胰酶化和钙化对异体滑膜内肌腱与骨隧道愈合作用研究
发布时间：2018-07-22 14:19
【摘要】：第一章：对比研究狗趾深屈肌腱、前交叉韧带、跟腱和髌腱之间的差异 目的：本实验的主要目的为对比研究狗趾深屈肌腱、前交叉韧带、跟腱和髌腱之间在生物力学和组织学上的差异。 方法：20只狗后腿用于获取趾深屈肌腱(N=20)、前交叉韧带(N=20)、跟腱中间束(N=20)和带1/3髌腱的骨-髌腱-骨(N=20)。利用摩擦力测试、压痕实验、组织学和免疫组织化学等方法以评估之间的差异。 结果：摩擦力测试发现,第1个循环时,与髌腱组(336.62±110.87mN)和跟腱组(147.89±54.73mmN)相比,趾深屈肌腱组(47.24±14.40mN)和前交叉韧带组(51.98±12.10mN)摩擦力显著较小(P0.05)。第5个循环时,与髌腱组(414.29±104.49mN)和跟腱组(260.11±99.11mN)相比,趾深屈肌腱组(88.73±18.33mN)和前交叉韧带组(126.88±50.83mN)摩擦力亦显著较小(P0.05)。第10个循环时,与髌腱组(442.12±106.69mN)相比,趾深屈肌腱组(132.53±26.08mN)、前交叉韧带组(196.69±84.80mmN)和跟腱组(311.89±124.78mN)摩擦力显著较小(P0.05)。第100个循环时,与髌腱组(571.06±132.50mN)相比,趾深屈肌腱组(397.26±39.98mN)和前交叉韧带组(396.53±90.59mN)摩擦力显著较小(P0.05)。压痕实验发现,与髌腱组抗压模量(2.85±0.43MPa)相比,趾深屈肌腱组(4.11±0.34MPa)、前交叉韧带组(3.54±0.32MPa)和跟腱组(4.71±0.38MPa)的抗压模量均显著较高(P0.05)。组织学检测发现趾深屈肌腱和前交叉韧带的腱外膜光滑,由单一肌腱外膜细胞覆盖；而髌腱和跟腱表面相对粗糙,腱旁组织清晰可见。Lubricin在趾深屈肌腱和前交叉韧带的表面有大量的表达,在细胞外基质和胶原纤维间也有少量表达。在跟腱的腱旁组织可见Lubricin表达,但肌腱表面Lubricin表达较少,腱旁组织和肌腱组织之间可见Lubricin表达,在肌腱的细胞外基质和胶原纤维间也有少量表达。在髌腱的腱旁组织、肌腱的细胞外基质和胶原纤维间可见少量Lubricin表达。 结论：趾深屈肌腱和前交叉韧带具有类似的表面摩擦力,且显著低于跟腱和髌腱,这些特点与肌腱Lubricin表达特点相吻合。 第二章：滑膜内肌腱体外钙化过程优化 目的：骨腱界面愈合是临床上最富有挑战性的问题。本研究的主要目的就是优化肌腱钙化的条件,为下一步的钙化肌腱体内研究做准备,并为促进骨腱界面愈合提供新的思路。 方法：60根趾深屈肌腱随机分成5组：1)正常肌腱组(Normal);2)不提取和无胎球蛋白钙化组(CaP)；3)提取和无胎球蛋白钙化组(CaPEXT);4)不提取和有胎球蛋白钙化组(CaPFetuin)和5)提取和有胎球蛋白钙化组(CaPEXTFetuin).提取组为在3%磷酸氢二钠溶液中缓慢搅拌并连续提取3天。胎球蛋白组除了使用普通中性钙盐和磷酸盐溶液外,钙化溶液还含有胎球蛋白。这些样本使用组织学、扫描电镜、压痕实验和缝线拔出测试等方法进行检测。 结果：CaP组肌腱表面可见到少量钙磷结晶附着。CaPEXT组肌腱可以见到少量磷酸钙渗透到肌腱内部,但深度很小。与CaP组和CaPEXT组肌腱钙化相比,CaPFetuin组肌腱有更深的磷酸钙进入肌腱内部,但是钙化在肌腱组织内深度仍然有限。然而磷酸钙在CaPEXTFetuin组肌腱内部渗透的更深,可达200μm。钙盐和磷酸盐含量测定发现,与CaPEXTFetuin组中的钙离子和磷酸根含量(4.82±0.59μmol和2.83±0.37gmol)相比,正常肌腱组(0.18±0.1μmol和0.024±0.01μmol)、CaP组(1.08±0.3μmol和0.65±0.21μmol)、CaPEXT组(1.6±0.45μmol和0.96±0.27μmol)和CaPFetuin组(3.27±0.35μmol和1.9±0.2μmol)中的钙离子和磷酸根含量显著较低(P0.05)。在CaP和CaPEXT组,可以见到有大量的钙磷结晶沉淀在肌腱表面上。然而,在CaPFetuin和CaPEXTFetuin组,并没有见到明显钙磷结晶沉淀在肌腱表面上,胶原纤维大小与正常肌腱的胶原纤维大小没有看到明显差异,钙化主要存在于肌腱的胶原纤维内。压痕实验发现,与正常对照组肌腱的抗压模量(4.34±0.59MPa)相比,CaP组(3.43±0.2MPa)、CaPEXT组(3.12±0.2MPa)、CaPFetuin组(3.56±0.31MPa)和CaPEXTFetuin组(3.33±0.18MPa)肌腱的抗压模量均显著降低(P0.05)。缝线拉出测试的最大拉断负荷在五组之间均无显著差异,其中正常组(14.11±4.07N)、CaP组(13.98±4.27N)、CaPEXT(14.3±4.32N)、CaPFetuin组(15.7±4.05N)和CaPEXTFetuin组(16.57±4.56N)(P0.05)。结论：磷酸氢二钠提取与胎球蛋白增强作用联合能够显著提高肌腱的钙化程度并在肌腱内形成了梯度的钙化区。这些发现可能对改善腱-骨愈合和促进骨腱链接点损伤后梯度界面再生有重要意义。但是还需要进一步的动物体内研究来证明这种新颖的钙化肌腱对骨腱愈合的作用。 第三章：胰酶化和钙化对同种异体滑膜内肌腱与骨愈合的作用 目的：本研究意在制备一种经过胰蛋白酶消化和钙化的同种异体滑膜内肌腱,并将其植入兔胫骨近端骨隧道模型,研究其对腱-骨界面愈合作用。 方法：8根趾深屈肌腱(FDP)用于优化胰蛋白酶消化时间。24根趾深屈肌腱随机分为对照组(N=12)和胰蛋白酶化钙化组(N=12)。其中每组4根肌腱用于体外评估胰蛋白酶化钙化效果。每组其余8根肌腱移植到兔胫骨近端骨隧道模型。通过组织学和生物力学等方法来检测胰蛋白酶化钙化技术对腱-骨界面愈合的作用。 结果：经过胰蛋白酶消化10分钟的肌腱,肌腱表面没有Lubricin的表达,只在肌腱细胞外基质有少量的Lubricin表达。因此本研究采用10分钟胰蛋白酶消化时间作为最佳消化时间。组织学检测发现胰蛋白酶化钙化组肌腱在钙化和未钙化之间有明显的界限。远端1cm部分肌腱表面的钙化表现为梯度钙化,而其他部分并没有见到肌腱钙化的现象。Lubricin免疫组化染色发现远端1cm部分肌腱表面没有Lubricin的表达,只在肌腱细胞外基质有少量的Lubricin表达,而肌腱的其他部分可以在其表面和细胞外基质上见到Lubricin表达。组织学检测骨隧道内肌腱部分,在正常对照组中可见到有一层薄的纤维瘢痕组织在肌腱与骨隧道界面形成。然而在胰蛋白酶化钙化组中,可以见到明显厚的纤维瘢痕组织在肌腱与骨隧道界面形成。更重要的是,胰蛋白酶化钙化组可以见到有明显的新骨在肌腱移植物内形成。与胰蛋白酶化钙化组相比,正常对照组的腱骨界面可以见到更多的空隙区域。此外,正常对照组和胰蛋白酶化钙化组均可见到大量单核细胞在肌腱内部浸润。正常对照组(5.76±3.21N)和胰蛋白酶化钙化组(5.21±4.21N)之间在最大拉断负荷上没有显著差异。此外,正常对照组(1.93±1.18N/M)和胰蛋白酶化钙化组(2.43±2.16N/M)在线性刚度上也没有显著差异。 结论：胰蛋白酶消化技术和钙化技术可能对同种异体滑膜内肌腱与骨隧道的整合,以及软骨和骨形成有促进作用。
[Abstract]:Chapter 1 : Compare the difference between the deep flexor tendon , the anterior cruciate ligament , the Achilles tendon and the patellar tendon of the dog .
Objective : To compare the biomechanical and histological differences between the deep flexor tendon , the anterior cruciate ligament , the Achilles tendon and the patellar tendon of the dog .
Methods : The hind legs of 20 dogs were used for the acquisition of deep flexor tendon ( N = 20 ) , anterior cruciate ligament ( N = 20 ) , middle tendon of Achilles tendon ( N = 20 ) and bone - patellar tendon - bone with 1 / 3 patellar tendon ( N = 20 ) . Friction test , indentation experiment , histology and immunohistochemistry were used to assess the difference .
Results : The frictional force of the flexor tendon group ( 47.24 卤 14.40 mN ) and the anterior cruciate ligament group ( 51.98 卤 12.10 mN ) was significantly smaller than that of the patellar tendon group ( 144.29 卤 10.38 mN ) and the Achilles tendon group ( P < 0.01 ) . At the 10th cycle , compared with the patellar tendon group ( 414.29 卤 10.34 MPa ) , the anterior cruciate ligament group ( 3.54 卤 0.32MPa ) and the anterior cruciate ligament group ( 4.71 卤 0.38MPa ) were significantly smaller ( P0.05 ) .
The surface of the tendon and the anterior cruciate ligament is very rough , and the tissue of the tendon is clearly visible . There is a small expression between the extracellular matrix and the collagen fiber .
Conclusion : Deep flexor tendon and anterior cruciate ligament have similar surface friction , and are significantly lower than those of Achilles tendon and patellar tendon . These characteristics are in agreement with the expression of tendon and tendon .
Chapter 2 : Optimization of in vitro calcification process of tendon in synovial membrane
Objective : The main aim of this study is to optimize the condition of tendon calcification , to prepare for the next step of calcification tendon in vivo , and to provide a new idea for promoting the healing of bone tendon .
Methods : 60 toe deep flexor tendons were randomly divided into 5 groups : 1 ) Normal tendon group ( Normal ) ; 2 ) No extraction and non - fetal globulin calcification group ;
3 ) Extraction and non - fetal globulin calcification group ( CaPEXT ) ; 4 ) extraction and presence of fetal globulin calcification group ( CaPEXTFOin ) . The extraction group was slowly stirred in 3 % disodium hydrogen phosphate solution and extracted continuously for 3 days . In addition to using common neutral calcium salt and phosphate solution , the calcification solution also contained fetal globulin . These samples were tested using methods such as histology , scanning electron microscope , indentation test and suture withdrawal test .
Results : There was no significant difference between Ca ~ ( 2 + ) and phosphate ( P < 0.05 ) in CaPEXT arm and CaPEXT arm . However , there was no significant difference between Ca ~ ( 2 + ) and phosphate ( 3.27 卤 0 . 31MPa ) , CaPEXT ( 3.27 卤 0 . 31MPa ) , CaPEXT ( 3.27 卤 0 . 31MPa ) and CaPEXTFOin group ( 3.33 卤 0 . 18MPa ) .
Chapter 3 : Effect of pancreatin and calcification on the healing of tendon and bone in allogeneic synovium
Objective : The aim of this study was to prepare a kind of allograft synovial tendon which was digested with trypsin and calcified , and implanted into rabbit proximal bone tunnel model to study its effect on the healing of tendon - bone interface .
Methods : Eight toe deep flexor tendons ( FDP ) were used to optimize the trypsinization time . 24 toe deep flexor tendons were randomly divided into control group ( N = 12 ) and trypsinization calcification group ( N = 12 ) .
Results : There was no significant difference between the tendon and non - calcification of the tendon in the normal control group . There was no significant difference between the normal control group ( 5.76 卤 3.21N ) and the trypsinized calcification group ( 5.21 卤 4.21N ) .
Conclusion : The technique of trypsin digestion and calcification may play a role in the integration of tendon and bone tunnel , and the formation of cartilage and bone .
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R686
本文编号：2137740
[Abstract]:Chapter 1 : Compare the difference between the deep flexor tendon , the anterior cruciate ligament , the Achilles tendon and the patellar tendon of the dog .
Objective : To compare the biomechanical and histological differences between the deep flexor tendon , the anterior cruciate ligament , the Achilles tendon and the patellar tendon of the dog .
Methods : The hind legs of 20 dogs were used for the acquisition of deep flexor tendon ( N = 20 ) , anterior cruciate ligament ( N = 20 ) , middle tendon of Achilles tendon ( N = 20 ) and bone - patellar tendon - bone with 1 / 3 patellar tendon ( N = 20 ) . Friction test , indentation experiment , histology and immunohistochemistry were used to assess the difference .
Results : The frictional force of the flexor tendon group ( 47.24 卤 14.40 mN ) and the anterior cruciate ligament group ( 51.98 卤 12.10 mN ) was significantly smaller than that of the patellar tendon group ( 144.29 卤 10.38 mN ) and the Achilles tendon group ( P < 0.01 ) . At the 10th cycle , compared with the patellar tendon group ( 414.29 卤 10.34 MPa ) , the anterior cruciate ligament group ( 3.54 卤 0.32MPa ) and the anterior cruciate ligament group ( 4.71 卤 0.38MPa ) were significantly smaller ( P0.05 ) .
The surface of the tendon and the anterior cruciate ligament is very rough , and the tissue of the tendon is clearly visible . There is a small expression between the extracellular matrix and the collagen fiber .
Conclusion : Deep flexor tendon and anterior cruciate ligament have similar surface friction , and are significantly lower than those of Achilles tendon and patellar tendon . These characteristics are in agreement with the expression of tendon and tendon .
Chapter 2 : Optimization of in vitro calcification process of tendon in synovial membrane
Objective : The main aim of this study is to optimize the condition of tendon calcification , to prepare for the next step of calcification tendon in vivo , and to provide a new idea for promoting the healing of bone tendon .
Methods : 60 toe deep flexor tendons were randomly divided into 5 groups : 1 ) Normal tendon group ( Normal ) ; 2 ) No extraction and non - fetal globulin calcification group ;
3 ) Extraction and non - fetal globulin calcification group ( CaPEXT ) ; 4 ) extraction and presence of fetal globulin calcification group ( CaPEXTFOin ) . The extraction group was slowly stirred in 3 % disodium hydrogen phosphate solution and extracted continuously for 3 days . In addition to using common neutral calcium salt and phosphate solution , the calcification solution also contained fetal globulin . These samples were tested using methods such as histology , scanning electron microscope , indentation test and suture withdrawal test .
Results : There was no significant difference between Ca ~ ( 2 + ) and phosphate ( P < 0.05 ) in CaPEXT arm and CaPEXT arm . However , there was no significant difference between Ca ~ ( 2 + ) and phosphate ( 3.27 卤 0 . 31MPa ) , CaPEXT ( 3.27 卤 0 . 31MPa ) , CaPEXT ( 3.27 卤 0 . 31MPa ) and CaPEXTFOin group ( 3.33 卤 0 . 18MPa ) .
Chapter 3 : Effect of pancreatin and calcification on the healing of tendon and bone in allogeneic synovium
Objective : The aim of this study was to prepare a kind of allograft synovial tendon which was digested with trypsin and calcified , and implanted into rabbit proximal bone tunnel model to study its effect on the healing of tendon - bone interface .
Methods : Eight toe deep flexor tendons ( FDP ) were used to optimize the trypsinization time . 24 toe deep flexor tendons were randomly divided into control group ( N = 12 ) and trypsinization calcification group ( N = 12 ) .
Results : There was no significant difference between the tendon and non - calcification of the tendon in the normal control group . There was no significant difference between the normal control group ( 5.76 卤 3.21N ) and the trypsinized calcification group ( 5.21 卤 4.21N ) .
Conclusion : The technique of trypsin digestion and calcification may play a role in the integration of tendon and bone tunnel , and the formation of cartilage and bone .
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R686
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