快速成形制备的PLGA支架—胶原凝胶复合体构建组织工程软骨的实验研究
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摘要
各种原因造成的软骨缺损是临床上常见的问题,由于软骨的组织学和生物学特性限制了其自身的修复能力,目前尚缺乏有效的治疗措施。组织工程技术的发展为软骨缺损的修复提供了新的手段,利用具有特定空间结构的支架材料引导软骨细胞的增殖和基质分泌,最终形成类似正常软骨组织的结构,并行使功能。
近年来软骨组织工程支架的研究侧重于对材料表面的设计以提高材料的生物性能。支架表面特性对细胞/支架复合起重要作用。目前有很多方法,如碱处理、等离子放电和表面改性等都可提高材料表面的生物相容性。在支架材料的研究中,复合材料是目前研究的热点,即将两种或两种以上具有互补理化性质的生物相容性可降解材料,按一定比例和方式组合,可设计出结构与性能优化的三维支架材料。以弥补单用人工合成或天然生物材料的缺陷。
本研究的目的是构建具有理想空间结构的复合支架材料,即运用快速成形技术制备的PLGA支架材料结合胶原凝胶,探讨其作为组织工程支架材料的可行性。
本研究包括以下几部分内容:
1制备具有生物活性的PLGA支架材料
将聚酯PLGA(LA/GA=50/50)充分溶解于1, 4-二氧六环有机溶剂形成无色透明液体。采用清华大学激光快速成形中心开发研制的低温挤出成形设备-生物材料快速成形机TissFormTM,经计算机辅助设计(CAD)结合低温挤出成形工艺,在低温成形室中固化堆积,得到冷冻的三维材料。低温快速成型的方法所制备的载体框架的孔隙结构、孔隙率、力学性能符合软骨组织工程载体框架的要求,可以作为软骨组织工程的支架材料。
2兔关节软骨细胞分离培养及生物活性检测
运用酶消化的方法获得软骨细胞,并观察原代及传代培养的细胞形态学变化。用甲苯胺蓝及免疫组化的方法鉴定软骨细胞。成功建立体外培养兔关节软骨细胞的实验方法。原代培养的软骨细胞呈多角形,传代3次后出现去分化。形态学、免疫组织化学染色显示细胞培养3代以内可以保持表型的稳定。本文建立的体外培养关节软骨细胞的方法简单可行。体外培养的第2代兔软骨细胞表型稳定,细胞增殖活力良好,适用于实验研究。
3 PLGA及胶原改性材料与软骨细胞相容性的观察通过对原始及改性材料与软骨细胞相容性的观察,检测支架材料的生物活性。包括亲水性的测定,细胞计数,扫描电镜。实验证实,改性后材料亲水性及细胞黏附率较改性前支架均有显著性提高。
4胶原凝胶包埋软骨细胞复合快速成形PLGA支架的接种方法
通过将胶原凝胶包埋的软骨细胞接种到快速成形的PLGA材料上并进行体外培养观察,细胞计数检测细胞粘附情况,倒置显微镜观察细胞在支架内分布的均一性。结果显示,90%以上的细胞能够有效、均匀接种于支架材料上,所以,胶原凝胶包埋软骨细胞三维接种能有效提高种子细胞的接种效率,防止细胞流失。
5裸鼠体内成软骨实验
通过将胶原凝胶包埋的软骨细胞接种到快速成形PLGA材料的复合物植入裸鼠体内于不同时间点进行大体观察,组织学染色检查软骨形成情况。大体观察,有类软骨样组织形成,所形成的组织形态保持良好,具有一定的厚度,未见收缩。组织学结果显示软骨细胞在体内生长增殖良好,随时间延长材料逐渐降解软骨组织逐渐成熟。
本实验证明低温快速成形技术制备的PLGA支架经胶原改性后具有良好的亲水性、降解性和生物相容性。凝胶包埋细胞接种支架材料的方法能够均匀、高效的将细胞固定于多孔材料中,提高细胞接种效率。同时,凝胶包埋的软骨细胞接种到快速成形的PLGA材料的复合物具有良好的成软骨能力。所以,胶原凝胶包埋快速成型PLGA支架材料可作为载体复合细胞进行软骨组织工程的研究。
Cartilage defects are a major problem in orthopedic surgery.Because the cartilage tissue itself lacks a blood supply to support repair and remodeling,once damaged,the cartilage has little capacity for spontaneous healing.Most of the current therapies to repair damaged cartilage have limititions.Progress in tissue engineering provided a new concept for tissue regeneration and transplantation.The scaffold can provide suitable spatial structure for cell proliferation and matrix secreting,thus facilitate tissue forming.
Since the scaffold surface properties play an important role in determining how cells respond to the biomaterials,the development of tissue engineering has recently focoused on the surface design of biodegradable scaffolds.Several approaches,such as alkali hydrolysis treatment,plasma treatment,ion irradiation and surface coating have been developed to improve the bioactivity of the surface.
The complex scaffolds were composed of two or more biomaterials to consist a 3-D scaffold with suitable structure and function. It makes up the disadvantages of using artificial synthetical material or natural materials alone. The purpose of this study was to develop a new complex scaffold for cartilage tissue engineering,which owns the advantages of high cell seeding efficiency,good ability to support chondrogenesis as well as adequate physical properities.rapid prototyping PLGA porous scaffold was chosed as framework, then chondrocytes suspended in collagen solution was seed in framework,and gelling process was performed.We believe rapid prototyping PLGA-collagen gel complex could act as a new scaffold for cartilage regeneration.
This study includes the following parts:
1 Fabricating PLGA scaffold with LDM techonology The structure of scaffold, porosity and mechanical property can meet with the requirement of scaffold for tissue-engineered cartilage.
2 Isolation of chondrocytes and identification of cell phenotype. Articular chondrocytes were isolated from the cartilages with the methods of enzymatic digestion.The morphological changes and growth feature of primary cultured and subcultured chondrocytes were observed under the inverted microscope each day. Toluidine blue staining and immunocytochemist ry were used to indentify the chondrocytes. The primarily cultured chondrocytes were in olygonal shape, and became dedifferentiation after 3 passages. The chondrocytes maintained the morphology and immunochemical staining pattern within the first 3 passages. The method used in this work for isolation and culture of chondrocytes is simple and feasible. The chondrocytes cultured in viro maintained the specific chondrocytes phenotype in the first 3 passages. The growth of the second passage was robust and may be suitable for most experiment.
3 The compatibility of original scaffold and hybrid scaffold.
After examing water absorption capability of the scaffold, chondrocytes proliferation and differentiation on the scaffold were calculated and the results showed that rapid prototyping PLGA coating with collagen had a good biocompatibility for the attatchment and proliferation of the chondrocyte. It might be ideal biomaterials in tissue-engineered cartilage field.
4 Seeding cell methods of seeding chondrocytes into scaffolds
Mixture chondrocytes with collagen solution was incorporated into a PLGA 3D scaffold, and made into gel in the scaffold, then cultured invitro.Attachment of chondrocytes were evaluated by cell count.Distribution of chondrocytes into 3D scaffolds was observed by phase microscope.The results showed More than 90 percent of seeding chondrocytes were able to incorporated into PLGA scaffolds efficiently and uniformly, and prevent cell loss.
5 cartilage tissue formation in nude mice
The complexes of seeding of chondrocytes encapsulated in collagen gel into RP PLGA scaffolds were transplanted into the back of nude mice. The specimens harvested at the 4th week,8th week and 12th week were analyzed by observation, histology and tested the quantification of glycosaminoglycan in neo-cartilage.The allograft of chondrocytes proliferated successfully in the back of the nude mice, new cartilage could be seen by the end of 12 weeks and the scaffolds have suitable degradation。The cartilage formed with the scaffold degrading.
Our research has demonstrated that the attachment , proliferation ,differentiation of chondrocytes on rapid prototyping PLGA coating with collagen.The methods of gel encapsulated cells incorporated to rapid prototyping PLGA can fix cells in scaffold uniformly and efficiently. Meanwhile the complex of gel encapsulated cells incorporated to rapid prototyping PLGA has the capability of cartilaginous tissue forming.Rapid prototyping PLGA-collagen gel was suitable scaffold to carry cells in the research of tissue-engineered cartilage.
近年来软骨组织工程支架的研究侧重于对材料表面的设计以提高材料的生物性能。支架表面特性对细胞/支架复合起重要作用。目前有很多方法,如碱处理、等离子放电和表面改性等都可提高材料表面的生物相容性。在支架材料的研究中,复合材料是目前研究的热点,即将两种或两种以上具有互补理化性质的生物相容性可降解材料,按一定比例和方式组合,可设计出结构与性能优化的三维支架材料。以弥补单用人工合成或天然生物材料的缺陷。
本研究的目的是构建具有理想空间结构的复合支架材料,即运用快速成形技术制备的PLGA支架材料结合胶原凝胶,探讨其作为组织工程支架材料的可行性。
本研究包括以下几部分内容:
1制备具有生物活性的PLGA支架材料
将聚酯PLGA(LA/GA=50/50)充分溶解于1, 4-二氧六环有机溶剂形成无色透明液体。采用清华大学激光快速成形中心开发研制的低温挤出成形设备-生物材料快速成形机TissFormTM,经计算机辅助设计(CAD)结合低温挤出成形工艺,在低温成形室中固化堆积,得到冷冻的三维材料。低温快速成型的方法所制备的载体框架的孔隙结构、孔隙率、力学性能符合软骨组织工程载体框架的要求,可以作为软骨组织工程的支架材料。
2兔关节软骨细胞分离培养及生物活性检测
运用酶消化的方法获得软骨细胞,并观察原代及传代培养的细胞形态学变化。用甲苯胺蓝及免疫组化的方法鉴定软骨细胞。成功建立体外培养兔关节软骨细胞的实验方法。原代培养的软骨细胞呈多角形,传代3次后出现去分化。形态学、免疫组织化学染色显示细胞培养3代以内可以保持表型的稳定。本文建立的体外培养关节软骨细胞的方法简单可行。体外培养的第2代兔软骨细胞表型稳定,细胞增殖活力良好,适用于实验研究。
3 PLGA及胶原改性材料与软骨细胞相容性的观察通过对原始及改性材料与软骨细胞相容性的观察,检测支架材料的生物活性。包括亲水性的测定,细胞计数,扫描电镜。实验证实,改性后材料亲水性及细胞黏附率较改性前支架均有显著性提高。
4胶原凝胶包埋软骨细胞复合快速成形PLGA支架的接种方法
通过将胶原凝胶包埋的软骨细胞接种到快速成形的PLGA材料上并进行体外培养观察,细胞计数检测细胞粘附情况,倒置显微镜观察细胞在支架内分布的均一性。结果显示,90%以上的细胞能够有效、均匀接种于支架材料上,所以,胶原凝胶包埋软骨细胞三维接种能有效提高种子细胞的接种效率,防止细胞流失。
5裸鼠体内成软骨实验
通过将胶原凝胶包埋的软骨细胞接种到快速成形PLGA材料的复合物植入裸鼠体内于不同时间点进行大体观察,组织学染色检查软骨形成情况。大体观察,有类软骨样组织形成,所形成的组织形态保持良好,具有一定的厚度,未见收缩。组织学结果显示软骨细胞在体内生长增殖良好,随时间延长材料逐渐降解软骨组织逐渐成熟。
本实验证明低温快速成形技术制备的PLGA支架经胶原改性后具有良好的亲水性、降解性和生物相容性。凝胶包埋细胞接种支架材料的方法能够均匀、高效的将细胞固定于多孔材料中,提高细胞接种效率。同时,凝胶包埋的软骨细胞接种到快速成形的PLGA材料的复合物具有良好的成软骨能力。所以,胶原凝胶包埋快速成型PLGA支架材料可作为载体复合细胞进行软骨组织工程的研究。
Cartilage defects are a major problem in orthopedic surgery.Because the cartilage tissue itself lacks a blood supply to support repair and remodeling,once damaged,the cartilage has little capacity for spontaneous healing.Most of the current therapies to repair damaged cartilage have limititions.Progress in tissue engineering provided a new concept for tissue regeneration and transplantation.The scaffold can provide suitable spatial structure for cell proliferation and matrix secreting,thus facilitate tissue forming.
Since the scaffold surface properties play an important role in determining how cells respond to the biomaterials,the development of tissue engineering has recently focoused on the surface design of biodegradable scaffolds.Several approaches,such as alkali hydrolysis treatment,plasma treatment,ion irradiation and surface coating have been developed to improve the bioactivity of the surface.
The complex scaffolds were composed of two or more biomaterials to consist a 3-D scaffold with suitable structure and function. It makes up the disadvantages of using artificial synthetical material or natural materials alone. The purpose of this study was to develop a new complex scaffold for cartilage tissue engineering,which owns the advantages of high cell seeding efficiency,good ability to support chondrogenesis as well as adequate physical properities.rapid prototyping PLGA porous scaffold was chosed as framework, then chondrocytes suspended in collagen solution was seed in framework,and gelling process was performed.We believe rapid prototyping PLGA-collagen gel complex could act as a new scaffold for cartilage regeneration.
This study includes the following parts:
1 Fabricating PLGA scaffold with LDM techonology The structure of scaffold, porosity and mechanical property can meet with the requirement of scaffold for tissue-engineered cartilage.
2 Isolation of chondrocytes and identification of cell phenotype. Articular chondrocytes were isolated from the cartilages with the methods of enzymatic digestion.The morphological changes and growth feature of primary cultured and subcultured chondrocytes were observed under the inverted microscope each day. Toluidine blue staining and immunocytochemist ry were used to indentify the chondrocytes. The primarily cultured chondrocytes were in olygonal shape, and became dedifferentiation after 3 passages. The chondrocytes maintained the morphology and immunochemical staining pattern within the first 3 passages. The method used in this work for isolation and culture of chondrocytes is simple and feasible. The chondrocytes cultured in viro maintained the specific chondrocytes phenotype in the first 3 passages. The growth of the second passage was robust and may be suitable for most experiment.
3 The compatibility of original scaffold and hybrid scaffold.
After examing water absorption capability of the scaffold, chondrocytes proliferation and differentiation on the scaffold were calculated and the results showed that rapid prototyping PLGA coating with collagen had a good biocompatibility for the attatchment and proliferation of the chondrocyte. It might be ideal biomaterials in tissue-engineered cartilage field.
4 Seeding cell methods of seeding chondrocytes into scaffolds
Mixture chondrocytes with collagen solution was incorporated into a PLGA 3D scaffold, and made into gel in the scaffold, then cultured invitro.Attachment of chondrocytes were evaluated by cell count.Distribution of chondrocytes into 3D scaffolds was observed by phase microscope.The results showed More than 90 percent of seeding chondrocytes were able to incorporated into PLGA scaffolds efficiently and uniformly, and prevent cell loss.
5 cartilage tissue formation in nude mice
The complexes of seeding of chondrocytes encapsulated in collagen gel into RP PLGA scaffolds were transplanted into the back of nude mice. The specimens harvested at the 4th week,8th week and 12th week were analyzed by observation, histology and tested the quantification of glycosaminoglycan in neo-cartilage.The allograft of chondrocytes proliferated successfully in the back of the nude mice, new cartilage could be seen by the end of 12 weeks and the scaffolds have suitable degradation。The cartilage formed with the scaffold degrading.
Our research has demonstrated that the attachment , proliferation ,differentiation of chondrocytes on rapid prototyping PLGA coating with collagen.The methods of gel encapsulated cells incorporated to rapid prototyping PLGA can fix cells in scaffold uniformly and efficiently. Meanwhile the complex of gel encapsulated cells incorporated to rapid prototyping PLGA has the capability of cartilaginous tissue forming.Rapid prototyping PLGA-collagen gel was suitable scaffold to carry cells in the research of tissue-engineered cartilage.
引文
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