骨AECM的制备、立体构筑、成分分析及Triton x-100残留量的测定
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摘要
目的
在骨组织工程中大量生物降解和可吸收材料，已经进行了广泛研究。细胞外支架是提供细胞粘附，增生和分化的基础，也是将生长因子固定于细胞组分上并维持其三维空间结构，允许新的组织生成的支撑物。虽然，一些人对脱细胞膜的细胞骨架进行了描述，但迄今为止还没有见到较理想的脱细胞骨细胞外基质的制备及骨细胞外基质骨陷窝计量检测的详细报道．本实验进行脱细胞骨基质制备，去除细胞成分，并进行形态学观察，为骨组织工程的新型天然支架材料提供依据。同时对脱细胞基质的成分和实验中所使用的除垢剂Triton x-100的残留量进行了检测。
材料与方法
1．脱细胞兔骨细胞外基质的制备：
用戊巴比妥钠将兔麻醉，取双侧胫骨，修整成片状，剔除骨膜，PBS彻底清洗。向广口瓶中加入Tris-HCl缓冲液，缓冲液内加入蛋白酶抑制剂，放入骨片，4℃下恒温震荡。然后把瓶内液体换成
内含 3畅 Triton x河 的 Tis－HCI（PH7．4）缓冲液洞样也加人
上述蛋白酶抑制剂，4℃恒温震荡后，用蒸馏水连续冲洗。之后加
入 DNas。和 RNase混合液室温下消化。再次向瓶中加人内含 3％
TitonxJ 的TiS－HCI缓冲液，4t下恒温震荡。蒸馏水充分
冲洗后，将制备的脱细胞的骨片放人TiS－HCI（PH7．4）缓冲液
中，4℃下保存备用。脱细胞的骨片用Von Ebnel氏脱钙液脱钙 15
天。
2．组织学检测
脱钙后脱细胞的基质用10％福尔马林固定，常规石蜡包埋，
切片后进行HE染色、Mallory－Heidenhain氏快速一步法染色、阿
利新蓝染色，光镜观察。
3．扫描电镜
将没有脱钙骨片用1．25％戊H醛一4％多聚甲醛固定扫描电
镜观察。
4．骨陷窝的形态测量方法和测量指标
Mallory－Heidenhain氏快速一步法染色的正常和脱细胞骨组
织各取10张标本，每个标本随机取10个视野，我们使用LUZEX
－F实时图像分析系统在400倍普通光镜观察下，测量骨陷窝密
度，并对500个骨陷窝的表面积、平均周长、长径、短径、骨陷窝间
平均距离进行了检测。
5．链霉素亲生物素一过氧化物酶法染色u－P）
脱细胞细胞外基质经脱钙后石蜡切片，常规二甲苯脱蜡；梯度
酒精水化；通过免疫组化方法标记纤维连结蛋白和层连蛋白。以
PBS置换一抗作为空白对照。
6．免疫组织化学电镜观察
脱细胞细胞外基质经免疫组化S－P染色后俄酸固定；逐级
丙酮脱水；Epon浸透包埋。超薄切片后 JEM－1200EX Electron
nlC］SSOpe观察。
·2·
7．反相高效液相色谱（Rp－HPLC）检测 Titon x刁00残留量
高效液相色谱柱（SPhPriSOOb C18）5卜m、15nun X 6ruml．D．；流
动相：乙睛／水（体积比 1：l人流速：二 1llHniln；温度：40 t；检测波
长：28Onm；进样量：5pl。朋S为空白对照，队 sp＊M＊ 血J卜V
ITriton x－100为对照品，在上述色谱条件下检测，3min出峰。
取脱细胞骨基质称重，将其钳碎研磨至糊状，加人PBS混匀，体积
到6Inl，取0．SInl样品加人甲醇0．SInl后1，0009上0min／t离心，
取上清液。分别取 0·SInl样品加人 0．spl、lpl、sgl Titon x－100
后加人甲醇0．sail后10009在 广t离心，取上清液。在上述色
谱条件下检测。
结 果
一、脱细胞骨细胞外基质形态学观察和计量学检测结果
1．大体观察
与新鲜胜骨相比，经脱细胞处理的兔胜骨脱细胞细胞外基质
外观颜色更浅淡，更自一些；我们同时也使用不同浓度的除垢剂和
脱细胞时间进行了脱细胞处理，证明 30天组并用3％Titon x－
100脱细胞效果最好，光镜和电镜观察下脱细胞彻底。
2．组织学检测
HE染色、Mallory－Heidenhain氏快速一步法染色、阿利新蓝
染色：光镜下，脱细胞骨基质胶原纤维排列整齐，椭圆形骨陷窝内
空虚，无骨细胞核及其他结构。
3．扫描电镜观察
正常胜骨扫描电镜观察骨陷窝中骨细胞清晰可见。脱细胞胜
骨扫描电镜观察仅见骨基质结构，骨陷窝空虚，骨陷窝内壁光滑规
则，无任何细胞残留物。
4．骨陷窝的形态计量学结果：我们对骨陷窝的密度和一些组
·3·
织形态学参数做了测量。这些参数对测算骨细胞的大小有重要意
义，根据体视学原理估计单位体积骨组织合细胞数，进而对工程骨
的构建提供实验依据。
二、在脱细胞细胞外基质中纤维连结蛋白和层连蛋白的检测
S－P法染色结果：纤维连结蛋白抗体和层连蛋白抗体的染色
阳性部位在细胞外基质，清晰棕色为阳性，以细胞外基质无棕色或
背景一致浅棕色为阴性。在脱细胞细胞外基质的骨陷窝周边部染
色较深，呈筛网状、线状分布，为清晰棕色。阴性对照组未见棕色。
免疫组织化学电镜观察结果：在细胞外基质骨陷窝周边部弥
散存在阳性标记，电子密度较高的DAB反应产物呈四块，网状分
布。基质内可见网状纤维结构，胶原纤维排列整齐。
三、RP－HPLC分析Titon x－100残留量
对照品出峰时
Objective
In bone tissue engineering a great deal of biodegraded and ab-sorbable material has been researched widely. The scaffold provides the base of cell adhesion, growth and differentiation, also is the structure that fixes growth factors in cell components and maintains three dimension spatial configuration to create new tissue. Although someone has narrated cell framework of acellular membrane, no ideal preparation of AECM or histomorphometry of bone lacunas have been reported in details. This experiment prepares ACEM by removing cell components and carries morphologic study to provide foundation for new natural supporting material of bone tissue engineering. Simultaneously, the components of AECM and remnant of detergent are determined.
Methods
1. Rabbit AECM preparation:
After anesthetized by sodium pentobarbital, two lateral tibias of
rabbit were taken down. After have been incised into patches and gotten rid of periosteum, they were washed by PBS. Bone patches were put into jar containing Tris - HCI buffer and proteinase inhibitors. After at 4℃ constant temperature stirring, The samples were then immersed in Tris - HCI buffer containing 3% Triton x - 100 and proteinase inhibitors, After 4℃ constant temperature stirring, distilled water washed bone patches and the samples were digested by DNase and RNase at 31℃. Then the samples were immersed into Tris -HCl buffer containing 3% Triton x - 100 and washed by distilled water again. Finally the samples were reserved in Tris - HCl buffer at 4℃. When needed, AECM were put into Von Ebnel liquid for 30 days.
2. Histology
First AECM was fixed by 10% formalin for 24 hours and paraffin embedded tissue, staining with hematoxylin and eosin, Mallory- Hei-denhain dyeing or Alcian blue. Under light microscope, AECM was examined
3. Scanning electron microscope
AECM were prepared in 1. 25% glutaraldhyde and observed by Scanning electron microscope.
4. Histomorphometry and parameters of bone lacunas Choosing 10 samples randomly and 10 eyeshot per sample staining by Mallory - Heidenhain rapid one - step dyeing, we measured density, perimeter, wide, length of bone lacunas and distance between lacunas under light microscope by LUZEX - F real - time image analyzing system.
5.S-P
After dimethylbenzene dewaxing AECM and dehydration through a graded series of alcohols, the expression of LN and FN were detec-
ted by immunochemohistology. The samples of PBS exchanging antibodies were regarded blank comparison.
6. Immuno - electron microscopic technique
After S - P dyeing of AECM, fixation by osmic acid and hydra-tion through a graded series of acetone; Epon embedded samples; After ultrathin section the samples were observed under JEM - 1200EX Electron Microscope.
7. Determination of Triton x - 100 by Rp - HPLC
High - performance ehromatogram column ( Spherisorb c18 ) 5jxm,15mm x 6mm I. D. ; flow phase: acetonitrile /water( 1:1; v/v) ; flow - rate; Iml/min; room temperature: 40Tl; wave length: 280nm; sample-. 5 ul. Blank comparison was PBS. 0. 5ul,1ul,5ul Triton x -100 were put into 0. 5ml PBS buffer as comparison samples. Then join 0. 5ml methanol separately. Determine Triton x - 100 with Rp -HPLC. In 3min apex comes out. Weighing and rubbing AECM, PBS mixes with scraps. After centrifugation, take up - clear liquid and Determine Triton x - 100 with Rp - HPLC
Results
1. Morphologic studies and histomorphologic survey of AECM
( 1 ) Bulk observation
Comparing with flesh tibia, appearance of AECM is more shallow and whiter. We also use different concentration of Triton x - 100 and time of cell extraction and Prove 30 days of cell extraction and 3% triton x -100 to be most effective. Cell component isnt found under light and electron microscope.
( 2 ) Histology
HE, Mallory - Heidenhain rapid one - step dyeing,Alcian dyeing : Under light microscope, collagen fibers of arrange uniformly. It' s blankness in ellipse bone lacunas and there is no nucleus and other configuration.
( 3 ) Osteocytes are in focus under Scanning
在骨组织工程中大量生物降解和可吸收材料，已经进行了广泛研究。细胞外支架是提供细胞粘附，增生和分化的基础，也是将生长因子固定于细胞组分上并维持其三维空间结构，允许新的组织生成的支撑物。虽然，一些人对脱细胞膜的细胞骨架进行了描述，但迄今为止还没有见到较理想的脱细胞骨细胞外基质的制备及骨细胞外基质骨陷窝计量检测的详细报道．本实验进行脱细胞骨基质制备，去除细胞成分，并进行形态学观察，为骨组织工程的新型天然支架材料提供依据。同时对脱细胞基质的成分和实验中所使用的除垢剂Triton x-100的残留量进行了检测。
材料与方法
1．脱细胞兔骨细胞外基质的制备：
用戊巴比妥钠将兔麻醉，取双侧胫骨，修整成片状，剔除骨膜，PBS彻底清洗。向广口瓶中加入Tris-HCl缓冲液，缓冲液内加入蛋白酶抑制剂，放入骨片，4℃下恒温震荡。然后把瓶内液体换成
内含 3畅 Triton x河 的 Tis－HCI（PH7．4）缓冲液洞样也加人
上述蛋白酶抑制剂，4℃恒温震荡后，用蒸馏水连续冲洗。之后加
入 DNas。和 RNase混合液室温下消化。再次向瓶中加人内含 3％
TitonxJ 的TiS－HCI缓冲液，4t下恒温震荡。蒸馏水充分
冲洗后，将制备的脱细胞的骨片放人TiS－HCI（PH7．4）缓冲液
中，4℃下保存备用。脱细胞的骨片用Von Ebnel氏脱钙液脱钙 15
天。
2．组织学检测
脱钙后脱细胞的基质用10％福尔马林固定，常规石蜡包埋，
切片后进行HE染色、Mallory－Heidenhain氏快速一步法染色、阿
利新蓝染色，光镜观察。
3．扫描电镜
将没有脱钙骨片用1．25％戊H醛一4％多聚甲醛固定扫描电
镜观察。
4．骨陷窝的形态测量方法和测量指标
Mallory－Heidenhain氏快速一步法染色的正常和脱细胞骨组
织各取10张标本，每个标本随机取10个视野，我们使用LUZEX
－F实时图像分析系统在400倍普通光镜观察下，测量骨陷窝密
度，并对500个骨陷窝的表面积、平均周长、长径、短径、骨陷窝间
平均距离进行了检测。
5．链霉素亲生物素一过氧化物酶法染色u－P）
脱细胞细胞外基质经脱钙后石蜡切片，常规二甲苯脱蜡；梯度
酒精水化；通过免疫组化方法标记纤维连结蛋白和层连蛋白。以
PBS置换一抗作为空白对照。
6．免疫组织化学电镜观察
脱细胞细胞外基质经免疫组化S－P染色后俄酸固定；逐级
丙酮脱水；Epon浸透包埋。超薄切片后 JEM－1200EX Electron
nlC］SSOpe观察。
·2·
7．反相高效液相色谱（Rp－HPLC）检测 Titon x刁00残留量
高效液相色谱柱（SPhPriSOOb C18）5卜m、15nun X 6ruml．D．；流
动相：乙睛／水（体积比 1：l人流速：二 1llHniln；温度：40 t；检测波
长：28Onm；进样量：5pl。朋S为空白对照，队 sp＊M＊ 血J卜V
ITriton x－100为对照品，在上述色谱条件下检测，3min出峰。
取脱细胞骨基质称重，将其钳碎研磨至糊状，加人PBS混匀，体积
到6Inl，取0．SInl样品加人甲醇0．SInl后1，0009上0min／t离心，
取上清液。分别取 0·SInl样品加人 0．spl、lpl、sgl Titon x－100
后加人甲醇0．sail后10009在 广t离心，取上清液。在上述色
谱条件下检测。
结 果
一、脱细胞骨细胞外基质形态学观察和计量学检测结果
1．大体观察
与新鲜胜骨相比，经脱细胞处理的兔胜骨脱细胞细胞外基质
外观颜色更浅淡，更自一些；我们同时也使用不同浓度的除垢剂和
脱细胞时间进行了脱细胞处理，证明 30天组并用3％Titon x－
100脱细胞效果最好，光镜和电镜观察下脱细胞彻底。
2．组织学检测
HE染色、Mallory－Heidenhain氏快速一步法染色、阿利新蓝
染色：光镜下，脱细胞骨基质胶原纤维排列整齐，椭圆形骨陷窝内
空虚，无骨细胞核及其他结构。
3．扫描电镜观察
正常胜骨扫描电镜观察骨陷窝中骨细胞清晰可见。脱细胞胜
骨扫描电镜观察仅见骨基质结构，骨陷窝空虚，骨陷窝内壁光滑规
则，无任何细胞残留物。
4．骨陷窝的形态计量学结果：我们对骨陷窝的密度和一些组
·3·
织形态学参数做了测量。这些参数对测算骨细胞的大小有重要意
义，根据体视学原理估计单位体积骨组织合细胞数，进而对工程骨
的构建提供实验依据。
二、在脱细胞细胞外基质中纤维连结蛋白和层连蛋白的检测
S－P法染色结果：纤维连结蛋白抗体和层连蛋白抗体的染色
阳性部位在细胞外基质，清晰棕色为阳性，以细胞外基质无棕色或
背景一致浅棕色为阴性。在脱细胞细胞外基质的骨陷窝周边部染
色较深，呈筛网状、线状分布，为清晰棕色。阴性对照组未见棕色。
免疫组织化学电镜观察结果：在细胞外基质骨陷窝周边部弥
散存在阳性标记，电子密度较高的DAB反应产物呈四块，网状分
布。基质内可见网状纤维结构，胶原纤维排列整齐。
三、RP－HPLC分析Titon x－100残留量
对照品出峰时
Objective
In bone tissue engineering a great deal of biodegraded and ab-sorbable material has been researched widely. The scaffold provides the base of cell adhesion, growth and differentiation, also is the structure that fixes growth factors in cell components and maintains three dimension spatial configuration to create new tissue. Although someone has narrated cell framework of acellular membrane, no ideal preparation of AECM or histomorphometry of bone lacunas have been reported in details. This experiment prepares ACEM by removing cell components and carries morphologic study to provide foundation for new natural supporting material of bone tissue engineering. Simultaneously, the components of AECM and remnant of detergent are determined.
Methods
1. Rabbit AECM preparation:
After anesthetized by sodium pentobarbital, two lateral tibias of
rabbit were taken down. After have been incised into patches and gotten rid of periosteum, they were washed by PBS. Bone patches were put into jar containing Tris - HCI buffer and proteinase inhibitors. After at 4℃ constant temperature stirring, The samples were then immersed in Tris - HCI buffer containing 3% Triton x - 100 and proteinase inhibitors, After 4℃ constant temperature stirring, distilled water washed bone patches and the samples were digested by DNase and RNase at 31℃. Then the samples were immersed into Tris -HCl buffer containing 3% Triton x - 100 and washed by distilled water again. Finally the samples were reserved in Tris - HCl buffer at 4℃. When needed, AECM were put into Von Ebnel liquid for 30 days.
2. Histology
First AECM was fixed by 10% formalin for 24 hours and paraffin embedded tissue, staining with hematoxylin and eosin, Mallory- Hei-denhain dyeing or Alcian blue. Under light microscope, AECM was examined
3. Scanning electron microscope
AECM were prepared in 1. 25% glutaraldhyde and observed by Scanning electron microscope.
4. Histomorphometry and parameters of bone lacunas Choosing 10 samples randomly and 10 eyeshot per sample staining by Mallory - Heidenhain rapid one - step dyeing, we measured density, perimeter, wide, length of bone lacunas and distance between lacunas under light microscope by LUZEX - F real - time image analyzing system.
5.S-P
After dimethylbenzene dewaxing AECM and dehydration through a graded series of alcohols, the expression of LN and FN were detec-
ted by immunochemohistology. The samples of PBS exchanging antibodies were regarded blank comparison.
6. Immuno - electron microscopic technique
After S - P dyeing of AECM, fixation by osmic acid and hydra-tion through a graded series of acetone; Epon embedded samples; After ultrathin section the samples were observed under JEM - 1200EX Electron Microscope.
7. Determination of Triton x - 100 by Rp - HPLC
High - performance ehromatogram column ( Spherisorb c18 ) 5jxm,15mm x 6mm I. D. ; flow phase: acetonitrile /water( 1:1; v/v) ; flow - rate; Iml/min; room temperature: 40Tl; wave length: 280nm; sample-. 5 ul. Blank comparison was PBS. 0. 5ul,1ul,5ul Triton x -100 were put into 0. 5ml PBS buffer as comparison samples. Then join 0. 5ml methanol separately. Determine Triton x - 100 with Rp -HPLC. In 3min apex comes out. Weighing and rubbing AECM, PBS mixes with scraps. After centrifugation, take up - clear liquid and Determine Triton x - 100 with Rp - HPLC
Results
1. Morphologic studies and histomorphologic survey of AECM
( 1 ) Bulk observation
Comparing with flesh tibia, appearance of AECM is more shallow and whiter. We also use different concentration of Triton x - 100 and time of cell extraction and Prove 30 days of cell extraction and 3% triton x -100 to be most effective. Cell component isnt found under light and electron microscope.
( 2 ) Histology
HE, Mallory - Heidenhain rapid one - step dyeing,Alcian dyeing : Under light microscope, collagen fibers of arrange uniformly. It' s blankness in ellipse bone lacunas and there is no nucleus and other configuration.
( 3 ) Osteocytes are in focus under Scanning
引文
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2. Goldstein SA, Patil PV, Moalli MR. Perspectives on tissue engineering of bone. Clin Orthop 1999, (367 Suppl) :S419-23.
3. Sims CD, Butler PE, Cao YL, Casanova R, Randolph MA, Black A, Vacanti CA, Yaremchuk MJ. Tissue engineered neocartilage using plasma derived polymer substrates and chondrocytes. Plast Reconstr Surg 1998, 101(6) :1580-5.
4．柏树令，包伟珂，王军．心脏线粒体及其固定装置在心衰时的形态学改变．中国医科大学学报 1999，28(3)：174-175．
5. Nela Angelova, David Hunkeler. Rationalizing the design of polymeric biomaterials. Trends biotechnol 1999, (17) :409-421.
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