果胶/聚乙烯醇复合水凝胶人工髓核假体的基础研究
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摘要
研究背景
80%的人在一生中曾有过下腰痛困扰,而椎间盘退行性病变是下腰痛常见的病因。对于有症状的椎间盘退变性疾病原则上采用“阶梯疗法”进行治疗,随着椎间盘退变的加剧,治疗阶梯随之上升,治疗费用和副作用相应增加,故宜在椎间盘退变的早期对其进行椎间关节成形(Spine Arthroplasty),以期保留和重建脊柱运动单元(Functional segment unit,FSU)的生理功能。髓核置换是采用人工髓核假体重建前柱、早期治疗椎间盘退变性疾的一种椎间关节成形技术,具有恢复椎间高度、保留运动等功能,成为脊柱外科研究热点之一。
PDN假体(Prosthetic Disc Nucleus)是较早进入临床的亲水性髓核假体,早期能恢复椎间高度、有效维持运动功能,改善临床症状;但是在中长期随访时发现软骨终板损伤(60.0%)、假体下沉(49.2%)等并发症,其主要原因是PDN假体材料过硬、表面积过小导致软骨终板应力集中。因此,人工髓核假体的研制应选用弹性模量适宜的水凝胶、其形状应与软骨终板匹配。
本课题在聚乙烯醇(PVA)水凝胶的基础上,复合仙人掌的提炼物果胶,制备果胶/聚乙烯醇复合(Pectin/PVA Composite,CoPP)水凝胶,为成形人工髓核假体的研制提供一种弹性模量适宜的新型水凝胶;并优化设计CoPP水凝胶人工髓核假体的外形与尺寸,实现假体与软骨终板的面面接触、合理传载,为CoPP水凝胶人工髓核假体的研发提供实验依据。
研究目的
1、探索CoPP水凝胶的制备方法,并表征其微观结构,评价其溶胀性能。
2、评价溶胀平衡CoPP水凝胶纵向加载时的静态生物力学性能。
3、评价CoPP水凝胶的体外急性细胞毒性及植入动物体内后的局部炎症反应,完成CoPP水凝胶的基本生物相容性评价。
4、探索腰椎间盘MRI图像重建髓核三维模型的方法,并完成国人CoPP人工髓核假体三维形状、尺寸的优化设计。
研究方法
1、选定适宜质量比例的XPV-MC300果胶、PVA-124聚乙烯醇及蒸馏水,经过溶胀、低频振荡加热、冻融,制备成圆柱状PVA水凝胶试件12个、CoPP水凝胶试件24个,随机分为两组,分别观察冻干(Freeze Drying,FD)及无水乙醇(Absolute Ethanol,AE)脱水对PVA、CoPP水凝胶质量脱水率(D_(RM))、体积脱水率(D_(RV))的影响。采用红外光谱仪(FTIR)、差示扫描量热仪(DSC)、X射线衍射仪(XRD)、扫描电镜(SEM),对其微观结构进行表征。经冻干、无水乙醇脱水至恒重的PVA与CoPP水凝胶试件共8个,在37℃、0.10MPa下,分别浸泡于生理盐水(NS)及含10%小牛血清的高糖DMEM细胞培养液中,观察其质量溶胀率(S_(RM))、体积溶胀率(S_(RV)),并按照公式lg(S_(RM))=lg(k)+n lg(t),计算PVA、CoPP水凝胶的溶胀速率常数(k)、扩散特征指数(n)等溶胀动力学参数。采用重复测量数据的方差分析,比较质量脱水率、体积脱水率在不同时间的差异,采用独立样本t检验比较相同时间冻干与无水乙醇脱水方法、PVA与CoPP水凝胶之间的差异。
2、将溶胀平衡的圆柱状CoPP、PVA水凝胶试件各4个,在MTS-858 Mini Bionix材料试验机上纵向预加载5次,加载速率5N/s,回零后加载至600N,持续30min后快速卸载,实时记录力、位移、时间;并测量加载前、卸载后24小时内的质量、尺寸。计算以下指标:①将第6次应力、应变行线性拟合,所得一次项系数即为弹性模量;②将第5次加载-卸载数据,利用Origin7.5软件按照横坐标为0～55%、纵坐标为0～2.0MPa(长度比1.25)制出应力-应变曲线,以统一格式导出图像,用Metlab7.0软件计算滞后环面积及上升曲线与横坐标间的面积,两者的比值即为能量吸收率;③外力达到600N时应变为S_C,持续加载30min,每隔5min的应变与S_C相比,即为归一化蠕变量;④卸载后的质量、体积占加载前试件原始质量、体积的百分比,即为归一化质量与体积百分比。采用独立样本t检验,比较CoPP、PVA水凝胶两者的弹性模量、能量吸收率;采用重复测量数据的方差分析,比较加载前、卸载后即刻、24小时之间的归一化质量及体积百分比;采用独立样本t检验比较PVA与CoPP水凝胶之间归一化质量及体积百分比的差异。
3、选用NCTC L929小鼠成纤维细胞进行体外急性细胞毒性试验,分为正常组、阴性对照组(PVA/FD、PVA/AE)、实验组(CoPP/FD、CoPP/AE)、阳性对照组(0.64%苯酚),定期施加每组的干预因素,观察细胞生长状态,于培养2、4、7d测定OD值、计算相对增殖率(RGR)。此外,选用30只SD大鼠(雌雄各半,体重为182g±13g),在左、右臀肌内分别植入经无水乙醇脱水的CoPP、PVA水凝胶(10mm*3mm*3mm,长*宽*厚),观察手术切口及一般情况,于术后1周、4周、12周取材,观察大体标本及经HE染色的组织切片,采用GB/T 161752-1996中炎症细胞反应标准和纤维囊腔分级标准,进行组织反应程度分级,并观察光镜下CoPP水凝胶的微观结构。
4、在接受GE Signa Excite 3.0磁共振仪常规检查腰部MRI的患者中,除外创伤、炎症、畸形及前中柱肿瘤等疾患,选取无阳性体征、年龄居于20～50岁、T2加权扫描中椎间盘退变不超过Pfirrmann 2级与Modic分型不超过1级的患者。行层厚1mm～1.5mm、层间距0mm的冠状位或矢状位T2序列扫描,获得DICOM 3.0 MRI图像。将图像导入Mimics10.01软件,完成图像的准确分割,通过Calculate 3D计算,重建髓核的三维模型,分别测量MRI图像、三维模型的髓核矢径、横径、高度及倾斜角。依据重建的髓核三维模型,设计CoPP水凝胶人工髓核假体的形状;结合体积脱水率、溶胀率的计算公式,计算出CoPP水凝胶人工髓核假体制备时的体积(V_(pre))、脱水状态植入时的体积(V_(imp))与人体髓核体积(V_(np))之间的换算系数,将上述两个系数的立方根与人体髓核的矢径、横径、高度相乘,计算出CoPP人工髓核假体植入时的理想尺寸,据此设计出CoPP水凝胶人工髓核假体植入时的型号、尺寸。
结果
1、聚乙烯醇(25%)、果胶(5%)的水溶液,经过溶胀、低频振荡加热、冻融,成功制备出圆柱状CoPP水凝胶试件。冻干脱水破坏水凝胶的外形,而无水乙醇脱水不破坏水凝胶外形,但是冻干的脱水率显著大于无水乙醇的脱水率。25%PVA水凝胶的质量脱水率、体积脱水率,显著大于同种脱水方法下的CoPP水凝胶。CoPP水凝胶的FTIR谱图、XRD图是PVA和果胶两者的叠加;CoPP水凝胶的DSC曲线中有两个熔融峰,分别在182.6℃和225.1℃处,后者与PVA粉末的结晶熔融峰对应;经冻干、无水乙醇脱水CoPP水凝胶的断面,在扫描电镜下均可见孔隙形成,孔径在10μm～50μm之间,其内含有颗粒;而同样条件下的PVA水凝胶断面,未发现明显的孔隙形成。CoPP水凝胶的扩散特征指数(n)接近0.5,CoPP水凝胶的溶胀速率常数(k)、以及溶胀平衡时的质量溶胀率(197.38%～252.49%)、体积溶胀率(241.47%～308.93%),均显著大于PVA水凝胶。
2、在纵向加载-卸载试验过程中,CoPP水凝胶、PVA水凝胶试件均未出现破坏,其应力-应变曲线可分为相对平缓的起始段、相对陡峭的上升段,呈非线性表现。CoPP水凝胶全段应力.应变曲线拟合后的弹性模量为(2.76±0.09)MPa,与成形髓核假体适宜的3 MPa接近;显著小于PVA水凝胶的(4.89±0.61)MPa。在加载-卸载的应力-应变曲线中,CoPP、PVA水凝胶均出现应变滞后于应力现象,形成滞后环,其对应的能量吸收率,CoPP水凝胶为12.50%±1.47%,PVA水凝胶为8.57%±1.78%,两者差异显著。持续加载30min,CoPP、PVA水凝胶均达到蠕变平衡,其归一化蠕变量分别为1.076±0.005、1.105±0.030。卸载后即刻,CoPP水凝胶归一化质量百分比为(97.67±0.98)%,显著小于PVA水凝胶的(99.15±0.18)%。卸载后24小时,CoPP、PVA水凝胶归一化质量百分比、体积百分比,与加载前无差异。
3、阴性对照组(PVA/FD、PVA/AE)、实验组(CoPP/FD、CoPP/AE)中的L929细胞,呈长梭形,与正常对照组同时贴壁,圆形细胞均小于10%,胞浆内可见离散颗粒,极个别细胞出现溶解,随着培养时间的延长,均出现接触性生长抑制。阳性对照组中的L929细胞几乎全部为圆形、体积减小,无贴壁,无明显细胞增殖。CoPP组RGR均大于75%,PVA组RGR超过80%,细胞毒性小于1级;阳性对照组RGR小于24%、细胞毒性为4～5级。所有SD大鼠于术后2～6小时苏醒、开始活动,无死亡,手术切口无红肿、渗出、化脓等炎症表现,均一期愈合。在植入1周时,PVA、CoPP水凝胶的体积较植入时明显增大、在肌袋内可活动,肌肉色泽鲜红、局部无脓性分泌物。病理切片显示:PVA、CoPP组局部组织的炎性细胞反应为Ⅳ级、未形成完整囊腔;CoPP水凝胶呈嗜碱性、染色较深,孔隙较多、孔径大,PVA水凝胶近中性、染色浅,孔隙少、孔径小。在植入4周时,PVA、CoPP水凝胶在肌袋内基本无活动,局部组织结构无异常,炎性细胞反应为Ⅰ级,囊腔反应为Ⅳ级;PVA、CoPP水凝胶的染色、结构无变化。在植入12周时,PVA、CoPP水凝胶的体积、外观与前2次取材时相似,炎性细胞及囊腔反应均为Ⅰ级;CoPP水凝胶断面仍为嗜碱性深染,其边缘连续、无明显缺损;孔隙丰富,孔隙间的连接与第1周、4周相近,孔内仍可见微小颗粒。
4、本实验获取的薄层MRI图像,采自一名34岁女性,冠状位脂肪抑制T2序列扫描(OCor fS,TE82.7ms,TR4000ms),包括腰1～骶1椎间盘前、后缘,层厚1.2mm、层间距0mm的连续扫描,像素大小为0.625mm,获得44张DICOM3.0格式的MRI图像。MRI图像上椎间盘为Pfirrmann 1～2级;采用Mimics软件分割图像理想,重建的腰1～骶1髓核模型,其侧视为椭圆,由扁平逐渐变成高耸穹隆状,外形由铁饼状向橄榄球状过渡。MRI图像上髓核矢径、横径与高度,自上而下呈逐渐增大趋势,以横径为明显;所有髓核的平均矢径、横径与高度分别为(20.8±1.5)mm、(34.7±3.5)mm、(9.2±1.1)mm,与3D髓核模型的测量结果相近。CoPP水凝胶假体以完全脱水状态植入,其体积为人体髓核的25.90%～29.29%,其尺寸为人体髓核的63.74%～66.41%。本实验设计出CoPP水凝胶人工髓核假体为近似橄榄球状的椭圆球体,其侧面为椭圆型,分小号(S)、中号(M_1、M_2)及大号(L)四种,其矢径为13 mm～14mm、横径为20 mm～24mm,高度为5.5 mm～7.0mm。
结论
1、采用低频振荡、反复冻融可制备果胶/聚乙烯醇复合(CoPP)水凝胶,适宜采用无水乙醇脱水方法脱水。CoPP水凝胶具有一定的结晶度,存在两种结晶区,为多孔结构。CoPP水凝胶具有良好的再水化特性,水分子是以菲克扩散模式渗入,CoPP水凝胶具有溶胀程度大、溶胀速率快的特点,比单一材料的PVA水凝胶具有更好的溶胀性能。
2、CoPP水凝胶是一种粘弹性材料,相对于PVA水凝胶而言,具有软而韧、缓冲载荷能力较强、蠕变特性等特点,其弹性模量与成形髓核假体适宜的模量接近:具有较好的渗透性,利于椎间隙内物质转运,适宜于成形替代椎间盘髓核组织。
3、CoPP水凝胶的急性细胞毒性不超过1级,在SD大鼠体内组织反应轻,未发生降解,其基本生物相容性良好,可进一步补充慢性毒性、遗传毒性、致癌毒性,完善其生物相容性评价。
4、采用腰椎间盘薄层MRI图像,通计算机图像处理技术可成功重建髓核的三维模型。设计出的CoPP水凝胶人工髓核假体近似橄榄球状,与软骨终板形状匹配,有望合理传载;其所占空间较小,利于球窝器械把持,将有望通过后路椎间盘镜系统微创植入椎间隙。
Backgrounds
About 80%of the population will experience low back pain during adulthood, which is commonly in due to degenerative disc diseases(DDD).Staging therapies of symptomatic degenerative disc diseases should be tailored to grade of degeneration in principle,following with increasing cost,risk and collateral damage.Therefore,disc degeneration should be intervened by spine arthroplasty in the early stage to preserve and restore physical roles of intervertebral disc in the functional spinal uint(FSU). Nucleus pulposus replacement is a modality of spine arthroplasty for early DDD to restore height of disc and motion of FSU by reconstructing anterior column with prosthetic implant,and has become an attractive spot in spinal surgery studies.
The Prosthetic Disc Nucleus(abbr.PDN) was the hydrophilic prosthesis of nucleus pulposus and early applied to clinic with good outcome in the short term follow-up,however there were considerable damages of cartilaginous endplates (60.0%) and subsidence of prosthesis(49.2%) in the four-year-follow-up.The stress concentration of cartilaginous endplate was responsible for damages and subsidence, which resulted from the higher elastic modulus of materials and a wedge shape or rectangle.Therefore,prosthetic nucleus pulposus should be made of the hydrogel with suitable elastic modulus,whose shape should match with cartilaginous endplate.
Based on the poly(vinyl alcohol)(abbr.PVA) hydrogel,compounding pectin extracted from the cactus with PVA,this paper is to develop a new kind of composite hydrogel,the pectin/PVA composite(abbr.CoPP) hydrogel,whose elastic modulus will be suitable to make prosthetic nucleus pulposus.What's more,this paper is to optimally design the shape and size of prosthetic nucleus pulposus made of CoPP hydrogel in order to avoid stress concentration of cartilaginous endplate.Therefore,it is the new CoPP hydrogel and optimal design of prosthesis that is to provide basic study for development of prosthetic nucleus pulposus made of CoPP hydrogel.
Objectives
1.To explore preparation methods of pectin/poly(vinyl alcohol) composite(CoPP) hydrogel,characterize its microstructures and evaluate the swelling properties.
2.To evaluate biomechanical properties of CoPP hydrogel compressed statically at the swelling equilibration.
3.To evaluate biocompatibility of CoPP hydrogel for nucleus pulpous prosthesis, including in vitro cytotoxicity and in vivo inflammatory reaction.
4.To explore reconstructive methods of three-dimensional nuclues pulposus derived from lamellar MR imaging,and to optimally design shape and size of prosthetic nucleus pulposus made of CoPP hydrogel for Chinese people.
Methods
1.The mixture of XPV-MC300 pectin,PVA-124 and distilled water with appropriate mass percentage was given swelling,heating by low frequency oscillation and freezing thawing to prepare cylindrical test pieces of CoPP hydrogel and PVA hydrogel.The PVA hydrogel tests(n=12) and CoPP hydrogel tests(n=24) were randomly divided into two groups and dehydrated by vacuum freeze-dry(abbr. FD) and anhydrous alcohol(abbr.AE),respectively,whose mass and volume dehydrant ratio were compared by variance analysis of repeated measure data and dehydrant ratio were compared by variance analysis of repeated measure data and between two dehydrant methods and two hydrogels compared by independent sample t test.The mierostructures of CoPP hydrogel were characterized by FTIR, DSC,XRD and SEM.Eight dehydrated test pieces of PVA and CoPP hydrogel were selected and soaked into normal saline and high glucose DMEM solution with 10%calf serum at 37℃and 0.10MPa(1 atm).The swelling ratio of mass and volume were observed.Furthermore,the diffusion characteristic index(n) and swelling velocity constant(k) of CoPP were calculated according to the equation: Ig(S_(RM)) = 1g(k) + n 1g(t).
2.Four cylindrical test pieces of CoPP hydrogel and PVA hydrogel were prepared and compressed lengthways statically by MTS-858 Mini Bionix material test machine at the swelling equilibration.After 5 circles of preload,whose maximal load was 500N with 5N/s loading rate,600N was applied and kept for 30min.The force,displacement and time were recorded at real time.The masses and sizes of test pieces were measured before loading and during 24 hours after unloading. Based on above data,these indexes were calculated:①elastic modulus was the coefficient of line fitting between stress and strain of 6th loading;②absorption ratio of power was the relative value of areas between hysteresis loop and under the buildup curve,calculated by Metlab7.0 software according to the stress-strain curve of the fifth loading and unloading,in the same X-axis strain(0～55%), Y-axis stress(0～2.0MPa) and 1.25 ratio of X-axis/Y-axis drawn by Origin7.5;③normalization creep was the ratio of creep between every other 5min with continous load(600N) and at the beginning of 600 N;④normalization mass and volume percentage were the ratio of mass and volume after unloading relative to them before loading.The elastic modulus and absorption ratio of power between CoPP and PVA hydrogels were compared by independent sample t test,and normalization mass and volume percentage were compared by variance analysis of repeated measure data and independent sample t test.
3.The in vitro cytotoxicity of CoPP hydrogel was tested by NCTC L929 cell lines, which were divided into four groups:normal,negative control(PVA/FD and PVA/AE),experiment(CoPP/FD and CoPP/AE) and positive control(0.64% phenol),regularly intervened by each treatment.The growth status of cells were observed regularly,OD values of each group were measured at 2nd,4th and 7th day and then relative growth rates(abbr.RGR) were calculated.There were thirty SD rats(15 male and 15 female,body mass 182g±13g) for inflammatory reaction in vivo.CoPP and PVA hydrogels(10mm*3mm*3mm,Length*Width* Thickness) were implanted into left and right intramuscular of gluteus of SD rats,respectively. Postoperative wounds and general status of rats were observed,and local muscles containing with hydrogels were taken and sectioned at 1st,4th,and 12th week.The macroscopic specimen and microscopic reaction of inflammatory were observed and evaluated according to the standard of GB/ T 161752-1996,including the microstructure of CoPP hydrogel with light microscope.
4.The lamellar lumbar MRI of normal Chinese subject was screened from subjects accepting lumbar standard scan by GE Signa Excite 3.0.Trauma,inflammation, deformity and tumor of anterior and middle column of spine were excluded.The criteria of inclusions were at age of 20～50 years old,disc degeneration not exceeding Pfirrmann 2,degeneration of endplate not exceeding Modic 1.The selected subject was scanned in T2 serials to get DICOM 3.0 MR imaging of lumbar in sagittal or coronal view,with 1mm～1.5mm thickness and 0 mm space. These DICOM images of lumbar MR were imported into the Mimics10.01 software.After accurate segmentation of nucleus pulposus in the imaging,the three-dimension(3D) models were reconstructed by Calculate 3D function of Mimics.The sagittal diameter,transverse diameter,height and slipping angle of nucleus pulposus were measured in MR imaging and 3D model,respectively.The shape of prosthetic nucleus pulposus made of CoPP hydrogel was designed according to the reconstructive 3D model of nucleus pulposus.According to equation of volume dehydrant ratio and volume swelling ratio of CoPP hydrogel, the volume conversion coefficents of preparation(V_(pre)) and dehydrated implant (V_(imp)) relative to human nucleus pulposus were calculated.The expected sizes of CoPP hydrogel prosthesis were multiplying results between the cube roots of volume conversion coefficents and anatomic sizes of human nucleus pulposus.On basis of expected sizes,shapes and sizes of prosthetic nucleus pulposus made of CoPP hydrogel were optimally designed.
Results
1.CoPP hydrogel was successfully prepared from the water solution of PVA(25%) and pectin(5%) by swelling,heating with low frequency oscillation and freezing thawing,whose cylindrical shapes can be maintained not by vacuum freeze-dry but by anhydrous alcohol in the dehydrating course.However,the dehydrant ratios of vacuum freeze-dry hydrogels were significantly superior to them of anhydrous alcohol hydrogels.The dehydrant ratios of PVA hydrogel were significantly superior to them of CoPP hydrogel in the same dehydrant course. The FTIR and XRD graph of CoPP hydrogel were superposition of PVA and pectin,respectively.There were two melt apexes in DSC graph of CoPP hydrogel, at the temperature of 182.6℃and 225.1℃,whose posterior apex was corresponding to the melt apex of PVA powder.The section of CoPP hydrogel were full of 10μm-50μm pores with granules;however,there was no pore in the section of PVA hydrogel.The diffusion characteristic index(n) of CoPP hydrogel was near to 0.5,and the swelling velocity constant(k),the swelling ratio of mass (197.38%～252.49%) and swelling ratio of volume(241.47%～308.93%) of CoPP hydrogel at the swelling equilibration were superior to those of PVA hydrogel.
2.There was not any test pieces of CoPP and PVA hydrogels destroyed by loading and unloading.All curvatures of stress-strain consisted two parts:the first was mild starting,and the posterior was steep rise,which were nonlinear.The elastic modulus of CoPP hydrogel was(2.76±0.09) MPa,near to 3MPa which is optimal elastic modulus of preformed nucleus prosthesis,and was significantly inferior to that of PVA hydrogel(4.89±0.61) MPa.There was also significant difference of absorption ratio of power between CoPP hydrogel(12.50±1.47)%and PVA hydrogel(8.57±1.78)%.Normalization creeps of CoPP and PVA hydrogels under 30 min continuous loads were 1.076±0.005 and 1.105±0.030,respectively.The normalization mass percentage of CoPP hydrogel was(97.67%±0.98)%, significantly inferior to that of PVA hydrogel(99.15±0.18)%.At time of 24 hours after unloading,there was no significance of the normalization percentage of mass and volume between two kinds of hydrogels.
3.The L929 cells of negative group(PVA/FD and PVA/AE) and experiment group (CoPP/FD and CoPP/AE) were long fusiform adhered to the wall at the same time relative to normal group,and occurred contact growth inhibiting.Less 10%of cells were circular with discrete grain in negative and experimental groups.RGRs of CoPP group were over 75%,RGRs of PVA group were over 80%,and cytotoxicity of both groups was grade 0～1.On the contrast,RGRs of positive group were inferior to 24%and cytotoxicity was over grade 4.All of experimental rats waked up and began walking during 2～4 hours after operation,without death, and wounds healed primarily without red swelling,exudation or suppuration.At week 1,PVA and CoPP hydrogels were surrounded by bright and red muscles without exudation or suppuration,movable and were larger than those of implants. The sections of CoPP hydrogel were basophilia,stained blue and porous,however, sections of PVA hydrogel were close to neutrophilia,stained undertint red,and pligoporous with smaller pore.The inflammatory reactions of local tissue were grade 4 of inflammatory cell infiltrate(ICI) without integrate cyst.At week 4,the PVA and CoPP hydrogels were also surrounded by normal muscle,however,were not movable.The inflammatory reactions of local tissue were grade 1 of ICI and grade 4 of cyst reaction.At week 12,volumes and shapes of PVA and CoPP hydrogels were similar to that at 1st and 4th week,and both ICI and cyst of the local tissue were grade 1.Sections of CoPP hydrogel were also basophilia,stained blue,whose edge were consistent without defect.There were full of pores with micrograins at 12th week,which were similar to that at 1st and 4th week.
4.Lamellar lumbar MR images of 34 years old woman in this paper were scanned in coronal vies with 1.2mm thickness and 0 mm space by T2 serials(OCor fs, TE82.7ms,TR4000ms),including anterior and posterior margin of invertebral discs from L1 to S1.There were 44 sheets of DICOM3.0 images of MR,whose pixel size was 0.625mm.The disc degeneration of lumbar MRI was Pfirrmann 1～2,whose nucleus pulposus was segmentated well.3D models of nucleus pulposus were successfully reconstructed from lamellar lumbar MR images, whose lateral views were ellipse from fiat to rear fomix,and whose shapes were from discs-form to rugby-form.The sagittal diameter,transverse diameter,height were increasing from upper to lower,especially transverse diameter.The average of sagittal diameter,transverse diameter and height in MR images were (20.8±1.5)mm,(34.7±3.5)mm and(9.2±1.1)mm,which were similar to them of 3D models.The dehydrated CoPP hydrogel prosthesis would be implanted,whose volume was 25.90%～29.29%and whose size was 63.74%～66.41%of human nucleus pulposus.The CoPP hydrogel prosthesis were ellipsoid near to rugby,with four sizes(S,M_1,M_2 and L),whose sagittal diameters were 13mm～14mm, transverse diameters were 20mm～24mm and heights were 5.5mm～7.0mm.
Conclusions
1.It is successful for swelling,heating with low frequency oscillation and freezing thawing to prepare CoPP hydrogel,and it is proper to be dehydrated by anhydrous alcohol.CoPP hydrogel is porous and with determinate crystallization,including two kinds of crystallization zones.CoPP hydrogel has good rehydration with Fick model diffusion of water,and are faster and larger than those of PVA hydrogel.
2.CoPP hydrogel is a kind of viscoelastic material,which is softer and more tenacious,has better absorption shock of load,whose elastic modulus is near to the optimal elastic modulus of preformed prosthetic nucleus pulposus,has better permeability of substance in contrast to PVA hydrogel,and is suitable to made preformed prosthesis of lumbar nucleus pulposus.
3.The cytotoxicity of CoPP hydrogel in vitro is 0～1 grade,the inflammatory reaction of CoPP hydrogel in vivo is mild,and then the basic biocompatibility of CoPP hydrogel is good and there is no degradation of CoPP hydrogel.The genotoxicity,carcinogencity and reproductive toxicity of CoPP hydrogel need evaluating in the future.
4.3D model of nucleus pulposus can be successfully reconstructed from lamellar lumbar MR images.The CoPP hydrogel prosthesis of nucleus pulposus is ellipsoid near to rugby,matching with the shape of cartilaginous endplate,which is helpful to reasonable transmission of load.It is smaller volumes that help CoPP hydrogel prosthesis to be held with ball-and-socket instrument and minimally invasively implanted by posterior diskoscope.
80%的人在一生中曾有过下腰痛困扰,而椎间盘退行性病变是下腰痛常见的病因。对于有症状的椎间盘退变性疾病原则上采用“阶梯疗法”进行治疗,随着椎间盘退变的加剧,治疗阶梯随之上升,治疗费用和副作用相应增加,故宜在椎间盘退变的早期对其进行椎间关节成形(Spine Arthroplasty),以期保留和重建脊柱运动单元(Functional segment unit,FSU)的生理功能。髓核置换是采用人工髓核假体重建前柱、早期治疗椎间盘退变性疾的一种椎间关节成形技术,具有恢复椎间高度、保留运动等功能,成为脊柱外科研究热点之一。
PDN假体(Prosthetic Disc Nucleus)是较早进入临床的亲水性髓核假体,早期能恢复椎间高度、有效维持运动功能,改善临床症状;但是在中长期随访时发现软骨终板损伤(60.0%)、假体下沉(49.2%)等并发症,其主要原因是PDN假体材料过硬、表面积过小导致软骨终板应力集中。因此,人工髓核假体的研制应选用弹性模量适宜的水凝胶、其形状应与软骨终板匹配。
本课题在聚乙烯醇(PVA)水凝胶的基础上,复合仙人掌的提炼物果胶,制备果胶/聚乙烯醇复合(Pectin/PVA Composite,CoPP)水凝胶,为成形人工髓核假体的研制提供一种弹性模量适宜的新型水凝胶;并优化设计CoPP水凝胶人工髓核假体的外形与尺寸,实现假体与软骨终板的面面接触、合理传载,为CoPP水凝胶人工髓核假体的研发提供实验依据。
研究目的
1、探索CoPP水凝胶的制备方法,并表征其微观结构,评价其溶胀性能。
2、评价溶胀平衡CoPP水凝胶纵向加载时的静态生物力学性能。
3、评价CoPP水凝胶的体外急性细胞毒性及植入动物体内后的局部炎症反应,完成CoPP水凝胶的基本生物相容性评价。
4、探索腰椎间盘MRI图像重建髓核三维模型的方法,并完成国人CoPP人工髓核假体三维形状、尺寸的优化设计。
研究方法
1、选定适宜质量比例的XPV-MC300果胶、PVA-124聚乙烯醇及蒸馏水,经过溶胀、低频振荡加热、冻融,制备成圆柱状PVA水凝胶试件12个、CoPP水凝胶试件24个,随机分为两组,分别观察冻干(Freeze Drying,FD)及无水乙醇(Absolute Ethanol,AE)脱水对PVA、CoPP水凝胶质量脱水率(D_(RM))、体积脱水率(D_(RV))的影响。采用红外光谱仪(FTIR)、差示扫描量热仪(DSC)、X射线衍射仪(XRD)、扫描电镜(SEM),对其微观结构进行表征。经冻干、无水乙醇脱水至恒重的PVA与CoPP水凝胶试件共8个,在37℃、0.10MPa下,分别浸泡于生理盐水(NS)及含10%小牛血清的高糖DMEM细胞培养液中,观察其质量溶胀率(S_(RM))、体积溶胀率(S_(RV)),并按照公式lg(S_(RM))=lg(k)+n lg(t),计算PVA、CoPP水凝胶的溶胀速率常数(k)、扩散特征指数(n)等溶胀动力学参数。采用重复测量数据的方差分析,比较质量脱水率、体积脱水率在不同时间的差异,采用独立样本t检验比较相同时间冻干与无水乙醇脱水方法、PVA与CoPP水凝胶之间的差异。
2、将溶胀平衡的圆柱状CoPP、PVA水凝胶试件各4个,在MTS-858 Mini Bionix材料试验机上纵向预加载5次,加载速率5N/s,回零后加载至600N,持续30min后快速卸载,实时记录力、位移、时间;并测量加载前、卸载后24小时内的质量、尺寸。计算以下指标:①将第6次应力、应变行线性拟合,所得一次项系数即为弹性模量;②将第5次加载-卸载数据,利用Origin7.5软件按照横坐标为0～55%、纵坐标为0～2.0MPa(长度比1.25)制出应力-应变曲线,以统一格式导出图像,用Metlab7.0软件计算滞后环面积及上升曲线与横坐标间的面积,两者的比值即为能量吸收率;③外力达到600N时应变为S_C,持续加载30min,每隔5min的应变与S_C相比,即为归一化蠕变量;④卸载后的质量、体积占加载前试件原始质量、体积的百分比,即为归一化质量与体积百分比。采用独立样本t检验,比较CoPP、PVA水凝胶两者的弹性模量、能量吸收率;采用重复测量数据的方差分析,比较加载前、卸载后即刻、24小时之间的归一化质量及体积百分比;采用独立样本t检验比较PVA与CoPP水凝胶之间归一化质量及体积百分比的差异。
3、选用NCTC L929小鼠成纤维细胞进行体外急性细胞毒性试验,分为正常组、阴性对照组(PVA/FD、PVA/AE)、实验组(CoPP/FD、CoPP/AE)、阳性对照组(0.64%苯酚),定期施加每组的干预因素,观察细胞生长状态,于培养2、4、7d测定OD值、计算相对增殖率(RGR)。此外,选用30只SD大鼠(雌雄各半,体重为182g±13g),在左、右臀肌内分别植入经无水乙醇脱水的CoPP、PVA水凝胶(10mm*3mm*3mm,长*宽*厚),观察手术切口及一般情况,于术后1周、4周、12周取材,观察大体标本及经HE染色的组织切片,采用GB/T 161752-1996中炎症细胞反应标准和纤维囊腔分级标准,进行组织反应程度分级,并观察光镜下CoPP水凝胶的微观结构。
4、在接受GE Signa Excite 3.0磁共振仪常规检查腰部MRI的患者中,除外创伤、炎症、畸形及前中柱肿瘤等疾患,选取无阳性体征、年龄居于20～50岁、T2加权扫描中椎间盘退变不超过Pfirrmann 2级与Modic分型不超过1级的患者。行层厚1mm～1.5mm、层间距0mm的冠状位或矢状位T2序列扫描,获得DICOM 3.0 MRI图像。将图像导入Mimics10.01软件,完成图像的准确分割,通过Calculate 3D计算,重建髓核的三维模型,分别测量MRI图像、三维模型的髓核矢径、横径、高度及倾斜角。依据重建的髓核三维模型,设计CoPP水凝胶人工髓核假体的形状;结合体积脱水率、溶胀率的计算公式,计算出CoPP水凝胶人工髓核假体制备时的体积(V_(pre))、脱水状态植入时的体积(V_(imp))与人体髓核体积(V_(np))之间的换算系数,将上述两个系数的立方根与人体髓核的矢径、横径、高度相乘,计算出CoPP人工髓核假体植入时的理想尺寸,据此设计出CoPP水凝胶人工髓核假体植入时的型号、尺寸。
结果
1、聚乙烯醇(25%)、果胶(5%)的水溶液,经过溶胀、低频振荡加热、冻融,成功制备出圆柱状CoPP水凝胶试件。冻干脱水破坏水凝胶的外形,而无水乙醇脱水不破坏水凝胶外形,但是冻干的脱水率显著大于无水乙醇的脱水率。25%PVA水凝胶的质量脱水率、体积脱水率,显著大于同种脱水方法下的CoPP水凝胶。CoPP水凝胶的FTIR谱图、XRD图是PVA和果胶两者的叠加;CoPP水凝胶的DSC曲线中有两个熔融峰,分别在182.6℃和225.1℃处,后者与PVA粉末的结晶熔融峰对应;经冻干、无水乙醇脱水CoPP水凝胶的断面,在扫描电镜下均可见孔隙形成,孔径在10μm～50μm之间,其内含有颗粒;而同样条件下的PVA水凝胶断面,未发现明显的孔隙形成。CoPP水凝胶的扩散特征指数(n)接近0.5,CoPP水凝胶的溶胀速率常数(k)、以及溶胀平衡时的质量溶胀率(197.38%～252.49%)、体积溶胀率(241.47%～308.93%),均显著大于PVA水凝胶。
2、在纵向加载-卸载试验过程中,CoPP水凝胶、PVA水凝胶试件均未出现破坏,其应力-应变曲线可分为相对平缓的起始段、相对陡峭的上升段,呈非线性表现。CoPP水凝胶全段应力.应变曲线拟合后的弹性模量为(2.76±0.09)MPa,与成形髓核假体适宜的3 MPa接近;显著小于PVA水凝胶的(4.89±0.61)MPa。在加载-卸载的应力-应变曲线中,CoPP、PVA水凝胶均出现应变滞后于应力现象,形成滞后环,其对应的能量吸收率,CoPP水凝胶为12.50%±1.47%,PVA水凝胶为8.57%±1.78%,两者差异显著。持续加载30min,CoPP、PVA水凝胶均达到蠕变平衡,其归一化蠕变量分别为1.076±0.005、1.105±0.030。卸载后即刻,CoPP水凝胶归一化质量百分比为(97.67±0.98)%,显著小于PVA水凝胶的(99.15±0.18)%。卸载后24小时,CoPP、PVA水凝胶归一化质量百分比、体积百分比,与加载前无差异。
3、阴性对照组(PVA/FD、PVA/AE)、实验组(CoPP/FD、CoPP/AE)中的L929细胞,呈长梭形,与正常对照组同时贴壁,圆形细胞均小于10%,胞浆内可见离散颗粒,极个别细胞出现溶解,随着培养时间的延长,均出现接触性生长抑制。阳性对照组中的L929细胞几乎全部为圆形、体积减小,无贴壁,无明显细胞增殖。CoPP组RGR均大于75%,PVA组RGR超过80%,细胞毒性小于1级;阳性对照组RGR小于24%、细胞毒性为4～5级。所有SD大鼠于术后2～6小时苏醒、开始活动,无死亡,手术切口无红肿、渗出、化脓等炎症表现,均一期愈合。在植入1周时,PVA、CoPP水凝胶的体积较植入时明显增大、在肌袋内可活动,肌肉色泽鲜红、局部无脓性分泌物。病理切片显示:PVA、CoPP组局部组织的炎性细胞反应为Ⅳ级、未形成完整囊腔;CoPP水凝胶呈嗜碱性、染色较深,孔隙较多、孔径大,PVA水凝胶近中性、染色浅,孔隙少、孔径小。在植入4周时,PVA、CoPP水凝胶在肌袋内基本无活动,局部组织结构无异常,炎性细胞反应为Ⅰ级,囊腔反应为Ⅳ级;PVA、CoPP水凝胶的染色、结构无变化。在植入12周时,PVA、CoPP水凝胶的体积、外观与前2次取材时相似,炎性细胞及囊腔反应均为Ⅰ级;CoPP水凝胶断面仍为嗜碱性深染,其边缘连续、无明显缺损;孔隙丰富,孔隙间的连接与第1周、4周相近,孔内仍可见微小颗粒。
4、本实验获取的薄层MRI图像,采自一名34岁女性,冠状位脂肪抑制T2序列扫描(OCor fS,TE82.7ms,TR4000ms),包括腰1～骶1椎间盘前、后缘,层厚1.2mm、层间距0mm的连续扫描,像素大小为0.625mm,获得44张DICOM3.0格式的MRI图像。MRI图像上椎间盘为Pfirrmann 1～2级;采用Mimics软件分割图像理想,重建的腰1～骶1髓核模型,其侧视为椭圆,由扁平逐渐变成高耸穹隆状,外形由铁饼状向橄榄球状过渡。MRI图像上髓核矢径、横径与高度,自上而下呈逐渐增大趋势,以横径为明显;所有髓核的平均矢径、横径与高度分别为(20.8±1.5)mm、(34.7±3.5)mm、(9.2±1.1)mm,与3D髓核模型的测量结果相近。CoPP水凝胶假体以完全脱水状态植入,其体积为人体髓核的25.90%～29.29%,其尺寸为人体髓核的63.74%～66.41%。本实验设计出CoPP水凝胶人工髓核假体为近似橄榄球状的椭圆球体,其侧面为椭圆型,分小号(S)、中号(M_1、M_2)及大号(L)四种,其矢径为13 mm～14mm、横径为20 mm～24mm,高度为5.5 mm～7.0mm。
结论
1、采用低频振荡、反复冻融可制备果胶/聚乙烯醇复合(CoPP)水凝胶,适宜采用无水乙醇脱水方法脱水。CoPP水凝胶具有一定的结晶度,存在两种结晶区,为多孔结构。CoPP水凝胶具有良好的再水化特性,水分子是以菲克扩散模式渗入,CoPP水凝胶具有溶胀程度大、溶胀速率快的特点,比单一材料的PVA水凝胶具有更好的溶胀性能。
2、CoPP水凝胶是一种粘弹性材料,相对于PVA水凝胶而言,具有软而韧、缓冲载荷能力较强、蠕变特性等特点,其弹性模量与成形髓核假体适宜的模量接近:具有较好的渗透性,利于椎间隙内物质转运,适宜于成形替代椎间盘髓核组织。
3、CoPP水凝胶的急性细胞毒性不超过1级,在SD大鼠体内组织反应轻,未发生降解,其基本生物相容性良好,可进一步补充慢性毒性、遗传毒性、致癌毒性,完善其生物相容性评价。
4、采用腰椎间盘薄层MRI图像,通计算机图像处理技术可成功重建髓核的三维模型。设计出的CoPP水凝胶人工髓核假体近似橄榄球状,与软骨终板形状匹配,有望合理传载;其所占空间较小,利于球窝器械把持,将有望通过后路椎间盘镜系统微创植入椎间隙。
Backgrounds
About 80%of the population will experience low back pain during adulthood, which is commonly in due to degenerative disc diseases(DDD).Staging therapies of symptomatic degenerative disc diseases should be tailored to grade of degeneration in principle,following with increasing cost,risk and collateral damage.Therefore,disc degeneration should be intervened by spine arthroplasty in the early stage to preserve and restore physical roles of intervertebral disc in the functional spinal uint(FSU). Nucleus pulposus replacement is a modality of spine arthroplasty for early DDD to restore height of disc and motion of FSU by reconstructing anterior column with prosthetic implant,and has become an attractive spot in spinal surgery studies.
The Prosthetic Disc Nucleus(abbr.PDN) was the hydrophilic prosthesis of nucleus pulposus and early applied to clinic with good outcome in the short term follow-up,however there were considerable damages of cartilaginous endplates (60.0%) and subsidence of prosthesis(49.2%) in the four-year-follow-up.The stress concentration of cartilaginous endplate was responsible for damages and subsidence, which resulted from the higher elastic modulus of materials and a wedge shape or rectangle.Therefore,prosthetic nucleus pulposus should be made of the hydrogel with suitable elastic modulus,whose shape should match with cartilaginous endplate.
Based on the poly(vinyl alcohol)(abbr.PVA) hydrogel,compounding pectin extracted from the cactus with PVA,this paper is to develop a new kind of composite hydrogel,the pectin/PVA composite(abbr.CoPP) hydrogel,whose elastic modulus will be suitable to make prosthetic nucleus pulposus.What's more,this paper is to optimally design the shape and size of prosthetic nucleus pulposus made of CoPP hydrogel in order to avoid stress concentration of cartilaginous endplate.Therefore,it is the new CoPP hydrogel and optimal design of prosthesis that is to provide basic study for development of prosthetic nucleus pulposus made of CoPP hydrogel.
Objectives
1.To explore preparation methods of pectin/poly(vinyl alcohol) composite(CoPP) hydrogel,characterize its microstructures and evaluate the swelling properties.
2.To evaluate biomechanical properties of CoPP hydrogel compressed statically at the swelling equilibration.
3.To evaluate biocompatibility of CoPP hydrogel for nucleus pulpous prosthesis, including in vitro cytotoxicity and in vivo inflammatory reaction.
4.To explore reconstructive methods of three-dimensional nuclues pulposus derived from lamellar MR imaging,and to optimally design shape and size of prosthetic nucleus pulposus made of CoPP hydrogel for Chinese people.
Methods
1.The mixture of XPV-MC300 pectin,PVA-124 and distilled water with appropriate mass percentage was given swelling,heating by low frequency oscillation and freezing thawing to prepare cylindrical test pieces of CoPP hydrogel and PVA hydrogel.The PVA hydrogel tests(n=12) and CoPP hydrogel tests(n=24) were randomly divided into two groups and dehydrated by vacuum freeze-dry(abbr. FD) and anhydrous alcohol(abbr.AE),respectively,whose mass and volume dehydrant ratio were compared by variance analysis of repeated measure data and dehydrant ratio were compared by variance analysis of repeated measure data and between two dehydrant methods and two hydrogels compared by independent sample t test.The mierostructures of CoPP hydrogel were characterized by FTIR, DSC,XRD and SEM.Eight dehydrated test pieces of PVA and CoPP hydrogel were selected and soaked into normal saline and high glucose DMEM solution with 10%calf serum at 37℃and 0.10MPa(1 atm).The swelling ratio of mass and volume were observed.Furthermore,the diffusion characteristic index(n) and swelling velocity constant(k) of CoPP were calculated according to the equation: Ig(S_(RM)) = 1g(k) + n 1g(t).
2.Four cylindrical test pieces of CoPP hydrogel and PVA hydrogel were prepared and compressed lengthways statically by MTS-858 Mini Bionix material test machine at the swelling equilibration.After 5 circles of preload,whose maximal load was 500N with 5N/s loading rate,600N was applied and kept for 30min.The force,displacement and time were recorded at real time.The masses and sizes of test pieces were measured before loading and during 24 hours after unloading. Based on above data,these indexes were calculated:①elastic modulus was the coefficient of line fitting between stress and strain of 6th loading;②absorption ratio of power was the relative value of areas between hysteresis loop and under the buildup curve,calculated by Metlab7.0 software according to the stress-strain curve of the fifth loading and unloading,in the same X-axis strain(0～55%), Y-axis stress(0～2.0MPa) and 1.25 ratio of X-axis/Y-axis drawn by Origin7.5;③normalization creep was the ratio of creep between every other 5min with continous load(600N) and at the beginning of 600 N;④normalization mass and volume percentage were the ratio of mass and volume after unloading relative to them before loading.The elastic modulus and absorption ratio of power between CoPP and PVA hydrogels were compared by independent sample t test,and normalization mass and volume percentage were compared by variance analysis of repeated measure data and independent sample t test.
3.The in vitro cytotoxicity of CoPP hydrogel was tested by NCTC L929 cell lines, which were divided into four groups:normal,negative control(PVA/FD and PVA/AE),experiment(CoPP/FD and CoPP/AE) and positive control(0.64% phenol),regularly intervened by each treatment.The growth status of cells were observed regularly,OD values of each group were measured at 2nd,4th and 7th day and then relative growth rates(abbr.RGR) were calculated.There were thirty SD rats(15 male and 15 female,body mass 182g±13g) for inflammatory reaction in vivo.CoPP and PVA hydrogels(10mm*3mm*3mm,Length*Width* Thickness) were implanted into left and right intramuscular of gluteus of SD rats,respectively. Postoperative wounds and general status of rats were observed,and local muscles containing with hydrogels were taken and sectioned at 1st,4th,and 12th week.The macroscopic specimen and microscopic reaction of inflammatory were observed and evaluated according to the standard of GB/ T 161752-1996,including the microstructure of CoPP hydrogel with light microscope.
4.The lamellar lumbar MRI of normal Chinese subject was screened from subjects accepting lumbar standard scan by GE Signa Excite 3.0.Trauma,inflammation, deformity and tumor of anterior and middle column of spine were excluded.The criteria of inclusions were at age of 20～50 years old,disc degeneration not exceeding Pfirrmann 2,degeneration of endplate not exceeding Modic 1.The selected subject was scanned in T2 serials to get DICOM 3.0 MR imaging of lumbar in sagittal or coronal view,with 1mm～1.5mm thickness and 0 mm space. These DICOM images of lumbar MR were imported into the Mimics10.01 software.After accurate segmentation of nucleus pulposus in the imaging,the three-dimension(3D) models were reconstructed by Calculate 3D function of Mimics.The sagittal diameter,transverse diameter,height and slipping angle of nucleus pulposus were measured in MR imaging and 3D model,respectively.The shape of prosthetic nucleus pulposus made of CoPP hydrogel was designed according to the reconstructive 3D model of nucleus pulposus.According to equation of volume dehydrant ratio and volume swelling ratio of CoPP hydrogel, the volume conversion coefficents of preparation(V_(pre)) and dehydrated implant (V_(imp)) relative to human nucleus pulposus were calculated.The expected sizes of CoPP hydrogel prosthesis were multiplying results between the cube roots of volume conversion coefficents and anatomic sizes of human nucleus pulposus.On basis of expected sizes,shapes and sizes of prosthetic nucleus pulposus made of CoPP hydrogel were optimally designed.
Results
1.CoPP hydrogel was successfully prepared from the water solution of PVA(25%) and pectin(5%) by swelling,heating with low frequency oscillation and freezing thawing,whose cylindrical shapes can be maintained not by vacuum freeze-dry but by anhydrous alcohol in the dehydrating course.However,the dehydrant ratios of vacuum freeze-dry hydrogels were significantly superior to them of anhydrous alcohol hydrogels.The dehydrant ratios of PVA hydrogel were significantly superior to them of CoPP hydrogel in the same dehydrant course. The FTIR and XRD graph of CoPP hydrogel were superposition of PVA and pectin,respectively.There were two melt apexes in DSC graph of CoPP hydrogel, at the temperature of 182.6℃and 225.1℃,whose posterior apex was corresponding to the melt apex of PVA powder.The section of CoPP hydrogel were full of 10μm-50μm pores with granules;however,there was no pore in the section of PVA hydrogel.The diffusion characteristic index(n) of CoPP hydrogel was near to 0.5,and the swelling velocity constant(k),the swelling ratio of mass (197.38%～252.49%) and swelling ratio of volume(241.47%～308.93%) of CoPP hydrogel at the swelling equilibration were superior to those of PVA hydrogel.
2.There was not any test pieces of CoPP and PVA hydrogels destroyed by loading and unloading.All curvatures of stress-strain consisted two parts:the first was mild starting,and the posterior was steep rise,which were nonlinear.The elastic modulus of CoPP hydrogel was(2.76±0.09) MPa,near to 3MPa which is optimal elastic modulus of preformed nucleus prosthesis,and was significantly inferior to that of PVA hydrogel(4.89±0.61) MPa.There was also significant difference of absorption ratio of power between CoPP hydrogel(12.50±1.47)%and PVA hydrogel(8.57±1.78)%.Normalization creeps of CoPP and PVA hydrogels under 30 min continuous loads were 1.076±0.005 and 1.105±0.030,respectively.The normalization mass percentage of CoPP hydrogel was(97.67%±0.98)%, significantly inferior to that of PVA hydrogel(99.15±0.18)%.At time of 24 hours after unloading,there was no significance of the normalization percentage of mass and volume between two kinds of hydrogels.
3.The L929 cells of negative group(PVA/FD and PVA/AE) and experiment group (CoPP/FD and CoPP/AE) were long fusiform adhered to the wall at the same time relative to normal group,and occurred contact growth inhibiting.Less 10%of cells were circular with discrete grain in negative and experimental groups.RGRs of CoPP group were over 75%,RGRs of PVA group were over 80%,and cytotoxicity of both groups was grade 0～1.On the contrast,RGRs of positive group were inferior to 24%and cytotoxicity was over grade 4.All of experimental rats waked up and began walking during 2～4 hours after operation,without death, and wounds healed primarily without red swelling,exudation or suppuration.At week 1,PVA and CoPP hydrogels were surrounded by bright and red muscles without exudation or suppuration,movable and were larger than those of implants. The sections of CoPP hydrogel were basophilia,stained blue and porous,however, sections of PVA hydrogel were close to neutrophilia,stained undertint red,and pligoporous with smaller pore.The inflammatory reactions of local tissue were grade 4 of inflammatory cell infiltrate(ICI) without integrate cyst.At week 4,the PVA and CoPP hydrogels were also surrounded by normal muscle,however,were not movable.The inflammatory reactions of local tissue were grade 1 of ICI and grade 4 of cyst reaction.At week 12,volumes and shapes of PVA and CoPP hydrogels were similar to that at 1st and 4th week,and both ICI and cyst of the local tissue were grade 1.Sections of CoPP hydrogel were also basophilia,stained blue,whose edge were consistent without defect.There were full of pores with micrograins at 12th week,which were similar to that at 1st and 4th week.
4.Lamellar lumbar MR images of 34 years old woman in this paper were scanned in coronal vies with 1.2mm thickness and 0 mm space by T2 serials(OCor fs, TE82.7ms,TR4000ms),including anterior and posterior margin of invertebral discs from L1 to S1.There were 44 sheets of DICOM3.0 images of MR,whose pixel size was 0.625mm.The disc degeneration of lumbar MRI was Pfirrmann 1～2,whose nucleus pulposus was segmentated well.3D models of nucleus pulposus were successfully reconstructed from lamellar lumbar MR images, whose lateral views were ellipse from fiat to rear fomix,and whose shapes were from discs-form to rugby-form.The sagittal diameter,transverse diameter,height were increasing from upper to lower,especially transverse diameter.The average of sagittal diameter,transverse diameter and height in MR images were (20.8±1.5)mm,(34.7±3.5)mm and(9.2±1.1)mm,which were similar to them of 3D models.The dehydrated CoPP hydrogel prosthesis would be implanted,whose volume was 25.90%～29.29%and whose size was 63.74%～66.41%of human nucleus pulposus.The CoPP hydrogel prosthesis were ellipsoid near to rugby,with four sizes(S,M_1,M_2 and L),whose sagittal diameters were 13mm～14mm, transverse diameters were 20mm～24mm and heights were 5.5mm～7.0mm.
Conclusions
1.It is successful for swelling,heating with low frequency oscillation and freezing thawing to prepare CoPP hydrogel,and it is proper to be dehydrated by anhydrous alcohol.CoPP hydrogel is porous and with determinate crystallization,including two kinds of crystallization zones.CoPP hydrogel has good rehydration with Fick model diffusion of water,and are faster and larger than those of PVA hydrogel.
2.CoPP hydrogel is a kind of viscoelastic material,which is softer and more tenacious,has better absorption shock of load,whose elastic modulus is near to the optimal elastic modulus of preformed prosthetic nucleus pulposus,has better permeability of substance in contrast to PVA hydrogel,and is suitable to made preformed prosthesis of lumbar nucleus pulposus.
3.The cytotoxicity of CoPP hydrogel in vitro is 0～1 grade,the inflammatory reaction of CoPP hydrogel in vivo is mild,and then the basic biocompatibility of CoPP hydrogel is good and there is no degradation of CoPP hydrogel.The genotoxicity,carcinogencity and reproductive toxicity of CoPP hydrogel need evaluating in the future.
4.3D model of nucleus pulposus can be successfully reconstructed from lamellar lumbar MR images.The CoPP hydrogel prosthesis of nucleus pulposus is ellipsoid near to rugby,matching with the shape of cartilaginous endplate,which is helpful to reasonable transmission of load.It is smaller volumes that help CoPP hydrogel prosthesis to be held with ball-and-socket instrument and minimally invasively implanted by posterior diskoscope.
引文
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