载骨形态发生蛋白2活性肽纳米仿生骨基质材料成骨能力和修复骨缺损的实验研究
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摘要
目的构建BMP-2活性肽/PLGA-[ASP-PEG]仿生骨基质材料,评价BMP-2活性肽对大鼠骨髓基质干细胞(BMSC)的粘附、增殖及诱导成骨分化的影响。
方法对聚乳酸-乙醇酸共聚物(PLGA)改性,制成PLGA-[ASP-PEG]多元共聚物,同时用固相合成法合成寡肽BMP-2活性肽(P24),将其与PLGA-[ASP-PEG]共价结合,构建BMP-2活性肽(P24)/ PLGA-[ASP-PEG]仿生骨基质支架材料。实验分三组,将大鼠BMSC与不同材料复合培养。A组为PLGA-[ASP-PEG]仿生骨基质材料,B组为PLGA-[ASP-PEG]材料,C组为单纯的PLGA材料。流式细胞仪(FCM)对培养细胞性质进行鉴定。沉淀法测定细胞与材料到粘附率。四甲基偶氮唑蓝微量酶反应比色法(MTT)、考马斯亮蓝法和扫描电镜(SEM)检测BMSC体外增殖活性,成骨诱导标志物碱性磷酸酶(ALP)活性,骨钙蛋白(OCN)含量,ALP染色和钙结节染色了解BMSC成骨分化情况。
结果FCM证实分离培养的细胞为BMSC。细胞粘附率检测显示,BMSC在P24/ PLGA-[ASP-PEG]材料表面和PLGA-[ASP-PEG]材料表面的粘附性能和增殖能力明显高于PLGA表面,差异有统计学意义(P<0.05)。与SEM、细胞生长曲线和细胞蛋白质含量测定结果一致。ALP活性和OCN含量检测显示:P24诱导的A组ALP活性和OCN含量明显高于B组和C组,ALP染色和钙结节染色:P24诱导的A组出现阳性表现的时间最早。
结论PLGA-[ASP-PEG]能促进BMSC在骨基质材料表面的粘附、增殖并能较好地保持细胞的形态,PLGA-[ASP-PEG]材料及其降解产物不影响BMSC的成骨分化, P24能体外诱导BMSC向成骨方向分化,具有与BMP-2类似的骨诱导活性。
第二部分BMP-2活性肽体外定向诱导大鼠BMSC向成骨方向分化的剂量依赖性研究
目的探讨自行合成的寡肽P24对大鼠BMSC体外定向诱导成骨分化的能力,评价P24的成骨诱导活性及体外诱导成骨的最佳剂量。
方法取4周龄SD大鼠分离培养BMSC,传至第3代时改用成骨诱导培养基,培养基中分别加入浓度为300、200、100、50和0μg/ml的P24,并依次记为A、B、C、D和E组。观察细胞的形态学改变,检测细胞的ALP活性和OCN含量,实时荧光定量RT-PCR检测成骨标志物基因Ⅰ胶原(COL-Ⅰ)、骨桥蛋白(OPN)及OCN mRNA的表达。
结果倒置相差显微镜下观察,用成骨诱导培养基培养3～4d,培养细胞形态由长梭形转变为短梭形或多角形。随着培养基中P24浓度的增加,细胞发生成骨样改变的时间提前。ALP活性和OCN含量检测显示,A和B组ALP活性和OCN含量较其他三组增加明显,差异有显著性(P<0.05),组间比较,差异无显著性(P>0.05)。实时荧光定量RT-PCR检测显示培养14d,成骨诱导标志物COL-Ⅰ、OPN和OCN mRNA在各组均有表达。A组和B组的Ct值明显大于C组和D组,差异有显著性(P<0.05),但A组和B组之间差异无显著性(P>0.05)。
结论P24体外能有效地诱导BMSC向成骨方向分化,在PLGA-[ASP-PEG]材料体系中,最适合的P24体外诱导剂量认为是200μg/ml。
第三部分BMP-2活性肽/PLGA-[ASP-PEG]仿生骨基质材料异位成骨的实验研究
目的评价P24/PLGA-[ASP-PEG]仿生骨基质材料的异位成骨能力。
方法成年雄性SD大鼠48只,随机分成三组,分别在大鼠背部双侧竖脊肌浅层肌袋中植入不同材料。A组:P24/ PLGA-[ASP-PEG]仿生骨基质材料;B组:单纯PLGA-[ASP-PEG]材料;C组:单纯的明胶海绵材料。X线、CT+三维成像观察植入物成骨情况,不同时间点病理切片,行组织学观察,western blot检测植入区成骨标志物Col-I及OPN蛋白的表达,实时荧光定量RT-PCR检测Col-I及OPN mRNA的表达。
结果X线、CT+三维成像:8周时,A组可见明显的成骨表现,B组偶见成骨样影像,C组未见新骨表现。组织学观察:A组8周骨小梁明显增粗增宽,表面形成类骨密质的结构,可见大量新骨形成,在新骨外周部有骨小梁及新生血管。B组8周时植入区纤维组织增生,偶见软骨细胞及少量成骨细胞。C组8周明胶海绵吸收,呈现肌肉组织表现,未见新骨形成。实时荧光定量RT-PCR:各组8周均有Col-I mRNA和OPN mRNA的表达, A组中Col-I mRNA和OPN mRNA为高表达。与western blot检测结果一致。
结论构建的P24/PLGA-[ASP-PEG]仿生骨基质材料能够诱导异位成骨,P24具有与天然BMP-2类似的异位诱导成骨能力和骨诱导活性,其成骨过程以软骨内化骨为主。
第四部分BMP-2活性肽/PLGA-[ASP-PEG]仿生骨基质材料修复兔股骨缺损的实验研究
目的探讨P24/PLGA-[ASP-PEG]仿生骨基质材料修复兔股骨中段临界大小骨缺损的可行性。
方法36只新西兰大白兔制备成股骨中段15mm的临界大小骨缺损模型。按实验设计分为三组。A组:植入P24/ PLGA-[ASP-PEG]仿生骨基质材料,B组植入PLGA-[ASP-PEG]材料;C组,植入单纯的明胶海绵。5孔钢板螺钉内固定。X线观察,组织学观察、修复组织ALP测定以及生物力学测试,比较各组材料修复骨缺损的能力。
结果X线显示及评价:A组8周骨痂与宿主骨界限模糊,骨痂密度不均匀,皮质骨轮廓出现,骨缺损已基本消失,有典型的骨愈合影像;12周骨痂开始塑性,新生骨皮质结构清晰,与断端骨自然连接,髓腔有再通趋势。按照X线骨缺损愈合分级,16周时4级以上为9只(优良率64.28%)。B组12周缺损区有少量骨痂生成,断端吸收变细,髓腔开始闭锁,缺损区依然存在。X线骨缺损愈合分级0级为5只(占62.5%)。C组:12周骨缺损区表现为骨不连影像,髓腔闭锁,钢板有松动迹象。X线骨缺损愈合分级均为0级。组织学观察:A组:8周时成骨细胞活跃,新骨大量增生,相互融合生长,呈编织骨和骨小梁结构,骨小梁钙化形成板层股。12周时编织骨增厚密集,呈典型的板层骨结构,骨细胞趋于成熟,有血管形成和长入,不规则骨髓腔出现。B组:8周纤维组织增生,材料部分吸收。12周植入区两端与宿主骨交界处,新骨增生,可见骨小梁和板层骨。植入区中央材料大部分吸收,可见大量的纤维组织,偶见软骨细胞。C组:12周缺损区被纤维结缔组织填充,骨端增生不活跃,髓腔封闭。生物力学测试:A组最大抗弯曲负荷122.14±12.83N,与同组正常骨参照值相比,修复比率为87.42%。B组最大抗弯曲负荷38.38±8.09N,与同组正常骨参照值相比,修复比率为26.89%。二者具有统计学差异性(P<0.05)。
结论体外构建的P24/PLGA-[ASP-PEG]仿生骨基质材料是一种理想的组织工程支架材料,能诱导启动典型的软骨内化骨过程,促进骨缺损的修复。
Objective In this research, a new biomimetic bone tissue engineering scaffold material, BMP-2-derived peptide P24 / PLGA-[ASP-PEG] composite was constructed. The effect of P24 to the adhesion, proliferation and osteodifferentiation of bone mesenchymal stem cells (BMSCs) were investigated.
Methods PLGA was modified with polyethylene glycol (PEG) and asparagic acid (ASP), and tri-block polymer PLGA-[ASP-PEG] material was prepared. Then peptide P24 was introduced into PLGA-(PEG-ASP)n scaffolds by cross-linkers. Thus, BMP-2-derived peptide P24 / PLGA-[ASP-PEG] composite was synthesized. BMSCs were cultured on three kinds of material respectively, one was P24 / PLGA-[ASP-PEG] biomimetic material (Group A), one was PLGA-[ASP-PEG] material (Group B), the other was simple PLGA material (Group C). The lineage specificity of the cells on these materials were detected by Flow cytometry(FCM). The cell adhesion was assessed by precipitation method. The proliferative ability of BMSCs were measured by MTT assay, Coomassie brilliant blue staining and scanning electron microscope(SEM). And the measure of alkaline phosphatase(ALP) activity and osteocalcin(OCN) level and ALP staining and calcium tubercle staining were performed to assess the cells’differentiation towards osteoblasts.
Results FCM confirmed the cells that isolated and cultured were BMSCs. The adhesive ratio measure suggested that adhesion and proliferation of BMSCs on the surface of P24 / PLGA-[ASP-PEG] is much higher than the control group (P<0.05). These were coincident with the cells’growth curve and the result of total protein assay and SEM. The ALP activity and osteocalcin(OCN) level of BMSCs in P24 / PLGA-[ASP-PEG] group were significant higher than in PLGA-[ASP-PEG] group and PLGA group. And in the assessment of ALP staining and calcium tubercle staining, positive staining was expressed firstly in P24 / PLGA-[ASP-PEG] group.
Conclusion The PLGA-[ASP-PEG] material was confirmed to improve BMSCs’adhesion and proliferation, and maintain their morphology. The material and its degradation product didn’t impact BMSCs’osteodifferentiation. And the BMP-2 derived peptide P24 could induce BMSCs’differentiation towards osteoblasts, its activity was equal to BMP-2.
PartⅡDose-dependent of BMP-2-derived Peptied on Osteogentc Induction of BMSCs in vitro
Objective To evaluate the capability of synthesized BMP2-derived peptide P24 on osteogenic induction of BMSCs, and investigate its dose-dependent and optimal dose in vitro.
Methods BMSCs were separated from 4-week-old SD rats and cultured with normal medium. At 3rd generation the medium was changed to osteogenetic medium which contained different dose of BMP2-derived peptide. The concentrations of BMP2-derived peptide contained in medium were 300、200、100、50 and 0μg/ml, respectively. These groups were labeled with A～E by turns. The change of cell morphology was observed with microscopy. ALP activities and OCN levels were taken at different points of time. The mRNA expression level of several osteogenetic marker such as Type I collagen(Col-I), osteopontin(OPN) and osteocalcin(OCN) were measured using real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) technique to detect the osteoinductivity of different concentration of BMP2-derived peptide,.
Results Under inverted phase contrast microscope, MSCs had changed their shape from long fusiform to short fusiform or polygon with the osteogenetic medium for 3~4 days. With the increase of the concentration of BMP2-derived peptide in medium, the time of MSCs changed their shape into osteoblast-like had advanced accordingly. The detection of ALP activities and OCN levels suggested that group A and group B were significant higher than the rest three groups (P<0.05). But there was no statistical significance between group A and group B(P>0.05). FQ-PCR suggested that after cultured for 14d, Col-I, OPN and OCN were expressed in each group. And the value of cycle threshold (Ct) of Group A and Group B were significant higher than the rest three groups(P<0.05). But there has no statistical significance between group A and group B(P>0.05).
Conclusion BMP2-derived peptide can induce BMSCs to differentiate into osteoblasts effectively. And among the PLGA-[ASP-PEG] material system, the optimal inductive concentration of P24 was 200μg/ml.
PartⅢEctopic osteogenesis of BMP-2-derived peptide combined with PLGA-[ASP-PEG] scaffold
Objective Evaluated the ectopic osteogenetic capacity of synthesis BMP2-derived peptide combined with poly (lactic acid / glycolic acid / asparagic acid-co-polyethylene glycol) (PLGA-[ASP-PEG]) by animal experiment.
Methods A total of 48 SD rats were allocated into three groups randomly. Three kinds of materials were respectively implanted into each side of superficial layer of erector spinae. Group A: BMP2-derived peptide P24 / PLGA-[ASP-PEG] complex, Group B: simple PLGA-[ASP-PEG], Groups C: simple gelatin sponge . The bone formation of the implants was detected by X-ray and CT three-dimensional imaging, further assessed by histological sections in different points of time. The osteogenic marker, Col-I and OPN were detected by western blot at protein level and by RT-PCR at mRNA level.
Results X-ray and CT three-dimensional imaging showed obvious osteogenesis in group A, occasional osteogenesis in group B, and no bone formation in group C in the 8th week. Histological examination showed at 8th week, in group A, bone trabeculae were found to be robust and wide with some compact bone-like tissue formed on their surface, and plenty of new bone formation was found, some bone trabeculae and new vessels located around them; and the material was absorbed totally. In group B, fibroplasia was found around implant and chondrocytes and osteoblasts could be seen occasionally; and in Group C, implanted gelation sponge was absorbed, the area displayed muscle tissue-like appearance, no new bone structure could be found. Real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) suggested that Col-I mRNA and OPN mRNA were expressed at each groups at 8th week, and in group A the express levels were higher than others. Western blot obtained the same consequence.
Conclusion BMP2-derived peptide P24 could induce ectopic osteogenesis, and its ectopic osteogenetic capacity and osteoinductivity were equal to nature BMP-2. Os endochondrale acted predominantly during osteogenesis.
PartⅣAccelerated bone regeneration with a novel synthetic BMP-2-derived bioactive oligopeptide combined with a nano-biomimetic PLGA-[ASP-PEG] scaffold
Objective Furtherly investigate the osteogenetic capacity of biomimetic materials modified with a novel peptide P24 derived from Bone Morphogenetic Protein-2 combined with PLGA-[ASP-PEG] polymer by using them to repair critical-sized bone defect.
Methods A total of 36 new zealand white rabbits were allocated into three groups randomly. A 1.5cm unilateral segmental critical-sized bone defect was created in the left femoral diaphysis in all rabbits. Three kinds of materials were used respectively. P24 combined with PLGA-[ASP-PEG] scaffold were implanted in group A, PLGA-[ASP-PEG] without P24 were implanted in group B, and simple gelatin sponge were implanted in group C. Five-hole plates and screws were applied to perform internal fixation routinely. Macroscopic observation, X-ray, histological investigation, ALP activity of repaired tissue and biomechanics measure were taken to evaluate the bone repair capability of each kind of material.
Results Radiographic findings showed: In group A, at 8th week, the boundary between bone callus and host bone became obscure, bone callus appeared uneven in density, the profile of os integumentale was displayed, bone defect almost disappeared, typical symbol of bone healing was observed; at 12th week, bone callus moulding was found, the texture of new os integumentale became clear, and them united with the broken end naturally, and the medullary cavity was trend to be recanalization. According to the standardized radiographic scales of bone defect repair, 9 rabbits were assessed as Grade 4 or above (fine ratio was 64.28%). In group B, at 12th week, a little bone callus was formed in the defect area, the broken ends were absorbed and became small, the medullary cavity began to be atresic, and the defect was still presented. There were 5 rabbits that be assessed as Grade 0 (62.5%). In group C, at 12th week, ununion symbol was still observed, atresic medullary cavity and loosing plate were be found. All of the rabbits were assessed as grade 0. Histological observations showed: In group A, in 8th week, active osteoblasts could be found, plenty of new bone tissue was formed and fused with each other, and seemed like woven bone and bone trabecula, lamellar bone was formed by calcification of bone trabeculae. At 12th week, woven bone tissue became thick and intensive, and turned into typical lamellar bone, osteocytes were trend to be mature, and with angiopoiesis and its invasion, irregular medullary cavities were formed. In group B, at 8th week, fibroplasia could be found and the material was absorbed partly.; at 12th week, new bone formation was found in the juncture of implant and host bone tissue, and bone trabeculae and lamellar bone were appeared. The middle of material was absorbed subtotally, massive fibrous tissue could be seen, and chondrocytes were be occasionally seen. In group C, at 12th week, the defect area was filled by fibrous connective tissue, hyperplasy in broken ends were inconspicuous, and medullary cavities were blocked. Biomechanics measure suggested the ultimate bending load of group A was 122.14±12.83N, which reparative ratio was 87.42% when compared with the reference value of normal bone in the same group; and in group B, the ultimate bending load was 38.38±8.09N, the reparative ratio of it was 26.89% when compared with the reference value of normal bone in the same group. And there was statistical difference between group A and B (p<0.05).
Conclusion This novel biomimetic material P24/PLGA-[ASP-PEG] is an ideal scaffold for bone tissue engineering, which can trigger typical os endochondrale procession and improve repair of bone defect.
方法对聚乳酸-乙醇酸共聚物(PLGA)改性,制成PLGA-[ASP-PEG]多元共聚物,同时用固相合成法合成寡肽BMP-2活性肽(P24),将其与PLGA-[ASP-PEG]共价结合,构建BMP-2活性肽(P24)/ PLGA-[ASP-PEG]仿生骨基质支架材料。实验分三组,将大鼠BMSC与不同材料复合培养。A组为PLGA-[ASP-PEG]仿生骨基质材料,B组为PLGA-[ASP-PEG]材料,C组为单纯的PLGA材料。流式细胞仪(FCM)对培养细胞性质进行鉴定。沉淀法测定细胞与材料到粘附率。四甲基偶氮唑蓝微量酶反应比色法(MTT)、考马斯亮蓝法和扫描电镜(SEM)检测BMSC体外增殖活性,成骨诱导标志物碱性磷酸酶(ALP)活性,骨钙蛋白(OCN)含量,ALP染色和钙结节染色了解BMSC成骨分化情况。
结果FCM证实分离培养的细胞为BMSC。细胞粘附率检测显示,BMSC在P24/ PLGA-[ASP-PEG]材料表面和PLGA-[ASP-PEG]材料表面的粘附性能和增殖能力明显高于PLGA表面,差异有统计学意义(P<0.05)。与SEM、细胞生长曲线和细胞蛋白质含量测定结果一致。ALP活性和OCN含量检测显示:P24诱导的A组ALP活性和OCN含量明显高于B组和C组,ALP染色和钙结节染色:P24诱导的A组出现阳性表现的时间最早。
结论PLGA-[ASP-PEG]能促进BMSC在骨基质材料表面的粘附、增殖并能较好地保持细胞的形态,PLGA-[ASP-PEG]材料及其降解产物不影响BMSC的成骨分化, P24能体外诱导BMSC向成骨方向分化,具有与BMP-2类似的骨诱导活性。
第二部分BMP-2活性肽体外定向诱导大鼠BMSC向成骨方向分化的剂量依赖性研究
目的探讨自行合成的寡肽P24对大鼠BMSC体外定向诱导成骨分化的能力,评价P24的成骨诱导活性及体外诱导成骨的最佳剂量。
方法取4周龄SD大鼠分离培养BMSC,传至第3代时改用成骨诱导培养基,培养基中分别加入浓度为300、200、100、50和0μg/ml的P24,并依次记为A、B、C、D和E组。观察细胞的形态学改变,检测细胞的ALP活性和OCN含量,实时荧光定量RT-PCR检测成骨标志物基因Ⅰ胶原(COL-Ⅰ)、骨桥蛋白(OPN)及OCN mRNA的表达。
结果倒置相差显微镜下观察,用成骨诱导培养基培养3～4d,培养细胞形态由长梭形转变为短梭形或多角形。随着培养基中P24浓度的增加,细胞发生成骨样改变的时间提前。ALP活性和OCN含量检测显示,A和B组ALP活性和OCN含量较其他三组增加明显,差异有显著性(P<0.05),组间比较,差异无显著性(P>0.05)。实时荧光定量RT-PCR检测显示培养14d,成骨诱导标志物COL-Ⅰ、OPN和OCN mRNA在各组均有表达。A组和B组的Ct值明显大于C组和D组,差异有显著性(P<0.05),但A组和B组之间差异无显著性(P>0.05)。
结论P24体外能有效地诱导BMSC向成骨方向分化,在PLGA-[ASP-PEG]材料体系中,最适合的P24体外诱导剂量认为是200μg/ml。
第三部分BMP-2活性肽/PLGA-[ASP-PEG]仿生骨基质材料异位成骨的实验研究
目的评价P24/PLGA-[ASP-PEG]仿生骨基质材料的异位成骨能力。
方法成年雄性SD大鼠48只,随机分成三组,分别在大鼠背部双侧竖脊肌浅层肌袋中植入不同材料。A组:P24/ PLGA-[ASP-PEG]仿生骨基质材料;B组:单纯PLGA-[ASP-PEG]材料;C组:单纯的明胶海绵材料。X线、CT+三维成像观察植入物成骨情况,不同时间点病理切片,行组织学观察,western blot检测植入区成骨标志物Col-I及OPN蛋白的表达,实时荧光定量RT-PCR检测Col-I及OPN mRNA的表达。
结果X线、CT+三维成像:8周时,A组可见明显的成骨表现,B组偶见成骨样影像,C组未见新骨表现。组织学观察:A组8周骨小梁明显增粗增宽,表面形成类骨密质的结构,可见大量新骨形成,在新骨外周部有骨小梁及新生血管。B组8周时植入区纤维组织增生,偶见软骨细胞及少量成骨细胞。C组8周明胶海绵吸收,呈现肌肉组织表现,未见新骨形成。实时荧光定量RT-PCR:各组8周均有Col-I mRNA和OPN mRNA的表达, A组中Col-I mRNA和OPN mRNA为高表达。与western blot检测结果一致。
结论构建的P24/PLGA-[ASP-PEG]仿生骨基质材料能够诱导异位成骨,P24具有与天然BMP-2类似的异位诱导成骨能力和骨诱导活性,其成骨过程以软骨内化骨为主。
第四部分BMP-2活性肽/PLGA-[ASP-PEG]仿生骨基质材料修复兔股骨缺损的实验研究
目的探讨P24/PLGA-[ASP-PEG]仿生骨基质材料修复兔股骨中段临界大小骨缺损的可行性。
方法36只新西兰大白兔制备成股骨中段15mm的临界大小骨缺损模型。按实验设计分为三组。A组:植入P24/ PLGA-[ASP-PEG]仿生骨基质材料,B组植入PLGA-[ASP-PEG]材料;C组,植入单纯的明胶海绵。5孔钢板螺钉内固定。X线观察,组织学观察、修复组织ALP测定以及生物力学测试,比较各组材料修复骨缺损的能力。
结果X线显示及评价:A组8周骨痂与宿主骨界限模糊,骨痂密度不均匀,皮质骨轮廓出现,骨缺损已基本消失,有典型的骨愈合影像;12周骨痂开始塑性,新生骨皮质结构清晰,与断端骨自然连接,髓腔有再通趋势。按照X线骨缺损愈合分级,16周时4级以上为9只(优良率64.28%)。B组12周缺损区有少量骨痂生成,断端吸收变细,髓腔开始闭锁,缺损区依然存在。X线骨缺损愈合分级0级为5只(占62.5%)。C组:12周骨缺损区表现为骨不连影像,髓腔闭锁,钢板有松动迹象。X线骨缺损愈合分级均为0级。组织学观察:A组:8周时成骨细胞活跃,新骨大量增生,相互融合生长,呈编织骨和骨小梁结构,骨小梁钙化形成板层股。12周时编织骨增厚密集,呈典型的板层骨结构,骨细胞趋于成熟,有血管形成和长入,不规则骨髓腔出现。B组:8周纤维组织增生,材料部分吸收。12周植入区两端与宿主骨交界处,新骨增生,可见骨小梁和板层骨。植入区中央材料大部分吸收,可见大量的纤维组织,偶见软骨细胞。C组:12周缺损区被纤维结缔组织填充,骨端增生不活跃,髓腔封闭。生物力学测试:A组最大抗弯曲负荷122.14±12.83N,与同组正常骨参照值相比,修复比率为87.42%。B组最大抗弯曲负荷38.38±8.09N,与同组正常骨参照值相比,修复比率为26.89%。二者具有统计学差异性(P<0.05)。
结论体外构建的P24/PLGA-[ASP-PEG]仿生骨基质材料是一种理想的组织工程支架材料,能诱导启动典型的软骨内化骨过程,促进骨缺损的修复。
Objective In this research, a new biomimetic bone tissue engineering scaffold material, BMP-2-derived peptide P24 / PLGA-[ASP-PEG] composite was constructed. The effect of P24 to the adhesion, proliferation and osteodifferentiation of bone mesenchymal stem cells (BMSCs) were investigated.
Methods PLGA was modified with polyethylene glycol (PEG) and asparagic acid (ASP), and tri-block polymer PLGA-[ASP-PEG] material was prepared. Then peptide P24 was introduced into PLGA-(PEG-ASP)n scaffolds by cross-linkers. Thus, BMP-2-derived peptide P24 / PLGA-[ASP-PEG] composite was synthesized. BMSCs were cultured on three kinds of material respectively, one was P24 / PLGA-[ASP-PEG] biomimetic material (Group A), one was PLGA-[ASP-PEG] material (Group B), the other was simple PLGA material (Group C). The lineage specificity of the cells on these materials were detected by Flow cytometry(FCM). The cell adhesion was assessed by precipitation method. The proliferative ability of BMSCs were measured by MTT assay, Coomassie brilliant blue staining and scanning electron microscope(SEM). And the measure of alkaline phosphatase(ALP) activity and osteocalcin(OCN) level and ALP staining and calcium tubercle staining were performed to assess the cells’differentiation towards osteoblasts.
Results FCM confirmed the cells that isolated and cultured were BMSCs. The adhesive ratio measure suggested that adhesion and proliferation of BMSCs on the surface of P24 / PLGA-[ASP-PEG] is much higher than the control group (P<0.05). These were coincident with the cells’growth curve and the result of total protein assay and SEM. The ALP activity and osteocalcin(OCN) level of BMSCs in P24 / PLGA-[ASP-PEG] group were significant higher than in PLGA-[ASP-PEG] group and PLGA group. And in the assessment of ALP staining and calcium tubercle staining, positive staining was expressed firstly in P24 / PLGA-[ASP-PEG] group.
Conclusion The PLGA-[ASP-PEG] material was confirmed to improve BMSCs’adhesion and proliferation, and maintain their morphology. The material and its degradation product didn’t impact BMSCs’osteodifferentiation. And the BMP-2 derived peptide P24 could induce BMSCs’differentiation towards osteoblasts, its activity was equal to BMP-2.
PartⅡDose-dependent of BMP-2-derived Peptied on Osteogentc Induction of BMSCs in vitro
Objective To evaluate the capability of synthesized BMP2-derived peptide P24 on osteogenic induction of BMSCs, and investigate its dose-dependent and optimal dose in vitro.
Methods BMSCs were separated from 4-week-old SD rats and cultured with normal medium. At 3rd generation the medium was changed to osteogenetic medium which contained different dose of BMP2-derived peptide. The concentrations of BMP2-derived peptide contained in medium were 300、200、100、50 and 0μg/ml, respectively. These groups were labeled with A～E by turns. The change of cell morphology was observed with microscopy. ALP activities and OCN levels were taken at different points of time. The mRNA expression level of several osteogenetic marker such as Type I collagen(Col-I), osteopontin(OPN) and osteocalcin(OCN) were measured using real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) technique to detect the osteoinductivity of different concentration of BMP2-derived peptide,.
Results Under inverted phase contrast microscope, MSCs had changed their shape from long fusiform to short fusiform or polygon with the osteogenetic medium for 3~4 days. With the increase of the concentration of BMP2-derived peptide in medium, the time of MSCs changed their shape into osteoblast-like had advanced accordingly. The detection of ALP activities and OCN levels suggested that group A and group B were significant higher than the rest three groups (P<0.05). But there was no statistical significance between group A and group B(P>0.05). FQ-PCR suggested that after cultured for 14d, Col-I, OPN and OCN were expressed in each group. And the value of cycle threshold (Ct) of Group A and Group B were significant higher than the rest three groups(P<0.05). But there has no statistical significance between group A and group B(P>0.05).
Conclusion BMP2-derived peptide can induce BMSCs to differentiate into osteoblasts effectively. And among the PLGA-[ASP-PEG] material system, the optimal inductive concentration of P24 was 200μg/ml.
PartⅢEctopic osteogenesis of BMP-2-derived peptide combined with PLGA-[ASP-PEG] scaffold
Objective Evaluated the ectopic osteogenetic capacity of synthesis BMP2-derived peptide combined with poly (lactic acid / glycolic acid / asparagic acid-co-polyethylene glycol) (PLGA-[ASP-PEG]) by animal experiment.
Methods A total of 48 SD rats were allocated into three groups randomly. Three kinds of materials were respectively implanted into each side of superficial layer of erector spinae. Group A: BMP2-derived peptide P24 / PLGA-[ASP-PEG] complex, Group B: simple PLGA-[ASP-PEG], Groups C: simple gelatin sponge . The bone formation of the implants was detected by X-ray and CT three-dimensional imaging, further assessed by histological sections in different points of time. The osteogenic marker, Col-I and OPN were detected by western blot at protein level and by RT-PCR at mRNA level.
Results X-ray and CT three-dimensional imaging showed obvious osteogenesis in group A, occasional osteogenesis in group B, and no bone formation in group C in the 8th week. Histological examination showed at 8th week, in group A, bone trabeculae were found to be robust and wide with some compact bone-like tissue formed on their surface, and plenty of new bone formation was found, some bone trabeculae and new vessels located around them; and the material was absorbed totally. In group B, fibroplasia was found around implant and chondrocytes and osteoblasts could be seen occasionally; and in Group C, implanted gelation sponge was absorbed, the area displayed muscle tissue-like appearance, no new bone structure could be found. Real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) suggested that Col-I mRNA and OPN mRNA were expressed at each groups at 8th week, and in group A the express levels were higher than others. Western blot obtained the same consequence.
Conclusion BMP2-derived peptide P24 could induce ectopic osteogenesis, and its ectopic osteogenetic capacity and osteoinductivity were equal to nature BMP-2. Os endochondrale acted predominantly during osteogenesis.
PartⅣAccelerated bone regeneration with a novel synthetic BMP-2-derived bioactive oligopeptide combined with a nano-biomimetic PLGA-[ASP-PEG] scaffold
Objective Furtherly investigate the osteogenetic capacity of biomimetic materials modified with a novel peptide P24 derived from Bone Morphogenetic Protein-2 combined with PLGA-[ASP-PEG] polymer by using them to repair critical-sized bone defect.
Methods A total of 36 new zealand white rabbits were allocated into three groups randomly. A 1.5cm unilateral segmental critical-sized bone defect was created in the left femoral diaphysis in all rabbits. Three kinds of materials were used respectively. P24 combined with PLGA-[ASP-PEG] scaffold were implanted in group A, PLGA-[ASP-PEG] without P24 were implanted in group B, and simple gelatin sponge were implanted in group C. Five-hole plates and screws were applied to perform internal fixation routinely. Macroscopic observation, X-ray, histological investigation, ALP activity of repaired tissue and biomechanics measure were taken to evaluate the bone repair capability of each kind of material.
Results Radiographic findings showed: In group A, at 8th week, the boundary between bone callus and host bone became obscure, bone callus appeared uneven in density, the profile of os integumentale was displayed, bone defect almost disappeared, typical symbol of bone healing was observed; at 12th week, bone callus moulding was found, the texture of new os integumentale became clear, and them united with the broken end naturally, and the medullary cavity was trend to be recanalization. According to the standardized radiographic scales of bone defect repair, 9 rabbits were assessed as Grade 4 or above (fine ratio was 64.28%). In group B, at 12th week, a little bone callus was formed in the defect area, the broken ends were absorbed and became small, the medullary cavity began to be atresic, and the defect was still presented. There were 5 rabbits that be assessed as Grade 0 (62.5%). In group C, at 12th week, ununion symbol was still observed, atresic medullary cavity and loosing plate were be found. All of the rabbits were assessed as grade 0. Histological observations showed: In group A, in 8th week, active osteoblasts could be found, plenty of new bone tissue was formed and fused with each other, and seemed like woven bone and bone trabecula, lamellar bone was formed by calcification of bone trabeculae. At 12th week, woven bone tissue became thick and intensive, and turned into typical lamellar bone, osteocytes were trend to be mature, and with angiopoiesis and its invasion, irregular medullary cavities were formed. In group B, at 8th week, fibroplasia could be found and the material was absorbed partly.; at 12th week, new bone formation was found in the juncture of implant and host bone tissue, and bone trabeculae and lamellar bone were appeared. The middle of material was absorbed subtotally, massive fibrous tissue could be seen, and chondrocytes were be occasionally seen. In group C, at 12th week, the defect area was filled by fibrous connective tissue, hyperplasy in broken ends were inconspicuous, and medullary cavities were blocked. Biomechanics measure suggested the ultimate bending load of group A was 122.14±12.83N, which reparative ratio was 87.42% when compared with the reference value of normal bone in the same group; and in group B, the ultimate bending load was 38.38±8.09N, the reparative ratio of it was 26.89% when compared with the reference value of normal bone in the same group. And there was statistical difference between group A and B (p<0.05).
Conclusion This novel biomimetic material P24/PLGA-[ASP-PEG] is an ideal scaffold for bone tissue engineering, which can trigger typical os endochondrale procession and improve repair of bone defect.
引文
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