组织工程化犬气管的生物力学研究
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摘要
目的:
各种原因如肿瘤、外伤、战伤等造成的气管缺损在临床上较为常见,当缺损较大,难以直接吻合修复时,气管代替物就成为重建气管的理想手段,目前组织工程化气管因其在免疫排斥、材料来源等方面的优势逐渐为人重视。同时移植后的气管塌陷问题也一直在困扰着人们,气管的塌陷除了免疫、血运等方面的因素外,去抗原的处理过程会不会对其力学支撑性能有影响呢?本实验的目的在于检测不同方法去抗原处理后的脱细胞气管基质的张开角和残余应变,来推断其是否存在力学性能的改变。为气管的组织工程化研究提供力学方面参考。
方法:
本实验取用犬甲状软骨下第5-10气管环,共分为三组:A组为深低温冷冻组,B组为新鲜气管组,C组为改良Courtman法气管黏膜脱细胞组。冷冻气管组:气管经程序降温后,先放入-196℃的液氮中冷冻保存6周,再复温后检测;改良的Courtman方法气管黏膜脱细胞处理移植组:先经非离子型表面活性剂TritonX-100、酶制剂联合处理黏膜脱细胞后再检测。新鲜组:取材后于4℃D-Hanks内保存,并尽快检测。检测指标及方法:选取直径及形态相近的气管段,着色笔分别标明气管软骨部分和肌肉部分的中点,垂直于气管纵轴方向,将相邻软骨环之间的韧带剪断,使之成为无载荷状态的独立气管环,按顺序盛放在装有生理盐水的培养皿中,将培养皿放在扫描仪上直接扫描,从而将无载荷状态气管环的几何信息数字化存储在计算机中。取得信息后,将气管环自扫描仪上取下,从气管环肌肉部分中点剪开,从而得到零应力状态下气管扇形体,将其放回培养皿中,静置20分钟,待其形态稳定后,再放回扫描仪进行扫描,从而取得零应力状态下气管环的数字几何信息。最后,运用专用图像分析软件处理扫描所得图像,测得无载荷状态气管环和零应力状态气管扇形体的内、外壁弧长以及张开角等几何尺寸。
结果:
经统计分析,结果(1)新鲜气管组(B组)张开角为101.379±12.290度,大于采用深低温冷冻法脱细胞组(A组)的张开角42.879±12.295度(P＜0.01);(2)B组张开角大于采用改良Courtman法脱细胞组(C组)的张开角72.211±10.487度(P＜0.01);(3)C组张开角大于A组张开角(P＜0.01),均有统计学差异。由此可初步推断,采用冷冻方法进行脱细胞处理对气管的生物力学性能会产生较明显负面影响,而采用改良Courtman法进行脱细胞处理对气管力学性能的负面影响较前者来说要相对小一些。
根据公式计算出各组气管标本的残余应变。对内壁残余应变E_i进行方差分析,可认为各组均数相等,即三组气管标本内壁残余应变无统计学差异(F=1.91,P=0.1628,按α=0.0500水准)。对外壁残余应变E_o进行方差分析,得F=7.52,P=0.0018,按α=0.0500水准,认为各组总体均数不相等。进而对E_o采用三组之间两两比较的Q检验进行统计,结果A组E_0(0.158±0.059)小于B组E_o(0.259±0.080)且有统计学差异(Q=5.3402,p＜0.01),B组E_o大于C组E_o(0.187±0.072)且有统计学差异(Q=3.6547,p＜0.05),而A、C组E_o之间无统计学差异(Q=1.4761,P＞0.05)。即新鲜组气管外壁残余应变大于另外两组脱细胞气管基质,且差异具有统计学意义,而两种不同方法脱细胞气管基质的外壁残余应并没有统计学意义上的差异。
结论:
改良Courtman气管脱细胞法构建的组织工程气管基质张开角明显大于冷冻法处理后的气管基质张开角,这说明采用改良Courtman气管脱细胞法构建组织工程气管过程中,气管力学性能变化较小,即改良Courtman气管脱细胞法构建的组织工程气管基质抗压缩能力优于冷冻法处理组的气管基质。
Objective:Tracheal defects caused by wounds,tumors or battlefield hurt are tough problems for clinic doctors.While the end-to-end suture can not be used in repairing the large defect,tracheal substitute became the only effective way to reconstruct the form and function of airway.Tissue-engineered trachea has become more and more hot due to its advantages in the area of immunologic rejection and material resource.The problem that trachea grafts will sink after transplantation is still exist.Whether the process of making tracheal acellular matrix will decrease biomechanical property besides the immunological rejection and the revascularization of the tracheal allografts is still unknown.The aim of this experiment is to detect the opening angle and residual strain of acellular matrix of canine trachea under unload and zero stress situation,in order to find the basic mechanical property change of tissue engineered trachea,and provide the basic mechanical reference for tracheal tissue engineering.
Method:The 5~(th)-10~(th)trachea rings below thyroid cartilage of canine were chosen,and were divided into three groups.Group A:cryopreserved tracheal acellular matrix(n=14),Group B:fresh trachea(n=14),Group C:acellular matrix treated with modified Courtman to remove epithelium of tracheal mucosa(n=12).Group A:The acellular matrix were stored in liquid nitrogen(-196 degrees C)for six weeks and then rewarmed,Group B:fresh trachea stored in 4℃D-Hanks solution without any disposal.Group C:The acellular matrix were treated with non-ionic surfactants(NIS) (Triton X-100 and enzyme preparation)to remove epithelium of mucosa.
The index and method of detection:The trachea segments of similar diameter and shape were marked at the middle point of muscle and the junction between cartilage and muscle,then sniped the ligaments between cartilage tings to achieve unload single tracheal rings.These rings were put into culture capsule which contain normal saline and detected by scanner.After stored the digital messages into computer,cut off the muscle at the middle point of the tracheal rings to achieve the tracheal sector. Then they were placed for 20 minutes for stabilization,and detected by scanner.The pictures were analyzed by special software to achieve the opening angle and inner and outer arc length under unload and zero stress situation.
Results:The mean opening angle of Group B(fresh trachea)was 101.379±12.290 degree,of group A(cryopreserved tracheal acellular matrix)was 42.879±12.295 degree,of group C(acellular matrix treated with modified Courtman) was 72.211±10.487 degree.The opening angles between each group had statistical difference:A and B q=18.5742,P＜0.0010,B and C q=8.8979,P＜0.0010;A and C t=8.9476,P＜0.0010。This result showed that both two process of making tracheal acellular matrix will decrease the opening angle,the modified Courtman method might had a lighter influence than hypothermal method.
The variance analysis of outer residual strain showed that they had different means(P=0.0018,α=0.0500).The "q" test of independent sample between each group showed that residual strain of group B was bigger than A(q=5.3402,p＜0.01)(had statistical difference);residual strain of group B was bigger than group C(q=3.6547, p＜0.05)(had statistical difference),while group A and group C had no statistical difference(q=1.4761,P＞0.05).This result showed that fresh trachea had bigger residual strain than that of the other two acellular matrix groups,and there was no statistical difference between cryopreserved tracheal acellular matrix and acellular matrix treated with modified Courtman.
Conclusion:Fresh trachea has distinct larger opening angle than that of acellular matrix.This means in the process of removing cells from the tracheas,various physical and chemical factors can deduce the anti- compressibility of acellular trachea matrix,and the tissue engineered acellular matrix treated with modified Courtman has a better anti- compressibility than cryopreserved tracheal acellular matrix.
各种原因如肿瘤、外伤、战伤等造成的气管缺损在临床上较为常见,当缺损较大,难以直接吻合修复时,气管代替物就成为重建气管的理想手段,目前组织工程化气管因其在免疫排斥、材料来源等方面的优势逐渐为人重视。同时移植后的气管塌陷问题也一直在困扰着人们,气管的塌陷除了免疫、血运等方面的因素外,去抗原的处理过程会不会对其力学支撑性能有影响呢?本实验的目的在于检测不同方法去抗原处理后的脱细胞气管基质的张开角和残余应变,来推断其是否存在力学性能的改变。为气管的组织工程化研究提供力学方面参考。
方法:
本实验取用犬甲状软骨下第5-10气管环,共分为三组:A组为深低温冷冻组,B组为新鲜气管组,C组为改良Courtman法气管黏膜脱细胞组。冷冻气管组:气管经程序降温后,先放入-196℃的液氮中冷冻保存6周,再复温后检测;改良的Courtman方法气管黏膜脱细胞处理移植组:先经非离子型表面活性剂TritonX-100、酶制剂联合处理黏膜脱细胞后再检测。新鲜组:取材后于4℃D-Hanks内保存,并尽快检测。检测指标及方法:选取直径及形态相近的气管段,着色笔分别标明气管软骨部分和肌肉部分的中点,垂直于气管纵轴方向,将相邻软骨环之间的韧带剪断,使之成为无载荷状态的独立气管环,按顺序盛放在装有生理盐水的培养皿中,将培养皿放在扫描仪上直接扫描,从而将无载荷状态气管环的几何信息数字化存储在计算机中。取得信息后,将气管环自扫描仪上取下,从气管环肌肉部分中点剪开,从而得到零应力状态下气管扇形体,将其放回培养皿中,静置20分钟,待其形态稳定后,再放回扫描仪进行扫描,从而取得零应力状态下气管环的数字几何信息。最后,运用专用图像分析软件处理扫描所得图像,测得无载荷状态气管环和零应力状态气管扇形体的内、外壁弧长以及张开角等几何尺寸。
结果:
经统计分析,结果(1)新鲜气管组(B组)张开角为101.379±12.290度,大于采用深低温冷冻法脱细胞组(A组)的张开角42.879±12.295度(P＜0.01);(2)B组张开角大于采用改良Courtman法脱细胞组(C组)的张开角72.211±10.487度(P＜0.01);(3)C组张开角大于A组张开角(P＜0.01),均有统计学差异。由此可初步推断,采用冷冻方法进行脱细胞处理对气管的生物力学性能会产生较明显负面影响,而采用改良Courtman法进行脱细胞处理对气管力学性能的负面影响较前者来说要相对小一些。
根据公式计算出各组气管标本的残余应变。对内壁残余应变E_i进行方差分析,可认为各组均数相等,即三组气管标本内壁残余应变无统计学差异(F=1.91,P=0.1628,按α=0.0500水准)。对外壁残余应变E_o进行方差分析,得F=7.52,P=0.0018,按α=0.0500水准,认为各组总体均数不相等。进而对E_o采用三组之间两两比较的Q检验进行统计,结果A组E_0(0.158±0.059)小于B组E_o(0.259±0.080)且有统计学差异(Q=5.3402,p＜0.01),B组E_o大于C组E_o(0.187±0.072)且有统计学差异(Q=3.6547,p＜0.05),而A、C组E_o之间无统计学差异(Q=1.4761,P＞0.05)。即新鲜组气管外壁残余应变大于另外两组脱细胞气管基质,且差异具有统计学意义,而两种不同方法脱细胞气管基质的外壁残余应并没有统计学意义上的差异。
结论:
改良Courtman气管脱细胞法构建的组织工程气管基质张开角明显大于冷冻法处理后的气管基质张开角,这说明采用改良Courtman气管脱细胞法构建组织工程气管过程中,气管力学性能变化较小,即改良Courtman气管脱细胞法构建的组织工程气管基质抗压缩能力优于冷冻法处理组的气管基质。
Objective:Tracheal defects caused by wounds,tumors or battlefield hurt are tough problems for clinic doctors.While the end-to-end suture can not be used in repairing the large defect,tracheal substitute became the only effective way to reconstruct the form and function of airway.Tissue-engineered trachea has become more and more hot due to its advantages in the area of immunologic rejection and material resource.The problem that trachea grafts will sink after transplantation is still exist.Whether the process of making tracheal acellular matrix will decrease biomechanical property besides the immunological rejection and the revascularization of the tracheal allografts is still unknown.The aim of this experiment is to detect the opening angle and residual strain of acellular matrix of canine trachea under unload and zero stress situation,in order to find the basic mechanical property change of tissue engineered trachea,and provide the basic mechanical reference for tracheal tissue engineering.
Method:The 5~(th)-10~(th)trachea rings below thyroid cartilage of canine were chosen,and were divided into three groups.Group A:cryopreserved tracheal acellular matrix(n=14),Group B:fresh trachea(n=14),Group C:acellular matrix treated with modified Courtman to remove epithelium of tracheal mucosa(n=12).Group A:The acellular matrix were stored in liquid nitrogen(-196 degrees C)for six weeks and then rewarmed,Group B:fresh trachea stored in 4℃D-Hanks solution without any disposal.Group C:The acellular matrix were treated with non-ionic surfactants(NIS) (Triton X-100 and enzyme preparation)to remove epithelium of mucosa.
The index and method of detection:The trachea segments of similar diameter and shape were marked at the middle point of muscle and the junction between cartilage and muscle,then sniped the ligaments between cartilage tings to achieve unload single tracheal rings.These rings were put into culture capsule which contain normal saline and detected by scanner.After stored the digital messages into computer,cut off the muscle at the middle point of the tracheal rings to achieve the tracheal sector. Then they were placed for 20 minutes for stabilization,and detected by scanner.The pictures were analyzed by special software to achieve the opening angle and inner and outer arc length under unload and zero stress situation.
Results:The mean opening angle of Group B(fresh trachea)was 101.379±12.290 degree,of group A(cryopreserved tracheal acellular matrix)was 42.879±12.295 degree,of group C(acellular matrix treated with modified Courtman) was 72.211±10.487 degree.The opening angles between each group had statistical difference:A and B q=18.5742,P＜0.0010,B and C q=8.8979,P＜0.0010;A and C t=8.9476,P＜0.0010。This result showed that both two process of making tracheal acellular matrix will decrease the opening angle,the modified Courtman method might had a lighter influence than hypothermal method.
The variance analysis of outer residual strain showed that they had different means(P=0.0018,α=0.0500).The "q" test of independent sample between each group showed that residual strain of group B was bigger than A(q=5.3402,p＜0.01)(had statistical difference);residual strain of group B was bigger than group C(q=3.6547, p＜0.05)(had statistical difference),while group A and group C had no statistical difference(q=1.4761,P＞0.05).This result showed that fresh trachea had bigger residual strain than that of the other two acellular matrix groups,and there was no statistical difference between cryopreserved tracheal acellular matrix and acellular matrix treated with modified Courtman.
Conclusion:Fresh trachea has distinct larger opening angle than that of acellular matrix.This means in the process of removing cells from the tracheas,various physical and chemical factors can deduce the anti- compressibility of acellular trachea matrix,and the tissue engineered acellular matrix treated with modified Courtman has a better anti- compressibility than cryopreserved tracheal acellular matrix.
引文
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2、Pearson FG,Thompson DW,et al.Adenoid cystic carcinoma of the trachea.Ann Thorac Surg,1974;18:16-29.
3、Schuller DE,Parrish RT.Reconstruction of the larynx and trachea:Arch Otolaryngol Head Neck Surg.1988;114(3):278-286.
4、Bailey B J,Kosoy J.Observations in the development of tracheal prostheses and tracheal transplantation.Laryngoscope.1970;80(10):1553-1565.
5 Culld L,Lally EKPA,Mair EA,et al.Tracheal reconstruction with polytetrafluoroethylene grft in dogs.AnnThoracSurg,1990;50(6):899.
6 Balderman SC,Weinblatt G,Buffalo NY.Tracheal autograft revescularization.J Thorac Cardiovasc Surg,1987;94:434-41.
7、DeSagun EZ,Botts JL,Srivastava A,et al.Long-term outcome of xenogenic dermal matrix implantation in immunocompetent rats.J Surg Res,2001 Mar;96(1):96-106.
8、Allman A J,McPherson TB,Badylak SF,et al.Xenogeneic extracellular matrix grafts elicit a TH2-restricted immune response.Transplantation,2001 Jun 15;71(11):1631-1640.
9、冯元桢著.生物力学——运动、流动、应力和生长.邓善熙译.成都:四川教育出版社,1993.
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