可注射性纳米羟基磷灰石、壳聚糖复合材料与骨髓基质细胞生物相容性及其修复骨缺损的实验研究
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摘要
研究背景：
各种原因(创伤,肿瘤、感染等)引起的骨缺损修复一直是骨科的一大难题。目前主要的治疗方法有自体骨移植、异体骨移植。它们都有各自的缺点：自体骨移植的缺点有来源有限、供体部位易发生感染、畸形以及植骨功能的继发丧失等；异体骨移植有传播疾病、引起宿主免疫排斥反应等的危险。组织工程的兴起为骨缺损的修复提供了一条广阔的途径,寻找一种合适的骨缺损修复材料是近年来骨组织工程的研究热点。理想的骨组织工程材料要求具备以下几点：良好的生物相容性及降解性：骨传导性及诱导性；满意的机械强度；可塑形性；合适的孔径,保证新生骨组织的长入和营养物质的运输供应：支持骨细胞生长和功能分化的表面化学性质与微结构。单一的生物材料往往很难具有如此多的特性,因而不同材料之间复合越来越受到大家的关注,清华大学材料科学与工程系采用仿生学原理,制成一种可注射的塑形简便并且具有良好骨传导、骨诱导性的纳米羟基磷灰石／壳聚糖(nano-Hydroxyapatite/chitosan, nHA/CS)复合骨修复材料,孔隙率90%,孔径50-200μm。该材料具有较好的初始稳定性和强度,既可以原位成形固化,满足有支撑需要的骨缺损部位的修复,又可以任意塑形,对其他类型骨缺损部位进行原位填充。但该复合材料是否具良好的生物相容性,对机体细胞是否无害尚不确切,同时该材料对骨缺损的修复效果也待进一步动物实验证实。
研究目的：
1、采用密度梯度离心法获得原代骨髓基质细胞(Bone Marrow Stem Cells, BMSCs),体外将稳定的传至第3代的兔骨髓基质细胞与可注射性纳米羟基磷灰石／壳聚糖复合材料共同培养,观察该材料的生物相容性。
2、建立兔股骨髁上骨缺损模型,将可注射性羟基磷灰石／壳聚糖复合材料植入兔股骨髁上骨缺损内,观察该材料对骨缺损的修复效果。
时间及地点：本实验于2008-08至2009-11在南方医科大学珠江医院中心实验室完成。
研究方法：
1、纳米羟基磷灰石／壳聚糖复合材料生物相容性实验方法：
采用广泛认可的密度梯度离心法从2周龄健康新西兰大白兔股骨和胫骨骨髓中获取原代兔骨髓基质细胞,将稳定的传至第三代的兔骨髓基质细胞接种到纳米羟基磷灰石／壳聚糖复合材料上,体外复合培养,作为实验组,单纯骨髓基质细胞培养作为对照组。倒置相差显微镜、电镜对接种到材料上的细胞进行形态学及生长增殖状况进行观察,计数细胞接种后1,2,4 h在材料表面的黏附状况,并计算黏附率,流式细胞仪检测种植细胞的细胞周期,有无异倍体出现,并计算增殖指数,对接种细胞的遗传学行为进行评价。
2、纳米羟基磷灰石／壳聚糖复合材料骨缺损修复实验方法：
将24只体重为1.5-2.0kg的新西兰白兔,双侧股骨髁上建立直径为7mm,深度为10mm骨缺损,并随机分为4周组、8周组、12周组,每组8只。将可注射性纳米羟基磷灰石／壳聚糖复合材料植入一侧骨缺损作为实验组,另一侧植入单纯可注射壳聚糖材料作为对照组。分别于4、8、12周末①大体观察材料植入后动物的生命活动状况,并观察标本骨缺损修复愈合状况；②X线、CT横断面扫描进一步观察骨缺损的影像学修复效果,并根据CT值,对骨缺损愈合状况进行评估；③组织病理学切片观察骨缺损与材料交界部位新生骨形成,材料降解,骨缺损愈合的病理变化过程,④电镜检测纳米羟基磷灰石／壳聚糖复合材料侧骨缺损部位植入材料与自体骨结合状况及新骨形成,材料降解的连续过程。
研究结果：
1、纳米羟基磷灰石／壳聚糖复合材料生物相容性实验结果：
①倒置显微镜观察发现,梭形的骨髓基质细胞围绕在纳米羟基磷灰石／壳聚糖复合材料边缘生长,随着时间的递增,细胞数目逐渐增加,生长状况良好。
②电镜学检测发现骨髓基质细胞在纳米羟基磷灰石／壳聚糖复合材料表面上生长状况良好,从胞体伸出长短不等的伪足黏附材料上。
③黏附率测定结果表明,细胞接种到纳米羟基磷灰石／壳聚糖复合材料1 h时黏附率低于对照组(P<0.05),但接种2,4 h后两组黏附率差异无显著性意义(P>0.05)。
④流式细胞仪检测发现细胞接种后,两组均保持正常的分裂增殖速度(P>0.05)。材料组和对照组细胞皆为正常的二倍体细胞,未见异倍体细胞形成,纳米羟基磷灰石／壳聚糖复合材料对兔骨髓基质细胞的细胞周期影响不大。
2、纳米羟基磷灰石／壳聚糖复合材料骨缺损修复实验：
①大体观察：术后所有兔子生命活动状况均未见异常,观察期间内未出现肢体骨折现象。
实验组：4周后正常骨组织与材料之间的界限尚清晰;术后8周股骨髁上缺损区材料与周围宿主骨组织界限模糊,表面光滑,有光泽,按压移植部位有阻抗；术后12周骨缺损完全修复,色泽与周围无明显差异,按压移植部位阻抗与周边正常骨组织相似,高度约与正常骨组织相平。
对照组：对照组4周后材料和骨组织结合较为紧密,无明显缝隙,纤维软组织长入材料中；术后8周材料与骨组织界限模糊,表面有光泽但欠光滑,颜色稍暗,按压移植部位稍有阻抗；术后12周骨缺损未完全愈合,色泽与周围无明显差异,按压移植部位有一定的阻抗,有部分骨皮质缺损,纤维组织填充。
②X线检查结果：
实验组：4周时正常骨组织与材料之间存在一环形的透光带,髁上缺损可见；8周时正常骨组织与材料之间透光带模糊,缺损区域透光性稍低于周围宿主骨组织；12周时髁上骨缺损愈合,未见与周围骨组织有明显的透光性差异。
对照组：4周时股骨髁上骨缺损清晰可见,材料与周围组织之间存在透光带；8周时骨缺损较前减小,缺损区域透光性低于周围宿主骨组织；12周时骨缺损较前进一减小,但未完全愈合。
③CT检查：
实验组：4周正常骨组织与材料之间有明确的界限,骨缺损外层骨皮质不连续；8周时正常骨组织与材料界限模糊,外层骨皮质已有连接；12周时骨缺损完全修复,缺损区域于周围骨组织之间未见有明显差异。
对照组：4周正常骨组织与材料之间界限明显,并可见到缺损；8周时界限稍模糊,骨缺损较前有愈合,但外层骨皮质不连续；12周时骨缺损部分修复,仍有少部分骨皮质仍不连续。
CT值比较提示4、8、12周末实验组、对照组骨修复效果均存在差异(P<0.05),结合大体及影像学证据,实验组骨修复效果优于对照组。
④组织形态学：
实验组：4周时,材料与宿主骨交界有新骨形成,大量纤维骨痂形成,材料与骨组织生物形容性好,可见大量成骨细胞生长,此时已有部分材料降解；8周后,出现大量新生骨小梁,且骨小梁排列较整齐,并可见少量板层状样骨组织,材料较前有进一步降解；12周时,材料已完全降解,可见大量新生板层骨组织,哈佛氏系统形成,原缺损区被新生板层骨组织填充,骨组织相互连续。
对照组：4周时纤维软组织长入材料,并可见淋巴细胞等,宿主骨边缘可见少量纤维骨痂形成,并见材料降解；8周后见大量纤维骨痂形成,少量新生骨小梁形成,未见板层骨形成,材料较前进一步降解；12周后少量板层样骨组织形成,纤维组织填充原骨缺损区域,材料已完全降解。
⑤电镜学观察
电镜检查发现：实验组4周时,骨质与材料紧密结合,但交界面尚清晰；植入8周时,材料与周围宿主骨组织界限模糊；材料植入12周后,扫描电镜观察到材料已基本完全降解,骨缺损部位完全被板层骨所替代。
结论：①兔骨髓基质细胞能在可注射纳米羟基磷灰石／壳聚糖材料上正常生长、增殖、分化,说明该材料对细胞无毒性,具有良好的生物相容性。②可注射性纳米羟基磷灰石／壳聚糖复合材料骨缺损修复能力较单纯壳聚糖好,具有确实的骨缺损修复能力。
BACKGROUND:
Bone defects caused by variety of reasons (trauma, tumor, infection, etc.) has been a major orthopedic problem. The main treatment of bone defects were autologous bone graft, allogeneic bone graft. They all have their own drawbacks:the shortcomings of autogenous bone graft were the limited of resources, the infection of donor part, deformity and function loss, etc; allograft may transmitted diseases, and caused host immune rejection reaction. The emergence of tissue-engineered has provided a broad way to the bone defects,and found a suitable material for bone defects was a hotspot in bone tissue engineering research in recent years. An ideal materials should have:good biological compatibilty and degradation; bone conductivity and induced; mechanical strength; Plasticity; appropriate aperture to ensure that new bone tissue can growth in and the supplying of nutrient; the chemical surface and micro-structure that support differentiation of bone cell growth.A single material often did not have so many features, and thus the composite of different materials was increasingly, Materials Science and Engineering of QingHua University, using bionics principles, made a kind of injectable Plasticity composite which have good bone conduction, and bone-induced availability injectable nano-hydroxyapatite/ chitosan (nano-Hydroxyapatite/chitosan,nHA/CS) composite,90% porosity, pore size 50～200μm. The material has good initial stability and strength, which fit for the parts of Weight-bearing, but also fit for the parts of other types of bone defect filling, with the material degradation, the new bone formated, completed the conversed and shaped of the bones.However, the biocompatibility of the composite is poorly understood,and the effect to repair bone defect is unknown.
OBJECTIVE:
1、Obtaining primary bone marrow strom cells by density gradient centrifugation, The nHA/CS composite was co-cultured with the passage 3 cells of BMECs in vitro, To evaluate the biocompatibility of material.
2、Created bone defect model On the rabbit's femoral condyle, implanted injectable nHA/CS composite in bone defect, the material effect on bone defects repairing was observed.To study the repair of bone defect with injectable nHA/CS composite.
TIME AND SETTING:
The observation was performed at the Central Laboratory of Zhujiang Hospital, Southern Medical University from August,2008 to November,2009.
METHODS:
1、The Methods of Biological Compatibility Experiment:
Obtain primary bone marrow stem cells from the 2-week-old New Zealand rabbits femur and tibia bone marrow by density gradient centrifugation which was widely recognized, The nHA/CS composite was co-cultured with the passage 3 cells of BMECs.The BMECs group was served as the control group.Qbservated the growth and proliferation of cells which inoculated on the materials by contrast microscope, electron microscopy, cells adhered on the of surface of materials were counted after 1,2,4 h, and calculate the adhesion rate,cell cycle was observed by flow cytometry,and calculate the proliferative index of inoculated cells.
2、The Methods Bone Defected Repaired Experiment:
24 New Zealand white rabbits weight 1.5-2.0kg were established a defects on femoral with diameter of 7mm, a depth of 10mm. And divided into 4-week group, 8-week group,12-week group randomly. Injectable nHA/CS composite materials was implanted in the side of the bone defect as an experimental group, injectable CS materials was implanted on the other side as the control group.4,8,12 weeks later, observation were made as follow:①The activities of animal after implantation and the healing status of bone defect;②X ray, CT scan observated the repairing of bone defect and bone defect healing was assessed in accordance with CT score;③The material degradation, new bone formation, the pathological changes of bone defect healing process, were observed on the junction of bone and materials;④The continuous process of material degradation and new bone formation was observed by SEM..
RESULTS:
1、The Result of Biological Compatibility:
①Microscope:The BMSCs grown on edge of material, the number of cells was increasing with time, the growth was in good condition.
②SEM:The BMSCs grown well on the surface of nHA/CS, a lot of pseudopod from the cell body were found.
③Adhesion rate:The attachment rate in the nHA/CS group was lower than that of the control group at 1 hour (P< 0.05); however, there was no significant difference after 2 and 4 hours(P> 0.05). After seeding,
④Flow cytometry:After seeding, the proliferation of cell had no changes (P> 005). Normal diploid cells could be found in both groups, which indicated that nHA/CS had no influence to cell cycle.
2、nHA/CS materials for experimental bone defect repair:
①General observation:the rabbits were activity after operation,there were not rabbits occured fractured during the observation period in both group.
The nHA/CS group:The edge of normal bone and material was still clear 4 weeks later;And the boundary was blur 8 weeks later, the surface was smooth, there was a little impedance on defect; The bone defects are fully restored the 12 weeks later, the impedance was similar with nomoral bone.
The CS group:The materials was closely with bone tissue 4 weeks later, there was fibrous grown into materials; The boundary between material and bone tissue was blur 8 weeks later, the Surface was not smooth, there was little impedance on defect;Bone defect was not healed completely, some cortical defects, fibrous tissue filled after 12 weeks.
②X ray:
The nHA/CS group:A ring of light belt existed between the normal bone tissue and material, condyle defects can be seen 4 weeks later; The light belt between normal bone tissue and material was vague, the light transmittance in the defects slightly less than the host bone tissue 8 weeks later, The supracondylar bone defect was healed,there is no obvious difference between the bone tissue and bone defect in light transmission 12 weeks later.
The CS group:The bone defect on the femoral condyle was clearly, light belt was clearly between the material and the surrounding tissue 4 weeks later; The bone defect was reduction compared with the previous, light transmittance of defect area is slightly less than ambient host bone tissue8 weeks later, Bone defects was decreased compared with previous, but not completely healed 12 weeks later.
③CT examination:
The nHA/CS group:The line between normal bone tissue and material is clear 4 weeks later, outer cortical was discontinuous; The line was blurred 8 weeks later, outer cortical was continuous; Bone defect was healed 12weeks later.
The CS group:The bone defect was visible outer cortical was discontinuous 4 weeks later;Bone defect was smaller compared with the previous 8 weeks later; The bone defects,did not complete healed, some cortical discontinuity 12 weeks later.
CT Comparison prompted that there is differences between the experimental group and control group in effects of bone repairing (P<0.05);
④Histology
The nHA/CS group:The new bone formation at the junction of material and host bone 4 weeks later, a lot of new bone was formated;And there was lamellar bone tissue formatted 8 weeks later; Harvard's system formed, the original defect was filled with new lamellar bone tissue, bone tissue continuous 12 weeks later.
The CS group:The fibrous tissue growth into material, and lymphocytes can be seen around the material4 weeks later;A few of new bone was formed at the edge of host bone, new trabecular bone formation after 8 weeks later; A few of lamellar bone tissue formation 12 weeks later, fibrous tissue filled the defect.
⑤SEM:In the experimental group part of the material degraded while the new bone formatted 4 weeks later, the materials was replaced by new bone 12 weeks later.
CONCLUSION:
①The BMSCs can be grown, proliferatd, differentiated on the surface of nHA/CS which indicated that the material has good biocompatibility.
②The nHA/CS composite has better capacity on bone defect repairing compared with pure chitosan, played a good role in bone defect repair.
各种原因(创伤,肿瘤、感染等)引起的骨缺损修复一直是骨科的一大难题。目前主要的治疗方法有自体骨移植、异体骨移植。它们都有各自的缺点：自体骨移植的缺点有来源有限、供体部位易发生感染、畸形以及植骨功能的继发丧失等；异体骨移植有传播疾病、引起宿主免疫排斥反应等的危险。组织工程的兴起为骨缺损的修复提供了一条广阔的途径,寻找一种合适的骨缺损修复材料是近年来骨组织工程的研究热点。理想的骨组织工程材料要求具备以下几点：良好的生物相容性及降解性：骨传导性及诱导性；满意的机械强度；可塑形性；合适的孔径,保证新生骨组织的长入和营养物质的运输供应：支持骨细胞生长和功能分化的表面化学性质与微结构。单一的生物材料往往很难具有如此多的特性,因而不同材料之间复合越来越受到大家的关注,清华大学材料科学与工程系采用仿生学原理,制成一种可注射的塑形简便并且具有良好骨传导、骨诱导性的纳米羟基磷灰石／壳聚糖(nano-Hydroxyapatite/chitosan, nHA/CS)复合骨修复材料,孔隙率90%,孔径50-200μm。该材料具有较好的初始稳定性和强度,既可以原位成形固化,满足有支撑需要的骨缺损部位的修复,又可以任意塑形,对其他类型骨缺损部位进行原位填充。但该复合材料是否具良好的生物相容性,对机体细胞是否无害尚不确切,同时该材料对骨缺损的修复效果也待进一步动物实验证实。
研究目的：
1、采用密度梯度离心法获得原代骨髓基质细胞(Bone Marrow Stem Cells, BMSCs),体外将稳定的传至第3代的兔骨髓基质细胞与可注射性纳米羟基磷灰石／壳聚糖复合材料共同培养,观察该材料的生物相容性。
2、建立兔股骨髁上骨缺损模型,将可注射性羟基磷灰石／壳聚糖复合材料植入兔股骨髁上骨缺损内,观察该材料对骨缺损的修复效果。
时间及地点：本实验于2008-08至2009-11在南方医科大学珠江医院中心实验室完成。
研究方法：
1、纳米羟基磷灰石／壳聚糖复合材料生物相容性实验方法：
采用广泛认可的密度梯度离心法从2周龄健康新西兰大白兔股骨和胫骨骨髓中获取原代兔骨髓基质细胞,将稳定的传至第三代的兔骨髓基质细胞接种到纳米羟基磷灰石／壳聚糖复合材料上,体外复合培养,作为实验组,单纯骨髓基质细胞培养作为对照组。倒置相差显微镜、电镜对接种到材料上的细胞进行形态学及生长增殖状况进行观察,计数细胞接种后1,2,4 h在材料表面的黏附状况,并计算黏附率,流式细胞仪检测种植细胞的细胞周期,有无异倍体出现,并计算增殖指数,对接种细胞的遗传学行为进行评价。
2、纳米羟基磷灰石／壳聚糖复合材料骨缺损修复实验方法：
将24只体重为1.5-2.0kg的新西兰白兔,双侧股骨髁上建立直径为7mm,深度为10mm骨缺损,并随机分为4周组、8周组、12周组,每组8只。将可注射性纳米羟基磷灰石／壳聚糖复合材料植入一侧骨缺损作为实验组,另一侧植入单纯可注射壳聚糖材料作为对照组。分别于4、8、12周末①大体观察材料植入后动物的生命活动状况,并观察标本骨缺损修复愈合状况；②X线、CT横断面扫描进一步观察骨缺损的影像学修复效果,并根据CT值,对骨缺损愈合状况进行评估；③组织病理学切片观察骨缺损与材料交界部位新生骨形成,材料降解,骨缺损愈合的病理变化过程,④电镜检测纳米羟基磷灰石／壳聚糖复合材料侧骨缺损部位植入材料与自体骨结合状况及新骨形成,材料降解的连续过程。
研究结果：
1、纳米羟基磷灰石／壳聚糖复合材料生物相容性实验结果：
①倒置显微镜观察发现,梭形的骨髓基质细胞围绕在纳米羟基磷灰石／壳聚糖复合材料边缘生长,随着时间的递增,细胞数目逐渐增加,生长状况良好。
②电镜学检测发现骨髓基质细胞在纳米羟基磷灰石／壳聚糖复合材料表面上生长状况良好,从胞体伸出长短不等的伪足黏附材料上。
③黏附率测定结果表明,细胞接种到纳米羟基磷灰石／壳聚糖复合材料1 h时黏附率低于对照组(P<0.05),但接种2,4 h后两组黏附率差异无显著性意义(P>0.05)。
④流式细胞仪检测发现细胞接种后,两组均保持正常的分裂增殖速度(P>0.05)。材料组和对照组细胞皆为正常的二倍体细胞,未见异倍体细胞形成,纳米羟基磷灰石／壳聚糖复合材料对兔骨髓基质细胞的细胞周期影响不大。
2、纳米羟基磷灰石／壳聚糖复合材料骨缺损修复实验：
①大体观察：术后所有兔子生命活动状况均未见异常,观察期间内未出现肢体骨折现象。
实验组：4周后正常骨组织与材料之间的界限尚清晰;术后8周股骨髁上缺损区材料与周围宿主骨组织界限模糊,表面光滑,有光泽,按压移植部位有阻抗；术后12周骨缺损完全修复,色泽与周围无明显差异,按压移植部位阻抗与周边正常骨组织相似,高度约与正常骨组织相平。
对照组：对照组4周后材料和骨组织结合较为紧密,无明显缝隙,纤维软组织长入材料中；术后8周材料与骨组织界限模糊,表面有光泽但欠光滑,颜色稍暗,按压移植部位稍有阻抗；术后12周骨缺损未完全愈合,色泽与周围无明显差异,按压移植部位有一定的阻抗,有部分骨皮质缺损,纤维组织填充。
②X线检查结果：
实验组：4周时正常骨组织与材料之间存在一环形的透光带,髁上缺损可见；8周时正常骨组织与材料之间透光带模糊,缺损区域透光性稍低于周围宿主骨组织；12周时髁上骨缺损愈合,未见与周围骨组织有明显的透光性差异。
对照组：4周时股骨髁上骨缺损清晰可见,材料与周围组织之间存在透光带；8周时骨缺损较前减小,缺损区域透光性低于周围宿主骨组织；12周时骨缺损较前进一减小,但未完全愈合。
③CT检查：
实验组：4周正常骨组织与材料之间有明确的界限,骨缺损外层骨皮质不连续；8周时正常骨组织与材料界限模糊,外层骨皮质已有连接；12周时骨缺损完全修复,缺损区域于周围骨组织之间未见有明显差异。
对照组：4周正常骨组织与材料之间界限明显,并可见到缺损；8周时界限稍模糊,骨缺损较前有愈合,但外层骨皮质不连续；12周时骨缺损部分修复,仍有少部分骨皮质仍不连续。
CT值比较提示4、8、12周末实验组、对照组骨修复效果均存在差异(P<0.05),结合大体及影像学证据,实验组骨修复效果优于对照组。
④组织形态学：
实验组：4周时,材料与宿主骨交界有新骨形成,大量纤维骨痂形成,材料与骨组织生物形容性好,可见大量成骨细胞生长,此时已有部分材料降解；8周后,出现大量新生骨小梁,且骨小梁排列较整齐,并可见少量板层状样骨组织,材料较前有进一步降解；12周时,材料已完全降解,可见大量新生板层骨组织,哈佛氏系统形成,原缺损区被新生板层骨组织填充,骨组织相互连续。
对照组：4周时纤维软组织长入材料,并可见淋巴细胞等,宿主骨边缘可见少量纤维骨痂形成,并见材料降解；8周后见大量纤维骨痂形成,少量新生骨小梁形成,未见板层骨形成,材料较前进一步降解；12周后少量板层样骨组织形成,纤维组织填充原骨缺损区域,材料已完全降解。
⑤电镜学观察
电镜检查发现：实验组4周时,骨质与材料紧密结合,但交界面尚清晰；植入8周时,材料与周围宿主骨组织界限模糊；材料植入12周后,扫描电镜观察到材料已基本完全降解,骨缺损部位完全被板层骨所替代。
结论：①兔骨髓基质细胞能在可注射纳米羟基磷灰石／壳聚糖材料上正常生长、增殖、分化,说明该材料对细胞无毒性,具有良好的生物相容性。②可注射性纳米羟基磷灰石／壳聚糖复合材料骨缺损修复能力较单纯壳聚糖好,具有确实的骨缺损修复能力。
BACKGROUND:
Bone defects caused by variety of reasons (trauma, tumor, infection, etc.) has been a major orthopedic problem. The main treatment of bone defects were autologous bone graft, allogeneic bone graft. They all have their own drawbacks:the shortcomings of autogenous bone graft were the limited of resources, the infection of donor part, deformity and function loss, etc; allograft may transmitted diseases, and caused host immune rejection reaction. The emergence of tissue-engineered has provided a broad way to the bone defects,and found a suitable material for bone defects was a hotspot in bone tissue engineering research in recent years. An ideal materials should have:good biological compatibilty and degradation; bone conductivity and induced; mechanical strength; Plasticity; appropriate aperture to ensure that new bone tissue can growth in and the supplying of nutrient; the chemical surface and micro-structure that support differentiation of bone cell growth.A single material often did not have so many features, and thus the composite of different materials was increasingly, Materials Science and Engineering of QingHua University, using bionics principles, made a kind of injectable Plasticity composite which have good bone conduction, and bone-induced availability injectable nano-hydroxyapatite/ chitosan (nano-Hydroxyapatite/chitosan,nHA/CS) composite,90% porosity, pore size 50～200μm. The material has good initial stability and strength, which fit for the parts of Weight-bearing, but also fit for the parts of other types of bone defect filling, with the material degradation, the new bone formated, completed the conversed and shaped of the bones.However, the biocompatibility of the composite is poorly understood,and the effect to repair bone defect is unknown.
OBJECTIVE:
1、Obtaining primary bone marrow strom cells by density gradient centrifugation, The nHA/CS composite was co-cultured with the passage 3 cells of BMECs in vitro, To evaluate the biocompatibility of material.
2、Created bone defect model On the rabbit's femoral condyle, implanted injectable nHA/CS composite in bone defect, the material effect on bone defects repairing was observed.To study the repair of bone defect with injectable nHA/CS composite.
TIME AND SETTING:
The observation was performed at the Central Laboratory of Zhujiang Hospital, Southern Medical University from August,2008 to November,2009.
METHODS:
1、The Methods of Biological Compatibility Experiment:
Obtain primary bone marrow stem cells from the 2-week-old New Zealand rabbits femur and tibia bone marrow by density gradient centrifugation which was widely recognized, The nHA/CS composite was co-cultured with the passage 3 cells of BMECs.The BMECs group was served as the control group.Qbservated the growth and proliferation of cells which inoculated on the materials by contrast microscope, electron microscopy, cells adhered on the of surface of materials were counted after 1,2,4 h, and calculate the adhesion rate,cell cycle was observed by flow cytometry,and calculate the proliferative index of inoculated cells.
2、The Methods Bone Defected Repaired Experiment:
24 New Zealand white rabbits weight 1.5-2.0kg were established a defects on femoral with diameter of 7mm, a depth of 10mm. And divided into 4-week group, 8-week group,12-week group randomly. Injectable nHA/CS composite materials was implanted in the side of the bone defect as an experimental group, injectable CS materials was implanted on the other side as the control group.4,8,12 weeks later, observation were made as follow:①The activities of animal after implantation and the healing status of bone defect;②X ray, CT scan observated the repairing of bone defect and bone defect healing was assessed in accordance with CT score;③The material degradation, new bone formation, the pathological changes of bone defect healing process, were observed on the junction of bone and materials;④The continuous process of material degradation and new bone formation was observed by SEM..
RESULTS:
1、The Result of Biological Compatibility:
①Microscope:The BMSCs grown on edge of material, the number of cells was increasing with time, the growth was in good condition.
②SEM:The BMSCs grown well on the surface of nHA/CS, a lot of pseudopod from the cell body were found.
③Adhesion rate:The attachment rate in the nHA/CS group was lower than that of the control group at 1 hour (P< 0.05); however, there was no significant difference after 2 and 4 hours(P> 0.05). After seeding,
④Flow cytometry:After seeding, the proliferation of cell had no changes (P> 005). Normal diploid cells could be found in both groups, which indicated that nHA/CS had no influence to cell cycle.
2、nHA/CS materials for experimental bone defect repair:
①General observation:the rabbits were activity after operation,there were not rabbits occured fractured during the observation period in both group.
The nHA/CS group:The edge of normal bone and material was still clear 4 weeks later;And the boundary was blur 8 weeks later, the surface was smooth, there was a little impedance on defect; The bone defects are fully restored the 12 weeks later, the impedance was similar with nomoral bone.
The CS group:The materials was closely with bone tissue 4 weeks later, there was fibrous grown into materials; The boundary between material and bone tissue was blur 8 weeks later, the Surface was not smooth, there was little impedance on defect;Bone defect was not healed completely, some cortical defects, fibrous tissue filled after 12 weeks.
②X ray:
The nHA/CS group:A ring of light belt existed between the normal bone tissue and material, condyle defects can be seen 4 weeks later; The light belt between normal bone tissue and material was vague, the light transmittance in the defects slightly less than the host bone tissue 8 weeks later, The supracondylar bone defect was healed,there is no obvious difference between the bone tissue and bone defect in light transmission 12 weeks later.
The CS group:The bone defect on the femoral condyle was clearly, light belt was clearly between the material and the surrounding tissue 4 weeks later; The bone defect was reduction compared with the previous, light transmittance of defect area is slightly less than ambient host bone tissue8 weeks later, Bone defects was decreased compared with previous, but not completely healed 12 weeks later.
③CT examination:
The nHA/CS group:The line between normal bone tissue and material is clear 4 weeks later, outer cortical was discontinuous; The line was blurred 8 weeks later, outer cortical was continuous; Bone defect was healed 12weeks later.
The CS group:The bone defect was visible outer cortical was discontinuous 4 weeks later;Bone defect was smaller compared with the previous 8 weeks later; The bone defects,did not complete healed, some cortical discontinuity 12 weeks later.
CT Comparison prompted that there is differences between the experimental group and control group in effects of bone repairing (P<0.05);
④Histology
The nHA/CS group:The new bone formation at the junction of material and host bone 4 weeks later, a lot of new bone was formated;And there was lamellar bone tissue formatted 8 weeks later; Harvard's system formed, the original defect was filled with new lamellar bone tissue, bone tissue continuous 12 weeks later.
The CS group:The fibrous tissue growth into material, and lymphocytes can be seen around the material4 weeks later;A few of new bone was formed at the edge of host bone, new trabecular bone formation after 8 weeks later; A few of lamellar bone tissue formation 12 weeks later, fibrous tissue filled the defect.
⑤SEM:In the experimental group part of the material degraded while the new bone formatted 4 weeks later, the materials was replaced by new bone 12 weeks later.
CONCLUSION:
①The BMSCs can be grown, proliferatd, differentiated on the surface of nHA/CS which indicated that the material has good biocompatibility.
②The nHA/CS composite has better capacity on bone defect repairing compared with pure chitosan, played a good role in bone defect repair.
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