亲骨性BMP-2活性多肽及其仿生骨修复材料生物活性的实验研究

发布时间：2018-05-08 16:43

本文选题：BMP2 + 多肽　；参考：《华中科技大学》2013年博士论文

【摘要】：第一部分两种不同BMP2活性多肽的亲骨性评价目的对比研究两种不同BMP-2活性多肽的亲骨性,以探讨影响多肽亲骨性的相关因素。方法以固相合成法分别合成两种不同的BMP-2活性多肽P20和P24,在P24中增加设计聚天冬氨酸短肽和磷酸化丝氨酸活性位点。以纳米羟基磷灰石晶体模拟骨组织,采用经典的吸附法对两种多肽进行初步的体外骨吸附性能研究,分别在不同吸附时间和不同质量羟基磷灰石介质的条件下考察两种多肽对羟基磷灰石的吸附率。结果两种BMP-2活性多肽在30min内均能与羟基磷灰石迅速吸附,此后随时间延长吸附速度减缓,P20在60min时吸附率达到峰值,而P24在90min时达到峰值,为P20的两倍。两种多肽的吸附率随着羟基磷灰石质量的增多而升高,在5mg、10mg和15mg三种不同羟基磷灰石量时P24的吸附率均显著高于P20(P0.05),P24对15mg羟基磷灰石的吸附率几乎是同样条件下P20的3倍。结论通过加入聚天冬氨酸和磷酸化丝氨酸活性位点可显著增强BMP-2活性多肽的亲骨性,P24具有良好的亲骨性。亲骨性BMP-2活性多肽对羟基磷灰石的最佳吸附时间是90min,且其吸附率与羟基磷灰石质量呈正相关。第二部分携载不同BMP2活性多肽的仿生骨基质材料体外控释及其对MC3T3-E1细胞粘附、增殖及成骨分化能力的影响目的观察两种BMP-2活性多肽的体外释放动力学,评价不同多肽和仿生骨基质材料对小鼠MC3T3-E1前成骨细胞粘附、增殖和成骨分化能力的影响。方法将两种不同的BMP-2活性多肽P20和P24分别与TBC和nHAC/PLA两种载体材料复合,构建P20/TBC、P20/nHAC/PLA、P24/TBC和P24/nHAC/PLA仿生骨基质材料,同时以P24/可吸收胶原蛋白海绵缓释体为对照比较BMP-2活性多肽在各种支架材料中的释放动力学。为评价各仿生骨基质材料的生物学活性,将实验分为空白对照、P20和P24三组,每组另设TBC和nHAC/PLA两个亚组。分别将小鼠MC3T3-E1前成骨细胞种植在各组材料表面,分别测定细胞在材料表面的粘附率,采用MTT法检测MC3T3-E1细胞在材料上的增殖活性,通过检测碱性磷酸酶(ALP)活性来评价MC3T3-E1细胞在各组材料上的成骨分化活性。结果体外控释实验发现P24和P20无论在nHAC/PLA还是TBC材料中,均较P24在ACS材料中释放更加缓慢,同时P24较P20在两种材料中的缓释效果更突出。而细胞粘附率、MTT实验及ALP活性检测结果显示,MC3T3-E1细胞在所有多肽组仿生骨基质材料表面的粘附和增殖能力及ALP活性均显著高于空白组,而在P24组显著高于P20组,在P24/TBC组显著高于P24/nHAC/PLA组,组间差异均有统计学意义(P0.05)。结论TBC和nHAC/PLA均适合作为BMP-2活性多肽的缓释载体材料,且TBC尤为理想。亲骨性可明显增强P24在TBC和nHAC/PLA材料中的缓释性能,并能明显促进小鼠MC3T3-E1前成骨细胞在材料上的粘附、增殖和成骨分化。第三部分不同BMP2活性多肽／仿生骨基质材料复合物体内诱导异位成骨的实验研究目的比较P20和P24与TBC和nHAC/PLA构建的仿生骨基质材料的异位成骨能力,探讨亲骨性对多肽骨诱导活性的影响。方法将30只SD大鼠随机分为空白组、P20组及P24组,每组下设煅烧骨和nHAC/PLA两个亚组。每只大鼠制备双侧股四头肌肌袋模型,按实验分组分别于左侧放入TBC材料,右侧放入nHAC/PLA材料。植入材料后第2、8周分别做放射学检测(X-ray、3D-CT)并记录植入体CT骨密度值(HU)。术后2、8周分别处死大鼠取材,应用高分辨率Micro CT测定标本内羟基磷灰石总体积值(BV),然后标本行HE染色组织学检测。结果8周时X线检测可见P24组植入体密度均较2周时增高,P20及空白组密度变化不明显。CT扫描骨密度检测结果显示,8W时P20/TBC组、P24/TBC组植入体骨密度值较2W时明显增高,差异有统计学意义(P0.05)。而空白组、P20/nHAC/PLA组和P24/nHAC/PLA组植入体骨密度值较2W时仅略增高,差异无统计学意义(P0.05)。BV定量结果显示：在TBC组,2W和8W时P24组BV值均显著高于其他各组,P20组仅8W时显著高于对照组,差异有统计学意义(P0.05)。nHAC/PLA组在2W时各组间无显著性差异(P0.05),8W时P24组BV值显著高于其他各组,P20组显著高于空白组,差异有统计学意义(P0.05)。组织形态学观察2周时P24/TBC组植入体孔隙内可看到散在的类骨质形成。8周时P24/TBC组可见大量编织骨形成、有较多活跃的成骨细胞及新生血管结构。P24/nHAC/PLA组新生骨组织明显少于P24/TBC组。P20组仅见散在类骨质形成。空白组无明显成骨。结论煅烧骨和nHAC/PLA均是理想的骨组织工程支架材料,增加亲骨性后能显著增强BMP-2活性多肽与两种材料复合构建的仿生骨基质材料的异位成骨能力第四部分亲骨性BMP2活性多肽/煅烧骨复合支架材料修复犬桡骨临界性缺损的实验研究目的观察亲骨性BMP-2活性多肽P24/煅烧骨复合材料修复犬桡骨临界性骨缺损的能力,探讨其进一步应用的可行性。方法6只比格犬制备双侧桡骨中段20mm临界性骨缺损,按侧别随机分为4组,空白对照组骨缺损处不放置任何植入物,TBC组植入单纯TBC材料,P24组植入P24/TBC复合材料,BMP2组植入rhBMP2/TBC复合材料。术后12周和24周分别行X线检测,术后24周取材行CT扫描加三维重建并行组织学检测评价各组材料的骨修复能力。结果影像学及组织学检测结果均提示空白对照组骨缺损处无新骨形成；TBC组各时间点影像学检查均无明显骨痂形成,骨-材料界面无明显融合,组织学观察可见缺损内少许类骨质和编织骨形成,无板层骨结构；P24组在12W时骨-材料界面模糊,有少许骨痂形成,24W时较多骨痂形成,骨-材料界面已融为一体。组织学可见缺损内较多新骨形成,有大量板层骨连接成片,可见大量活跃成骨细胞,骨-材料界面完全融合。BMP2组结果和P24组相似。P24组和BMP2组新生骨面积百分比与TBC组比较有非常显著性差异(P0.01),P24组和BMP2组间无显著性差异(P0.05)。结论自行研制的P24/TBC复合支架材料具有良好的骨诱导活性,是一种理想的组织工程骨修复材料,值得进行进一步的临床前研究。
[Abstract]:Evaluation of the affinity of two different BMP2 active polypeptides in the first part

Objective To investigate the affinity of two different BMP - 2 active polypeptides in order to investigate the relative factors influencing the affinity of polypeptide .

Methods Two different BMP - 2 active polypeptides ( P20 and P24 ) were synthesized by solid - phase synthesis . In P24 , poly ( aspartic acid ) short peptide and phosphorylated Ser active site were designed . The bone tissue was simulated by nano hydroxyapatite crystal . The adsorption rate of the two polypeptides on hydroxyapatite was investigated under different adsorption time and different mass of hydroxyapatite medium .

Results The adsorption rate of the two kinds of BMP - 2 polypeptides increased with the increase of hydroxyapatite mass , and the adsorption rate of P24 to 15 mg hydroxyapatite was almost 3 times higher than that of P24 at 5 mg , 10 mg and 15 mg .

Conclusion The affinity of BMP - 2 active polypeptide can be significantly enhanced by the addition of poly - aspartic acid and phosphorylating serine . The best adsorption time of bone - like BMP - 2 active polypeptide on hydroxyapatite is 90 min , and its adsorption rate is positively correlated with the quality of hydroxyapatite .

In vitro controlled release of bionic bone matrix material carrying different BMP2 active polypeptides and its effect on cell adhesion , proliferation and osteogenic differentiation of MC3T3 - E1 cells

Objective To observe the in vitro release kinetics of two BMP - 2 active polypeptides , evaluate the effects of different polypeptides and biomimetic bone matrix materials on the adhesion , proliferation and osteogenic differentiation of MC3T3 - E1 in mice .

Methods Two kinds of different BMP - 2 active polypeptides were prepared by combining two different BMP - 2 active polypeptides , respectively , and the release kinetics of BMP - 2 active polypeptide in various scaffold materials was established by using P24 / TBSA as control .

Results In vitro controlled - release experiment , P24 and P24 were released more slowly than P24 in ACS material , while P24 was more prominent than that of P24 in both materials . The results of MTT assay and ALP activity showed that MC3T3 - E1 cells had higher adhesion and proliferation ability and ALP activity in all polypeptide groups than in blank group .

Conclusion The release properties of P24 can obviously enhance the release properties of P24 in the materials of TB3T3 - E1 , and can obviously promote the adhesion , proliferation and osteogenic differentiation of MC3T3 - E1 osteoblasts in mice .

Experimental study of ectopic osteogenesis induced by different BMP2 active polypeptide / bionic bone matrix composites in vitro

Objective To compare the ectopic osteogenesis of the bionic bone matrix material constructed by p20 and P24 with TBCs and nJs / PLA , and to investigate the influence of bone affinity on the osteoinductive activity of the polypeptide .

Methods Thirty SD rats were randomly divided into two groups : blank group , P20 group and P24 group . Each group was divided into two sub - groups of calcined bone and n \

Results Compared with control group , the values of bone mineral density were significantly higher than those in control group ( P0.05 ) .

Conclusion It is ideal bone tissue engineering scaffold material for both the calcined bone and nits / PLA , which can significantly enhance the ectopic osteogenesis of the bionic bone matrix material of BMP - 2 active polypeptide and two materials .

Experimental study on repairing the critical defect of canine radial bone with the fourth partial bone affinity BMP2 active polypeptide / calcined bone composite scaffold material

Objective To investigate the ability of bone morphogenetic protein - 2 ( BMP - 2 ) active polypeptide P24 / calcined bone composite to repair the critical bone defect in dogs .

Methods Six Beagle dogs were randomly divided into 4 groups , and no implants were placed in the blank control group . All the implants were implanted into the bone defect of the blank control group , the P24 / TBCs were implanted in the group P24 , the BMP2 group was implanted with the rhBMP2 / TBCs . After 12 weeks and 24 weeks after operation , the X - ray examination was performed , and the bone repair capacity of the materials was evaluated by CT scanning and three - dimensional reconstruction parallel histological examination at 24 weeks after operation .

Results The results of imaging and histological examination indicated that there was no new bone formation at the bone defect of the blank control group .
There was no obvious callus formation at all time points of the group , no obvious fusion was observed in the interface of the bone - material , the histological observation showed that there were little bone and braided bone formation in the defect , and no lamellar bone structure was observed .
There was no significant difference in bone - material interface between P24 group and BMP2 group ( P0.01 ) , there was no significant difference between P24 group and BMP2 group ( P0.05 ) .

Conclusion It is an ideal tissue engineering bone repairing material which is worthy of further pre - clinical research .

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R314

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