大鼠脱细胞脊髓支架制备及相关性能的实验研究
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摘要
背景
脊髓损伤近年来发病率有增高的趋势并呈现出高发生率,高致残率,高耗费,低死亡率的特点。组织工程学治疗脊髓损伤是目前研究的热点之一,它能整合治疗研究的各种有效策略,诱导轴突再生并提供良好的三维通道,减少胶质瘢痕和空洞形成。支架是组织工程研究中的关键,目前尚无与脊髓三维结构高度相似且在理化性能也与脊髓相似的支架材料。
近年来一种新的趋势是采用天然的脱细胞基质作为神经组织的修复材料,即用同种异体来源的材料,采用脱细胞技术制备的脱细胞基质,经过去细胞、部分或完全去除有机质或无机质、去抗原等处理,形成生物衍生材料(bio-derived material ),用于组织工程研究。理论上讲将脊髓组织进行脱细胞处理构建理想的组织工程支架是可行的,但目前尚无相关文献报道。
目的
通过采用物理冻融结合化学萃取方法初步构建大鼠脱细胞脊髓支架并优化脱细胞方案,应用HE染色、髓鞘染色及扫描电镜等方法评价制备脱细胞脊髓支架的可行性,然后通过对支架的成分分析、免疫原性分析、细胞毒性、组织相容性,血液相容性、全身毒性反应等检测评价脱细胞脊髓支架的生物安全性,并将神经干细胞与支架复合,观察细胞在支架材料上的生长情况,评价支架材料的细胞相容性,从而为构建理想的组织工程脊髓支架提供实验基础。
方法
1.脱细胞方案的确定:采用冻融结合化学萃取方法制备脱细胞脊髓支架,并应用HE染色、髓鞘染色及扫描电镜等方法检测脱细胞效果以确定最合适脱细胞方案。
2.脱细胞脊髓支架成分分析及免疫原性检测:应用免疫组织化学方案分析支架可能存在的成分;将脱细胞脊髓支架及同种异体脊髓组织分别进行大鼠椎旁皮下埋植后,于术后1w,2w,3w,4w周取材检测免疫排斥反应情况(免疫组织化学方法),评价免疫原性。
3.脱细胞脊髓支架细胞毒性及全身毒性研究:通过浸提液与细胞共培养评价细胞毒性;通过溶血试验、凝血时间测定评价血液相容性;通过热原试验、全身毒性反应评价支架的全身毒性。
4.脱细胞脊髓支架复合神经干细胞体外组织工程脊髓构建研究:将神经干细胞以不同浓度梯度接种在脱细胞脊髓支架上进行联合培养,观察细胞存活情况,比较不同接种密度上架细胞黏附率(Adhesive rate),绘制生长曲线。免疫组化结果用生物医学图像分析仪行平均光密度(AOD)分析,以重复资料的方差分析、t检验统计结果。
结果及结论
1、用冻融结合化学萃取法构建脱细胞脊髓组织工程支架是可行的,该支架具有良好的三维网状结构,脱细胞彻底,细胞外基质得到保存;
2.构建的脱细胞脊髓支架材料为三维网状结构,具有相互沟通的孔隙,其孔径为6-150μm,孔隙率平均为(93.6±1.5)%,含水能力平均为(58.2±1.6)%。初步显示出支架材料具有良好的结构性能;
3.构建的脱细胞脊髓支架材料含有Ⅳ型胶原、LN,FN等对细胞黏附、增殖、分化有利的活性成分,植入大鼠皮下后,未引发明显的炎性细胞浸润显示出支架具有极低的免疫原性;
4.构建的支架材料对体外NIH3T3细胞的生长、增殖及代谢无明显抑制作用,无致热原存留,无溶血作用,对凝血功能无影响,无急性全身毒性作用,初步显示出支架材料具有良好的血液相容性及生物安全性;
5.构建的支架材料与神经干细胞细胞构建细胞-支架复合体后神经干细胞能在支架内较长时间生存,显示出支架材料良好细胞相容性及广阔的应用前景。
Backgrounds :
Spinal cord injury usually results in devastating and permanent loss of function below the injured place. Because the CNS axons lack the ability for spontaneous regeneration--further compounded by chemical (myelin inhibitors) and physical (e.g. glial scar) barriers to regeneration after spinal cord injury, patients normally experience poor functional recovery.
Several methods have been investigated to overcome this hostile environment for regeneration and to promote partial functional recovery. Strategies to promote axonal extension through a site of injury include both the provision of nervous system growth factors and the implantation of substrates to support axon extension, such as cellular grafts. In general, however, the growth of axons is highly random and does not extend beyond the lesion site and into host tissue.Recently researchers have realized that it is difficult to achieve complete functional recovery relying on a single method. This has presented tissue engineering technology as an alternative strategy for the treatment of spinal cord injury. The scaffold can guide the linear growth of axons across a site of injury, in addition to providing neurotrophic and/or cellular support. This will help retain the native organization of regenerating axons across the lesion site and into distal host tissue, eventually increasing the probability of achieving function recovery.
Various natural and synthetic polymeric materials have been used to promote the functional recovery after spinal cord injury. Nevertheless, further improvements are needed for all previously-reported approaches, which mimic a native extracellular matrix. Acellular scaffolds are among the various materials that have been recently used in tissue reconstruction. Acellular scaffolds are the noncellular part of a tissue and consist of such proteins as collagen and carbohydrate structures secreted by resident cells. They can be transplanted without rejection and can provide a conductive environment for normal cellular attachment, migration, proliferation, differentiation and angiogenesis as well as a framework for tissue regeneration, since they are completely replaced by the host tissue. In the last few years, acellular matrices have been successfully used to substitute and repair skin, bladder, urethra, small bowel, cardiac valve, blood vessel, skeletal muscle,peripheral nervous defects, among others. In an attempt to mimic the regenerative capacity of the spinal cord graft, we have investigated acellular spinal cord that provides the physical pathway for axonal regeneration.
Objective:
The acellular spinal cord scaffold prepared by freeze thawing and chemical extraction medthod. The program of decellulated was made and optimized. The acellular spinal cord scaffold was estimated by HE stain and Immunostaining, Myelin Staining, Scanning Electron Microscopy Analysis. The biological safety of acellular spinal cord scaffold was detected by component analysis, immunogenicity analysis, cytotoxicity, histocompatibility, blood compatibility, general toxicity reaction, thus provide rationale for constructing the ideal tissue engineering scaffold of spinal cord.
Materials and Methods
1. The program of acellular spinal cord scaffold preparation: the acellular spinal cord scaffold prepared by freeze thawing and chemical extraction medthod. The effect of acellular spinal cord scaffold was estimated by HE stain and Immunostaining, Myelin Staining, Scanning Electron Microscopy Analysis,thus the optimized program was determined.
2.Component analysis and immunogenicity detection: Specimens were embedded in tissue-freezing medium, with temperature fixed at -20℃. Sections of 8 mm thick were obtained and routinely stained with immunostained for collagen typesⅣ, Laminin(LN), Fibronectin(FN).
A bilateral surgical approach was made to implant the acellular spinal cord scaffolds into the subcutaneous back skins of SD rat. Thereafter, a small skin incision was made (10 mm long), and a pocket was created through blunt dissection. The scaffolds were then implanted through these skin incisions subcutaneously into the mid-portion of the back areas. The incision was sewed using conventional cotton sutures. The tissue was obtained at 1、2、3 and 4w after the operation, with inflammatory reaction evaluated by HE stain. The immunogenicity of acelular scaffold was tested by immunohistochemical examining the intensity of CD4+ and CD8+ cells that infiltrated the allografts.
3. Cytotoxicity and general toxicity reaction investigation:
The cytotoxicity was tested through co-incubation of scaffolds with NIH 3T3 cells. The blood compatibility was detected by hemolysis test and clotting time.With the haemolysis rate <5%, the scaffold is qualified for becoming bio-tissue engineering materials. With the haemolysis rate≥5%,the scaffold has hemoclasis. The general toxicity was evaluated through pyrogen test and general toxic reaction.
4. The acellular spinal cord scaffold combinated neural stem cells to construct tissue engineering spinal cord: the neural stem cells was seeded in the scaffold by different concentration gradient and then co-cultured in incubator. The cell survival status was observed by microscope,then calculated the adhesive rate and drawed growth curve of cells.
Results and Conclusions
Macroscopic Observation
There was a large amount of white-colored floss secreting from spinal cord during decellulation. After being treated, the spinal cord scaffold became ivory-white and translucent, and yet still shaped as a circular cylinder. However, the diameter shrank to 2/3-4/5 of that of the original spinal cord and the strength decreased slightly, although the tenacity remained unchanged and the viscosity increased slightly.
HE Staining
Normal spinal cord has generous neurons ,glial cells and myelin sheaths. In cross section, a network of the extracellular matrix was seen in the scaffold. The cells, myelin and axons disappeared after the spinal cord was treated with the detergents TritonX-100 and deoxycholate. Typical network of empty tubes were viewed in longitudinal sections .
Myelin Staining
Normal myelin sheath of spinal cord is black and has regulation shape. In acellular spinal cord, either no myelin sheaths is observed or only a small quantity of myelin-sheath pieces is detected.
SEM Analysis
In the scaffold, the cells have been removed completely, although the extracellular matrix and the pore have remained to form three diamensional network structures.The pore and the channel of scaffold diameter was 6-150μm and 119±26μm respectively.
Immunohistochemistry
Positive reaction to LN,FN and IV collagen is seen in both normal spinal cord and acellular spinal cord.The staining is weaker in acellular scaffold than in normal spinal cord, which indicates that the majority of the extracellular matrix is preserved after the decellulation treatment of spinal cords.
Biocompatibility in Vitro
A haemolysis rate <5% qualifies scaffold as bio-tissue engineering materials. After scaffolds were co-incubated with NIH 3T3 cells for 72 h, the NIH 3T3 cells showed no signs of cytotoxicity (loss of adherence, nuclear condensation, and cell soma contraction) and cells proliferated normally compared to cells in control wells, expanding from approximately 50–100% confluency within 72 h.
Histocompatibility in Vivo
The lymphocyte, neutrophilic granulocyte and fibroblast were seen in control groups and experimental group animals. The degree of infiltration in experimental groups was significantly weaker than in control groups after 1 week of implantation. There was no obvious increase of infiltrated cell of implantation and the neutrophilic granulocyte had vanished after 4 weeks. However, there was multiplicity lymphocyte and neutrophilic granulocyte infiltrated in control groups after 4w of implantation.
There were sparing CD4+ and CD8+ leukomonocyte infiltration 1week and 2 weeks after implantation. Moreover, there were no obvious increase in experimental groups. There were massive CD4+ and CD8+ leukomonocyte infiltratration after implantation, and the staining intensity of positive cells were obviously stronger compared with the experimental groups.
Our study first showed that segments from the spinal cord of Sprague–Dawley rats can be successfully extracted to become acellular. The outcome of the extraction was monitored by morphological methods and immunohistochemistry. The extraction procedure involved the removal of myelin following the weil’s myelin staining, and cells, leaving largely intact ECM. Immunohistochemistry analysis revealed the presence of Laminin, Fibronectin and IV collagen in the ECM.
The scaffold of spinal cord is an emulated three-dimensional natural spinal cord which has fundamentally distinct and innate superiority over biological degradation materials. It is easy to obtain and is not confined by length or caliber. It can be isoloci transplantation and anatomy transplantation.
Acellular spinal cord scaffolds created in this study have a number of positive properties that can potentially support axonal regeneration after nervous system injury. The scaffolds are soft and flexible, containing linear guidance pores extending through their full length. Because the scaffolds are stable under physiological conditions, there is no risk of introducing toxic molecules to the site of injury. Based on current findings, we believe that extracted spinal cord could be used as allografts, with the possibility of becoming useful for spinal cord repair in the future. Ongoing work in vivo will test their ability to support axonal regeneration after spinal cord injury. The experiments of functional nerve recovery as well as microscopical (and morphometric) analysis will be evaluated in more detail.
脊髓损伤近年来发病率有增高的趋势并呈现出高发生率,高致残率,高耗费,低死亡率的特点。组织工程学治疗脊髓损伤是目前研究的热点之一,它能整合治疗研究的各种有效策略,诱导轴突再生并提供良好的三维通道,减少胶质瘢痕和空洞形成。支架是组织工程研究中的关键,目前尚无与脊髓三维结构高度相似且在理化性能也与脊髓相似的支架材料。
近年来一种新的趋势是采用天然的脱细胞基质作为神经组织的修复材料,即用同种异体来源的材料,采用脱细胞技术制备的脱细胞基质,经过去细胞、部分或完全去除有机质或无机质、去抗原等处理,形成生物衍生材料(bio-derived material ),用于组织工程研究。理论上讲将脊髓组织进行脱细胞处理构建理想的组织工程支架是可行的,但目前尚无相关文献报道。
目的
通过采用物理冻融结合化学萃取方法初步构建大鼠脱细胞脊髓支架并优化脱细胞方案,应用HE染色、髓鞘染色及扫描电镜等方法评价制备脱细胞脊髓支架的可行性,然后通过对支架的成分分析、免疫原性分析、细胞毒性、组织相容性,血液相容性、全身毒性反应等检测评价脱细胞脊髓支架的生物安全性,并将神经干细胞与支架复合,观察细胞在支架材料上的生长情况,评价支架材料的细胞相容性,从而为构建理想的组织工程脊髓支架提供实验基础。
方法
1.脱细胞方案的确定:采用冻融结合化学萃取方法制备脱细胞脊髓支架,并应用HE染色、髓鞘染色及扫描电镜等方法检测脱细胞效果以确定最合适脱细胞方案。
2.脱细胞脊髓支架成分分析及免疫原性检测:应用免疫组织化学方案分析支架可能存在的成分;将脱细胞脊髓支架及同种异体脊髓组织分别进行大鼠椎旁皮下埋植后,于术后1w,2w,3w,4w周取材检测免疫排斥反应情况(免疫组织化学方法),评价免疫原性。
3.脱细胞脊髓支架细胞毒性及全身毒性研究:通过浸提液与细胞共培养评价细胞毒性;通过溶血试验、凝血时间测定评价血液相容性;通过热原试验、全身毒性反应评价支架的全身毒性。
4.脱细胞脊髓支架复合神经干细胞体外组织工程脊髓构建研究:将神经干细胞以不同浓度梯度接种在脱细胞脊髓支架上进行联合培养,观察细胞存活情况,比较不同接种密度上架细胞黏附率(Adhesive rate),绘制生长曲线。免疫组化结果用生物医学图像分析仪行平均光密度(AOD)分析,以重复资料的方差分析、t检验统计结果。
结果及结论
1、用冻融结合化学萃取法构建脱细胞脊髓组织工程支架是可行的,该支架具有良好的三维网状结构,脱细胞彻底,细胞外基质得到保存;
2.构建的脱细胞脊髓支架材料为三维网状结构,具有相互沟通的孔隙,其孔径为6-150μm,孔隙率平均为(93.6±1.5)%,含水能力平均为(58.2±1.6)%。初步显示出支架材料具有良好的结构性能;
3.构建的脱细胞脊髓支架材料含有Ⅳ型胶原、LN,FN等对细胞黏附、增殖、分化有利的活性成分,植入大鼠皮下后,未引发明显的炎性细胞浸润显示出支架具有极低的免疫原性;
4.构建的支架材料对体外NIH3T3细胞的生长、增殖及代谢无明显抑制作用,无致热原存留,无溶血作用,对凝血功能无影响,无急性全身毒性作用,初步显示出支架材料具有良好的血液相容性及生物安全性;
5.构建的支架材料与神经干细胞细胞构建细胞-支架复合体后神经干细胞能在支架内较长时间生存,显示出支架材料良好细胞相容性及广阔的应用前景。
Backgrounds :
Spinal cord injury usually results in devastating and permanent loss of function below the injured place. Because the CNS axons lack the ability for spontaneous regeneration--further compounded by chemical (myelin inhibitors) and physical (e.g. glial scar) barriers to regeneration after spinal cord injury, patients normally experience poor functional recovery.
Several methods have been investigated to overcome this hostile environment for regeneration and to promote partial functional recovery. Strategies to promote axonal extension through a site of injury include both the provision of nervous system growth factors and the implantation of substrates to support axon extension, such as cellular grafts. In general, however, the growth of axons is highly random and does not extend beyond the lesion site and into host tissue.Recently researchers have realized that it is difficult to achieve complete functional recovery relying on a single method. This has presented tissue engineering technology as an alternative strategy for the treatment of spinal cord injury. The scaffold can guide the linear growth of axons across a site of injury, in addition to providing neurotrophic and/or cellular support. This will help retain the native organization of regenerating axons across the lesion site and into distal host tissue, eventually increasing the probability of achieving function recovery.
Various natural and synthetic polymeric materials have been used to promote the functional recovery after spinal cord injury. Nevertheless, further improvements are needed for all previously-reported approaches, which mimic a native extracellular matrix. Acellular scaffolds are among the various materials that have been recently used in tissue reconstruction. Acellular scaffolds are the noncellular part of a tissue and consist of such proteins as collagen and carbohydrate structures secreted by resident cells. They can be transplanted without rejection and can provide a conductive environment for normal cellular attachment, migration, proliferation, differentiation and angiogenesis as well as a framework for tissue regeneration, since they are completely replaced by the host tissue. In the last few years, acellular matrices have been successfully used to substitute and repair skin, bladder, urethra, small bowel, cardiac valve, blood vessel, skeletal muscle,peripheral nervous defects, among others. In an attempt to mimic the regenerative capacity of the spinal cord graft, we have investigated acellular spinal cord that provides the physical pathway for axonal regeneration.
Objective:
The acellular spinal cord scaffold prepared by freeze thawing and chemical extraction medthod. The program of decellulated was made and optimized. The acellular spinal cord scaffold was estimated by HE stain and Immunostaining, Myelin Staining, Scanning Electron Microscopy Analysis. The biological safety of acellular spinal cord scaffold was detected by component analysis, immunogenicity analysis, cytotoxicity, histocompatibility, blood compatibility, general toxicity reaction, thus provide rationale for constructing the ideal tissue engineering scaffold of spinal cord.
Materials and Methods
1. The program of acellular spinal cord scaffold preparation: the acellular spinal cord scaffold prepared by freeze thawing and chemical extraction medthod. The effect of acellular spinal cord scaffold was estimated by HE stain and Immunostaining, Myelin Staining, Scanning Electron Microscopy Analysis,thus the optimized program was determined.
2.Component analysis and immunogenicity detection: Specimens were embedded in tissue-freezing medium, with temperature fixed at -20℃. Sections of 8 mm thick were obtained and routinely stained with immunostained for collagen typesⅣ, Laminin(LN), Fibronectin(FN).
A bilateral surgical approach was made to implant the acellular spinal cord scaffolds into the subcutaneous back skins of SD rat. Thereafter, a small skin incision was made (10 mm long), and a pocket was created through blunt dissection. The scaffolds were then implanted through these skin incisions subcutaneously into the mid-portion of the back areas. The incision was sewed using conventional cotton sutures. The tissue was obtained at 1、2、3 and 4w after the operation, with inflammatory reaction evaluated by HE stain. The immunogenicity of acelular scaffold was tested by immunohistochemical examining the intensity of CD4+ and CD8+ cells that infiltrated the allografts.
3. Cytotoxicity and general toxicity reaction investigation:
The cytotoxicity was tested through co-incubation of scaffolds with NIH 3T3 cells. The blood compatibility was detected by hemolysis test and clotting time.With the haemolysis rate <5%, the scaffold is qualified for becoming bio-tissue engineering materials. With the haemolysis rate≥5%,the scaffold has hemoclasis. The general toxicity was evaluated through pyrogen test and general toxic reaction.
4. The acellular spinal cord scaffold combinated neural stem cells to construct tissue engineering spinal cord: the neural stem cells was seeded in the scaffold by different concentration gradient and then co-cultured in incubator. The cell survival status was observed by microscope,then calculated the adhesive rate and drawed growth curve of cells.
Results and Conclusions
Macroscopic Observation
There was a large amount of white-colored floss secreting from spinal cord during decellulation. After being treated, the spinal cord scaffold became ivory-white and translucent, and yet still shaped as a circular cylinder. However, the diameter shrank to 2/3-4/5 of that of the original spinal cord and the strength decreased slightly, although the tenacity remained unchanged and the viscosity increased slightly.
HE Staining
Normal spinal cord has generous neurons ,glial cells and myelin sheaths. In cross section, a network of the extracellular matrix was seen in the scaffold. The cells, myelin and axons disappeared after the spinal cord was treated with the detergents TritonX-100 and deoxycholate. Typical network of empty tubes were viewed in longitudinal sections .
Myelin Staining
Normal myelin sheath of spinal cord is black and has regulation shape. In acellular spinal cord, either no myelin sheaths is observed or only a small quantity of myelin-sheath pieces is detected.
SEM Analysis
In the scaffold, the cells have been removed completely, although the extracellular matrix and the pore have remained to form three diamensional network structures.The pore and the channel of scaffold diameter was 6-150μm and 119±26μm respectively.
Immunohistochemistry
Positive reaction to LN,FN and IV collagen is seen in both normal spinal cord and acellular spinal cord.The staining is weaker in acellular scaffold than in normal spinal cord, which indicates that the majority of the extracellular matrix is preserved after the decellulation treatment of spinal cords.
Biocompatibility in Vitro
A haemolysis rate <5% qualifies scaffold as bio-tissue engineering materials. After scaffolds were co-incubated with NIH 3T3 cells for 72 h, the NIH 3T3 cells showed no signs of cytotoxicity (loss of adherence, nuclear condensation, and cell soma contraction) and cells proliferated normally compared to cells in control wells, expanding from approximately 50–100% confluency within 72 h.
Histocompatibility in Vivo
The lymphocyte, neutrophilic granulocyte and fibroblast were seen in control groups and experimental group animals. The degree of infiltration in experimental groups was significantly weaker than in control groups after 1 week of implantation. There was no obvious increase of infiltrated cell of implantation and the neutrophilic granulocyte had vanished after 4 weeks. However, there was multiplicity lymphocyte and neutrophilic granulocyte infiltrated in control groups after 4w of implantation.
There were sparing CD4+ and CD8+ leukomonocyte infiltration 1week and 2 weeks after implantation. Moreover, there were no obvious increase in experimental groups. There were massive CD4+ and CD8+ leukomonocyte infiltratration after implantation, and the staining intensity of positive cells were obviously stronger compared with the experimental groups.
Our study first showed that segments from the spinal cord of Sprague–Dawley rats can be successfully extracted to become acellular. The outcome of the extraction was monitored by morphological methods and immunohistochemistry. The extraction procedure involved the removal of myelin following the weil’s myelin staining, and cells, leaving largely intact ECM. Immunohistochemistry analysis revealed the presence of Laminin, Fibronectin and IV collagen in the ECM.
The scaffold of spinal cord is an emulated three-dimensional natural spinal cord which has fundamentally distinct and innate superiority over biological degradation materials. It is easy to obtain and is not confined by length or caliber. It can be isoloci transplantation and anatomy transplantation.
Acellular spinal cord scaffolds created in this study have a number of positive properties that can potentially support axonal regeneration after nervous system injury. The scaffolds are soft and flexible, containing linear guidance pores extending through their full length. Because the scaffolds are stable under physiological conditions, there is no risk of introducing toxic molecules to the site of injury. Based on current findings, we believe that extracted spinal cord could be used as allografts, with the possibility of becoming useful for spinal cord repair in the future. Ongoing work in vivo will test their ability to support axonal regeneration after spinal cord injury. The experiments of functional nerve recovery as well as microscopical (and morphometric) analysis will be evaluated in more detail.
引文
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