的修复能力，了解rhBMPZ剂量与骨缺损修复效果的关系。方法：以犬挠
骨 2刀cm骨缺损为实验模型，分别植入含 0、l、2、4mg nMP2的复合材料，
、通过大体观察及影像学、组织学、骨密度检查，评价骨缺损的修复效果。结果：
术后12周骨缺损的修复率是空白组0只用只，ling组4只用只，Zing组8只用
只，4mg组 7只用只；术后 24周的骨缺损修复率与 12周时相似，不同之处是
骨痴得到改建。术后口周时 1、2、4mg组的骨密度值分别为正常值的 42．2％、
sl．8％、79．5％，术后24周时分别为正常值的61．4％、90．9％、88．6％。结论：（1）
充填型活性人工植骨材料在犬体内有良好的成骨活性；①2、4mg组对犬挠骨
ZCm骨缺损有良好的修复作用，ICg组也有一定的修复作用：q）植骨材料对骨
3
第四军医大学博士学位论文
一
缺损的修复效果在一定的范围内与植入的rhBMP－2量成正相关。
二、大段型活性人工植骨材料的研制及相关研究
1．大段活性人工骨的制造
目的：制备具有一定强度和仿生结构的大段型活性人工植骨材料。方法：
以聚左旋乳酸和磷酸三钙粉末为原料，在溶剂作用下按7：3的比例制成浆料，
通过材料成形机制备成冷冻状态的三维立体框架结构，于冻干机中抽真空，溶
剂升华后形成常温下的PLLA·CTCP 三维立体框架载体材料。负压法复合
几旧MP－2，冻干后形成活性人工骨。排液法检测材料的孔隙率，扫描电镜观察
材料的空隙大小和nMP－2的复合效果，万能材料实验机测量材料的力学强度。
结果：制备出柱状、套
The treatment of bone defection is one of the most important subjects in orthopaedics. Because traditional methods such as autografting and allografting have some shortcomings, people have been searching the bone substitutes. In this study, using rhBMP-2 as bone growth factor, the filling type of bioactive bone substitute and the large segmental bioactive bone substitute were made. And then, a series of animal experiments were done to test the bone forming activity and the bone defect repairing ability of these two types of bone substitutes, so as to provide some theoretical basis for them using in the future.
I The preparation of the filling type of bioactive bone substitutes
and related research
1 The experimental research of rhBMP-2 in inducing ectopic bone formation Objective: To test the activity of rhBMP-2 in this study in inducing ectopic bone formation, and to understand its activity when it compounding with the carrier. Methods: Three materials were implanted into mice's thigh muscle pouches, which are gelatin, rhBMP-2 and the composite of rhBMP-2 and gelatin. The effect of ectopic bone formation was evaluated by radiography and histology in 3 days, 1
week, 2 and 3 weeks after operation, and the weight of new bone inducing by a unit rhBMP-2 was calculated in 3 weeks of postoperation to compare the activity of bone formation. Results: There was new bone formed in the rhBMP-2 group and the composite group in 2-3 weeks of postoperation, but in the control group there was only inflammatory reaction. The weight of new bone induced by a unit dose of rhBMP-2 in 3 weeks of postoperation in composite group is 8.8 times of that in rhBMP-2 group. Conclusions: The rhBMP-2 has a very good activity in inducing ectopic bone formation; the gelatin as a carrier could amplify the activity about 8.8 times.
2 The preparation of three filling type of bioactive bone substitutes and the experimental study using them to repair defects in long bone of the rabbits
Objective: Combining gelatin ?hydroxyapatite (Gel ?HA) with rhBMP-2, lecithin with rhBMP-2, and chitin with rhBMP-2 to prepare three filling type of bioactive bone substitutes; to observe their repairing ability to bone defect, and to compare the effect of carriers to the bone forming ability of rhBMP-2. Methods: A 1.5 cm defect was made in middle-superior segment of rabbit's radius, then the three substitutes were implanted into the defect as three experiment groups, and nothing was implanted in the fourth group which is a control group. The repairing effect was evaluated by radiograph in 4, 8 and 12 weeks of postoperation, and by histology and BMD (Bone Mineral Density) in 12 weeks of postoperation. Results: Radiographically, in the rhBMP-2/Gel ?HA and rhBMP-2/Lec groups, all defects were connected by callus in 12 weeks; in the rhBMP-2/Chi group, there formed a soft tissue mass, and the defect was not repaired; in the control group, there was no callus formation and the defect was not repaired. Histologically, in the rhBMP-2/Gel ?HA and the rhBMP-2/Lec groups, the defects were connected by callus, which was composed by outer's lamellar bone and center's myeloid tissue;
the soft tissue in the defect site in rhBMP-2/Chi group was a inflammation mass wrapped by fibrous tissue; in the control group, the defect was connected by fibrous tissue. The BMD of the new callus in the rhBMP-2/Gel ?HA group and the rhBMP-2/Lec group were 55.5% and 48.1% of that of the normal radius in 12 weeks of postoperation. About degradation time, the lecithin is about 1 week, and the Gel ?HA is about 2 weeks. Conclusions: The rhBMP-2 has positive activity of inducing bone formation. Lecithin and Gel ?HA are good carriers of the rhBMP-2, and relatively, Gel ?HA has a better slowly-releasing effect. Chitin in this experiment is not suitable to be a carrier of rhBMP-2 for its foreign body reaction in the rabbit. 3 The preparation of a filling type of bioactive bone substitutes and the experimental study using them to repair defects in long bone of the dogs
Objectiv
一、充填型活性人工植骨材料的研制及相关研究
1．rhBMP-2异位诱导成骨活性的实验
目的：检验本研究所使用的利用原核生物表达的rhBMP-2的成骨活性，了解其复合载体以后的成骨能力。方法：实验设三组，单纯rhBMP-2组、rhBMP-2·明胶复合组及单纯明胶组，将材料植入小鼠股部肌袋，术后3天、1、2、3周通过大体观察及影像学和组织学检查，评价异位成骨效果；计算术后3周两个实验组中单位剂量rhBMP-2的诱导成骨量，比较成骨活性。结果：对照组仅出现炎症反应，无新骨形成。rhBMP-2组和rhBMP-2·明胶复合体组于术后2～3周形成小梁骨；术后3周复合体组单位剂量rhBMP-2的诱导成骨量是rhBMP-2组的8.8倍。结论：rhBMP-2有良好的异位诱导成骨活性；明胶作为载体可使rhBMP-2的异位诱导成果活性增大8.8倍。
2．三种充填型活性植骨材料的制备及修复兔长骨骨缺损的实验研究
第四军医大学博士学位论文
一
目的：将hBMP－二分别与明胶·羟基磷灰石、卵磷脂、甲壳素复合形成三
种充填型活性植骨材料，通过兔挠骨1．scm骨缺损模型观察三种植骨材料修复
骨缺损的能力，比较不同载体材料对n］．－－MP－2成骨作用的影响。方法：将含等
量f］．－MP－2的三种充填型活性植骨材料植入兔挠骨缺损中，并设空白对照组，
通过影像学、组织学、骨密度检查，评价骨缺损的修复效果。结果：术后12周
时，明胶·羟基磷灰石组和卵磷脂组的骨缺损全部得到修复，两组骨痴密度分
别为正常值的H＊％和陀．1％：甲壳素组形成被纤维组织包裹的包块，无新骨形
成，缺损未修复；空白组无骨俪形成，缺损未修复；载体材料的降解情况是明
胶·羟基磷灰石2周内降解，卵磷脂1周内降解，甲壳素术后12周仍有材料残
留。结论：ti］．－－MP－2在兔体内有肯定的诱导成骨作用。明胶·羟基磷灰石和卵
磷脂都是hBMP－2的良好载体，形成植骨材料后对骨缺损都有良好的修复效果，
相比较而言，明胶·羟基磷灰石的缓释时间更长，复合hBMP－2后对骨缺损的
修复效果更好。本实验所用的甲壳素在兔体内可以引起异物反应，不适合作为
rbBMPZ的载体c
3．充填型活性植骨材料修复犬长骨骨缺损的实验研究
目的：将hBto－2与明胶、羟基磷灰石、卵磷脂复合形成充填型活性植骨
材料，并通过犬挠骨缺损修复实验观察该充填型活性人工植骨材料对犬长骨骨
缺损的修复能力，了解rhBMPZ剂量与骨缺损修复效果的关系。方法：以犬挠
骨 2刀cm骨缺损为实验模型，分别植入含 0、l、2、4mg nMP2的复合材料，
、通过大体观察及影像学、组织学、骨密度检查，评价骨缺损的修复效果。结果：
术后12周骨缺损的修复率是空白组0只用只，ling组4只用只，Zing组8只用
只，4mg组 7只用只；术后 24周的骨缺损修复率与 12周时相似，不同之处是
骨痴得到改建。术后口周时 1、2、4mg组的骨密度值分别为正常值的 42．2％、
sl．8％、79．5％，术后24周时分别为正常值的61．4％、90．9％、88．6％。结论：（1）
充填型活性人工植骨材料在犬体内有良好的成骨活性；①2、4mg组对犬挠骨
ZCm骨缺损有良好的修复作用，ICg组也有一定的修复作用：q）植骨材料对骨
3
第四军医大学博士学位论文
一
缺损的修复效果在一定的范围内与植入的rhBMP－2量成正相关。
二、大段型活性人工植骨材料的研制及相关研究
1．大段活性人工骨的制造
目的：制备具有一定强度和仿生结构的大段型活性人工植骨材料。方法：
以聚左旋乳酸和磷酸三钙粉末为原料，在溶剂作用下按7：3的比例制成浆料，
通过材料成形机制备成冷冻状态的三维立体框架结构，于冻干机中抽真空，溶
剂升华后形成常温下的PLLA·CTCP 三维立体框架载体材料。负压法复合
几旧MP－2，冻干后形成活性人工骨。排液法检测材料的孔隙率，扫描电镜观察
材料的空隙大小和nMP－2的复合效果，万能材料实验机测量材料的力学强度。
结果：制备出柱状、套
The treatment of bone defection is one of the most important subjects in orthopaedics. Because traditional methods such as autografting and allografting have some shortcomings, people have been searching the bone substitutes. In this study, using rhBMP-2 as bone growth factor, the filling type of bioactive bone substitute and the large segmental bioactive bone substitute were made. And then, a series of animal experiments were done to test the bone forming activity and the bone defect repairing ability of these two types of bone substitutes, so as to provide some theoretical basis for them using in the future.
I The preparation of the filling type of bioactive bone substitutes
and related research
1 The experimental research of rhBMP-2 in inducing ectopic bone formation Objective: To test the activity of rhBMP-2 in this study in inducing ectopic bone formation, and to understand its activity when it compounding with the carrier. Methods: Three materials were implanted into mice's thigh muscle pouches, which are gelatin, rhBMP-2 and the composite of rhBMP-2 and gelatin. The effect of ectopic bone formation was evaluated by radiography and histology in 3 days, 1
week, 2 and 3 weeks after operation, and the weight of new bone inducing by a unit rhBMP-2 was calculated in 3 weeks of postoperation to compare the activity of bone formation. Results: There was new bone formed in the rhBMP-2 group and the composite group in 2-3 weeks of postoperation, but in the control group there was only inflammatory reaction. The weight of new bone induced by a unit dose of rhBMP-2 in 3 weeks of postoperation in composite group is 8.8 times of that in rhBMP-2 group. Conclusions: The rhBMP-2 has a very good activity in inducing ectopic bone formation; the gelatin as a carrier could amplify the activity about 8.8 times.
2 The preparation of three filling type of bioactive bone substitutes and the experimental study using them to repair defects in long bone of the rabbits
Objective: Combining gelatin ?hydroxyapatite (Gel ?HA) with rhBMP-2, lecithin with rhBMP-2, and chitin with rhBMP-2 to prepare three filling type of bioactive bone substitutes; to observe their repairing ability to bone defect, and to compare the effect of carriers to the bone forming ability of rhBMP-2. Methods: A 1.5 cm defect was made in middle-superior segment of rabbit's radius, then the three substitutes were implanted into the defect as three experiment groups, and nothing was implanted in the fourth group which is a control group. The repairing effect was evaluated by radiograph in 4, 8 and 12 weeks of postoperation, and by histology and BMD (Bone Mineral Density) in 12 weeks of postoperation. Results: Radiographically, in the rhBMP-2/Gel ?HA and rhBMP-2/Lec groups, all defects were connected by callus in 12 weeks; in the rhBMP-2/Chi group, there formed a soft tissue mass, and the defect was not repaired; in the control group, there was no callus formation and the defect was not repaired. Histologically, in the rhBMP-2/Gel ?HA and the rhBMP-2/Lec groups, the defects were connected by callus, which was composed by outer's lamellar bone and center's myeloid tissue;
the soft tissue in the defect site in rhBMP-2/Chi group was a inflammation mass wrapped by fibrous tissue; in the control group, the defect was connected by fibrous tissue. The BMD of the new callus in the rhBMP-2/Gel ?HA group and the rhBMP-2/Lec group were 55.5% and 48.1% of that of the normal radius in 12 weeks of postoperation. About degradation time, the lecithin is about 1 week, and the Gel ?HA is about 2 weeks. Conclusions: The rhBMP-2 has positive activity of inducing bone formation. Lecithin and Gel ?HA are good carriers of the rhBMP-2, and relatively, Gel ?HA has a better slowly-releasing effect. Chitin in this experiment is not suitable to be a carrier of rhBMP-2 for its foreign body reaction in the rabbit. 3 The preparation of a filling type of bioactive bone substitutes and the experimental study using them to repair defects in long bone of the dogs
Objectiv
引文
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