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
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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(P<0.05),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(P<0.05)。
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(P<0.05)。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(P>0.05)。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(P<0.05)。nHAC/PLA组在2W时各组间无显著性差异(P>0.05),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(P<0.05)。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(P<0.01),P24组和BMP2组间无显著性差异(P>0.05)。
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。
Part Ⅰ Evaluation on Bone-Seeking Properties of two Different synthetic oligopeptides derived from BMP-2
Objective This paper aims to compare the bone-seeking properties of two different synthetic oligopeptides derived from BMP-2, so as to explore the relevant factors to influence the bone-seeking properties of the BMP2-related oligopeptide.
Method Two different oligopeptides P20and P24were synthesized by solid phase synthesis process, and the P24contained a repeating sequence of Asp (aspartic acid) and phosphorylated serine. The nano-HAP crystals were used to simulate bone tissues, then the bone-seeking properties of two different oligopeptides were preliminary studied through traditional hydroxyapatite binding assay so as to determine the adsorption rates of these two peptides under different absorption time with different HAP quantity.
Results Both oligopeptides could achieve rapid adsorption of HAP within30min, then their adsorption speed decreased with the increase of time, P20achieved its peak of adsorption rate at60min, P24achieved its peak of adsorption rate which was twice as much as that of P20at90min; the adsorption rates of both oligopeptides increased along with the increase in the quantity of HAP, and the adsorption rate of P24was significantly higher than that of P20for the different quantity of HAP such as5mg,10mg or15mg.(P<0.05), the adsorption rate of P24to15mg HAP was nearly3times as much as that of P20.
Conclusion The addition of the active sites of repeating sequence of Asp and phosphorylated serine can significantly enhance the bone-seeking properties of the P24, and P24has a better bone-seeking property. The best adsorption time of the bone-seeking oligopeptides is90min, and their adsorption rates were positively correlated to the HAP quantity.
Part Ⅱ Adhesion, Proliferation and Osteogenic Differentiation of MC3T3-E1cells on Controlled Release Biomimetic Bone Matrix Materials Loaded with Different BMP2-related oligopeptide
Objective This paper aims to investigate the release kinetics of two kinds of BMP-2-related oligopeptide in vitro, so as to evaluate the effect of the different biomimetic bone matrix materials loaded with BMP-2-related oligopeptides on the adhesion, proliferation, and osteogenic differentiation of MC3T3-E1cell.
Methods Two different BMP-2-related oligopeptides P20and P24were loaded within ture bone ceramics (TBC) or nHAC/PLA scaffold materials respectively, so as to construct4composites (P20/TBC, P20/nHAC/PLA, P24/TBC and P24/nHAC/PLA), and the P24/Absorbable collagen sponges(ACS) were used as control material. Then their in vitro release kinetics were measured. To evaluate the biologic activity of these materials, they were divided into3groups including Control, P20and P24group, and each group was divided into TBC and nHAC/PLA subgroup. The MC3T3-E1cells were cultured on six kinds of materials respectively. The cell adhesion rate were assessed by precipitation method. The proliferative ability of MC3T3-E1cells were determined by MTT assay. And the measure of alkaline phosphatase (ALP) activity were performed to assess the differentiation towards osteoblasts of MC3T3-E1cells
Results The in vitro experiment showed that the release of P24or P20in nHAC/PLA or TBC scaffold material was slower than that of P24in ACS, and the release characteristics of P24was more striking than that of P20in TBC or nHAC/PLA material. While the cell adhesion rate, results of MTT experiments and ALP activity determination suggested that adhesion and proliferation of MC3T3-E1cells on the surfaces of all biomaterials containing BMP-2-related oligopeptides were higher than those on the surface of control groups, and those in P24group were significantly higher than those in P20group, and those in P24/TBC group were significantly higher than the P24/nHAC/PLA group (P<0.05).
Conclusion TBC and nHAC/PLA scaffold materials are suitable as controlled release carrier for BMP-2-related oligopeptide delivery, and TBC material is particularly ideal. The bone-seeking properties of P24can markedly enhance the performance of the sustained-release of P24in the TBC and nHAC/PLA scaffold materials, and can significantly promote the MC3T3-E1cells adhesion, proliferation, and osteodifferentiation on the biomaterials.
Part Ⅲ The effect of different BMP-2-related oligopeptides on ectopic bone formation in sintered bone or nHAC/PLA scaffold.
Objective This paper aims to compare the ectopic osteogenesis capacities of two different BMP-2-related oligopeptides, P20and P24, combined with sintered bone (TBC) or nano-hydroxyapatite-collagen PLA (nHAC/PLA) scaffold material respectively, in order to explore the osteoinductive activity of bone-seeking BMP-2-related oligopeptide.
Methods A total of30Sprague Dawley rats were divided into control group, P20group, and P24group randomly, each group was divided into TBC and nHAC/PLA subgroup (each n=10). Each rat was processed to prepare bilateral quadriceps muscle pouches model. Subsequently, the TBC material was implanted in left side and the nHAC/PLA material was implanted in the right side according to the experimental group. Radiographic and computer tomographic examinations were performed at2and8weeks post implantation, so as to evaluate the bone density. At2and8weeks after surgery the implanted materials were explanted, and then the total volume of the HAP in specimens (BV) were determined by high-resolution Micro CT, further histological examination were taken to determine the bone formation.
Results The X-ray detection at8th weeks showed the densities of implants in P24groups were higher than those at2th weeks, the changes in densities of implants of P20and the control groups were not significant. The bone density results showed that, the density of implants in P20/TBC groups, P24/TBC groups at8weeks were significantly higher than those at2th week (P<0.05). While the density of implants in control groups, P20/nHAC/PLA groups, P24/nHAC/PLA groups were slightly higher than those at2th week (P>0.05). BV quantitative results showed:in TBC groups, the BV value of P24groups at2weeks or8weeks was significantly higher than that in other groups, while the that the BV value of P20only at8th week was significantly higher than that in control groups (P<0.05); in nHAC/PLA groups, the BV values of P20and P24groups at2th week were slightly higher than that in other groups(P>0.05), the BV value of P24groups at8th week was significantly higher than that in other groups (P<0.05), and the BV value of P20groups at8th week was significantly higher than that in control groups (P<0.05). The observation of histomorphology showed that sporadic osteoid could be observed in P24/TBC groups at2weeks and a large number of woven bone formation and many active osteoblasts and neovascularization structures could be seen in P24/TBC groups at8th week. Significantly less woven bone formation was seen for the P24/nHAC/PLA groups at8weeks compared to the P24/TBC groups. There were only sporadic osteoid could be seen in the P20groups; and in the control groups, no new bone structure could be found.
Conclusion Both sintered bone and nHAC/PLA are ideal candidate for bone tissue engineering scaffold materials; the ectopic osteogenetic capacity and osteoinductivity of BMP-2-related oligopeptide was significantly improved by modified with bone-seeking moieties when coupled with TBC or nHAC/PLA scaffold material.
Part IV Experimental Study on Repair of Critical-size Defects in Canine Radius Using Bone-Seeking BMP-2-related Oligopeptide Combined with Sintered Bone Scaffold Material
Objective To investigate the efficacy of bone-seeking BMP-2-related oligopeptide/sintered bone (TBC) composite scaffold material for enhancing healing of critical-size defects in canine radius, so as to explore the feasibility of their further applications.
Methods A total of6beagle dogs were processed to prepare the critical-size defects (20mm) in middle section of bilateral radials, these dogs were divided into4groups according to their preparation sides randomly:there was no implant placing into the control bone defect group, pure TBC material was implanted into TBC group, the bone-seeking P24/TBC material was implanted into P24group and the rhBMP2/T material was implanted into BMP2group. After12weeks and24weeks of implantation, the X-ray detection was performed for them.3D-CT and histomorphometric evaluations were performed to evaluate the bone repair capability of each kind of material at24weeks after implantation.
Results The results of the radiographic and histomorphometric examination suggested that no new bone was found in the control group. In TBC group, the radiographic examination result at each time point showed that no callus could be found obviously and no obvious fusion was seen in bone-material interface; A little osteoid and woven bone, but no lamellar bone structure were observed in defects histomorphologically. In P24group, the bone-material interface was unclear and there was a little callus formation at12week; There was much callus formation with good osteointegrated at bone-material interface at24week. Histomorphometric examination showed much new bone formation, a large number of inteconnected lamellar bones, numerous active osteoblasts can be found, the bone-material interface had been integrated. The results of BMP2group were similar to those of P24group. And the differences between the new bone area percentage of P24group or BMP2group and that of TBC group were extremely significant (P<0.01), while the differences between that of P24group and that of BMP2group were not significant (P>0.05).
Conclusion The bone-seeking P24/TBC composite scaffold material has good osteoinductive activity, it's an ideal bone repair material for tissue engineering and deserve further pre-clinical research.
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(P<0.05),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(P<0.05)。
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(P<0.05)。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(P>0.05)。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(P<0.05)。nHAC/PLA组在2W时各组间无显著性差异(P>0.05),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(P<0.05)。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(P<0.01),P24组和BMP2组间无显著性差异(P>0.05)。
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。
Part Ⅰ Evaluation on Bone-Seeking Properties of two Different synthetic oligopeptides derived from BMP-2
Objective This paper aims to compare the bone-seeking properties of two different synthetic oligopeptides derived from BMP-2, so as to explore the relevant factors to influence the bone-seeking properties of the BMP2-related oligopeptide.
Method Two different oligopeptides P20and P24were synthesized by solid phase synthesis process, and the P24contained a repeating sequence of Asp (aspartic acid) and phosphorylated serine. The nano-HAP crystals were used to simulate bone tissues, then the bone-seeking properties of two different oligopeptides were preliminary studied through traditional hydroxyapatite binding assay so as to determine the adsorption rates of these two peptides under different absorption time with different HAP quantity.
Results Both oligopeptides could achieve rapid adsorption of HAP within30min, then their adsorption speed decreased with the increase of time, P20achieved its peak of adsorption rate at60min, P24achieved its peak of adsorption rate which was twice as much as that of P20at90min; the adsorption rates of both oligopeptides increased along with the increase in the quantity of HAP, and the adsorption rate of P24was significantly higher than that of P20for the different quantity of HAP such as5mg,10mg or15mg.(P<0.05), the adsorption rate of P24to15mg HAP was nearly3times as much as that of P20.
Conclusion The addition of the active sites of repeating sequence of Asp and phosphorylated serine can significantly enhance the bone-seeking properties of the P24, and P24has a better bone-seeking property. The best adsorption time of the bone-seeking oligopeptides is90min, and their adsorption rates were positively correlated to the HAP quantity.
Part Ⅱ Adhesion, Proliferation and Osteogenic Differentiation of MC3T3-E1cells on Controlled Release Biomimetic Bone Matrix Materials Loaded with Different BMP2-related oligopeptide
Objective This paper aims to investigate the release kinetics of two kinds of BMP-2-related oligopeptide in vitro, so as to evaluate the effect of the different biomimetic bone matrix materials loaded with BMP-2-related oligopeptides on the adhesion, proliferation, and osteogenic differentiation of MC3T3-E1cell.
Methods Two different BMP-2-related oligopeptides P20and P24were loaded within ture bone ceramics (TBC) or nHAC/PLA scaffold materials respectively, so as to construct4composites (P20/TBC, P20/nHAC/PLA, P24/TBC and P24/nHAC/PLA), and the P24/Absorbable collagen sponges(ACS) were used as control material. Then their in vitro release kinetics were measured. To evaluate the biologic activity of these materials, they were divided into3groups including Control, P20and P24group, and each group was divided into TBC and nHAC/PLA subgroup. The MC3T3-E1cells were cultured on six kinds of materials respectively. The cell adhesion rate were assessed by precipitation method. The proliferative ability of MC3T3-E1cells were determined by MTT assay. And the measure of alkaline phosphatase (ALP) activity were performed to assess the differentiation towards osteoblasts of MC3T3-E1cells
Results The in vitro experiment showed that the release of P24or P20in nHAC/PLA or TBC scaffold material was slower than that of P24in ACS, and the release characteristics of P24was more striking than that of P20in TBC or nHAC/PLA material. While the cell adhesion rate, results of MTT experiments and ALP activity determination suggested that adhesion and proliferation of MC3T3-E1cells on the surfaces of all biomaterials containing BMP-2-related oligopeptides were higher than those on the surface of control groups, and those in P24group were significantly higher than those in P20group, and those in P24/TBC group were significantly higher than the P24/nHAC/PLA group (P<0.05).
Conclusion TBC and nHAC/PLA scaffold materials are suitable as controlled release carrier for BMP-2-related oligopeptide delivery, and TBC material is particularly ideal. The bone-seeking properties of P24can markedly enhance the performance of the sustained-release of P24in the TBC and nHAC/PLA scaffold materials, and can significantly promote the MC3T3-E1cells adhesion, proliferation, and osteodifferentiation on the biomaterials.
Part Ⅲ The effect of different BMP-2-related oligopeptides on ectopic bone formation in sintered bone or nHAC/PLA scaffold.
Objective This paper aims to compare the ectopic osteogenesis capacities of two different BMP-2-related oligopeptides, P20and P24, combined with sintered bone (TBC) or nano-hydroxyapatite-collagen PLA (nHAC/PLA) scaffold material respectively, in order to explore the osteoinductive activity of bone-seeking BMP-2-related oligopeptide.
Methods A total of30Sprague Dawley rats were divided into control group, P20group, and P24group randomly, each group was divided into TBC and nHAC/PLA subgroup (each n=10). Each rat was processed to prepare bilateral quadriceps muscle pouches model. Subsequently, the TBC material was implanted in left side and the nHAC/PLA material was implanted in the right side according to the experimental group. Radiographic and computer tomographic examinations were performed at2and8weeks post implantation, so as to evaluate the bone density. At2and8weeks after surgery the implanted materials were explanted, and then the total volume of the HAP in specimens (BV) were determined by high-resolution Micro CT, further histological examination were taken to determine the bone formation.
Results The X-ray detection at8th weeks showed the densities of implants in P24groups were higher than those at2th weeks, the changes in densities of implants of P20and the control groups were not significant. The bone density results showed that, the density of implants in P20/TBC groups, P24/TBC groups at8weeks were significantly higher than those at2th week (P<0.05). While the density of implants in control groups, P20/nHAC/PLA groups, P24/nHAC/PLA groups were slightly higher than those at2th week (P>0.05). BV quantitative results showed:in TBC groups, the BV value of P24groups at2weeks or8weeks was significantly higher than that in other groups, while the that the BV value of P20only at8th week was significantly higher than that in control groups (P<0.05); in nHAC/PLA groups, the BV values of P20and P24groups at2th week were slightly higher than that in other groups(P>0.05), the BV value of P24groups at8th week was significantly higher than that in other groups (P<0.05), and the BV value of P20groups at8th week was significantly higher than that in control groups (P<0.05). The observation of histomorphology showed that sporadic osteoid could be observed in P24/TBC groups at2weeks and a large number of woven bone formation and many active osteoblasts and neovascularization structures could be seen in P24/TBC groups at8th week. Significantly less woven bone formation was seen for the P24/nHAC/PLA groups at8weeks compared to the P24/TBC groups. There were only sporadic osteoid could be seen in the P20groups; and in the control groups, no new bone structure could be found.
Conclusion Both sintered bone and nHAC/PLA are ideal candidate for bone tissue engineering scaffold materials; the ectopic osteogenetic capacity and osteoinductivity of BMP-2-related oligopeptide was significantly improved by modified with bone-seeking moieties when coupled with TBC or nHAC/PLA scaffold material.
Part IV Experimental Study on Repair of Critical-size Defects in Canine Radius Using Bone-Seeking BMP-2-related Oligopeptide Combined with Sintered Bone Scaffold Material
Objective To investigate the efficacy of bone-seeking BMP-2-related oligopeptide/sintered bone (TBC) composite scaffold material for enhancing healing of critical-size defects in canine radius, so as to explore the feasibility of their further applications.
Methods A total of6beagle dogs were processed to prepare the critical-size defects (20mm) in middle section of bilateral radials, these dogs were divided into4groups according to their preparation sides randomly:there was no implant placing into the control bone defect group, pure TBC material was implanted into TBC group, the bone-seeking P24/TBC material was implanted into P24group and the rhBMP2/T material was implanted into BMP2group. After12weeks and24weeks of implantation, the X-ray detection was performed for them.3D-CT and histomorphometric evaluations were performed to evaluate the bone repair capability of each kind of material at24weeks after implantation.
Results The results of the radiographic and histomorphometric examination suggested that no new bone was found in the control group. In TBC group, the radiographic examination result at each time point showed that no callus could be found obviously and no obvious fusion was seen in bone-material interface; A little osteoid and woven bone, but no lamellar bone structure were observed in defects histomorphologically. In P24group, the bone-material interface was unclear and there was a little callus formation at12week; There was much callus formation with good osteointegrated at bone-material interface at24week. Histomorphometric examination showed much new bone formation, a large number of inteconnected lamellar bones, numerous active osteoblasts can be found, the bone-material interface had been integrated. The results of BMP2group were similar to those of P24group. And the differences between the new bone area percentage of P24group or BMP2group and that of TBC group were extremely significant (P<0.01), while the differences between that of P24group and that of BMP2group were not significant (P>0.05).
Conclusion The bone-seeking P24/TBC composite scaffold material has good osteoinductive activity, it's an ideal bone repair material for tissue engineering and deserve further pre-clinical research.
引文
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