异种骨移植材料的制备与实验研究
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摘要
第一章异种骨移植材料的制备与生物学特性研究
目的:异种骨移植可以解决临床上自体骨与同种骨来源不足的问题,目前常用的处理方法虽可有效降低其免疫原性,但破坏了骨诱导活性与生物力学性能。α-半乳糖抗原(α-Gal)是主要的异种抗原,本研究拟采用α-半乳糖苷酶消化猪骨组织,旨在探索一种既能去除其抗原性,又保留其力学性能与成骨活性的异种骨材料处理方法。
方法:(1)猪骨组织的酶消化:取市售新鲜猪骨,清洗去除血液与骨髓组织,酒精脱脂,分别采用酶活性为15U/ml、30U/ml、60U/ml的α-半乳糖苷酶37℃下消化8、16h,彻底清洗;(2)骨组织中α-Gal抗原相对含量的检测:采用ELISA法检测各组酶消化猪骨组织中α-Gal相对含量:猪骨组织粉碎,称取25mg,加抗α-Gal抗原的M86抗体200μl,37℃下孵育1h,充分洗3次后加辣根过氧化物酶标记的羊抗鼠IgM抗体200μl,37℃下孵育1h,洗5次,加底物液孵育,终止反应,测490nm处OD值;(3)猪骨与戊二醛的交联:根据ELISA法检测结果筛选出一组较为合理的酶消化条件,将该组猪骨经戊二醛交联处理,检测交联后猪骨α-Gal抗原相对含量,并与未经酶消化的新鲜猪骨、酶消化猪骨比较,根据结果确定制备方法;(4)猪骨组织的形态学观察:对制备的猪骨进行大体观察、扫描电镜观察,用Smile View软件测量其腔隙的直径;(5)猪骨的力学性能检测:猪松质骨制备成10mm×10mm×20mm规格骨块,按优化筛选的方法酶消化,用电子万能材料试验机检测其抗压强度与弹性模量,以观察酶消化对异种骨材料力学性能的影响。
采用SPSS11.5统计软件进行统计分析,酶消化猪骨组织α-Gal抗原相对含量采用析因设计的方差分析,不同骨组织中α-Gal相对含量、猪骨抗压性能实验采用多样本均数单向方差分析(One-way ANOVA),组间多重比较方差齐性时采用SNK法,方差不齐时采用Dunnet's T3法,检验水准为α=0.05。
结果:(1)猪骨组织的酶消化:不同活性的α-半乳糖苷酶消化不同时间的猪骨组织中α-gal抗原含量不同,酶消化一定时间后OD值趋于稳定,优化筛选酶消化条件为酶活性30 U/ml的α-半乳糖苷酶消化8h;(2)猪骨与戊二醛的交联:30U/ml消化8h组与交联组的α-Gal抗原相对含量差异不显著,而与新鲜猪骨有显著性差异(P＜0.05)。(3)猪骨的形态学观察:扫描电镜观察表明制备的猪骨为呈三维网状多孔结构,其腔隙大小在150μm-600μm之间,骨小梁上分布有孔径在10μm-100μm的小孔;(4)力学性能检测:各试验组材料的抗压强度、弹性模量均没有统计学上的差异,酶消化对力学性能的无显著影响。
结论:优化筛选较为合理的猪源性异种骨的酶消化方法为酶活性30U/ml的α-半乳糖苷酶37℃下消化8h,制备方法有效去除了α-Gal抗原,对猪骨材料的形态学结构与力学性能无明显影响。
第二章异种骨移植材料的生物学评价
目的:采用体外细胞毒性实验与动物体内实验方法,评价所制备的猪骨材料的生物相容性。
方法:(1)兔骨髓间充质干细胞(BMSCs)培养:取乳兔四肢骨骨髓,原代培养8-10天后传代培养,用台盼蓝染色方法判定细胞活力,取F1、2、3代细胞,流式细胞仪检测表面抗原CD44阳性率。(2)体外细胞毒性试验:采用MTT法检测猪源性异种骨对BMSCs生长增殖的影响。经酶消化的5mm×5mm×5mm猪源性异种骨小块,参照中华人民共和国国家标准《医疗器械生物学评价》,按1g猪骨:10ml浸提液的比例,无菌密闭容器内10%胎牛血清(FBS)L-DMEM培养液37℃浸取72h,取上清液加作为实验组培养基,以含10%FBS的L-DMEM培养液为对照。以F3代BMSCs为实验细胞,制成细胞浓度约1×10~5/ml的细胞悬液,5%CO_2培养箱37℃培养24h,按组别分别以异种骨浸提液与对照培养液培养,每隔24h取5孔,MTT法检测吸光值。(3)猪骨与兔BMSCs的复合培养:将F3代兔BMSCs消化成浓度为1×10~5/ml左右的细胞悬液,接种子5mm×5mm×5mm的经酶消化的猪骨小块上,加10%FBS的L-DMEM培养基至液面没过骨块,置于5%CO_2培养箱37℃培养,分别于培养的1w,2w,3w,倒置相差显微镜下观察材料周围细胞生长情况,取材后2.5%戊二醛固定3h,脱水,干燥,喷金,扫描电镜下观察细胞的形态与生长情况。(4)动物体内植入局部刺激反应试验:参照国家标准GB/T 16886.6-1997进行异种骨植入材料局部反应评价。Wistar鼠18只,体重150g左右,麻醉,于脊柱两侧皮下及股部肌袋内分别植入规格为5mm×5mm×5mm的猪骨小块与人骨小块。术后大体观察动物饮食、活动、切口反应等情况。分别于术后1、4、12周处死6只大鼠,取出植入块及其周围包裹的组织,行大体观察、组织学观察。
采用SPSS11.5统计软件进行统计分析,体外细胞毒性实验BMSCs的OD值采用析因设计的方差分析,同一时间点两组数据比较采用独立样本t检验,检验水准为α=0.05。
结果:(1)BMSCs的培养:传代培养的BMSCs形态均一,生长旺盛,F1-F3代细胞成活率均在95%左右,F1代、F2代、F3代细胞CD44阳性率分别为93.9%、95.1%、95.8%。(2)体外细胞毒性试验:相同时间实验组与对照组间OD值无显著性差异(F=0.070,P=0.7910),猪骨材料对BMSCs的生长与增殖无明显影响,表明其无细胞毒性。(3)猪骨与BMSCs复合培养:随着培养时间延长,材料周围细胞逐渐聚集,细胞状态正常;扫描电镜下观察,细胞在猪骨材料表面紧密贴附生长,细胞呈多边形、梭形,有较多的伪足向猪骨材料表面伸出,细胞形态正常,功能活跃,表明该材料细胞相容性良好。(4)动物体内植入后的局部刺激反应试验:皮下与肌内植入后1w,纤维组织包裹并长入植入的猪骨腔隙内,伴有较明显的炎性细胞浸润,4w时,炎症反应减轻,12w时,植入的猪骨腔隙内由疏松的结缔组织充填,炎症反应消失,与周围正常组织中的无明显区别,表明猪骨材料具有良好的组织相容性。
结论:酶消化法所制备的猪源性异种骨移植材料具有良好的生物相容性。
第三章异种骨移植材料的骨诱导活性研究
目的:采用免疫抑制大鼠为实验动物,以异位成骨的方式,通过组织学观察、碱性磷酸酶活性测定,评价所制备异种骨的骨诱导活性。
方法:实验组材料为α-半乳糖苷酶消化去抗原的猪皮质骨,0.6N的HCl脱矿,以脱矿后经高温高压处理灭活其成骨活性物质的猪骨为阴性对照。Wistar大鼠24只,体重约150-180g,腹腔注射地塞米松,剂量为每次0.25mg/100g体重,每3天一次,造成免疫抑制。大鼠麻醉后,两侧股部肌袋分别植入实验组材料和对照样品,缝合肌肉皮肤。术后3、4、5、6w各处死6只大鼠,取出植入材料,固定,包埋,行组织学观察;将植入物称重,研磨粉碎,加2ml生理盐水提取,3000rpm离心20min,取上清液,测碱性磷酸酶(ALP)活性。
用SPSS11.5统计软件对ALP活性检测结果进行统计分析,采用析因设计资料的方差分析,同一时间点两组数据比较采用独立样本t检验,检验水准为α=0.05。
结果:组织学观察显示,实验组植入材料3w时被纤维组织包裹,其吸收腔内可见有间充质细胞开始长入;可见有间充质细胞转变形成软骨细胞,并开始分泌形成软骨基质;4w时植入材料内有较多软骨组织形成;5w时形成成熟的软软骨组织;6w时,成骨更为活跃,植入材料内有更多的成熟软骨组织形成,在其边缘吸收腔隙内有类骨质形成;术后3-6w,对照组样品显示吸收,但未见成骨相关的细胞,也无软骨或骨组织形成。ALP活性测定结果显示,不同时间点的ALP活性差异显著(F=321.084,P=0.000),实验组与对照组间差异显著(F=484.330,P=0.000),同一取材时间点实验组ALP活性均比对照组高(P＜0.05)。
结论:采用α-半乳糖苷酶消化去抗原的猪源性异种骨保留了良好的骨诱导活性,可以作为一种潜在的骨移植替代材料,来弥补临床上自体骨与同种骨材料来源的不足。
第四章异种骨修复兔桡骨节段性骨缺损的实验研究
目的:利用兔桡骨节段性骨缺损模型,观察与评价所制备猪源性异种骨移植材料对兔节段性骨缺损的修复,为临床应用提供实验依据。
方法:家兔24只,体重2-2.5 kg,以双侧桡骨为实验对象。家兔静脉注射3%戊巴比妥钠麻醉,用牙科钻截断桡骨干中段,造成双侧桡骨约12mm长的节段性骨缺损,按组植入相应骨移植材料。分别于第4、8、12周分批处死动物8只,取尺桡骨全段。实验分组:根据植入材料不同分组,每组每个时间点4例,A组为空白对照组,节段性骨缺损处不植入任何材料,直接缝合,作为阴性对照;B组为异种骨组,植入酶处理方法制备的去抗原猪骨;C组为兔同种骨组,植入兔松质骨;D组为自体骨组,将取下的兔桡骨骨段用无菌生理盐水清洗后,植于其对侧骨缺损处。大体观察:观察植入材料与宿主骨融合、局部成骨和周围软组织分布、炎症反应等情况;X线检查:拍桡骨正位X线片观察,并进行评分。组织学观察:标本用10%福尔马林缓冲液固定,50%甲酸脱钙,逐级脱水,透明,石蜡包埋,切片,HE染色,组织学观察。
数据结果采用SPSS11.5统计软件包进行统计分析,X线评分采用析因设计资料的方差分析,方差齐性时组间多重比较采用LSD法,不满足方差齐性要求时,组间多重比较采用Dunnet's T3法,统计学显著水平为α=0.05。
结果:大体观察:术后各组动物前肢均有不同程度肿胀,1w恢复正常活动。所有动物切口均Ⅰ期愈合,状态良好,未见明显并发症。X线观察:术后4w,空白对照组骨缺损清晰可见,缺损处两侧宿主骨断端阴影锐利,有微量骨痂形成;异种骨组骨缺损区呈现雾状影,宿主骨断端有少量低密度骨痂形成;兔同种骨组截骨端有少量骨痂形成,缺损区呈雾状阴影;自体骨组植入骨密度无明显变化,截骨线清晰稍显模糊,有少量骨痂形成。术后8w,空白对照组骨缺损仍清晰可见,缺损处截骨端有少量骨痂形成,尺骨侧可见有少量骨痂形成;异种骨组植入骨与宿主骨桥接处稍显模糊,缺损区雾状影填充;兔同种骨组截骨端与缺损区尺骨表面形成骨痂,缺损区密度比骨干低;自体骨组宿主骨骨干周围形成较多的骨痂,与植入骨连接,桥接处呈现高密度影,界线模糊。术后12w,空白对照组骨缺损仍清晰可见,缺损区变小,骨不愈合。异种骨组植入骨与宿主骨界线模糊,密度较骨干低;兔同种骨组植入骨与宿主骨形成连接,连接处界线模糊,骨缺损区密度较均匀;自体骨组植入骨与宿主骨形成连续性连接,骨髓腔再通。X线评分:术后不同取材时间,异种骨组与兔同种骨组之间骨缺损修复无显著差异(4w、8w、12w的P值分别为0.574,1.000,0.805),而与空白对照组与自体骨组之间分别存在着显著差异(P＜0.05);各组X线评分都随着时间的延长而升高,不同时间的X线评分有显著性差异(F=75.077,P=0.000)。组织学观察:空白对照组骨缺损区由纤维组织填充,成骨不活跃;异种骨组截骨端新生的骨组织穿插长入植骨材料的腔隙,植入骨显示吸收,逐渐有新骨形成,12w时可见有软骨细胞与软骨组织连接植入骨与宿主骨;兔同种骨组植入材料显示吸收,截骨端逐渐有新生骨穿插长入植入骨,12w时植入骨上可见有新生类骨质开始形成;自体骨组成骨活跃,植入骨逐渐被新生骨组织取代,至12w时骨髓腔再通。
结论:α-半乳糖苷酶消化的猪源性异种骨用于修复兔桡骨节段性骨缺损时,取得了与兔同种骨一致的修复效果,本方法所制备的异种骨可作为一种骨移植替代材料。
Chapter 1:Preparation of Porcine Bone and Experimental Study on its Biological Properties
Objective:To develop a feasible method to prepare deantigenated porcine bone through enzyme treatment,which has no effect on the osteoinductivity and mechanic properties of the xenogeneic bone graft produced thereby.
Methods:(1)Enzyme treatment of porcine bone:Xenogeneic bone was prepared from flesh porcine bone by a series treatment includingα-Galactosidase of different activity digesting for 8 or 16 hours at 37℃,washing,freezedrying and irradiation at a does of 25kGy.(2)Determination of theα-Gal(α-gal epitopes):The concentration ofα-Gal within porcine bone tissue were tested by ELISA in order to choose a suitable processing way for deantigenation.(3)Crosslinking of glutaraldehyde with the poricine bone:Crosslinking of glutaraldehyde with the poricine bone prepared by the selected group was performed and concentration ofα-Gal was defined and compared with that of fresh porcine bone,enzyme treated porcine bone and human bone.The processing programe then was decided according to the testing result.(3) Morphological observation:Gross observation and SEM observation were performed and diameters of the caves of the porcine cancellous bone were measured.(4)Mechanic properties testing:The compressive properties of porcine bone at different stage of processing were tested to evaluate the effects of different treatment on its mechanic property.
Sstatistical analysis was performed with SPSS11.5 software package and the data were presented with(Mean±Standard Deviation).The data were tested with variance analysis,being significant when P＜0.05.
Results:(1) Enzyme treatment of porcine bone:The OD value of different groups in ELISA test were significantly different.The selected enzyme treatment was digesting byα-Galactosidase with an activity of 30U/ml for 8 hours at 37℃.(2) Crosslinking of glutaraldehyde with the poricine bone:No statistic significance was found among the OD values of cross-linked porcine bone and human bone,while there were significant difference between fresh porcine bone and other groups. According to the results,the finally processing methods wasα-Galactosidase of a 30U/ml activity digesting for 8 hours.(3) Morphological observation:The porous structure of prepared porcine cancellous bone was similar to that of human cancellous bone.The diameters of the caves were between 150μm -600μm,while the diameters of small pore in the trabecula were between 10μm-100μm.(4) Mechanic testing of porcine bone:Strength and elastic module of different groups had no statistical difference (P＜0.05) for the compressive test,indicating that different processing methods had no significant effect on the mechanical property of the porcine bone.
Conclusion:The antigen of porcine bone was removed effectively byα-Galactosidasse digesting,while the morphological structure and mechanic properties of the porcine bone remained
Chapter 2 Biological Evaluation of the Prepared Porcine Bone Graft
Objective:To evaluate the biocompatibility of the prepared porcine bone by in vitro cytotoxity experiment and in vivo animal experiment.
Methods:(1) Culture of rabbit BMSCs:Rabbit BMSCs were obtained as the experimental cells and the activity and content of CD44 surface antigen of the BMSCs were tested.(2)In vitro cytotoxity experiment:The leaching liquor of porcine bone was made by immersing the bone into L-DMEM medium with a scale of 10ml medium per gram bone materials at 37℃for 72 hours in a sterilized condition and L-DMEM medium which contained 10%FBS served as the control medium.200μl of BMSCs suspension with a cell concentration of 1×10~5/ml were seeded to the and cultured for 24 hours.Then the culture medium was removed and 200μl leaching liquor or control culturing medium were added according to groups respectively. Then cells were cultured at 37℃and 5%CO_2.The medium were exchanged every 3 days according to groups.Every 24 hours,5 wells of each group were selected and 20μl 5mg/ml MTT solution was added to each well.150μl DMSO was added after cultured for 4 hours.The 96-well plate was slightly shaken for about 10 minutes and the OD value at 490 nm was determined.(3)Co-culturing of the porcine bone and rabbit BMSCs:The prepared porcine bone,sized 5mm×5mm×5mm,were placed in the 12-well plate and 150μl BMSCs suspension of a concentration of 1×10~5/ml was seeded to the surface of the porcine bone and 10%FBS L-DMEM was added to immerse the porcine bone.Inverted phase contrast microscope observation and SEM observation were performed at 1,2,3 weeks.(4)Biological evaluation in vivo:18 Wistar mice,weighted 150-180g,were anesthetized.Subcutaneous and intramuscular implantation of prepared porcine bone and human bone pieces was performed.6 of the mice were killed each time respectively at 1,4,12 weeks and gross observation and histological observation were performed.
Results:(1) Culture of rabbit BMSCs:The cultured BMSCs were in a good condition.About 95%of F1 to F3 generation cells was active.93.9%of F1 generation,95.1%of F2 generation and 95.8%of F3 generation cells contained CD44 surface antigen.(2) In vitro cytotoxity experiment:The OD values of study group and control group at different time had no significant difference.(3) Co-culturing of porcine bone and BMSCs:Postoperatively 1-3 weeks,Polygon shaped and fusiform shaped BMSCs with a well condition adhered tightly to the surface of the porcine bone.Lots of pseudopodia from BMSCs reached the surface of the porcine bone.
Conclusion:The enzyme degested porcine bone had no obviously effect on the growth of BMSCs and showed good cell affinity when co-cultured with BMSCs. The local implantation also showed the prepared porcine bone contains good tissue compatibility.The porcine bone contained good biocompatibility and could be safe for clinic use.
Chapter 3 Study on the Osteoinductivity of the Prepared Porcine Bone Graft
Objective:To evaluate the osteoinductivity of the prepared porcine graft by histological observation and ALP activity determination in an ectopic bone formation model,using immuno-suppressed Wistar rats as experimental animal.
Methods:Samples of study group were enzyme-treated porcine bone, demineralized with 0.6N hydrochloric acid.The control group samples were the demineralized porcine bone,which were treated with autoclave for 30 minutes to destroy the activity of bone formation material.All samples were freezedried and irradiated at a does of 20kGy.Dexamethasone was intraperitoneally injected into Wistar rats in order to suppress their imunno-response.Intramuscular bone transplantation was performed by placing the demineralized porcine bone and control samples between bluntly dissected muscle groups of hind limbs of the rats. Histological observation was performed at different time post-operatively.ALP activity was tested postoperatively to determine the osteoinductivity of the demineralized porcine bone implants.
Result:The study group:Mesenchymal cells migrated into the absorpted lacuna and chondrocytes could be seen within the porcine bone implants at 3 weeks postoperatively;more cartilage presented at 4 weeks;adult cartilage was formed within the implants at 5 weeks;at 6 weeks,bone formation was more active and lots of cartilage within the implants and osteoid in the absorbed lacuna could be seen.No bone formation could be found in the control group 3-6 weeks postoperatively.ALP activity of the experimental group was obviously higher than that of the control group 3-6 weeks postoperatively.
Conclusion:The prepared porcine bone contained good osteoinductivity and could be a potential bone substitute for clinic implantation.
Chapter 4 Experimental Study of the Prepared Porcine Bone for Bone Reparation in a Radial Segmental Defect of Rabbit
Objective:To investigate the reparation of segmental bone defect by the prepared porcine bone and provide experimental reference for potential clinic use.
Methods 24 rabbits,weighted 2-2.5kg,were anesthetized and a 12mm-long bone defect was made by cutting the middle shaft of both side radii.Grafts were implanted according to groups:(A)Control group:no implantation was performed to the defect;(B) Xenogeneic bone group:the prepared porcine bone were implanted;(C) Allogeneic bone group:rabbit allogeneic cancellous bone were implanted;(D) Autograft group:the cut bone were implanted to the opposite side radius.8 animals were sacrificed(4 graft for each group were obtained) for each time respectively at 4, 8,12 weeks postoperatively.Generally and X-ray,histological observation were performed to evaluate the reparation of the segmental defect..
Results:Gross observation:All the animals were in good status and no detectable complications were found.X-ray observation:Postoperatively 4 weeks, little callus was formed and the defect was clear in the control group;bone defect was filled with shadow with a lower density than that of the rabbit radius and ulna,small quantity of callus was formed in the xenogeneic bone group;allogeneic bone group was similar to the porcine bone group;a little callus was formed in auto graft group. 8 weeks post-operationally,bone defect was clear and small quantity of callus was formed in control group;bone defect was filled with a lower density shadow and the bridge area was not very sharp in xenogeneic bone group;callus was formed and high-density shadow could be seen along the ulna in allogeneic bone group;the auto graft and the rabbit radius was connected with blurry shadow.12 weeks postoperatively,callus was formed in the end of the radius and the marrow cavity was blocked and bone non-union was formed in the control group;the defect was filled with low-density shadow and the bridge area was blurry in porcine bone group and allogeneic bone group;recanalization of the marrow cavity was formed between the graft and the radius in auto graft group.Score of X-ray of xenogeneic bone group ahc no significant difference while the difference of the xenogeneic bone group with control group or autograft group are both significant.Histological observation:the defect of control group was filled with connective tissue without active bone formation.postoperatively 4 weeks,bone defect of control group was filled with fibrous tissue which contained lots of lymphocytes;bone defect was filled with connective tissue and lots of lymphocytes could be seen in porcine bone group and rabbit allogeneic bone group;chondrocytes and osteoid grown between the radius and the auto graft in auto graft group.Postoperatively 8 weeks,the defect of control group was filled with fibrous tissue which contained lymphocytes;bone absorption and cement line could be seen in the xenogeneic bone group and rabbit allogeneic bone group;the autograft was connected with the rabbit radius shaft with active bone formation and absoption within the graft.Postoperatively 12 weeks,the defect of control group were filled with connective tissue;small quantity of new bone formation could be seen in xenogeneic bone group and rabbit allogeneic bone group; the autograft was mainly substituted by newly formed bone with active bone formation.
Conclusion The prepared porcine xenogeneic bone cobtained a cure effect similar to that of rabbit allogeneic bone in a rabbit radial segmental defect model, hence it may be a potential good bone substitute for bone implantation.
目的:异种骨移植可以解决临床上自体骨与同种骨来源不足的问题,目前常用的处理方法虽可有效降低其免疫原性,但破坏了骨诱导活性与生物力学性能。α-半乳糖抗原(α-Gal)是主要的异种抗原,本研究拟采用α-半乳糖苷酶消化猪骨组织,旨在探索一种既能去除其抗原性,又保留其力学性能与成骨活性的异种骨材料处理方法。
方法:(1)猪骨组织的酶消化:取市售新鲜猪骨,清洗去除血液与骨髓组织,酒精脱脂,分别采用酶活性为15U/ml、30U/ml、60U/ml的α-半乳糖苷酶37℃下消化8、16h,彻底清洗;(2)骨组织中α-Gal抗原相对含量的检测:采用ELISA法检测各组酶消化猪骨组织中α-Gal相对含量:猪骨组织粉碎,称取25mg,加抗α-Gal抗原的M86抗体200μl,37℃下孵育1h,充分洗3次后加辣根过氧化物酶标记的羊抗鼠IgM抗体200μl,37℃下孵育1h,洗5次,加底物液孵育,终止反应,测490nm处OD值;(3)猪骨与戊二醛的交联:根据ELISA法检测结果筛选出一组较为合理的酶消化条件,将该组猪骨经戊二醛交联处理,检测交联后猪骨α-Gal抗原相对含量,并与未经酶消化的新鲜猪骨、酶消化猪骨比较,根据结果确定制备方法;(4)猪骨组织的形态学观察:对制备的猪骨进行大体观察、扫描电镜观察,用Smile View软件测量其腔隙的直径;(5)猪骨的力学性能检测:猪松质骨制备成10mm×10mm×20mm规格骨块,按优化筛选的方法酶消化,用电子万能材料试验机检测其抗压强度与弹性模量,以观察酶消化对异种骨材料力学性能的影响。
采用SPSS11.5统计软件进行统计分析,酶消化猪骨组织α-Gal抗原相对含量采用析因设计的方差分析,不同骨组织中α-Gal相对含量、猪骨抗压性能实验采用多样本均数单向方差分析(One-way ANOVA),组间多重比较方差齐性时采用SNK法,方差不齐时采用Dunnet's T3法,检验水准为α=0.05。
结果:(1)猪骨组织的酶消化:不同活性的α-半乳糖苷酶消化不同时间的猪骨组织中α-gal抗原含量不同,酶消化一定时间后OD值趋于稳定,优化筛选酶消化条件为酶活性30 U/ml的α-半乳糖苷酶消化8h;(2)猪骨与戊二醛的交联:30U/ml消化8h组与交联组的α-Gal抗原相对含量差异不显著,而与新鲜猪骨有显著性差异(P＜0.05)。(3)猪骨的形态学观察:扫描电镜观察表明制备的猪骨为呈三维网状多孔结构,其腔隙大小在150μm-600μm之间,骨小梁上分布有孔径在10μm-100μm的小孔;(4)力学性能检测:各试验组材料的抗压强度、弹性模量均没有统计学上的差异,酶消化对力学性能的无显著影响。
结论:优化筛选较为合理的猪源性异种骨的酶消化方法为酶活性30U/ml的α-半乳糖苷酶37℃下消化8h,制备方法有效去除了α-Gal抗原,对猪骨材料的形态学结构与力学性能无明显影响。
第二章异种骨移植材料的生物学评价
目的:采用体外细胞毒性实验与动物体内实验方法,评价所制备的猪骨材料的生物相容性。
方法:(1)兔骨髓间充质干细胞(BMSCs)培养:取乳兔四肢骨骨髓,原代培养8-10天后传代培养,用台盼蓝染色方法判定细胞活力,取F1、2、3代细胞,流式细胞仪检测表面抗原CD44阳性率。(2)体外细胞毒性试验:采用MTT法检测猪源性异种骨对BMSCs生长增殖的影响。经酶消化的5mm×5mm×5mm猪源性异种骨小块,参照中华人民共和国国家标准《医疗器械生物学评价》,按1g猪骨:10ml浸提液的比例,无菌密闭容器内10%胎牛血清(FBS)L-DMEM培养液37℃浸取72h,取上清液加作为实验组培养基,以含10%FBS的L-DMEM培养液为对照。以F3代BMSCs为实验细胞,制成细胞浓度约1×10~5/ml的细胞悬液,5%CO_2培养箱37℃培养24h,按组别分别以异种骨浸提液与对照培养液培养,每隔24h取5孔,MTT法检测吸光值。(3)猪骨与兔BMSCs的复合培养:将F3代兔BMSCs消化成浓度为1×10~5/ml左右的细胞悬液,接种子5mm×5mm×5mm的经酶消化的猪骨小块上,加10%FBS的L-DMEM培养基至液面没过骨块,置于5%CO_2培养箱37℃培养,分别于培养的1w,2w,3w,倒置相差显微镜下观察材料周围细胞生长情况,取材后2.5%戊二醛固定3h,脱水,干燥,喷金,扫描电镜下观察细胞的形态与生长情况。(4)动物体内植入局部刺激反应试验:参照国家标准GB/T 16886.6-1997进行异种骨植入材料局部反应评价。Wistar鼠18只,体重150g左右,麻醉,于脊柱两侧皮下及股部肌袋内分别植入规格为5mm×5mm×5mm的猪骨小块与人骨小块。术后大体观察动物饮食、活动、切口反应等情况。分别于术后1、4、12周处死6只大鼠,取出植入块及其周围包裹的组织,行大体观察、组织学观察。
采用SPSS11.5统计软件进行统计分析,体外细胞毒性实验BMSCs的OD值采用析因设计的方差分析,同一时间点两组数据比较采用独立样本t检验,检验水准为α=0.05。
结果:(1)BMSCs的培养:传代培养的BMSCs形态均一,生长旺盛,F1-F3代细胞成活率均在95%左右,F1代、F2代、F3代细胞CD44阳性率分别为93.9%、95.1%、95.8%。(2)体外细胞毒性试验:相同时间实验组与对照组间OD值无显著性差异(F=0.070,P=0.7910),猪骨材料对BMSCs的生长与增殖无明显影响,表明其无细胞毒性。(3)猪骨与BMSCs复合培养:随着培养时间延长,材料周围细胞逐渐聚集,细胞状态正常;扫描电镜下观察,细胞在猪骨材料表面紧密贴附生长,细胞呈多边形、梭形,有较多的伪足向猪骨材料表面伸出,细胞形态正常,功能活跃,表明该材料细胞相容性良好。(4)动物体内植入后的局部刺激反应试验:皮下与肌内植入后1w,纤维组织包裹并长入植入的猪骨腔隙内,伴有较明显的炎性细胞浸润,4w时,炎症反应减轻,12w时,植入的猪骨腔隙内由疏松的结缔组织充填,炎症反应消失,与周围正常组织中的无明显区别,表明猪骨材料具有良好的组织相容性。
结论:酶消化法所制备的猪源性异种骨移植材料具有良好的生物相容性。
第三章异种骨移植材料的骨诱导活性研究
目的:采用免疫抑制大鼠为实验动物,以异位成骨的方式,通过组织学观察、碱性磷酸酶活性测定,评价所制备异种骨的骨诱导活性。
方法:实验组材料为α-半乳糖苷酶消化去抗原的猪皮质骨,0.6N的HCl脱矿,以脱矿后经高温高压处理灭活其成骨活性物质的猪骨为阴性对照。Wistar大鼠24只,体重约150-180g,腹腔注射地塞米松,剂量为每次0.25mg/100g体重,每3天一次,造成免疫抑制。大鼠麻醉后,两侧股部肌袋分别植入实验组材料和对照样品,缝合肌肉皮肤。术后3、4、5、6w各处死6只大鼠,取出植入材料,固定,包埋,行组织学观察;将植入物称重,研磨粉碎,加2ml生理盐水提取,3000rpm离心20min,取上清液,测碱性磷酸酶(ALP)活性。
用SPSS11.5统计软件对ALP活性检测结果进行统计分析,采用析因设计资料的方差分析,同一时间点两组数据比较采用独立样本t检验,检验水准为α=0.05。
结果:组织学观察显示,实验组植入材料3w时被纤维组织包裹,其吸收腔内可见有间充质细胞开始长入;可见有间充质细胞转变形成软骨细胞,并开始分泌形成软骨基质;4w时植入材料内有较多软骨组织形成;5w时形成成熟的软软骨组织;6w时,成骨更为活跃,植入材料内有更多的成熟软骨组织形成,在其边缘吸收腔隙内有类骨质形成;术后3-6w,对照组样品显示吸收,但未见成骨相关的细胞,也无软骨或骨组织形成。ALP活性测定结果显示,不同时间点的ALP活性差异显著(F=321.084,P=0.000),实验组与对照组间差异显著(F=484.330,P=0.000),同一取材时间点实验组ALP活性均比对照组高(P＜0.05)。
结论:采用α-半乳糖苷酶消化去抗原的猪源性异种骨保留了良好的骨诱导活性,可以作为一种潜在的骨移植替代材料,来弥补临床上自体骨与同种骨材料来源的不足。
第四章异种骨修复兔桡骨节段性骨缺损的实验研究
目的:利用兔桡骨节段性骨缺损模型,观察与评价所制备猪源性异种骨移植材料对兔节段性骨缺损的修复,为临床应用提供实验依据。
方法:家兔24只,体重2-2.5 kg,以双侧桡骨为实验对象。家兔静脉注射3%戊巴比妥钠麻醉,用牙科钻截断桡骨干中段,造成双侧桡骨约12mm长的节段性骨缺损,按组植入相应骨移植材料。分别于第4、8、12周分批处死动物8只,取尺桡骨全段。实验分组:根据植入材料不同分组,每组每个时间点4例,A组为空白对照组,节段性骨缺损处不植入任何材料,直接缝合,作为阴性对照;B组为异种骨组,植入酶处理方法制备的去抗原猪骨;C组为兔同种骨组,植入兔松质骨;D组为自体骨组,将取下的兔桡骨骨段用无菌生理盐水清洗后,植于其对侧骨缺损处。大体观察:观察植入材料与宿主骨融合、局部成骨和周围软组织分布、炎症反应等情况;X线检查:拍桡骨正位X线片观察,并进行评分。组织学观察:标本用10%福尔马林缓冲液固定,50%甲酸脱钙,逐级脱水,透明,石蜡包埋,切片,HE染色,组织学观察。
数据结果采用SPSS11.5统计软件包进行统计分析,X线评分采用析因设计资料的方差分析,方差齐性时组间多重比较采用LSD法,不满足方差齐性要求时,组间多重比较采用Dunnet's T3法,统计学显著水平为α=0.05。
结果:大体观察:术后各组动物前肢均有不同程度肿胀,1w恢复正常活动。所有动物切口均Ⅰ期愈合,状态良好,未见明显并发症。X线观察:术后4w,空白对照组骨缺损清晰可见,缺损处两侧宿主骨断端阴影锐利,有微量骨痂形成;异种骨组骨缺损区呈现雾状影,宿主骨断端有少量低密度骨痂形成;兔同种骨组截骨端有少量骨痂形成,缺损区呈雾状阴影;自体骨组植入骨密度无明显变化,截骨线清晰稍显模糊,有少量骨痂形成。术后8w,空白对照组骨缺损仍清晰可见,缺损处截骨端有少量骨痂形成,尺骨侧可见有少量骨痂形成;异种骨组植入骨与宿主骨桥接处稍显模糊,缺损区雾状影填充;兔同种骨组截骨端与缺损区尺骨表面形成骨痂,缺损区密度比骨干低;自体骨组宿主骨骨干周围形成较多的骨痂,与植入骨连接,桥接处呈现高密度影,界线模糊。术后12w,空白对照组骨缺损仍清晰可见,缺损区变小,骨不愈合。异种骨组植入骨与宿主骨界线模糊,密度较骨干低;兔同种骨组植入骨与宿主骨形成连接,连接处界线模糊,骨缺损区密度较均匀;自体骨组植入骨与宿主骨形成连续性连接,骨髓腔再通。X线评分:术后不同取材时间,异种骨组与兔同种骨组之间骨缺损修复无显著差异(4w、8w、12w的P值分别为0.574,1.000,0.805),而与空白对照组与自体骨组之间分别存在着显著差异(P＜0.05);各组X线评分都随着时间的延长而升高,不同时间的X线评分有显著性差异(F=75.077,P=0.000)。组织学观察:空白对照组骨缺损区由纤维组织填充,成骨不活跃;异种骨组截骨端新生的骨组织穿插长入植骨材料的腔隙,植入骨显示吸收,逐渐有新骨形成,12w时可见有软骨细胞与软骨组织连接植入骨与宿主骨;兔同种骨组植入材料显示吸收,截骨端逐渐有新生骨穿插长入植入骨,12w时植入骨上可见有新生类骨质开始形成;自体骨组成骨活跃,植入骨逐渐被新生骨组织取代,至12w时骨髓腔再通。
结论:α-半乳糖苷酶消化的猪源性异种骨用于修复兔桡骨节段性骨缺损时,取得了与兔同种骨一致的修复效果,本方法所制备的异种骨可作为一种骨移植替代材料。
Chapter 1:Preparation of Porcine Bone and Experimental Study on its Biological Properties
Objective:To develop a feasible method to prepare deantigenated porcine bone through enzyme treatment,which has no effect on the osteoinductivity and mechanic properties of the xenogeneic bone graft produced thereby.
Methods:(1)Enzyme treatment of porcine bone:Xenogeneic bone was prepared from flesh porcine bone by a series treatment includingα-Galactosidase of different activity digesting for 8 or 16 hours at 37℃,washing,freezedrying and irradiation at a does of 25kGy.(2)Determination of theα-Gal(α-gal epitopes):The concentration ofα-Gal within porcine bone tissue were tested by ELISA in order to choose a suitable processing way for deantigenation.(3)Crosslinking of glutaraldehyde with the poricine bone:Crosslinking of glutaraldehyde with the poricine bone prepared by the selected group was performed and concentration ofα-Gal was defined and compared with that of fresh porcine bone,enzyme treated porcine bone and human bone.The processing programe then was decided according to the testing result.(3) Morphological observation:Gross observation and SEM observation were performed and diameters of the caves of the porcine cancellous bone were measured.(4)Mechanic properties testing:The compressive properties of porcine bone at different stage of processing were tested to evaluate the effects of different treatment on its mechanic property.
Sstatistical analysis was performed with SPSS11.5 software package and the data were presented with(Mean±Standard Deviation).The data were tested with variance analysis,being significant when P＜0.05.
Results:(1) Enzyme treatment of porcine bone:The OD value of different groups in ELISA test were significantly different.The selected enzyme treatment was digesting byα-Galactosidase with an activity of 30U/ml for 8 hours at 37℃.(2) Crosslinking of glutaraldehyde with the poricine bone:No statistic significance was found among the OD values of cross-linked porcine bone and human bone,while there were significant difference between fresh porcine bone and other groups. According to the results,the finally processing methods wasα-Galactosidase of a 30U/ml activity digesting for 8 hours.(3) Morphological observation:The porous structure of prepared porcine cancellous bone was similar to that of human cancellous bone.The diameters of the caves were between 150μm -600μm,while the diameters of small pore in the trabecula were between 10μm-100μm.(4) Mechanic testing of porcine bone:Strength and elastic module of different groups had no statistical difference (P＜0.05) for the compressive test,indicating that different processing methods had no significant effect on the mechanical property of the porcine bone.
Conclusion:The antigen of porcine bone was removed effectively byα-Galactosidasse digesting,while the morphological structure and mechanic properties of the porcine bone remained
Chapter 2 Biological Evaluation of the Prepared Porcine Bone Graft
Objective:To evaluate the biocompatibility of the prepared porcine bone by in vitro cytotoxity experiment and in vivo animal experiment.
Methods:(1) Culture of rabbit BMSCs:Rabbit BMSCs were obtained as the experimental cells and the activity and content of CD44 surface antigen of the BMSCs were tested.(2)In vitro cytotoxity experiment:The leaching liquor of porcine bone was made by immersing the bone into L-DMEM medium with a scale of 10ml medium per gram bone materials at 37℃for 72 hours in a sterilized condition and L-DMEM medium which contained 10%FBS served as the control medium.200μl of BMSCs suspension with a cell concentration of 1×10~5/ml were seeded to the and cultured for 24 hours.Then the culture medium was removed and 200μl leaching liquor or control culturing medium were added according to groups respectively. Then cells were cultured at 37℃and 5%CO_2.The medium were exchanged every 3 days according to groups.Every 24 hours,5 wells of each group were selected and 20μl 5mg/ml MTT solution was added to each well.150μl DMSO was added after cultured for 4 hours.The 96-well plate was slightly shaken for about 10 minutes and the OD value at 490 nm was determined.(3)Co-culturing of the porcine bone and rabbit BMSCs:The prepared porcine bone,sized 5mm×5mm×5mm,were placed in the 12-well plate and 150μl BMSCs suspension of a concentration of 1×10~5/ml was seeded to the surface of the porcine bone and 10%FBS L-DMEM was added to immerse the porcine bone.Inverted phase contrast microscope observation and SEM observation were performed at 1,2,3 weeks.(4)Biological evaluation in vivo:18 Wistar mice,weighted 150-180g,were anesthetized.Subcutaneous and intramuscular implantation of prepared porcine bone and human bone pieces was performed.6 of the mice were killed each time respectively at 1,4,12 weeks and gross observation and histological observation were performed.
Results:(1) Culture of rabbit BMSCs:The cultured BMSCs were in a good condition.About 95%of F1 to F3 generation cells was active.93.9%of F1 generation,95.1%of F2 generation and 95.8%of F3 generation cells contained CD44 surface antigen.(2) In vitro cytotoxity experiment:The OD values of study group and control group at different time had no significant difference.(3) Co-culturing of porcine bone and BMSCs:Postoperatively 1-3 weeks,Polygon shaped and fusiform shaped BMSCs with a well condition adhered tightly to the surface of the porcine bone.Lots of pseudopodia from BMSCs reached the surface of the porcine bone.
Conclusion:The enzyme degested porcine bone had no obviously effect on the growth of BMSCs and showed good cell affinity when co-cultured with BMSCs. The local implantation also showed the prepared porcine bone contains good tissue compatibility.The porcine bone contained good biocompatibility and could be safe for clinic use.
Chapter 3 Study on the Osteoinductivity of the Prepared Porcine Bone Graft
Objective:To evaluate the osteoinductivity of the prepared porcine graft by histological observation and ALP activity determination in an ectopic bone formation model,using immuno-suppressed Wistar rats as experimental animal.
Methods:Samples of study group were enzyme-treated porcine bone, demineralized with 0.6N hydrochloric acid.The control group samples were the demineralized porcine bone,which were treated with autoclave for 30 minutes to destroy the activity of bone formation material.All samples were freezedried and irradiated at a does of 20kGy.Dexamethasone was intraperitoneally injected into Wistar rats in order to suppress their imunno-response.Intramuscular bone transplantation was performed by placing the demineralized porcine bone and control samples between bluntly dissected muscle groups of hind limbs of the rats. Histological observation was performed at different time post-operatively.ALP activity was tested postoperatively to determine the osteoinductivity of the demineralized porcine bone implants.
Result:The study group:Mesenchymal cells migrated into the absorpted lacuna and chondrocytes could be seen within the porcine bone implants at 3 weeks postoperatively;more cartilage presented at 4 weeks;adult cartilage was formed within the implants at 5 weeks;at 6 weeks,bone formation was more active and lots of cartilage within the implants and osteoid in the absorbed lacuna could be seen.No bone formation could be found in the control group 3-6 weeks postoperatively.ALP activity of the experimental group was obviously higher than that of the control group 3-6 weeks postoperatively.
Conclusion:The prepared porcine bone contained good osteoinductivity and could be a potential bone substitute for clinic implantation.
Chapter 4 Experimental Study of the Prepared Porcine Bone for Bone Reparation in a Radial Segmental Defect of Rabbit
Objective:To investigate the reparation of segmental bone defect by the prepared porcine bone and provide experimental reference for potential clinic use.
Methods 24 rabbits,weighted 2-2.5kg,were anesthetized and a 12mm-long bone defect was made by cutting the middle shaft of both side radii.Grafts were implanted according to groups:(A)Control group:no implantation was performed to the defect;(B) Xenogeneic bone group:the prepared porcine bone were implanted;(C) Allogeneic bone group:rabbit allogeneic cancellous bone were implanted;(D) Autograft group:the cut bone were implanted to the opposite side radius.8 animals were sacrificed(4 graft for each group were obtained) for each time respectively at 4, 8,12 weeks postoperatively.Generally and X-ray,histological observation were performed to evaluate the reparation of the segmental defect..
Results:Gross observation:All the animals were in good status and no detectable complications were found.X-ray observation:Postoperatively 4 weeks, little callus was formed and the defect was clear in the control group;bone defect was filled with shadow with a lower density than that of the rabbit radius and ulna,small quantity of callus was formed in the xenogeneic bone group;allogeneic bone group was similar to the porcine bone group;a little callus was formed in auto graft group. 8 weeks post-operationally,bone defect was clear and small quantity of callus was formed in control group;bone defect was filled with a lower density shadow and the bridge area was not very sharp in xenogeneic bone group;callus was formed and high-density shadow could be seen along the ulna in allogeneic bone group;the auto graft and the rabbit radius was connected with blurry shadow.12 weeks postoperatively,callus was formed in the end of the radius and the marrow cavity was blocked and bone non-union was formed in the control group;the defect was filled with low-density shadow and the bridge area was blurry in porcine bone group and allogeneic bone group;recanalization of the marrow cavity was formed between the graft and the radius in auto graft group.Score of X-ray of xenogeneic bone group ahc no significant difference while the difference of the xenogeneic bone group with control group or autograft group are both significant.Histological observation:the defect of control group was filled with connective tissue without active bone formation.postoperatively 4 weeks,bone defect of control group was filled with fibrous tissue which contained lots of lymphocytes;bone defect was filled with connective tissue and lots of lymphocytes could be seen in porcine bone group and rabbit allogeneic bone group;chondrocytes and osteoid grown between the radius and the auto graft in auto graft group.Postoperatively 8 weeks,the defect of control group was filled with fibrous tissue which contained lymphocytes;bone absorption and cement line could be seen in the xenogeneic bone group and rabbit allogeneic bone group;the autograft was connected with the rabbit radius shaft with active bone formation and absoption within the graft.Postoperatively 12 weeks,the defect of control group were filled with connective tissue;small quantity of new bone formation could be seen in xenogeneic bone group and rabbit allogeneic bone group; the autograft was mainly substituted by newly formed bone with active bone formation.
Conclusion The prepared porcine xenogeneic bone cobtained a cure effect similar to that of rabbit allogeneic bone in a rabbit radial segmental defect model, hence it may be a potential good bone substitute for bone implantation.
引文
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