复合环孢素A的同种异体骨与冻干骨、同系骨骨诱导性及免疫原性的比较
|
摘要
研究背景
长期以来,临床上的骨缺损修复,特别是大段骨缺损的修复,一直是外科医生面临的难题之一。解决方法主要有骨移植术及骨痂延长术。其中用于骨移植的材料主要有自体骨、同种异体骨、异种骨及人工骨四类。自体骨骨生成能力强、无免疫排斥等优点,但在骨缺损治疗过程中,自体骨具有明显的局限性和并发症,如来源有限、对供区有损伤、机会感染等,这些不利因素限制了其在临床上的应用；人工骨虽然传导性较好但骨诱导性较差、缺乏孔隙、颗粒较小、易分解、脆性大不耐压且相对缺乏生物活性等缺点,限制了它的应用。而同种异体骨自1880年Macewe做了人类历史上首例同种异体骨移植术,1908年Lexer最早实施了膝关节肿瘤切除后的同种异体骨移植术,1915年Albee出版了《骨移植外科》以来,同种异体骨移植逐渐成为骨科治疗的重要手段,它取材方便,数量形状不受限制,对已被破坏的骨折周围的内环境,有着恢复迅速、改善和重建血循环快捷并且其成骨性能及力学性能优于人工骨,而来源也不像自体骨那样受到限制等,诸多独特的优势和应用价值,进而使得同种异体骨在临床上应用广泛,逐渐成为骨科治疗的重要手段。但也存在一些目前难以彻底解决的问题,如免疫排斥、晚期感染、移植骨愈合缓慢等,同种异体骨移植愈合受它的生物机制和免疫机制两大原则支配,而免疫排斥反应是它的关键；免疫排斥反应的主要表现为异体骨周围炎症细胞浸润,再生血管变性闭塞,造成骨诱导和骨传导作用下降及广泛的骨吸收,引起骨延迟愈合、不愈合、骨质疏松、骨折等；异体骨是异体生物性材料,具有一定的免疫抗原性,免疫排斥反应是制约异体骨移植的主要因素。降低异体骨移植的免疫排斥反应是提高治疗效果和决定异体植骨成功与否的关键。早期异体骨移植的最大问题是疾病传播与免疫排斥反应,而移植排斥反应仍是导致新鲜同种异体骨移植失败的最主要原因。机体的免疫系统对于植入体内的非已成分(同种异体骨)会产生一系列免疫应答的防御行为,其中包括炎症反应、抗体产生、补体活化及各种细胞因子的表达等,因此在生物材料和医疗器械的生物学评价中引入免疫学评价的内容将对减小人体应用的风险提供必要的科学依据,同种异体骨是骨科最常用的骨植入材料之一,同时异体骨的免疫原性与异体骨移植修复骨缺损的临床效果密切相关。
早些时候人们曾尝试着用柳硫汞等化学方法的处理来降低异体骨的免疫原性和对其进行灭菌,但效果不佳。随着制备工艺的改进,人们逐渐倾向于用深低温冷冻法、冷冻干燥法及丫射线辐照来降低异体骨的免疫原性,其中丫射线辐照更兼具强大的灭菌功效。经过世界各国骨库几十年来的实践证明,上述方法能够很好地降低异体骨的免疫原性并有效地降低了因骨移植所致的疾病传播的风险,因而是目前最为常用的同种异体骨的制备方法。然而严格地讲,上述方法并不能完全消除同种异体骨的免疫原性,而经过上述方法处理后,同种异体骨的成骨性能及生物力学性能受到了明显的损害。致使目前为止临床上应用异体骨修复骨缺损的效果,特别是修复大段骨缺损的效果仍不能够令人满意。鉴于此本课题组对同种异体骨的制备方法进行新的探索。具体而言,就是在制备同种异体骨的过程中复合环孢菌素,通过在局部发挥环孢菌素强大的免疫抑制作用来代替深低温冷冻、冷冻干燥及γ射线辐照的降免疫原性作用,并用低温等离子体技术来代替γ射线辐照对异体骨进行灭菌,以期更好地抑制异体骨移植的免疫排斥反应,同时减轻制备过程中对异体骨成骨性能及力学性能的损害。另外,我们希望通过环孢菌素的局部应用来降低其药物副反应。
研究目的
为了进一步减轻同种异体骨的免疫原性,同时又不损害异体骨的机械强度,裴国献、陆海波等人采用新的方法制备了复合环孢素A的同种异体骨,并申请到了发明专利。在同种异体骨治疗大段骨缺损方面进行了有益的探索。但复合环孢素同种异体骨在制备过程中需要对供骨进行脱脂脱蛋白,一定程度上减轻了其免疫原性,但同时也造成了对同种异体骨生物学特性起重要作用的BMP及其他生物活性成分的大量丢失。因此有必要对其免疫原性及骨诱导性进行评估,通过对复合环孢素A并低温等离子体灭菌这一新的异体骨制备方法制备的同种异体骨与冻干同种异体骨及同系骨骨诱导性及免疫原性的比较,探讨复合环孢素A的同种异体骨在临床上应用的可能性。
研究方法
1、淋巴细胞二次刺激实验Alamarblue法和MTT法的选择
取Balb/c小鼠脾脏淋巴细胞,将分离得到的细胞原液稀释成浓度分别为2×105、1×105、0.5×105、0.25×105、0.125×105、0.0625×105cell／ml的细胞悬液。在96孔培养板上添加不同浓度细胞悬液200μl,重复6孔,并设置调零孔。Alamarblue法：铺板后每孔加入20μlAlamarblue。分别于培养0、2、4、6、20h后,用酶标仪在570nm(主波长)及600nm(参考波长)检测各个孔的吸光度值。按照说明书通过公式计算出Alamarblue的还原率。MTT法：细胞种板设计同AlamarBlue法。每空加入MTT20μl。培养4小时后,DMSO显色后,在490nm(主波长)及630nm(参考波长)检测各个孔的吸光度值。对Alamarblue法与MTT法、Alamarblue法与时间进行相关性分析。精密度测定：组内差异用变异系数CV表示。CV=组内各数据的标准差(s)／组内各数据的平均值(x)。
2、复合环孢素A的同种异体骨与冻干同种异体骨及同系骨骨诱导性及免疫原性的比较
复合环孢素A的同种异体骨的制备：取C57小鼠的髂骨,制成骨粒,脱脂脱蛋白后采用固体分散法复合复合环孢素A,然后采用低温等离子体灭菌。
冻干同种异体骨的制备：取C57小鼠的髂骨,制成骨粒,采用蒸馏水反复冲洗,在低温冷冻干燥机中冻干,采用γ射线辐照灭菌。
同系骨的制备：术中取Balb/c小鼠的髂骨,制成骨粒,生理盐水反复冲洗。
骨匀浆上清的制备：取C57小鼠及Balb/c小鼠的四肢骨骼蒸馏水反复冲洗,干燥后研磨成骨粉与细胞培养液混合制成骨粉悬液,在超净台中,经无菌滤器过滤,制得骨匀浆上清。
将制成的骨粒植入Balb/c小鼠左侧股直肌内制成股直肌袋模型。
分别在2、4、6、8w时取材,进行MASSON染色并进行碱性磷酸酶的检测。在4w时取各组小鼠脾脏,分离淋巴细胞,制成一定浓度的细胞悬液,加入对应的骨匀浆上清及Alamarblue指示剂,用酶标仪在570nm(主波长)及600hm(参考波长)检测各个孔的吸光度值。计算出各组的刺激指数。
3、用SPSS13.0对结果进行数据分析,实验结果采用x±s表示,多个样本均数比较采用析因设计方差分析的方法,单独比较采用one-way A N 0 V A或t检验分析方法。当p<0.05时,差异有统计学意义。
结果
1、淋巴细胞二次刺激实验Alamarblue法和MTT法的选择
细胞浓度在0.0625×105～2×105cell／ml之间时,0h(P>0.05)细胞浓度与还原率之间的相关关系不明显。2h(P<0.05)、4h(P<0.001)、6h(P<0.001)、20h(P<0.001)细胞浓度与还原率之间有显著的相关性。
不同的细胞浓度随着培养时间的延长,还原率也随之增加。最大细胞浓度20h的还原率仍未达100%。
Alamarblue法测得的还原率及MTT法测得的OD值均随着细胞浓度的增大而增加。对二者进行相关性分析,相关系数r=0.998,P<0.05。两种方法存在正线性相关关系,且相关关系密切。
细胞浓度为2×105时CV (Alamarblue)=0.0152, CV(MTT)=0.0487;细胞浓度为0625×105时CV (Alamarblue)=0.023, CV(MTT)=0.0369。
2复合环孢素A的同种异体骨与冻干同种异体骨及同系骨骨诱导性及免疫原性的比较
2.1淋巴细胞二次刺激实验
采用完全随机设计资料的方差分析分别对两个时间点三组淋巴细胞刺激指数进行分析。2w时,处理组间F=0.3228,P=0.7302>0.05,三组之间差异不显著。4w时,处理组间F=253.6085,P<0.001。三组间差异显著。三组间异质性进一步采用Bonferroni检验,复合环孢素A组与同系组差异不显著,冻干组与复合环孢素A组及同系组差异显著,冻干组淋巴细胞刺激指数较高。
2.2局部碱性磷酸酶检测
对4个时间点3组碱性磷酸酶数据进行两因素析因分析后发现,三组(F组间=25.1807；P<0.001)及各个时间点(F时间=239.5745；P<0.001)均存在统计学差异。进一步采用单向方差分析(one-way ANOVA)进行多样本均数间的比较发现,符合环孢素A组在2、4、6,8w的碱性磷酸酶含量统计学差异显著(F=76.9263,P<0.001)。同系组在2、4、6、8w的碱性磷酸酶含量统计学差异显著(F=60.7506,P<0.001)。冻干骨组在2,4,6,8 w的碱性磷酸酶含量统计学差异显著(F=132.4105,P<0.001)。2 w时三组间的碱性磷酸酶含量统计学差异不显著(F=10.5117,P<0.05).4 w时冻干组与复合环孢素A组、同系组间统计学差异显著,复合环孢素A组与同系组间差异不显著(F=0.0418,P<0.05)。6 w时冻干组与复合环孢素A组、同系组间统计学差异显著,复合环孢素A组与同系组间差异不显著(F=9.0822,P<0.05)。8 w时冻干组与复合环孢素A组、同系组间统计学差异显著,复合环孢素A组与同系组间差异不显著(F=5.7737,P<0.05)。
2.3局部组织学检测
通过MASSON染色,2、4、6、8w时,复合环孢素组与同系组的成成骨量均比冻干组高。三组在各个时间点植入骨粒周围均未发现炎症反应。
结论
1、淋巴细胞二次刺激实验Alamarblue法和MTT法的选择
在本研究中检测了不同浓度的BALB/C小鼠脾淋巴细胞在加入Alamarblue后不同时间所测得的吸光度值,并计算出相对应的还原率。并对加入指示剂4h后Alamarblue的还原率及MTT的OD值进行相关性分析。在加入Alamarblue后,不同细胞浓度的还原率随着作用时间的延长而增加,当细胞浓度为2×105cell/ml,作用20h后还原率为78.3%,仍未达到100%。不同的细胞浓度在作用时间为0h(P>0.05)条件下不同细胞浓度与还原率之间的线性相关关系不明显,2h(P<0.05)、4h(P<0.001)、6h(P<0.001)、20h(P<0.001)条件下不同细胞浓度与还原率之间正性线性相关关系明显。说明在细胞浓度在0.0625×105-2×105cell/ml时,加入Alamarblue后4、6、20h所得出的还原率均可间接反应出细胞数量,从而在使用小鼠脾细胞进行细胞增殖或细胞毒试验中反映细胞存活的情况。如果进行细胞增殖实验则推荐接种细胞密度为0.0625×105-0.5×105cell/ml,作用时间为4-6h。较高的细胞密度和较长的作用时间可能使所有检测的样本还原态的Alamarblue达到饱和状态。如果进行细胞毒性实验,则可采用本研究的细胞接种浓度及培养时间。
Alamarblue法与MTT法的结果高度相关。在高浓度时Alamarblue法与MTT法组内差异接近,均小于2%。低浓度时Alamarblue法与MTT法组内差异接近,均小于2%。说明在高浓度、低浓度时Alamarblue法与传统的MTT法相比稳性及重复性并无明显差异。
2复合环孢素A的同种异体骨与冻干同种异体骨及同系骨骨诱导性及免疫原性的比较
实验结果表明,术后2w,三组间碱性磷酸酶含量统计学上有显著差异。环孢素组与同系组的碱性磷酸酶含量比冻干组高,环孢素组与同系组含量差异不明显。组织学显示三组植入骨粒周围均没有显著的炎症细胞浸润。同系组植入骨粒骨小梁开始被吸收,条里变得模糊,冻干骨组植入骨粒骨小梁未被吸收,复合环孢素组植入骨粒的周围骨小梁开始被吸收。术后4w,三组间碱性磷酸酶含量统计学上有显著差异,复合环孢素组与同系组含量差异不明显。复合环孢素组与同系组的碱性磷酸酶含量比冻干组高。组织学表明同系组与复合环孢素组植入骨粒已经被吸收,可见大片新生的绿染的Ⅱ型胶原纤维,分布较均匀。冻干骨植入骨粒骨小梁大部分被吸收,新生胶原纤维较同系组及复合环孢素组少,主要以结缔纤维组织为主。术后6w,三组间碱性磷酸酶含量统计学上有显著差异。环孢素组与同系组的碱性磷酸酶含量比冻干组高,环孢素组与同系组含量差异不明显。组织学6w时,三组植入骨粒周围均没有炎症细胞浸润。环孢素组及同系组开始出现软骨内成骨,可见骨陷窝,及骨细胞。骨粒周围开始出现红染的胶原纤维,有层次感。冻干骨骨可见植入骨粒内出现新生的胶原纤维,排列杂乱。8w时三组间碱性磷酸酶含量统计学上有显著差异。环孢素组与同系组的碱性磷酸酶含量比冻干组高,环孢素组与同系组含量差异不明显。组织学8w时,同系组及复合环孢素组出现大片的红染的成熟胶原纤维,呈现板层排列,部分样本中间出现空腔,并含有髓腔内容物.冻干骨组可见成熟胶原纤维与新生胶原纤维交错分布,呈现红绿相间染色。
2w时各个组的淋巴细胞刺激指数三组间统计学差异不显著,4w三组间淋巴细胞刺激指数差异显著,同系组、复合环孢素组均低于冻干组,同系组与复合环孢素之间无显著差异。
可见复合在移植骨粒上的环孢素并低温等离子体灭菌,在局部抑制了T细胞的活化,为新骨的生成创造了良好的环境,避免长期全身应用环孢素所带来的副反应,同时减轻了力学方面的损害,有可能成为一种新的临床上治疗骨缺损的植骨材料。
BACKGROUND
Repairing bone defect, especially larger section defect, has been one of the major problem for orthopedists for a long time. Solutions are mainly bone grafting and bone callus lengthening。For the bone graft material which mainly autologous bone, allograft bone, xenograft bone and four types of artificial bone.Among these, autogenous bone material has the advantage of higher engraft capacity, less immunologic reject response and quicker healing. But it also has limitations and complications that can not be ignored, such as limited resource, secondary damage; increased secondary infection that restricted its clinical application. Artificial bone material also has not been used extensively because of its poor inductivity, insufficient pores, small particles, higher resolvability, friability and lower pressure resistance. On the other hand, allograft bone material has been chose as the favored material more and more frequently.1880, Macewen conducted the first allograft bone transplantation in human history.1908, Lexer conducted the first allograft bone transplantation after a knee tumorectomy; 1915, "Bone Transplant Surgery" written by Albee was published. Since then, allograft bone material has become the most important part of orthopedic clinical work. It has convenient resource, unlimited shape and quantity. And it can facilitate quicker rebuilding the circulation of damaged internal environment around bone defect. It has better bone-formation and dynamic capability than artificial bone material, broader resource than autogenous bone material. Therefore, allograft bone material has been more and more extensively used in clinic on account of all these outstanding advantages and clinical value. However, every thing has two faces, allograft bone material also has some unresolved problems, such as immunologic rejection, secondary infection and delayed engrafting ext.. Engraftment of allograft bone is under influence of biologic and especially immune mechanisms. Immunologic rejection manifest as inflammatory cells infiltration around graft, new vessels' degeneration and occlusion, and it cause decrease of inductivity and conductivity, as well as comprehensively bone absorption, then delaying of engraftment, failing engraftment, osteoporosis and bone fracture. Still, allograft bone material is biologic material; it has certain immune antigenic characteristics. Immunologic rejection is the key element limiting the engraftment. So, milder immunologic rejection of allograft bone transplantation is the pivot issue to improve prognosis and engraftment. Till today, immunologic response is still one of the paramount subjects in transplantation area. Along the progressing research about material's biocompatibility and emerging issues of clinical application of material and equipment, people come to realize the importance of interaction between human immune system and biologic material. The biggest problem in early allograft bone transplantation is infectious disease and immunologic rejection. Rejection to graft is still the most important reason failing engraftment presently. Patient's immune system generates a series of immunologic response caused by foreign element which was transplanted into patient's body. Immunologic responses include inflammatory reaction, antibodies generating, complements activation, and all kinds of cytokines expression. Introducing immunologic evaluation to biologic material and medical equipment evaluation will provide necessary scientific evidence to reduce the risk in clinical application. Allograft bone material is one of the most frequently used graft in orthopedic, its immunogenicity and the effect of bone defect repairing with allograft bone material has obvious relativity.
Earlier people had tried to use Sodium Mercurothiolate and other chemical methods to reduce allograft immunogenicity and sterilizing them, but to little avail. As the preparation process improvement, people gradually tend to use deep-cryogenic method, freeze-drying method and the y-ray irradiation to reduce the immunogenicity of allograft bone, in which y-ray irradiation has more powerful sterilization effect.After After several decades of practice,the bone bank all over the world has proved the above-mentioned method can reduce the immunogenicity of allogeneic bone and effectively reduces the result of bone graft due to the spread of disease risk, which is currently the most commonly used in allogeneic bone preparation methods. However, strictly speaking, the above method does not completely eliminate the immunogenicity of allogeneic bone, but after the above-mentioned method of treatment, allogeneic bone-forming properties and bio-mechanical properties of bone have been obvious damage. So in the clinical application,the effect of allograft bone to repair bone defects, in particular, to repair the effects of large bone defects still not satisfied. In view of this,the research group made new exploration of allogeneic bone preparation methods. In particular,we will compound cyclosporine in the process of preparation of allogeneic bone,which will a strong immune inhibition in the local. This approach will instead of deep hypothermia frozen, freeze-drying, and Y-ray irradiation in this respect. In order to better suppression of immune rejection,and reduce the damage of osteogenesis ability and mechanical properties of bone in the production process. In addition, we hope thatit will lower the side effects though the local application of cyclosporine.
OBJECTIVE
In the preparation process, Cyelosporine-impregnated Allograft Bone need to be skimmed and Deproteinized.It will mitigated the immune prototype, but also caused damage to the BMP and other bio-active ingredients which may play an important role in the biological characteristics of bone. So it is necessary to evaluate the immunogenicity and induction of Cyelosporine-impregnated Allograft Bone.In this article,we will disscuss the possibility for the the clinical application of Cyelosporine-impregnated Allograft Bone which is combined cyclosporine A and sterilize by low-temperature plasma through the comparision among Cyelosporine-impregnated Allograft Bone、freeze-dried bone and Homologous bone in osteoinductivity and immunogenicity.
METHODS
1 The choice of MTT and Alamarblue in lymphocyte stimulation test for the second time.
Separate the spleen cells of Balb/c mouse and dilute the cell original solution into a stock solution concentrations,2×105、1×105,0.5×105、0.25×105、0.125×105、0.0625×105cell/ml. In the 96-well culture plate add 200μl of cell suspension at different concentrations,each well repeat six times and set the zero holes.Method Alamarblue:Add 20μl Alamarblue for each well afther decking.Detect the absorbance values of each hole at 570nm (dominant wavelength) and 600nm (reference wavelength) after cultured 0、2、4、6、20h respectively, In accordance with the instructions, using the formula to calculate the reduction rate Alamarblue. Method MTT:decking with method Alamarblue, Add 20μl MTT for each well.Add DMSO after cultured for 4h,then detect the absorbance values of each hole at 570nm (dominant wavelength) and 600nm (reference wavelength). Precision Measurement: Group differences indicated by coefficient of variation CV. CV=group standard deviation of the data (s)/group of data within the average O.
2 Comparison of Osteoinductivity and mmunogenicity among Cyelosporine-impregnated Allograft Bone, freeze-dried bone allograft and homologous bone
Preparation of Cyelosporine-impregnated Allograft Bone:separate the iliac bone of C57 mouse,make them particles,skimmed and deprotained combined cyclosporine A in solid dispersion method, then sterilized in low-temperature plasma.
Preparation of freeze-dried bone:separate the iliac bone of C57 mouse, make them particles,rinsed with distilled water repeatedly freeze-dryed at freeze-drying machine,sterilized by y-ray irradiation.
Preparation of Homologous bone:separate the iliac bone of Balb/c mouse, make them particles before operation, washed with normal saline repeatly.
Preparation of Bone homogenate:take C57 mice and Balb/c mice limb bones, insed with distilled water repeatedly, dried and ground into bone meal,then add Cell culture medium.
Preparation of Rectus femoris bag Mode:implant the bone particles into bilateral rectus femoris of Balb/c mice.
Draw the materials at 2,4,6,8 weeks for alkaline phosphatase detect and MASSON staining.Operate the spleen cells of mice after 4 weeks,make a certain concentration of cell suspension,add the corresponding bone homogenate supernatant and Alamarblue indicator, detect the absorbance values of each hole at 570nm (dominant wavelength) and 600nm (reference wavelength), calculate the stimulation index of each group.
3 Analysis the dates with SPSS 13.0, the experimental results Said using x±s,compare different means in variance analysis method, when p<0.05, the difference was statistically significant.
RESULTS
1 The choice of MTT and Alamarblue in lymphocyte stimulation test for the second time.
When the concentration is between 0.0625×105～2×105cell/ml,the correlation between cell concentration and t the rate of reduction was not obvious at Oh (P>0.05) and 2h (P<0.05), the correlation between cell concentration and t the rate of reduction was significant relevance at 4h (P<0.001)、6h (P<0.001)、20h (P<0.001).
Different cell concentrations with the culture time prolonged, reduction rate is also increased.20h maximum cell density reduction rate has yet reached 100%.
Reduction rate of Alamarblue and OD value of MTT increases along with the cell concentration increased. The two pairs of correlation analysis, correlation coefficient r=0.998, P<0.05. There are two ways to positive linear correlation between the closely related and relevant.
The CV (Alamarblue)=0.0152, CV (MTT)=0.0180 when Cell concentration is 2×105 cell/ml and the CV (Alamarblue)=0.024, CV (MTT)=0.068 when cell concentration is 0625×105 cell/ml.
2 Comparison of Osteoinductivity and mmunogenicity among Cyelosporine-impregnated Allograft Bone, freeze-dried bone allograft and homologous bone
2.1 Secondary lymphocyte stimulation test
Analysis the lymphocyte stimulation index of the groups after 2,4 weeks. using ANOVA of completely random design data. F=0.3228, P=0.7302> 0.05 between the Treatment groups after 2 weeks, differences between the three groups is not significant.4w, the deal between the two groups F= 253.6085, P<0.001 between the Treatment groups after 4 weeks. Differences between the three groups is significant. The difference among the three groups for further use Bonferroni test.The group of Cyelosporine-impregnated Allograft Bone and the group of Homologous bone has no significant difference in lymphocyte stimulation index.the group of freeze-dried bone and the group of Cyelosporine-impregnated Allograft Bone has significant difference. Freeze-dried group has a higher lymphocyte stimulation index.
2.2 Detect alkaline phosphatase Locally
We find that F values of different groups (F between groups=25.1807; P<0.001) and different time (F time=239.5745; P<0.001) are significant difference through muti-way classification ANOVA.Compare different means in one-way ANOVA,we find that the Alkaline phosphatase level of Cyelosporine-impregnated Allograft Bone group at 2,4,6,8w has statistical difference (F=76.9263, P<0.001). Alkaline phosphatase level of Homologous bone group at 2,4,6,8w has statistical difference (F=60.7506，P<0.001). Alkaline phosphatase level of Freeze-dried bone group at 2,4,6,8w has statistical difference (F=132.4105, P<0.001). Alkaline phosphatase level of different groups has no statistical difference at 2 w (F=10.5117, P<0.05). Alkaline phosphatase level of different groups has significant statistical difference at 4 w (F=9.0822, P<0.05). Alkaline phosphatase level of different groups has significant statistical difference at 6 w (F=10.5117, P<0.05). Alkaline phosphatase level of different groups has significant statistical difference at 8 w (F=5.7737, P<0.05).
2.1 Histological staining
The amount of new bone of Cyelosporine-impregnated Allograft Bone group and Homologous bone group is higher than Freeze-dried group.Three groups were not found in inflammatory response around the implanted bone particles at different time points.
CONCLUSION
1 The choice of MTT and Alamarblue in lymphocyte stimulation test for the second time.
Alamarblue method and MTT method are highly correlated. At high concentrations Alamarblue method and MTT method group has similar Coefficient of variation,both is lower than 2%. At low concentrations Alamarblue method and MTT method group has similar Coefficient of variation,both is lower than 2%. Demonstrate Alamarblue has no difference at stability and reproducibility with traditional MTT whenever the concentration is high and low.
2 Comparison of Osteoinductivity and mmunogenicity among Cyelosporine-impregnated Allograft Bone, freeze-dried bone allograft and homologous bone
It is clear that the cyclosporine A around the bone particles depress the T-cell activation,and provide a good enciroment for the formation of new bone. So Cyelosporine-impregnated Allograft Bone has a stronger osteoinductive and a weaker immunogenicity.
长期以来,临床上的骨缺损修复,特别是大段骨缺损的修复,一直是外科医生面临的难题之一。解决方法主要有骨移植术及骨痂延长术。其中用于骨移植的材料主要有自体骨、同种异体骨、异种骨及人工骨四类。自体骨骨生成能力强、无免疫排斥等优点,但在骨缺损治疗过程中,自体骨具有明显的局限性和并发症,如来源有限、对供区有损伤、机会感染等,这些不利因素限制了其在临床上的应用；人工骨虽然传导性较好但骨诱导性较差、缺乏孔隙、颗粒较小、易分解、脆性大不耐压且相对缺乏生物活性等缺点,限制了它的应用。而同种异体骨自1880年Macewe做了人类历史上首例同种异体骨移植术,1908年Lexer最早实施了膝关节肿瘤切除后的同种异体骨移植术,1915年Albee出版了《骨移植外科》以来,同种异体骨移植逐渐成为骨科治疗的重要手段,它取材方便,数量形状不受限制,对已被破坏的骨折周围的内环境,有着恢复迅速、改善和重建血循环快捷并且其成骨性能及力学性能优于人工骨,而来源也不像自体骨那样受到限制等,诸多独特的优势和应用价值,进而使得同种异体骨在临床上应用广泛,逐渐成为骨科治疗的重要手段。但也存在一些目前难以彻底解决的问题,如免疫排斥、晚期感染、移植骨愈合缓慢等,同种异体骨移植愈合受它的生物机制和免疫机制两大原则支配,而免疫排斥反应是它的关键；免疫排斥反应的主要表现为异体骨周围炎症细胞浸润,再生血管变性闭塞,造成骨诱导和骨传导作用下降及广泛的骨吸收,引起骨延迟愈合、不愈合、骨质疏松、骨折等；异体骨是异体生物性材料,具有一定的免疫抗原性,免疫排斥反应是制约异体骨移植的主要因素。降低异体骨移植的免疫排斥反应是提高治疗效果和决定异体植骨成功与否的关键。早期异体骨移植的最大问题是疾病传播与免疫排斥反应,而移植排斥反应仍是导致新鲜同种异体骨移植失败的最主要原因。机体的免疫系统对于植入体内的非已成分(同种异体骨)会产生一系列免疫应答的防御行为,其中包括炎症反应、抗体产生、补体活化及各种细胞因子的表达等,因此在生物材料和医疗器械的生物学评价中引入免疫学评价的内容将对减小人体应用的风险提供必要的科学依据,同种异体骨是骨科最常用的骨植入材料之一,同时异体骨的免疫原性与异体骨移植修复骨缺损的临床效果密切相关。
早些时候人们曾尝试着用柳硫汞等化学方法的处理来降低异体骨的免疫原性和对其进行灭菌,但效果不佳。随着制备工艺的改进,人们逐渐倾向于用深低温冷冻法、冷冻干燥法及丫射线辐照来降低异体骨的免疫原性,其中丫射线辐照更兼具强大的灭菌功效。经过世界各国骨库几十年来的实践证明,上述方法能够很好地降低异体骨的免疫原性并有效地降低了因骨移植所致的疾病传播的风险,因而是目前最为常用的同种异体骨的制备方法。然而严格地讲,上述方法并不能完全消除同种异体骨的免疫原性,而经过上述方法处理后,同种异体骨的成骨性能及生物力学性能受到了明显的损害。致使目前为止临床上应用异体骨修复骨缺损的效果,特别是修复大段骨缺损的效果仍不能够令人满意。鉴于此本课题组对同种异体骨的制备方法进行新的探索。具体而言,就是在制备同种异体骨的过程中复合环孢菌素,通过在局部发挥环孢菌素强大的免疫抑制作用来代替深低温冷冻、冷冻干燥及γ射线辐照的降免疫原性作用,并用低温等离子体技术来代替γ射线辐照对异体骨进行灭菌,以期更好地抑制异体骨移植的免疫排斥反应,同时减轻制备过程中对异体骨成骨性能及力学性能的损害。另外,我们希望通过环孢菌素的局部应用来降低其药物副反应。
研究目的
为了进一步减轻同种异体骨的免疫原性,同时又不损害异体骨的机械强度,裴国献、陆海波等人采用新的方法制备了复合环孢素A的同种异体骨,并申请到了发明专利。在同种异体骨治疗大段骨缺损方面进行了有益的探索。但复合环孢素同种异体骨在制备过程中需要对供骨进行脱脂脱蛋白,一定程度上减轻了其免疫原性,但同时也造成了对同种异体骨生物学特性起重要作用的BMP及其他生物活性成分的大量丢失。因此有必要对其免疫原性及骨诱导性进行评估,通过对复合环孢素A并低温等离子体灭菌这一新的异体骨制备方法制备的同种异体骨与冻干同种异体骨及同系骨骨诱导性及免疫原性的比较,探讨复合环孢素A的同种异体骨在临床上应用的可能性。
研究方法
1、淋巴细胞二次刺激实验Alamarblue法和MTT法的选择
取Balb/c小鼠脾脏淋巴细胞,将分离得到的细胞原液稀释成浓度分别为2×105、1×105、0.5×105、0.25×105、0.125×105、0.0625×105cell／ml的细胞悬液。在96孔培养板上添加不同浓度细胞悬液200μl,重复6孔,并设置调零孔。Alamarblue法：铺板后每孔加入20μlAlamarblue。分别于培养0、2、4、6、20h后,用酶标仪在570nm(主波长)及600nm(参考波长)检测各个孔的吸光度值。按照说明书通过公式计算出Alamarblue的还原率。MTT法：细胞种板设计同AlamarBlue法。每空加入MTT20μl。培养4小时后,DMSO显色后,在490nm(主波长)及630nm(参考波长)检测各个孔的吸光度值。对Alamarblue法与MTT法、Alamarblue法与时间进行相关性分析。精密度测定：组内差异用变异系数CV表示。CV=组内各数据的标准差(s)／组内各数据的平均值(x)。
2、复合环孢素A的同种异体骨与冻干同种异体骨及同系骨骨诱导性及免疫原性的比较
复合环孢素A的同种异体骨的制备：取C57小鼠的髂骨,制成骨粒,脱脂脱蛋白后采用固体分散法复合复合环孢素A,然后采用低温等离子体灭菌。
冻干同种异体骨的制备：取C57小鼠的髂骨,制成骨粒,采用蒸馏水反复冲洗,在低温冷冻干燥机中冻干,采用γ射线辐照灭菌。
同系骨的制备：术中取Balb/c小鼠的髂骨,制成骨粒,生理盐水反复冲洗。
骨匀浆上清的制备：取C57小鼠及Balb/c小鼠的四肢骨骼蒸馏水反复冲洗,干燥后研磨成骨粉与细胞培养液混合制成骨粉悬液,在超净台中,经无菌滤器过滤,制得骨匀浆上清。
将制成的骨粒植入Balb/c小鼠左侧股直肌内制成股直肌袋模型。
分别在2、4、6、8w时取材,进行MASSON染色并进行碱性磷酸酶的检测。在4w时取各组小鼠脾脏,分离淋巴细胞,制成一定浓度的细胞悬液,加入对应的骨匀浆上清及Alamarblue指示剂,用酶标仪在570nm(主波长)及600hm(参考波长)检测各个孔的吸光度值。计算出各组的刺激指数。
3、用SPSS13.0对结果进行数据分析,实验结果采用x±s表示,多个样本均数比较采用析因设计方差分析的方法,单独比较采用one-way A N 0 V A或t检验分析方法。当p<0.05时,差异有统计学意义。
结果
1、淋巴细胞二次刺激实验Alamarblue法和MTT法的选择
细胞浓度在0.0625×105～2×105cell／ml之间时,0h(P>0.05)细胞浓度与还原率之间的相关关系不明显。2h(P<0.05)、4h(P<0.001)、6h(P<0.001)、20h(P<0.001)细胞浓度与还原率之间有显著的相关性。
不同的细胞浓度随着培养时间的延长,还原率也随之增加。最大细胞浓度20h的还原率仍未达100%。
Alamarblue法测得的还原率及MTT法测得的OD值均随着细胞浓度的增大而增加。对二者进行相关性分析,相关系数r=0.998,P<0.05。两种方法存在正线性相关关系,且相关关系密切。
细胞浓度为2×105时CV (Alamarblue)=0.0152, CV(MTT)=0.0487;细胞浓度为0625×105时CV (Alamarblue)=0.023, CV(MTT)=0.0369。
2复合环孢素A的同种异体骨与冻干同种异体骨及同系骨骨诱导性及免疫原性的比较
2.1淋巴细胞二次刺激实验
采用完全随机设计资料的方差分析分别对两个时间点三组淋巴细胞刺激指数进行分析。2w时,处理组间F=0.3228,P=0.7302>0.05,三组之间差异不显著。4w时,处理组间F=253.6085,P<0.001。三组间差异显著。三组间异质性进一步采用Bonferroni检验,复合环孢素A组与同系组差异不显著,冻干组与复合环孢素A组及同系组差异显著,冻干组淋巴细胞刺激指数较高。
2.2局部碱性磷酸酶检测
对4个时间点3组碱性磷酸酶数据进行两因素析因分析后发现,三组(F组间=25.1807；P<0.001)及各个时间点(F时间=239.5745；P<0.001)均存在统计学差异。进一步采用单向方差分析(one-way ANOVA)进行多样本均数间的比较发现,符合环孢素A组在2、4、6,8w的碱性磷酸酶含量统计学差异显著(F=76.9263,P<0.001)。同系组在2、4、6、8w的碱性磷酸酶含量统计学差异显著(F=60.7506,P<0.001)。冻干骨组在2,4,6,8 w的碱性磷酸酶含量统计学差异显著(F=132.4105,P<0.001)。2 w时三组间的碱性磷酸酶含量统计学差异不显著(F=10.5117,P<0.05).4 w时冻干组与复合环孢素A组、同系组间统计学差异显著,复合环孢素A组与同系组间差异不显著(F=0.0418,P<0.05)。6 w时冻干组与复合环孢素A组、同系组间统计学差异显著,复合环孢素A组与同系组间差异不显著(F=9.0822,P<0.05)。8 w时冻干组与复合环孢素A组、同系组间统计学差异显著,复合环孢素A组与同系组间差异不显著(F=5.7737,P<0.05)。
2.3局部组织学检测
通过MASSON染色,2、4、6、8w时,复合环孢素组与同系组的成成骨量均比冻干组高。三组在各个时间点植入骨粒周围均未发现炎症反应。
结论
1、淋巴细胞二次刺激实验Alamarblue法和MTT法的选择
在本研究中检测了不同浓度的BALB/C小鼠脾淋巴细胞在加入Alamarblue后不同时间所测得的吸光度值,并计算出相对应的还原率。并对加入指示剂4h后Alamarblue的还原率及MTT的OD值进行相关性分析。在加入Alamarblue后,不同细胞浓度的还原率随着作用时间的延长而增加,当细胞浓度为2×105cell/ml,作用20h后还原率为78.3%,仍未达到100%。不同的细胞浓度在作用时间为0h(P>0.05)条件下不同细胞浓度与还原率之间的线性相关关系不明显,2h(P<0.05)、4h(P<0.001)、6h(P<0.001)、20h(P<0.001)条件下不同细胞浓度与还原率之间正性线性相关关系明显。说明在细胞浓度在0.0625×105-2×105cell/ml时,加入Alamarblue后4、6、20h所得出的还原率均可间接反应出细胞数量,从而在使用小鼠脾细胞进行细胞增殖或细胞毒试验中反映细胞存活的情况。如果进行细胞增殖实验则推荐接种细胞密度为0.0625×105-0.5×105cell/ml,作用时间为4-6h。较高的细胞密度和较长的作用时间可能使所有检测的样本还原态的Alamarblue达到饱和状态。如果进行细胞毒性实验,则可采用本研究的细胞接种浓度及培养时间。
Alamarblue法与MTT法的结果高度相关。在高浓度时Alamarblue法与MTT法组内差异接近,均小于2%。低浓度时Alamarblue法与MTT法组内差异接近,均小于2%。说明在高浓度、低浓度时Alamarblue法与传统的MTT法相比稳性及重复性并无明显差异。
2复合环孢素A的同种异体骨与冻干同种异体骨及同系骨骨诱导性及免疫原性的比较
实验结果表明,术后2w,三组间碱性磷酸酶含量统计学上有显著差异。环孢素组与同系组的碱性磷酸酶含量比冻干组高,环孢素组与同系组含量差异不明显。组织学显示三组植入骨粒周围均没有显著的炎症细胞浸润。同系组植入骨粒骨小梁开始被吸收,条里变得模糊,冻干骨组植入骨粒骨小梁未被吸收,复合环孢素组植入骨粒的周围骨小梁开始被吸收。术后4w,三组间碱性磷酸酶含量统计学上有显著差异,复合环孢素组与同系组含量差异不明显。复合环孢素组与同系组的碱性磷酸酶含量比冻干组高。组织学表明同系组与复合环孢素组植入骨粒已经被吸收,可见大片新生的绿染的Ⅱ型胶原纤维,分布较均匀。冻干骨植入骨粒骨小梁大部分被吸收,新生胶原纤维较同系组及复合环孢素组少,主要以结缔纤维组织为主。术后6w,三组间碱性磷酸酶含量统计学上有显著差异。环孢素组与同系组的碱性磷酸酶含量比冻干组高,环孢素组与同系组含量差异不明显。组织学6w时,三组植入骨粒周围均没有炎症细胞浸润。环孢素组及同系组开始出现软骨内成骨,可见骨陷窝,及骨细胞。骨粒周围开始出现红染的胶原纤维,有层次感。冻干骨骨可见植入骨粒内出现新生的胶原纤维,排列杂乱。8w时三组间碱性磷酸酶含量统计学上有显著差异。环孢素组与同系组的碱性磷酸酶含量比冻干组高,环孢素组与同系组含量差异不明显。组织学8w时,同系组及复合环孢素组出现大片的红染的成熟胶原纤维,呈现板层排列,部分样本中间出现空腔,并含有髓腔内容物.冻干骨组可见成熟胶原纤维与新生胶原纤维交错分布,呈现红绿相间染色。
2w时各个组的淋巴细胞刺激指数三组间统计学差异不显著,4w三组间淋巴细胞刺激指数差异显著,同系组、复合环孢素组均低于冻干组,同系组与复合环孢素之间无显著差异。
可见复合在移植骨粒上的环孢素并低温等离子体灭菌,在局部抑制了T细胞的活化,为新骨的生成创造了良好的环境,避免长期全身应用环孢素所带来的副反应,同时减轻了力学方面的损害,有可能成为一种新的临床上治疗骨缺损的植骨材料。
BACKGROUND
Repairing bone defect, especially larger section defect, has been one of the major problem for orthopedists for a long time. Solutions are mainly bone grafting and bone callus lengthening。For the bone graft material which mainly autologous bone, allograft bone, xenograft bone and four types of artificial bone.Among these, autogenous bone material has the advantage of higher engraft capacity, less immunologic reject response and quicker healing. But it also has limitations and complications that can not be ignored, such as limited resource, secondary damage; increased secondary infection that restricted its clinical application. Artificial bone material also has not been used extensively because of its poor inductivity, insufficient pores, small particles, higher resolvability, friability and lower pressure resistance. On the other hand, allograft bone material has been chose as the favored material more and more frequently.1880, Macewen conducted the first allograft bone transplantation in human history.1908, Lexer conducted the first allograft bone transplantation after a knee tumorectomy; 1915, "Bone Transplant Surgery" written by Albee was published. Since then, allograft bone material has become the most important part of orthopedic clinical work. It has convenient resource, unlimited shape and quantity. And it can facilitate quicker rebuilding the circulation of damaged internal environment around bone defect. It has better bone-formation and dynamic capability than artificial bone material, broader resource than autogenous bone material. Therefore, allograft bone material has been more and more extensively used in clinic on account of all these outstanding advantages and clinical value. However, every thing has two faces, allograft bone material also has some unresolved problems, such as immunologic rejection, secondary infection and delayed engrafting ext.. Engraftment of allograft bone is under influence of biologic and especially immune mechanisms. Immunologic rejection manifest as inflammatory cells infiltration around graft, new vessels' degeneration and occlusion, and it cause decrease of inductivity and conductivity, as well as comprehensively bone absorption, then delaying of engraftment, failing engraftment, osteoporosis and bone fracture. Still, allograft bone material is biologic material; it has certain immune antigenic characteristics. Immunologic rejection is the key element limiting the engraftment. So, milder immunologic rejection of allograft bone transplantation is the pivot issue to improve prognosis and engraftment. Till today, immunologic response is still one of the paramount subjects in transplantation area. Along the progressing research about material's biocompatibility and emerging issues of clinical application of material and equipment, people come to realize the importance of interaction between human immune system and biologic material. The biggest problem in early allograft bone transplantation is infectious disease and immunologic rejection. Rejection to graft is still the most important reason failing engraftment presently. Patient's immune system generates a series of immunologic response caused by foreign element which was transplanted into patient's body. Immunologic responses include inflammatory reaction, antibodies generating, complements activation, and all kinds of cytokines expression. Introducing immunologic evaluation to biologic material and medical equipment evaluation will provide necessary scientific evidence to reduce the risk in clinical application. Allograft bone material is one of the most frequently used graft in orthopedic, its immunogenicity and the effect of bone defect repairing with allograft bone material has obvious relativity.
Earlier people had tried to use Sodium Mercurothiolate and other chemical methods to reduce allograft immunogenicity and sterilizing them, but to little avail. As the preparation process improvement, people gradually tend to use deep-cryogenic method, freeze-drying method and the y-ray irradiation to reduce the immunogenicity of allograft bone, in which y-ray irradiation has more powerful sterilization effect.After After several decades of practice,the bone bank all over the world has proved the above-mentioned method can reduce the immunogenicity of allogeneic bone and effectively reduces the result of bone graft due to the spread of disease risk, which is currently the most commonly used in allogeneic bone preparation methods. However, strictly speaking, the above method does not completely eliminate the immunogenicity of allogeneic bone, but after the above-mentioned method of treatment, allogeneic bone-forming properties and bio-mechanical properties of bone have been obvious damage. So in the clinical application,the effect of allograft bone to repair bone defects, in particular, to repair the effects of large bone defects still not satisfied. In view of this,the research group made new exploration of allogeneic bone preparation methods. In particular,we will compound cyclosporine in the process of preparation of allogeneic bone,which will a strong immune inhibition in the local. This approach will instead of deep hypothermia frozen, freeze-drying, and Y-ray irradiation in this respect. In order to better suppression of immune rejection,and reduce the damage of osteogenesis ability and mechanical properties of bone in the production process. In addition, we hope thatit will lower the side effects though the local application of cyclosporine.
OBJECTIVE
In the preparation process, Cyelosporine-impregnated Allograft Bone need to be skimmed and Deproteinized.It will mitigated the immune prototype, but also caused damage to the BMP and other bio-active ingredients which may play an important role in the biological characteristics of bone. So it is necessary to evaluate the immunogenicity and induction of Cyelosporine-impregnated Allograft Bone.In this article,we will disscuss the possibility for the the clinical application of Cyelosporine-impregnated Allograft Bone which is combined cyclosporine A and sterilize by low-temperature plasma through the comparision among Cyelosporine-impregnated Allograft Bone、freeze-dried bone and Homologous bone in osteoinductivity and immunogenicity.
METHODS
1 The choice of MTT and Alamarblue in lymphocyte stimulation test for the second time.
Separate the spleen cells of Balb/c mouse and dilute the cell original solution into a stock solution concentrations,2×105、1×105,0.5×105、0.25×105、0.125×105、0.0625×105cell/ml. In the 96-well culture plate add 200μl of cell suspension at different concentrations,each well repeat six times and set the zero holes.Method Alamarblue:Add 20μl Alamarblue for each well afther decking.Detect the absorbance values of each hole at 570nm (dominant wavelength) and 600nm (reference wavelength) after cultured 0、2、4、6、20h respectively, In accordance with the instructions, using the formula to calculate the reduction rate Alamarblue. Method MTT:decking with method Alamarblue, Add 20μl MTT for each well.Add DMSO after cultured for 4h,then detect the absorbance values of each hole at 570nm (dominant wavelength) and 600nm (reference wavelength). Precision Measurement: Group differences indicated by coefficient of variation CV. CV=group standard deviation of the data (s)/group of data within the average O.
2 Comparison of Osteoinductivity and mmunogenicity among Cyelosporine-impregnated Allograft Bone, freeze-dried bone allograft and homologous bone
Preparation of Cyelosporine-impregnated Allograft Bone:separate the iliac bone of C57 mouse,make them particles,skimmed and deprotained combined cyclosporine A in solid dispersion method, then sterilized in low-temperature plasma.
Preparation of freeze-dried bone:separate the iliac bone of C57 mouse, make them particles,rinsed with distilled water repeatedly freeze-dryed at freeze-drying machine,sterilized by y-ray irradiation.
Preparation of Homologous bone:separate the iliac bone of Balb/c mouse, make them particles before operation, washed with normal saline repeatly.
Preparation of Bone homogenate:take C57 mice and Balb/c mice limb bones, insed with distilled water repeatedly, dried and ground into bone meal,then add Cell culture medium.
Preparation of Rectus femoris bag Mode:implant the bone particles into bilateral rectus femoris of Balb/c mice.
Draw the materials at 2,4,6,8 weeks for alkaline phosphatase detect and MASSON staining.Operate the spleen cells of mice after 4 weeks,make a certain concentration of cell suspension,add the corresponding bone homogenate supernatant and Alamarblue indicator, detect the absorbance values of each hole at 570nm (dominant wavelength) and 600nm (reference wavelength), calculate the stimulation index of each group.
3 Analysis the dates with SPSS 13.0, the experimental results Said using x±s,compare different means in variance analysis method, when p<0.05, the difference was statistically significant.
RESULTS
1 The choice of MTT and Alamarblue in lymphocyte stimulation test for the second time.
When the concentration is between 0.0625×105～2×105cell/ml,the correlation between cell concentration and t the rate of reduction was not obvious at Oh (P>0.05) and 2h (P<0.05), the correlation between cell concentration and t the rate of reduction was significant relevance at 4h (P<0.001)、6h (P<0.001)、20h (P<0.001).
Different cell concentrations with the culture time prolonged, reduction rate is also increased.20h maximum cell density reduction rate has yet reached 100%.
Reduction rate of Alamarblue and OD value of MTT increases along with the cell concentration increased. The two pairs of correlation analysis, correlation coefficient r=0.998, P<0.05. There are two ways to positive linear correlation between the closely related and relevant.
The CV (Alamarblue)=0.0152, CV (MTT)=0.0180 when Cell concentration is 2×105 cell/ml and the CV (Alamarblue)=0.024, CV (MTT)=0.068 when cell concentration is 0625×105 cell/ml.
2 Comparison of Osteoinductivity and mmunogenicity among Cyelosporine-impregnated Allograft Bone, freeze-dried bone allograft and homologous bone
2.1 Secondary lymphocyte stimulation test
Analysis the lymphocyte stimulation index of the groups after 2,4 weeks. using ANOVA of completely random design data. F=0.3228, P=0.7302> 0.05 between the Treatment groups after 2 weeks, differences between the three groups is not significant.4w, the deal between the two groups F= 253.6085, P<0.001 between the Treatment groups after 4 weeks. Differences between the three groups is significant. The difference among the three groups for further use Bonferroni test.The group of Cyelosporine-impregnated Allograft Bone and the group of Homologous bone has no significant difference in lymphocyte stimulation index.the group of freeze-dried bone and the group of Cyelosporine-impregnated Allograft Bone has significant difference. Freeze-dried group has a higher lymphocyte stimulation index.
2.2 Detect alkaline phosphatase Locally
We find that F values of different groups (F between groups=25.1807; P<0.001) and different time (F time=239.5745; P<0.001) are significant difference through muti-way classification ANOVA.Compare different means in one-way ANOVA,we find that the Alkaline phosphatase level of Cyelosporine-impregnated Allograft Bone group at 2,4,6,8w has statistical difference (F=76.9263, P<0.001). Alkaline phosphatase level of Homologous bone group at 2,4,6,8w has statistical difference (F=60.7506，P<0.001). Alkaline phosphatase level of Freeze-dried bone group at 2,4,6,8w has statistical difference (F=132.4105, P<0.001). Alkaline phosphatase level of different groups has no statistical difference at 2 w (F=10.5117, P<0.05). Alkaline phosphatase level of different groups has significant statistical difference at 4 w (F=9.0822, P<0.05). Alkaline phosphatase level of different groups has significant statistical difference at 6 w (F=10.5117, P<0.05). Alkaline phosphatase level of different groups has significant statistical difference at 8 w (F=5.7737, P<0.05).
2.1 Histological staining
The amount of new bone of Cyelosporine-impregnated Allograft Bone group and Homologous bone group is higher than Freeze-dried group.Three groups were not found in inflammatory response around the implanted bone particles at different time points.
CONCLUSION
1 The choice of MTT and Alamarblue in lymphocyte stimulation test for the second time.
Alamarblue method and MTT method are highly correlated. At high concentrations Alamarblue method and MTT method group has similar Coefficient of variation,both is lower than 2%. At low concentrations Alamarblue method and MTT method group has similar Coefficient of variation,both is lower than 2%. Demonstrate Alamarblue has no difference at stability and reproducibility with traditional MTT whenever the concentration is high and low.
2 Comparison of Osteoinductivity and mmunogenicity among Cyelosporine-impregnated Allograft Bone, freeze-dried bone allograft and homologous bone
It is clear that the cyclosporine A around the bone particles depress the T-cell activation,and provide a good enciroment for the formation of new bone. So Cyelosporine-impregnated Allograft Bone has a stronger osteoinductive and a weaker immunogenicity.
引文
[1]Parikh S N. Bone graft substitutes:past, present, future.[J]. J Postgrad Med,2002, 48(2):142-148.
[2]den Boer F C, Wippermann B W, Blokhuis T J, et al. Healing of segmental bone defects with granular porous hydroxyapatite augmented with recombinant human osteogenic protein-1 or autologous bone marrow.[J]. J Orthop Res,2003,21(3): 521-528.
[3]W M. J Proc Roy Soc London [J]. J Proc Roy Soc London,1881,32:232-247.
[4]Mcauliffe J A. Bone graft substitutes.[J]. J Hand Ther,2003,16(2):180-187.
[5]Greenwald A S, Boden S D, Goldberg V M, et al. Bone-graft substitutes:facts, fictions, and applications.[J]. J Bone Joint Surg Am,2001,83-A Suppl 2 Pt 2: 98-103.
[6]邱贵兴孙世荃.同种异体骨植入材料的临床应用[J].中华骨科杂志,2004(10).
[7]彭吾训杨泽华王鑫.脱钙骨基质的临床应用与研究进展[J].昆明医学院学报,2003(04).
[8]Burchardt H. The biology of bone graft repair. [J]. Clin Orthop Relat Res, 1983(174):28-42.
[9]Nguyen H, Morgan D A, Forwood M R. Sterilization of allograft bone:is 25 kGy the gold standard for gamma irradiation?[J]. Cell Tissue Bank,2007,8(2): 81-91.
[10]Friedlaender G E. Bone allografts:the biological consequences of immunological events.[J]. J Bone Joint Surg Am,1991,73(8):1119-1122.
[11]Lance E M. Some observations on bone graft technology.[J]. Clin Orthop Relat Res,1985(200):114-124.
[12]Buckley S C, Stockley I, Hamer A J, et al. Irradiated allograft bone for acetabular revision surgery. Results at a mean of five years.[J]. J Bone Joint Surg Br,2005,87(3):310-313.
[13]Hamer A J, Strachan J R, Black M M, et al. Biochemical properties of cortical allograft bone using a new method of bone strength measurement. A comparison of fresh, fresh-frozen and irradiated bone.[J]. J Bone Joint Surg Br,1996,78(3): 363-368.
[14]Clatworthy M G, Ballance J, Brick G W, et al. The use of structural allograft for uncontained defects in revision total knee arthroplasty. A minimum five-year review.[J]. J Bone Joint Surg Am,2001,83-A(3):404-411.
[15]姚朗.细胞毒T淋巴细胞(CTL)活性测定方法进展[J].国外医学.临床生物化学与检验学分册,2002(02).
[16]Mattis A E, Bernhardt G, Lipp M, et al. Analyzing cytotoxic T lymphocyte activity:a simple and reliable flow cytometry-based assay.[J]. J Immunol Methods,1997,204(2):135-142.
[17]Derby E, Reddy V, Kopp W, et al. Three-color flow cytometric assay for the study of the mechanisms of cell-mediated cytotoxicity.[J]. Immunol Lett,2001, 78(1):35-39.
[18]Aubry J P, Blaecke A, Lecoanet-Henchoz S, et al. Annexin V used for measuring apoptosis in the early events of cellular cytotoxicity.[J]. Cytometry,1999,37(3): 197-204.
[19]Martin A, Morcillo N, Lemus D, et al. Multicenter study of MTT and resazurin assays for testing susceptibility to first-line anti-tuberculosis drugs.[J]. Int J Tuberc Lung Dis,2005,9(8):901-906.
[20]Hussain R F, Nouri A M, Oliver R T. A new approach for measurement of cytotoxicity using colorimetric assay.[J]. J Immunol Methods,1993,160(1): 89-96.
[21]盛大平徐元宏.alamarBlue在临床检验中的应用[J].国外医学.临床生物化学与检验学分册,2005(06).
[22]杨阳,刘宝瑞,钱晓萍.Alamar Blue法用于体外培养细胞活性检测的方法研究[J].现代肿瘤医学,2006(01).
[23]Xue M, Zhong M, Zhang Y, et al. [Effects of bovine plasma fibronectin on the proliferation of rat osteoblast in vitro][J]. Shanghai Kou Qiang Yi Xue,2003, 12(6):436-438.
[24]Nociari M M, Shalev A, Benias P, et al. A novel one-step, highly sensitive fluorometric assay to evaluate cell-mediated cytotoxicity.[J]. J Immunol Methods, 1998,213(2):157-167.
[25]Kwack K, Lynch R G. A new non-radioactive method for IL-2 bioassay.[J]. Mol Cells,2000,10(5):575-578.
[26].
[27]骨库技术与操作规范[J].中华骨科杂志,,24(10)：638-640.
[28]骨组织成分与Masson.三色染色反应的关系分析[J].中医正骨,,13(11)：5-6.
[29]黄长明王臻.骨形态发生蛋白在异体骨段移植愈合中的表达及临床意义[J].中国矫形外科杂志,2001(11).
[30]大段异体骨关节移植后长期转归的观察[J].中华外科杂志,,36(3)：169-171.
[31]Bertone A L, Pittman D D, Bouxsein M L, et al. Adenoviral-mediated transfer of human BMP-6 gene accelerates healing in a rabbit ulnar osteotomy model.[J]. J Orthop Res,2004,22(6):1261-1270.
[32]Mcgee M A, Findlay D M, Howie D W, et al. The use of OP-1 in femoral impaction grafting in a sheep model.[J]. J Orthop Res,2004,22(5):1008-1015.
[33]Tagil M, Jeppsson C, Wang J S, et al. No augmentation of morselized and impacted bone graft by OP-1 in a weight-bearing model.[J]. Acta Orthop Scand, 2003,74(6)：742-748.
[34]骨形态发生蛋白与骨缺损的治疗[J].实用骨科杂志,,8(4)：271-273.
[35]Yamaguchi A. [Recent advances in research on bone formation--BMP action and its mechanism][J]. Nippon Rinsho,2002,60 Suppl 3:40-47.
[36]Curtiss P J, Herndon C H. Immunological factors in homogenous-bone transplantation. I. Serological studies.[J]. J Bone Joint Surg Am,1956,38-A(1): 103-110.
[37]Bonfiglio M, Jeter W S, Smith C L. The immune concept:its relation to bone transplantation.[J]. Ann N Y Acad Sci,1955,59(3):417-433.
[38]Chalmers J. Transplantation immunity in bone homografting.[J]. J Bone Joint Surg Br,1959,41-B(1):160-179.
[39]Stevenson S, Horowitz M. The response to bone allografts.[J]. J Bone Joint Surg Am,1992,74(6):939-950.
[40]Kubler N, Reuther J, Kirchner T, et al. Osteoinductive, morphologic, and biomechanical properties of autolyzed, antigen-extracted, allogeneic human bone.[J]. J Oral Maxillofac Surg,1993,51(12):1346-1357.
[41]陆海波.复合环孢菌素同种异体骨的免疫学与组织学评价[J].中华创伤骨科杂志,2007,9(5)：442-447.
[42]Doi K, Akino T, Shigetomi M, et al. Revascularized intercalary bone allografts with short-term immunosuppression with cyclosporine in the canine.[J]. Plast Reconstr Surg,1998,101(3):793-801.
[43]Shigetomi M, Doi K, Kuwata N, et al. Experimental study on vascularized bone allografts for reconstruction of massive bone defects.[J]. Microsurgery,1994, 15(9):663-670.
[44]常青李守先巩性军张希全.环孢素A防治同种异体带瓣大血管移植后钙化的研究[J].山东大学学报(医学版),2005(08).
[45]环孢素A对脱钙骨基质植入物钙离子的影响[J].实用全科医学,,2(2)：97-98.
[46]徐忠烨唐文渊.环孢素A的神经保护作用[J].国外医学.神经病学神经外科学分册,2004(06).
[47]Yeo H, Beck L H, Mcdonald J M, et al. Cyclosporin A elicits dose-dependent biphasic effects on osteoblast differentiation and bone formation.[J]. Bone,2007, 40(6):1502-1516.
[48]Vm F G G. Bone and cartilage Allografts. Biology and Clinical Applications. Park Ridge, Illinois, The American Academy of Orthopaedic Surgeons[Z]. 1991.
[49]W M. J Proc Roy Soc London[Z].1881:32,232-247.
[50]Inskip H B V. Methods of bone transplantation[M].1983.445-466.
[51]E L. J Surg Gynecol Obster[J]. J Surg Gynecol Obster,1925,40:782-809.
[52]康焱廖威明.同种异体骨移植的研究和应用现状(文献综述)[J].国外医学.外科学分册,2004(04).
[53]Langlais F, Lambotte J C, Thomazeau H. Long-term results of hemipelvis reconstruction with allografts.[J]. Clin Orthop Relat Res,2001(388):178-186.
[54]黄长明福建厦门王臻童星杰福建厦门刘继中.大段异体骨移植治疗骨肿瘤[J].中华骨科杂志,2000(07).
[55]Clatworthy M G, Ballance J, Brick G W, et al. The use of structural allograft for uncontained defects in revision total knee arthroplasty. A minimum five-year review.[J]. J Bone Joint Surg Am,2001,83-A(3):404-411.
[56]姚长海侯树勋衷鸿宾李利章亚东.同种异体骨移植在骨肿瘤手术中的应用[J].中国矫形外科杂志,2003(21).
[57]Min K, Espinosa N, Bode B, et al. Total sacrectomy and reconstruction with structural allografts for neurofibrosarcoma of the sacrum. A case report.[J]. J Bone Joint Surg Am,2005,87(4):864-869.
[58]Myerson M S, Neufeld S K, Uribe J. Fresh-frozen structural allografts in the foot and ankle.[J]. J Bone Joint Surg Am,2005,87(1):113-120.
[59]Hartl A, Bitzan P, Wanivenhaus A, et al. Faster integration of human allograft bone than of the bovine substitute Lubboc:non-randomized evaluation of 20 cases with benign tumors or tumor-like conditions.[J]. Acta Orthop Scand,2004, 75(2):217-220.
[60]Giavaresi G, Torricelli P, Fornasari P M, et al. Blood vessel formation after soft-tissue implantation of hyaluronan-based hydrogel supplemented with copper ions.[J]. Biomaterials,2005,26(16):3001-3008.
[61]Manfrini M, Innocenti M, Ceruso M, et al. Original biological reconstruction of the hip in a 4-year-old girl.[J]. Lancet,2003,361(9352):140-142.
[62]王臻西安黄耀添西安胡蕴玉西安.人工关节假体复合大段同种异体骨移植重建肢体功能[J].中华外科杂志,1999(12).
[63]Anract P, Coste J, Vastel L, et al. [Proximal femoral reconstruction with megaprosthesis versus allograft prosthesis composite. A comparative study of functional results, complications and longevity in 41 cases][J]. Rev Chir Orthop Reparatrice Appar Mot,2000,86(3):278-288.
[64]周建生胡汝麒潘功平刘振华肖玉周.冷冻保存带血管异体骨移植治疗儿童骨缺损[J].中华显微外科杂志,1998(03).
[65]Chang D W, Weber K L. Segmental femur reconstruction using an intercalary allograft with an intramedullary vascularized fibula bone flap.[J]. J Reconstr Microsurg,2004,20(3):195-199.
[66]Bertone A L, Pittman D D, Bouxsein M L, et al. Adenoviral-mediated transfer of human BMP-6 gene accelerates healing in a rabbit ulnar osteotomy model.[J]. J Orthop Res,2004,22(6):1261-1270.
[67]Mcgee M A, Findlay D M, Howie D W, et al. The use of OP-1 in femoral impaction grafting in a sheep model.[J]. J Orthop Res,2004,22(5):1008-1015.
[68]Tagil M, Jeppsson C, Wang J S, et al. No augmentation of morselized and impacted bone graft by OP-1 in a weight-bearing model.[J]. Acta Orthop Scand, 2003,74(6):742-748.
[69]张贝利张伟滨邓廉夫.同种异体骨移植研究进展[J].国外医学.骨科学分册,2005(03).
[70]Burwell R G. Studies in the transplantation of bone. V. The capacity of fresh and treated homografts of bone to evoke transplantation immunity.1963. [J]. Clin Orthop Relat Res,1999(367 Suppl):S5-S11, S2-S4.
[71]Wieslander J, Heinegard D. Immunochemical analysis of cartilage proteoglycans. Antigenic determinants of substructures.[J]. Biochem J,1979,179(1):35-45.
[72]Rydberg L, Breimer M E, Brynger H, et al. ABO-incompatible kidney transplantation (A2 to O). Qualitative and semiquantitative studies of the humoral immune response against different blood group A antigens.[J]. Transplantation,1990,49(5):954-960.
[73]Stassen J G, van Dijk B A, van Horn J R, et al. No irregular erythrocyte antibodies observed after bone allografts in 144 patients.[J]. Acta Orthop Scand, 1993,64(3):354-356.
[74]Muscolo D L, Kawai S, Ray R D. Cellular and humoral immune response analysis of bone-allografted rats.[J]. J Bone Joint Surg Am,1976,58(6): 826-832.
[75]Hasegawa A, Ohara T, Hirayama N, et al. Reversible anuria associated with glomerular fibrin thrombi in ABO-incompatible renal transplants.[J]. Transplant Proc,1995,27(1):1024-1027.
[76]Bos G D, Goldberg V M, Powell A E, et al. The effect of histocompatibility matching on canine frozen bone allografts.[J]. J Bone Joint Surg Am,1983,65(1): 89-96.
[77]Bos G D, Goldberg V M, Zika J M, et al. Immune responses of rats to frozen bone allografts.[J]. J Bone Joint Surg Am,1983,65(2):239-246.
[78]Elves M W. Newer knowledge of the immunology of bone and cartilage. [J]. Clin Orthop Relat Res,1976(120):232-259.
[79]Langer F, Czitrom A, Pritzker K P, et al. The immunogenicity of fresh and frozen allogeneic bone.[J]. J Bone Joint Surg Am,1975,57(2):216-220.
[80]Lee M Y, Finn H A, Lazda V A, et al. Bone allografts are immunogenic and may preclude subsequent organ transplants.[J]. Clin Orthop Relat Res,1997(340): 215-219.
[81]Horowitz M C, Friedlaender G E. Immunologic aspects of bone transplantation. A rationale for future studies.[J]. Orthop Clin North Am,1987,18(2):227-233.
[82]Quattlebaum J B, Mellonig J T, Hensel N F. Antigenicity of freeze-dried cortical bone allograft in human periodontal osseous defects.[J]. J Periodontol,1988, 59(6):394-397.
[83]Turner D W, Mellonig J T. Antigenicity of freeze-dried bone allograft in periodontal osseous defects.[J]. J Periodontal Res,1981,16(1):89-99.
[84]Goldberg V M, Bos G D, Heiple K G, et al. Improved acceptance of frozen bone allografts in genetically mismatched dogs by immunosuppression.[J]. J Bone Joint Surg Am,1984,66(6):937-950.
[85]Voggenreiter G, Assenmacher S, Kreuzfelder E, et al. Immunosuppression with FK506 increases bone induction in demineralized isogeneic and xenogeneic bone matrix in the rat.[J]. J Bone Miner Res,2000,15(9):1825-1834.
[86]Gabl M, Pechlaner S, Lutz M, et al. Bilateral hand transplantation:bone healing under immunosuppression with tacrolimus, mycophenolate mofetil, and prednisolone.[J]. J Hand Surg Am,2004,29(6):1020-1027.
[87]Vossen M, Edelstein J, Majzoub R K, et al. Bone quality and healing in a swine vascularized bone allotransplantation model using cyclosporine-based immunosuppression therapy.[J]. Plast Reconstr Surg,2005,115(2):529-538.
[88]IKeda K, Shigetomi M, Ihara K, et al. Effects of cessation of immunosuppression on skeleton reconstructed by vascularized bone allograft in rats.[J]. J Orthop Res, 2004,22(2):388-394.
[89]Bray P W, Neligan P C, Bowen C V, et al. Survival of microvascular physeal allograft transplants following withdrawal of short-term postoperative immunosuppression.[J]. J Bone Joint Surg Am,2004,86-A(2):281-289.
[2]den Boer F C, Wippermann B W, Blokhuis T J, et al. Healing of segmental bone defects with granular porous hydroxyapatite augmented with recombinant human osteogenic protein-1 or autologous bone marrow.[J]. J Orthop Res,2003,21(3): 521-528.
[3]W M. J Proc Roy Soc London [J]. J Proc Roy Soc London,1881,32:232-247.
[4]Mcauliffe J A. Bone graft substitutes.[J]. J Hand Ther,2003,16(2):180-187.
[5]Greenwald A S, Boden S D, Goldberg V M, et al. Bone-graft substitutes:facts, fictions, and applications.[J]. J Bone Joint Surg Am,2001,83-A Suppl 2 Pt 2: 98-103.
[6]邱贵兴孙世荃.同种异体骨植入材料的临床应用[J].中华骨科杂志,2004(10).
[7]彭吾训杨泽华王鑫.脱钙骨基质的临床应用与研究进展[J].昆明医学院学报,2003(04).
[8]Burchardt H. The biology of bone graft repair. [J]. Clin Orthop Relat Res, 1983(174):28-42.
[9]Nguyen H, Morgan D A, Forwood M R. Sterilization of allograft bone:is 25 kGy the gold standard for gamma irradiation?[J]. Cell Tissue Bank,2007,8(2): 81-91.
[10]Friedlaender G E. Bone allografts:the biological consequences of immunological events.[J]. J Bone Joint Surg Am,1991,73(8):1119-1122.
[11]Lance E M. Some observations on bone graft technology.[J]. Clin Orthop Relat Res,1985(200):114-124.
[12]Buckley S C, Stockley I, Hamer A J, et al. Irradiated allograft bone for acetabular revision surgery. Results at a mean of five years.[J]. J Bone Joint Surg Br,2005,87(3):310-313.
[13]Hamer A J, Strachan J R, Black M M, et al. Biochemical properties of cortical allograft bone using a new method of bone strength measurement. A comparison of fresh, fresh-frozen and irradiated bone.[J]. J Bone Joint Surg Br,1996,78(3): 363-368.
[14]Clatworthy M G, Ballance J, Brick G W, et al. The use of structural allograft for uncontained defects in revision total knee arthroplasty. A minimum five-year review.[J]. J Bone Joint Surg Am,2001,83-A(3):404-411.
[15]姚朗.细胞毒T淋巴细胞(CTL)活性测定方法进展[J].国外医学.临床生物化学与检验学分册,2002(02).
[16]Mattis A E, Bernhardt G, Lipp M, et al. Analyzing cytotoxic T lymphocyte activity:a simple and reliable flow cytometry-based assay.[J]. J Immunol Methods,1997,204(2):135-142.
[17]Derby E, Reddy V, Kopp W, et al. Three-color flow cytometric assay for the study of the mechanisms of cell-mediated cytotoxicity.[J]. Immunol Lett,2001, 78(1):35-39.
[18]Aubry J P, Blaecke A, Lecoanet-Henchoz S, et al. Annexin V used for measuring apoptosis in the early events of cellular cytotoxicity.[J]. Cytometry,1999,37(3): 197-204.
[19]Martin A, Morcillo N, Lemus D, et al. Multicenter study of MTT and resazurin assays for testing susceptibility to first-line anti-tuberculosis drugs.[J]. Int J Tuberc Lung Dis,2005,9(8):901-906.
[20]Hussain R F, Nouri A M, Oliver R T. A new approach for measurement of cytotoxicity using colorimetric assay.[J]. J Immunol Methods,1993,160(1): 89-96.
[21]盛大平徐元宏.alamarBlue在临床检验中的应用[J].国外医学.临床生物化学与检验学分册,2005(06).
[22]杨阳,刘宝瑞,钱晓萍.Alamar Blue法用于体外培养细胞活性检测的方法研究[J].现代肿瘤医学,2006(01).
[23]Xue M, Zhong M, Zhang Y, et al. [Effects of bovine plasma fibronectin on the proliferation of rat osteoblast in vitro][J]. Shanghai Kou Qiang Yi Xue,2003, 12(6):436-438.
[24]Nociari M M, Shalev A, Benias P, et al. A novel one-step, highly sensitive fluorometric assay to evaluate cell-mediated cytotoxicity.[J]. J Immunol Methods, 1998,213(2):157-167.
[25]Kwack K, Lynch R G. A new non-radioactive method for IL-2 bioassay.[J]. Mol Cells,2000,10(5):575-578.
[26].
[27]骨库技术与操作规范[J].中华骨科杂志,,24(10)：638-640.
[28]骨组织成分与Masson.三色染色反应的关系分析[J].中医正骨,,13(11)：5-6.
[29]黄长明王臻.骨形态发生蛋白在异体骨段移植愈合中的表达及临床意义[J].中国矫形外科杂志,2001(11).
[30]大段异体骨关节移植后长期转归的观察[J].中华外科杂志,,36(3)：169-171.
[31]Bertone A L, Pittman D D, Bouxsein M L, et al. Adenoviral-mediated transfer of human BMP-6 gene accelerates healing in a rabbit ulnar osteotomy model.[J]. J Orthop Res,2004,22(6):1261-1270.
[32]Mcgee M A, Findlay D M, Howie D W, et al. The use of OP-1 in femoral impaction grafting in a sheep model.[J]. J Orthop Res,2004,22(5):1008-1015.
[33]Tagil M, Jeppsson C, Wang J S, et al. No augmentation of morselized and impacted bone graft by OP-1 in a weight-bearing model.[J]. Acta Orthop Scand, 2003,74(6)：742-748.
[34]骨形态发生蛋白与骨缺损的治疗[J].实用骨科杂志,,8(4)：271-273.
[35]Yamaguchi A. [Recent advances in research on bone formation--BMP action and its mechanism][J]. Nippon Rinsho,2002,60 Suppl 3:40-47.
[36]Curtiss P J, Herndon C H. Immunological factors in homogenous-bone transplantation. I. Serological studies.[J]. J Bone Joint Surg Am,1956,38-A(1): 103-110.
[37]Bonfiglio M, Jeter W S, Smith C L. The immune concept:its relation to bone transplantation.[J]. Ann N Y Acad Sci,1955,59(3):417-433.
[38]Chalmers J. Transplantation immunity in bone homografting.[J]. J Bone Joint Surg Br,1959,41-B(1):160-179.
[39]Stevenson S, Horowitz M. The response to bone allografts.[J]. J Bone Joint Surg Am,1992,74(6):939-950.
[40]Kubler N, Reuther J, Kirchner T, et al. Osteoinductive, morphologic, and biomechanical properties of autolyzed, antigen-extracted, allogeneic human bone.[J]. J Oral Maxillofac Surg,1993,51(12):1346-1357.
[41]陆海波.复合环孢菌素同种异体骨的免疫学与组织学评价[J].中华创伤骨科杂志,2007,9(5)：442-447.
[42]Doi K, Akino T, Shigetomi M, et al. Revascularized intercalary bone allografts with short-term immunosuppression with cyclosporine in the canine.[J]. Plast Reconstr Surg,1998,101(3):793-801.
[43]Shigetomi M, Doi K, Kuwata N, et al. Experimental study on vascularized bone allografts for reconstruction of massive bone defects.[J]. Microsurgery,1994, 15(9):663-670.
[44]常青李守先巩性军张希全.环孢素A防治同种异体带瓣大血管移植后钙化的研究[J].山东大学学报(医学版),2005(08).
[45]环孢素A对脱钙骨基质植入物钙离子的影响[J].实用全科医学,,2(2)：97-98.
[46]徐忠烨唐文渊.环孢素A的神经保护作用[J].国外医学.神经病学神经外科学分册,2004(06).
[47]Yeo H, Beck L H, Mcdonald J M, et al. Cyclosporin A elicits dose-dependent biphasic effects on osteoblast differentiation and bone formation.[J]. Bone,2007, 40(6):1502-1516.
[48]Vm F G G. Bone and cartilage Allografts. Biology and Clinical Applications. Park Ridge, Illinois, The American Academy of Orthopaedic Surgeons[Z]. 1991.
[49]W M. J Proc Roy Soc London[Z].1881:32,232-247.
[50]Inskip H B V. Methods of bone transplantation[M].1983.445-466.
[51]E L. J Surg Gynecol Obster[J]. J Surg Gynecol Obster,1925,40:782-809.
[52]康焱廖威明.同种异体骨移植的研究和应用现状(文献综述)[J].国外医学.外科学分册,2004(04).
[53]Langlais F, Lambotte J C, Thomazeau H. Long-term results of hemipelvis reconstruction with allografts.[J]. Clin Orthop Relat Res,2001(388):178-186.
[54]黄长明福建厦门王臻童星杰福建厦门刘继中.大段异体骨移植治疗骨肿瘤[J].中华骨科杂志,2000(07).
[55]Clatworthy M G, Ballance J, Brick G W, et al. The use of structural allograft for uncontained defects in revision total knee arthroplasty. A minimum five-year review.[J]. J Bone Joint Surg Am,2001,83-A(3):404-411.
[56]姚长海侯树勋衷鸿宾李利章亚东.同种异体骨移植在骨肿瘤手术中的应用[J].中国矫形外科杂志,2003(21).
[57]Min K, Espinosa N, Bode B, et al. Total sacrectomy and reconstruction with structural allografts for neurofibrosarcoma of the sacrum. A case report.[J]. J Bone Joint Surg Am,2005,87(4):864-869.
[58]Myerson M S, Neufeld S K, Uribe J. Fresh-frozen structural allografts in the foot and ankle.[J]. J Bone Joint Surg Am,2005,87(1):113-120.
[59]Hartl A, Bitzan P, Wanivenhaus A, et al. Faster integration of human allograft bone than of the bovine substitute Lubboc:non-randomized evaluation of 20 cases with benign tumors or tumor-like conditions.[J]. Acta Orthop Scand,2004, 75(2):217-220.
[60]Giavaresi G, Torricelli P, Fornasari P M, et al. Blood vessel formation after soft-tissue implantation of hyaluronan-based hydrogel supplemented with copper ions.[J]. Biomaterials,2005,26(16):3001-3008.
[61]Manfrini M, Innocenti M, Ceruso M, et al. Original biological reconstruction of the hip in a 4-year-old girl.[J]. Lancet,2003,361(9352):140-142.
[62]王臻西安黄耀添西安胡蕴玉西安.人工关节假体复合大段同种异体骨移植重建肢体功能[J].中华外科杂志,1999(12).
[63]Anract P, Coste J, Vastel L, et al. [Proximal femoral reconstruction with megaprosthesis versus allograft prosthesis composite. A comparative study of functional results, complications and longevity in 41 cases][J]. Rev Chir Orthop Reparatrice Appar Mot,2000,86(3):278-288.
[64]周建生胡汝麒潘功平刘振华肖玉周.冷冻保存带血管异体骨移植治疗儿童骨缺损[J].中华显微外科杂志,1998(03).
[65]Chang D W, Weber K L. Segmental femur reconstruction using an intercalary allograft with an intramedullary vascularized fibula bone flap.[J]. J Reconstr Microsurg,2004,20(3):195-199.
[66]Bertone A L, Pittman D D, Bouxsein M L, et al. Adenoviral-mediated transfer of human BMP-6 gene accelerates healing in a rabbit ulnar osteotomy model.[J]. J Orthop Res,2004,22(6):1261-1270.
[67]Mcgee M A, Findlay D M, Howie D W, et al. The use of OP-1 in femoral impaction grafting in a sheep model.[J]. J Orthop Res,2004,22(5):1008-1015.
[68]Tagil M, Jeppsson C, Wang J S, et al. No augmentation of morselized and impacted bone graft by OP-1 in a weight-bearing model.[J]. Acta Orthop Scand, 2003,74(6):742-748.
[69]张贝利张伟滨邓廉夫.同种异体骨移植研究进展[J].国外医学.骨科学分册,2005(03).
[70]Burwell R G. Studies in the transplantation of bone. V. The capacity of fresh and treated homografts of bone to evoke transplantation immunity.1963. [J]. Clin Orthop Relat Res,1999(367 Suppl):S5-S11, S2-S4.
[71]Wieslander J, Heinegard D. Immunochemical analysis of cartilage proteoglycans. Antigenic determinants of substructures.[J]. Biochem J,1979,179(1):35-45.
[72]Rydberg L, Breimer M E, Brynger H, et al. ABO-incompatible kidney transplantation (A2 to O). Qualitative and semiquantitative studies of the humoral immune response against different blood group A antigens.[J]. Transplantation,1990,49(5):954-960.
[73]Stassen J G, van Dijk B A, van Horn J R, et al. No irregular erythrocyte antibodies observed after bone allografts in 144 patients.[J]. Acta Orthop Scand, 1993,64(3):354-356.
[74]Muscolo D L, Kawai S, Ray R D. Cellular and humoral immune response analysis of bone-allografted rats.[J]. J Bone Joint Surg Am,1976,58(6): 826-832.
[75]Hasegawa A, Ohara T, Hirayama N, et al. Reversible anuria associated with glomerular fibrin thrombi in ABO-incompatible renal transplants.[J]. Transplant Proc,1995,27(1):1024-1027.
[76]Bos G D, Goldberg V M, Powell A E, et al. The effect of histocompatibility matching on canine frozen bone allografts.[J]. J Bone Joint Surg Am,1983,65(1): 89-96.
[77]Bos G D, Goldberg V M, Zika J M, et al. Immune responses of rats to frozen bone allografts.[J]. J Bone Joint Surg Am,1983,65(2):239-246.
[78]Elves M W. Newer knowledge of the immunology of bone and cartilage. [J]. Clin Orthop Relat Res,1976(120):232-259.
[79]Langer F, Czitrom A, Pritzker K P, et al. The immunogenicity of fresh and frozen allogeneic bone.[J]. J Bone Joint Surg Am,1975,57(2):216-220.
[80]Lee M Y, Finn H A, Lazda V A, et al. Bone allografts are immunogenic and may preclude subsequent organ transplants.[J]. Clin Orthop Relat Res,1997(340): 215-219.
[81]Horowitz M C, Friedlaender G E. Immunologic aspects of bone transplantation. A rationale for future studies.[J]. Orthop Clin North Am,1987,18(2):227-233.
[82]Quattlebaum J B, Mellonig J T, Hensel N F. Antigenicity of freeze-dried cortical bone allograft in human periodontal osseous defects.[J]. J Periodontol,1988, 59(6):394-397.
[83]Turner D W, Mellonig J T. Antigenicity of freeze-dried bone allograft in periodontal osseous defects.[J]. J Periodontal Res,1981,16(1):89-99.
[84]Goldberg V M, Bos G D, Heiple K G, et al. Improved acceptance of frozen bone allografts in genetically mismatched dogs by immunosuppression.[J]. J Bone Joint Surg Am,1984,66(6):937-950.
[85]Voggenreiter G, Assenmacher S, Kreuzfelder E, et al. Immunosuppression with FK506 increases bone induction in demineralized isogeneic and xenogeneic bone matrix in the rat.[J]. J Bone Miner Res,2000,15(9):1825-1834.
[86]Gabl M, Pechlaner S, Lutz M, et al. Bilateral hand transplantation:bone healing under immunosuppression with tacrolimus, mycophenolate mofetil, and prednisolone.[J]. J Hand Surg Am,2004,29(6):1020-1027.
[87]Vossen M, Edelstein J, Majzoub R K, et al. Bone quality and healing in a swine vascularized bone allotransplantation model using cyclosporine-based immunosuppression therapy.[J]. Plast Reconstr Surg,2005,115(2):529-538.
[88]IKeda K, Shigetomi M, Ihara K, et al. Effects of cessation of immunosuppression on skeleton reconstructed by vascularized bone allograft in rats.[J]. J Orthop Res, 2004,22(2):388-394.
[89]Bray P W, Neligan P C, Bowen C V, et al. Survival of microvascular physeal allograft transplants following withdrawal of short-term postoperative immunosuppression.[J]. J Bone Joint Surg Am,2004,86-A(2):281-289.