脊椎切除重建中不同截面积钛网内植骨生长情况的实验研究
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摘要
目的：建立一种腰椎切除脊柱融合动物模型,并观察椎体切除植入不同直径钛网后骨融合情况,对影响脊柱支撑体内骨生长情况有关因素进行分析,探寻在不影响骨愈合及生物力学情况下的最小的钛网与椎体截面积比,以指导临床在行腰椎切除融合术时选择合适直径钛网植入。自从Hibbs于1911年首次报道了脊柱融合的手术技术后,随着内固定技术及内固定材料的发展,脊柱融合技术已广泛应用于脊柱疾患的治疗。目前随着社会人口老龄化、结核发病率以及交通意外事故的增加,脊柱肿瘤、脊柱结核、脊柱爆裂性骨折等也随之增加,因此椎体切除内固定术也随之发展。一种新的骨替代材料,内固定材料及新技术等应用于人体之前,首先都需建立相应的动物脊柱融合模型进行各项指标的测试及研究,确定安全后才能进一步行临床试验。目前脊柱动物融合模型种类繁多,方式不一,其中小动物模型应用较多,大型动物相对较少,且融合效果及手术并发症不一。有报道应用新西兰白兔做动物脊柱融合模型死亡率和并发症达到20%以上,应用大型动物做脊柱融合模型会更为复杂。大型动物融合模型建立相对难度较大,应用山羊行脊柱融合模型目前较常见,但多限于颈椎,腰椎融合模型建立少见报道,难点在于手术复杂、时间较长,麻醉时间长,出血量较多,术后并发症多。动物椎体融合模型手术方式有三种,分别为前路、后路360°、后外侧入路融合。前路及后路360°造模对动物创伤较大,后外侧入路经腹膜外到达椎体,对动物创伤相对较小,并可在直视下切除椎体。为此我们探索一种有效的、重复性好的大型动物腰椎切除钛网植入融合模型。
一直以来,钛网常被用于治疗骨缺损,对钛网进行合理的剪裁与塑形后将其填充骨块,并移植于目标区域提供支撑作用,该技术明显提高了脊柱植骨融合率。但临床对椎体切除后植入钛网直径没有统一规范,造成许多问题,如植入后愈合率不高,强度不够,内固定取出后再骨折,或对愈合情况判断失误,未愈合即取出内固定等。一味选择粗钛网使钛网内填充的自体骨或异体骨骨量增多,导致供骨区并发症增多及给病人造成经济浪费。反之一味选择细钛网又可能导致骨愈合率不高或愈合强度不够、钛网易沉陷等,从而达不到坚强内固定的目的,造成手术失败。本实验通过观察腰椎切除脊柱融合动物模型椎间融合器的植骨融合情况,对影响脊柱支撑体内骨生长情况有关因素进行分析,探寻在不影响骨愈合及生物力学情况下钛网与椎体最小的截面积比,并分析有效评价钛网内骨融合情况的手段。
方法：我们以经后外侧腹膜外入路椎体切除钛网植入钢板内固定的方式建立腰椎切除脊柱融合动物模型。选取成熟山羊9只,雌雄不限,年龄1.5-3岁,体重35±2.5kg,山羊个体相近,以保证山羊腰椎体大小基本相同,随机分为三组,每组三只。三组均行单椎体切除,分别植入直径为10mm、12mm、16mm的钛网,10mm组为A组,12mm组为B组,16mm组为C组。术后饲养4个月。术后每月行X线检查,观察钛网位置及融合情况。羊处死后取脊柱标本行X线及三维CT检查,测量钛网-椎体接触面及钛网内不同部位CT值,观察骨愈合情况,并且根据三维CT结果计算钛网和椎体接触面截面积比值,以此来确定本实验探寻的钛网和椎体最小的截面积比。脊柱标本行前屈、后伸、侧弯、扭转等工况下生物力学检测,以评价骨愈合强度,并建立有限元模型进一步验证离体实验生物力学结果,及分析不同直径钛网植入后钛网、终板应力分布情况对融合情况的影响。标本行组织学切片观察接触面及钛网内植骨融合情况。分别于术前、术后第3天、5天、7天、2周、1个月抽静脉血,分离血清,然后用酶联免疫分析试剂盒测定骨形成调节细胞因子及代谢标志物(BMP-2、TGF-β、VEGF、BGP、BALP)血清值,观察这些指标在椎体切除并植入不同直径钛网后分泌、代谢情况,其出现高峰值时间及峰值大小与骨愈合情况是否有关,并观察这些指标之间有何关系。对各项检测结果进行统计学分析。
结果：成功构建了山羊腰椎体切除脊柱融合动物模型,实验组山羊均成活,一例切口感染,予以切口换药处理后痊愈,一例术后双后肢不全性瘫痪,3日后完全恢复。术后三维CT检查,测量钛网和椎体接触面截面积比及钛网和椎体接触面CT值,结果示10mm组钛网与椎体接触面截面积比是1/3,12mm组钛网与椎体接触面截面积比是1/2,16mm组钛网与椎体接触面面积比是2/3。钛网和椎体接触面CT值与相邻正常椎体CT值的比值结果示B组及C组无明显统计学差异,A组与B组及A组与C组统计学均有差异。B组及C组生物力学检测无明显统计学差异,A组与B组及A组与C组生物力学检测统计学有差异。组织学切片观察示B组及C组接触面处及钛网内骨小梁较A组致密、粗大,其中以C组生长最好,三组均显示距离接触面越远钛网内骨小梁越稀疏,且有无骨质区。三组血清学骨形成调节及代谢指标(BMP-2、TGF-β、VEGF、BGP、BALP)均于术后3天开始升高,且于5天—2周内达到峰值,随后开始下降,高峰值出现时间无统计学差异,但A组峰值较B组及C组均低,且A组的TGF-β、BGP和BALP高峰值出现时间较B组及C组早。
结论：本实验成功构建了山羊腰椎切除脊柱融合动物模型,且操作简单,重复性好,是一种较好的脊柱融合动物模型建立方法。腰椎椎体切除钛网植入时,钛网与椎体接触面比值应达到1/2,才对骨愈合时间及强度无明显影响。骨形成调节及代谢标志物高峰值出现时间及峰值大小与骨愈合强度有一定的相关性。三维CT检查可作为评价骨融合情况的首选手段。
Objective:To set up a kind of animal model of lumbar corpectomy spinal fusion, observe bone fusion conditions upon implantation of titanium cages of different diameter on vertebra resection, analyze relevant factors that impact graft of bones in spinal supporting, probe into the minimum area ratio of titanium cage section to vertebral section without impact to bone healing and biomechanical property, to guide selection of appropriate diameter titanium cage to be implanted in clinical lumbar corpectomy fusion operation.
Since spinal fusion operation technology was firstly reported by Hibbs in 1911, along with development of both internal fixation technology and internal fixation materials, spinal fusion has been widely applied in treatment of spinal diseases. In the wake of social population aging and increase of incidence of tuberculosis and traffic accident, spinal tumor, spinal tuberculosis and spinal bursting fracture follow the trend; therefore vertebra resection internal fixation keeps abreast. Prior to any new bone-replacement material, internal fixation and new technology etc. are introduced to human being, at first a corresponding animal spinal fusion model need to be set up to test and study all kinds of indices, and further clinical experiment is allowed after safety of the indices are confirmed.
At present there are a great variety of animal spinal fusion models with different patterns, among which small animal models are more applies than that of large animal models, and fusion effect and operation complication are different. It is reported that death rate and complication rate of New Zealand white rabbit as spinal fusion model is as much as 20%, while using large animal as spinal fusion model is more complicated. It is rather difficult to set up large animal fusion models, at present, goat is often used as spinal fusion model but is limited on cervical fusion while lumbar fusion is seldom reported as the difficult points are complicated and long time of operation, long anesthetic time, massive bleeding and more after-treatment complications. There are three patterns of animal spinal vertebra fusion model operation, namely anterior, posterior 360°and posterolateral approach. Anterior and posterior 360°model wound animals more largely, while posterolateral approach reaches vertebra via exterior of peritoneum that wounds animals relatively smaller as well as vertebra resection can be conducted by direct viewing. Therefore we probe into an effective large animal lumbar corpectomy titanium cage implantation fusion model with sound repetitiveness. For a long time, titanium cage is often used for treatment of bone defect; reasonable tailor and formation is made to titanium cage then bone block is filled, and it is implanted to targeted area to provide supporting. The technology apparently improves fusion rate of spinal implantation.
However there is not a uniform specification on diameter of titanium cage implanted in clinical vertebral resection, which caused a number of problems, for instance, low healing rate after implantation, insufficient strength, bone re-fracture after removing of interior fixation, or misjudgment on healing condition and removing interior fixation before completely healed. Blindly choosing thick titanium cages makes filled bone mass increase for either autogenous or allograft bone, which causes increase of complications in bone supply area as well as economic waste to patients. Vise versa blindly choosing thin cages may lead to low bone healing rate or insufficient strength, and the cages are easily sank, which fails to strength the interior fixation and causes operation failure. By virtue of observation of implanted bone fusion condition of animal model cage of lumbar corpectomy spinal fusion, this experiment analyzes relevant factors that impact growth of bones in spinal supporting, probes into the minimum area ratio of titanium cage section to vertebral section without impact bone healing and biomechanical property and analyzes measures that can evaluate bone fusion conditions in titanium cages effectively.
Methods:We set up a lumbar corpectomy spinal fusion animal model by means of titanium cage implantation plate interior fixation of posterolateral approach vertebral resection via posterolateral peritoneum. Choose nine mature goats no matter male or female at 1.5-3 years of age and 35±2.5 kg of weight; Individuality of each goat is similar to guarantee size of lumbar of goats are basically equal; the goats are divided into three groups at random with three goats in each group. All goats in three groups are single vertebral cut off and implanted diameter 10mm,12mm and 16mm titanium cages respectively; 10mm is group A,12mm is group B and 16mm is group C. The goats are feed four months after the operation and examined by X-ray on a monthly basis to observe location of the titanium cage and fusion condition. After the goats are slaughtered, their spinal samples are taken to examine by X-ray and three-dimension CT, measure CT values of contact area of titanium cage and the spine as well as CT values in different locations of the titanium cage, observe bone healing status and calculate area ratio of titanium cage section to vertebral section based on result of three-dimension CT to determine the minimum area ratio of titanium cage section to vertebral section, which is looked for in this experiment. The spine samples are taken biomechanical examination under conditions of flexion, extension, lateral and torsion to evaluate bone healing strength. In the meantime, finite element model is set up to further verify in vitro biomechanical result and analyze impact of titanium cage and lamina terminalis stress distribution to fusion of different diameter cages implantation. Samples are taken histological slice to observe contact area and implanted bone fusion status in titanium cage. Draw venous blood of the goats respectively before the operation and at the third, fifth, seventh day, two weeks and one month after the operation to separate serum, then use enzyme linked immuno assay kit to determine bone formation adjustment cytokine and metabolism markers (BMP-2、TGF-β、VEGF、BGP、BALP) serum value, observe secretion and metabolism conditions of the indices upon vertebral resection and implantation of titanium cages with different diameter, whether occurrence of peak value time and quantity of peak value are related with bone healing, as well as to observe relationship of the indices. Statistic analyses are made to all examination results.
Results:Successfully set up goat lumbar corpectomy spinal fusion animal model, all goats in experiment groups survive the operation, one goat is incision infected and heals after dressing on incision; one goat's both posterior limbs paralyze incompletely but thoroughly resume after three days. Examined by postoperative three-dimension CT and measured area ratio of titanium cage section to vertebral section and CT value of contact area of titanium cage and vertebra, the result shows that area ratio of titanium cage section to vertebral section of 10mm group is 1/3, area ratio of titanium cage section to vertebral section of 12mm group is 1/2 and area ratio of titanium cage section to vertebral section of 16mm group is 2/3. Result of ratio between CT value of contact area of titanium cage and vertebra and CT value of adjacent normal vertebra shows that there are no apparent statistic differences between group B and C, but there are statistic differences between group A and B, and group A and C. There are no apparent statistic differences on biomechanical examination between group B and C, but there are statistic differences on biomechanical examination between group A and B, and group A and C. Histological slice observation shows contact area and bone trabeculae of group B and group C are compact and thick than that of group A, among which graft of group B is the best; all three groups show that the further to the contact area, the bone trabeculae is thinner and without sclerotin area. Serology bone formation adjustment and metabolism indices (BMP-2、TGF-β、VEGF、BGP、BALP) of three groups all start to increase three days after the operation and reach peak value within five days to two weeks then decrease. There are no statistics differences on peak value occurrence time, but peak value of group A is lower than that of group B and group C, and occurrence time of peak value of TGF-β、BG and BALP of group A is earlier than that of group B and C.
Conclusion:The experiment successfully set up animal model of goat lumbar corpectomy spinal fusion with easy operation and sound repetitiveness, which is a relatively better way to set up spinal fusion animal model. At the time of lumbar corpectomy and titanium cage implantation, area ratio of titanium cage to vertebral section should reach 1/2 that are without apparent impact to bone healing time and strength. Occurrence time and scale of peak value of bone formation adjustment and metabolism markers have certain correlation with bone healing strength. Three-dimensional CT examination can be the first choice to evaluate bone fusion condition.
一直以来,钛网常被用于治疗骨缺损,对钛网进行合理的剪裁与塑形后将其填充骨块,并移植于目标区域提供支撑作用,该技术明显提高了脊柱植骨融合率。但临床对椎体切除后植入钛网直径没有统一规范,造成许多问题,如植入后愈合率不高,强度不够,内固定取出后再骨折,或对愈合情况判断失误,未愈合即取出内固定等。一味选择粗钛网使钛网内填充的自体骨或异体骨骨量增多,导致供骨区并发症增多及给病人造成经济浪费。反之一味选择细钛网又可能导致骨愈合率不高或愈合强度不够、钛网易沉陷等,从而达不到坚强内固定的目的,造成手术失败。本实验通过观察腰椎切除脊柱融合动物模型椎间融合器的植骨融合情况,对影响脊柱支撑体内骨生长情况有关因素进行分析,探寻在不影响骨愈合及生物力学情况下钛网与椎体最小的截面积比,并分析有效评价钛网内骨融合情况的手段。
方法：我们以经后外侧腹膜外入路椎体切除钛网植入钢板内固定的方式建立腰椎切除脊柱融合动物模型。选取成熟山羊9只,雌雄不限,年龄1.5-3岁,体重35±2.5kg,山羊个体相近,以保证山羊腰椎体大小基本相同,随机分为三组,每组三只。三组均行单椎体切除,分别植入直径为10mm、12mm、16mm的钛网,10mm组为A组,12mm组为B组,16mm组为C组。术后饲养4个月。术后每月行X线检查,观察钛网位置及融合情况。羊处死后取脊柱标本行X线及三维CT检查,测量钛网-椎体接触面及钛网内不同部位CT值,观察骨愈合情况,并且根据三维CT结果计算钛网和椎体接触面截面积比值,以此来确定本实验探寻的钛网和椎体最小的截面积比。脊柱标本行前屈、后伸、侧弯、扭转等工况下生物力学检测,以评价骨愈合强度,并建立有限元模型进一步验证离体实验生物力学结果,及分析不同直径钛网植入后钛网、终板应力分布情况对融合情况的影响。标本行组织学切片观察接触面及钛网内植骨融合情况。分别于术前、术后第3天、5天、7天、2周、1个月抽静脉血,分离血清,然后用酶联免疫分析试剂盒测定骨形成调节细胞因子及代谢标志物(BMP-2、TGF-β、VEGF、BGP、BALP)血清值,观察这些指标在椎体切除并植入不同直径钛网后分泌、代谢情况,其出现高峰值时间及峰值大小与骨愈合情况是否有关,并观察这些指标之间有何关系。对各项检测结果进行统计学分析。
结果：成功构建了山羊腰椎体切除脊柱融合动物模型,实验组山羊均成活,一例切口感染,予以切口换药处理后痊愈,一例术后双后肢不全性瘫痪,3日后完全恢复。术后三维CT检查,测量钛网和椎体接触面截面积比及钛网和椎体接触面CT值,结果示10mm组钛网与椎体接触面截面积比是1/3,12mm组钛网与椎体接触面截面积比是1/2,16mm组钛网与椎体接触面面积比是2/3。钛网和椎体接触面CT值与相邻正常椎体CT值的比值结果示B组及C组无明显统计学差异,A组与B组及A组与C组统计学均有差异。B组及C组生物力学检测无明显统计学差异,A组与B组及A组与C组生物力学检测统计学有差异。组织学切片观察示B组及C组接触面处及钛网内骨小梁较A组致密、粗大,其中以C组生长最好,三组均显示距离接触面越远钛网内骨小梁越稀疏,且有无骨质区。三组血清学骨形成调节及代谢指标(BMP-2、TGF-β、VEGF、BGP、BALP)均于术后3天开始升高,且于5天—2周内达到峰值,随后开始下降,高峰值出现时间无统计学差异,但A组峰值较B组及C组均低,且A组的TGF-β、BGP和BALP高峰值出现时间较B组及C组早。
结论：本实验成功构建了山羊腰椎切除脊柱融合动物模型,且操作简单,重复性好,是一种较好的脊柱融合动物模型建立方法。腰椎椎体切除钛网植入时,钛网与椎体接触面比值应达到1/2,才对骨愈合时间及强度无明显影响。骨形成调节及代谢标志物高峰值出现时间及峰值大小与骨愈合强度有一定的相关性。三维CT检查可作为评价骨融合情况的首选手段。
Objective:To set up a kind of animal model of lumbar corpectomy spinal fusion, observe bone fusion conditions upon implantation of titanium cages of different diameter on vertebra resection, analyze relevant factors that impact graft of bones in spinal supporting, probe into the minimum area ratio of titanium cage section to vertebral section without impact to bone healing and biomechanical property, to guide selection of appropriate diameter titanium cage to be implanted in clinical lumbar corpectomy fusion operation.
Since spinal fusion operation technology was firstly reported by Hibbs in 1911, along with development of both internal fixation technology and internal fixation materials, spinal fusion has been widely applied in treatment of spinal diseases. In the wake of social population aging and increase of incidence of tuberculosis and traffic accident, spinal tumor, spinal tuberculosis and spinal bursting fracture follow the trend; therefore vertebra resection internal fixation keeps abreast. Prior to any new bone-replacement material, internal fixation and new technology etc. are introduced to human being, at first a corresponding animal spinal fusion model need to be set up to test and study all kinds of indices, and further clinical experiment is allowed after safety of the indices are confirmed.
At present there are a great variety of animal spinal fusion models with different patterns, among which small animal models are more applies than that of large animal models, and fusion effect and operation complication are different. It is reported that death rate and complication rate of New Zealand white rabbit as spinal fusion model is as much as 20%, while using large animal as spinal fusion model is more complicated. It is rather difficult to set up large animal fusion models, at present, goat is often used as spinal fusion model but is limited on cervical fusion while lumbar fusion is seldom reported as the difficult points are complicated and long time of operation, long anesthetic time, massive bleeding and more after-treatment complications. There are three patterns of animal spinal vertebra fusion model operation, namely anterior, posterior 360°and posterolateral approach. Anterior and posterior 360°model wound animals more largely, while posterolateral approach reaches vertebra via exterior of peritoneum that wounds animals relatively smaller as well as vertebra resection can be conducted by direct viewing. Therefore we probe into an effective large animal lumbar corpectomy titanium cage implantation fusion model with sound repetitiveness. For a long time, titanium cage is often used for treatment of bone defect; reasonable tailor and formation is made to titanium cage then bone block is filled, and it is implanted to targeted area to provide supporting. The technology apparently improves fusion rate of spinal implantation.
However there is not a uniform specification on diameter of titanium cage implanted in clinical vertebral resection, which caused a number of problems, for instance, low healing rate after implantation, insufficient strength, bone re-fracture after removing of interior fixation, or misjudgment on healing condition and removing interior fixation before completely healed. Blindly choosing thick titanium cages makes filled bone mass increase for either autogenous or allograft bone, which causes increase of complications in bone supply area as well as economic waste to patients. Vise versa blindly choosing thin cages may lead to low bone healing rate or insufficient strength, and the cages are easily sank, which fails to strength the interior fixation and causes operation failure. By virtue of observation of implanted bone fusion condition of animal model cage of lumbar corpectomy spinal fusion, this experiment analyzes relevant factors that impact growth of bones in spinal supporting, probes into the minimum area ratio of titanium cage section to vertebral section without impact bone healing and biomechanical property and analyzes measures that can evaluate bone fusion conditions in titanium cages effectively.
Methods:We set up a lumbar corpectomy spinal fusion animal model by means of titanium cage implantation plate interior fixation of posterolateral approach vertebral resection via posterolateral peritoneum. Choose nine mature goats no matter male or female at 1.5-3 years of age and 35±2.5 kg of weight; Individuality of each goat is similar to guarantee size of lumbar of goats are basically equal; the goats are divided into three groups at random with three goats in each group. All goats in three groups are single vertebral cut off and implanted diameter 10mm,12mm and 16mm titanium cages respectively; 10mm is group A,12mm is group B and 16mm is group C. The goats are feed four months after the operation and examined by X-ray on a monthly basis to observe location of the titanium cage and fusion condition. After the goats are slaughtered, their spinal samples are taken to examine by X-ray and three-dimension CT, measure CT values of contact area of titanium cage and the spine as well as CT values in different locations of the titanium cage, observe bone healing status and calculate area ratio of titanium cage section to vertebral section based on result of three-dimension CT to determine the minimum area ratio of titanium cage section to vertebral section, which is looked for in this experiment. The spine samples are taken biomechanical examination under conditions of flexion, extension, lateral and torsion to evaluate bone healing strength. In the meantime, finite element model is set up to further verify in vitro biomechanical result and analyze impact of titanium cage and lamina terminalis stress distribution to fusion of different diameter cages implantation. Samples are taken histological slice to observe contact area and implanted bone fusion status in titanium cage. Draw venous blood of the goats respectively before the operation and at the third, fifth, seventh day, two weeks and one month after the operation to separate serum, then use enzyme linked immuno assay kit to determine bone formation adjustment cytokine and metabolism markers (BMP-2、TGF-β、VEGF、BGP、BALP) serum value, observe secretion and metabolism conditions of the indices upon vertebral resection and implantation of titanium cages with different diameter, whether occurrence of peak value time and quantity of peak value are related with bone healing, as well as to observe relationship of the indices. Statistic analyses are made to all examination results.
Results:Successfully set up goat lumbar corpectomy spinal fusion animal model, all goats in experiment groups survive the operation, one goat is incision infected and heals after dressing on incision; one goat's both posterior limbs paralyze incompletely but thoroughly resume after three days. Examined by postoperative three-dimension CT and measured area ratio of titanium cage section to vertebral section and CT value of contact area of titanium cage and vertebra, the result shows that area ratio of titanium cage section to vertebral section of 10mm group is 1/3, area ratio of titanium cage section to vertebral section of 12mm group is 1/2 and area ratio of titanium cage section to vertebral section of 16mm group is 2/3. Result of ratio between CT value of contact area of titanium cage and vertebra and CT value of adjacent normal vertebra shows that there are no apparent statistic differences between group B and C, but there are statistic differences between group A and B, and group A and C. There are no apparent statistic differences on biomechanical examination between group B and C, but there are statistic differences on biomechanical examination between group A and B, and group A and C. Histological slice observation shows contact area and bone trabeculae of group B and group C are compact and thick than that of group A, among which graft of group B is the best; all three groups show that the further to the contact area, the bone trabeculae is thinner and without sclerotin area. Serology bone formation adjustment and metabolism indices (BMP-2、TGF-β、VEGF、BGP、BALP) of three groups all start to increase three days after the operation and reach peak value within five days to two weeks then decrease. There are no statistics differences on peak value occurrence time, but peak value of group A is lower than that of group B and group C, and occurrence time of peak value of TGF-β、BG and BALP of group A is earlier than that of group B and C.
Conclusion:The experiment successfully set up animal model of goat lumbar corpectomy spinal fusion with easy operation and sound repetitiveness, which is a relatively better way to set up spinal fusion animal model. At the time of lumbar corpectomy and titanium cage implantation, area ratio of titanium cage to vertebral section should reach 1/2 that are without apparent impact to bone healing time and strength. Occurrence time and scale of peak value of bone formation adjustment and metabolism markers have certain correlation with bone healing strength. Three-dimensional CT examination can be the first choice to evaluate bone fusion condition.
引文
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