脱细胞组织工程血管支架材料的实验研究
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摘要
随着社会的发展,心血管疾病和创伤日益成为威胁人们健康及生命的重要因素。冠状动脉搭桥术、严重创伤致血管损伤和缺损、多种血管移植手术在外科中的应用以及整形外科游离皮瓣转移中血管蒂的长度不足等,使得血管移植材料的研究日益成为人们关注的热点。作为一种全新的技术,组织工程在提供合适的血管移植物研究方面取得了迅速的发展。组织工程血管是一种用组织工程学方法构建的具有良好生物相容性和力学特性的血管替代物,其基本的构件是血管支架材料和种子细胞,而血管支架的研究一直是目前血管组织工程研究领域的难点和重点。组织工程血管支架材料主要分为人工合成的生物可降解高分子聚合物和天然生物支架材料两大类。高分子可降解材料的应用目前虽然得到广泛的认可,但是由于材料价格较为昂贵,合成技术要求高,难以大规模应用。另外由于材料本身的性质影响,其对种子细胞的亲和性不足,构建的组织工程血管顺应性尚不理想,对血液动力学方面可能会有较大影响。而作为天然生物支架材料的脱细胞血管基质,由于其与细胞亲和力强,能为细胞生长、繁殖、分化提供近似体内组织发育的细胞外基质支架,且生物相容性、顺应性以及免疫排斥性低等特点都优于合成材料,因此在血管组织工程中得到越来越多的重视和应用。
目前国内外常用的主要脱细胞方法包括:机械法,酶消化法,化学去垢剂法等。以上几种方法各有利弊,物理方法对支架结构的破坏较小但脱细胞效果较差,而化学去垢剂方法脱细胞效果较好却容易破坏血管的胶原结构。另外,在利用化学去垢剂制备生物材料时,去垢剂的残留会导致脱细胞生物材料具有一定程度的细胞毒性。酶消化法制备脱细胞基质材料简单易行,但是高浓度的酶可引起支架胶原结构的破坏,而低浓度酶又无法完全去除动脉壁深部的细胞成份,脱细胞效果并不理想。因此寻找一种更高效和安全的脱细胞方法一直是脱细胞组织工程血管支架研究的一个热点。
同其它异体移植材料一样,脱细胞血管支架的研究应用也面临着传播供体潜在疾病以及在移植物处理应用过程中二次污染的问题。传统的生物材料灭菌方法主要有以环氧乙烷为代表的化学法和以钴60照射为代表的电离辐射法两种。但是这些方法都存在操作要求条件较高,过程较复杂以及可能降低材料的机械强度等缺点。因此,我们希望找到一种操作简便、灭菌效果确实、费用低廉、残留低且对材料的生物特性影响较小的脱细胞血管支架消毒方法。
目前仍没有足够的证据表明细胞能够长入脱细胞血管基质支架材料内部。因此,如何在保留脱细胞血管支架足够机械强度的前提下,提高脱细胞血管基质材料的疏松性和孔隙率,这也是目前组织工程脱细胞血管支架研究中一个亟待解决的难点。
最后,脱细胞血管材料制备后如何长期保存也是一个需要解决的问题,我们希望找到一种简单可行的储存技术来长期保存制备的脱细胞血管支架以达到临床和科研上“随取随用”的目的。
本实验采用天然血管作为组织工程血管支架的来源:
1.通过反复冻融及超高压处理,摸索上述处理与完全去除血管材料固有细胞所需核酸酶酶浓度的关系,以及对血管材料生物力学特性的影响,从而得到一种体外快速高效构建组织工程化小血管支架材料的方法。
2.在上述试验的基础上,我们将0.1%的过氧乙酸作为脱细胞血管支架的消毒剂,对经过氧乙酸处理的脱细胞血管支架进行组织学评估、超微结构观察、生物力学以及细胞毒性测定,以观察过氧乙酸对脱细胞血管支架材料生物相容性和生物力学特性的影响。
3.利用超声处理的空化效应,对制备的脱细胞血管支架进行不同强度及不同时间的超声处理。对经超声处理后支架材料的孔隙大小、生物相容性、细胞毒性、生物力学效应及与种子细胞亲和性进行评估,从而得出脱细胞血管材料基质疏松的最佳超声强度参数以及作用时间。
4.利用冷冻干燥技术,对制备好的脱细胞血管支架进行真空冷冻干燥处理。通过对经冻干处理并保存的脱细胞血管支架材料的各种生物学特性的分析,以判断冷冻干燥技术是否可以作为脱细胞生物支架材料长期保存的一种方法。
本研究结果表明:
1.采用反复冻融加超高压结合低浓度核酸酶消化、缓冲液冲洗的脱细胞方法可以在较短的时间内完全除去支架内细胞成份,且对材料的主要生物力学指标以及胶原结构无明显影响。相对于单一的超高压处理或反复冻融处理,反复冻融及超高压两种方法结合不仅能明显提高制备脱细胞血管支架的效率,其对于超高压设备参数的要求也明显降低,使得超高压生物处理这一过程在国产设备上即能完成。因此,其为今后脱细胞血管支架的快速高效制备提供一种新的思路和方法。
2.体外试验表明经过0.1%浓度过氧乙酸处理过的脱细胞血管材料,其细胞毒性、生物力学及胶原含量与未经过氧乙酸处理前相比没有明显的差异。因此应用0.1%浓度过氧乙酸作为组织工程血管支架制备过程中的消毒灭菌剂是可行的。
3.超声处理参数为强度300瓦,处理间隔1秒,处理时长1分钟时,脱细胞兔股动脉支架材料的疏松程度和材料的生物力学强度处于一个较均衡的水平。即材料疏松程度较未处理前明显提高,而生物力学强度无明显降低。因此,针对不同长度及厚度的血管材料,应用不同参数的超声处理,可以达到在不明显降低材料生物力学特性的前提下,使脱细胞血管基质材料结构明显疏松的目的。
4.经过冻干保存处理的脱细胞血管材料,其细胞毒性、生物力学以及其自身胶原结构与未经处理前相比没有明显的差异,冻干保存后的材料在体内依然具有良好的生物相容性。因此我们认为:冷冻干燥处理作为组织工程脱细胞血管支架此类管状生物材料的长期储存方法是可行的。
综上所述,通过系列的试验,我们找到了一种组织工程脱细胞血管支架材料快速制备、简便高效消毒及长时间储存的方法。同时我们也初步解决了脱细胞血管基质材料结构致密,种子细胞无法长入的问题,在组织工程脱细胞血管支架的研究上又前进了一步。
Cardiovascular diseases are prevalent and debilitating lesions that affect the quality of life among populations worldwide. Large numbers of patients suffers from diseases of the vascular system, resulting in a clear clinical need for developing functional arterial replacements. As a novel solution, tissue engineering has made significant progress toward the creation of vascular grafts for the repair of damaged or malformed vessels. It aims to address these lesions by integrating engineered, living substitutes with their native counterparts in vivo. For such a purpose, competent vascular scaffolding materials are essential. To date, two major categories of vascular scaffolding materials have been employed: synthetic polymers and natural collagen derivatives. Compared with synthetic polymers, natural acellular vascular scaffolds have the advantages of preserving ECM proteins important for cell attachment and the desired mechanical properties which made it the most intriguing materials used to create scaffolds for tissue engineering vascular applications.
Different decellularization methods have been developed to fabricate acellular vascular scaffolds for the purpose of tissue engineering blood vessel, mostly comprising physical methods、detergents and zymatic extraction methods. Recent studies revealed that vascular grafts that had been decellularized using detergents were more resistant to cellular in-growth than those treated with enzymatic extraction. Otherwise, there are also flaws of the enzymatic extraction method; long digestion time may cause a biomechanical damage of the biomaterials and short time treatment results in a residue of the original cells. Then finding an ideal way to produce acellular vascular scaffolds becomes an interesting subject.
As with any form of allografts, the risks of disease transmission from either the donor to recipient or from environmental contamination acquired during retrieval and processing of the graft must be considered. Historically, two methods have been commonly applied for the sterilisation of allograft biomaterials; chemical sterilisation utilising ethylene oxide gas and sterilisation with ionising radiation. Whilst both of these techniques have been demonstrated to be effective sterilisation procedures concerns have been raised about their potentially deleterious effects on important properties of the grafts. For these reasons, it is desirable to apply a high-level disinfection or sterilisation procedure to such grafts following retrieval and processing.
On the same time, it is also suggested that following acellular treatment, the structure of the acellular scaffold is still compact. How to open up the collagenous matrix and subsequently allow cells to enter and migrate into the scaffold of their own accord without altering the biological or mechanical properties of the scaffold, this will also be the subject for our works. Finally, we want to find out a preservation protocol after fabrication of the acellular vascullar scaffolds to meet the emergency request of the clinical and scientific research.
Object:
1.This study was to find a new way for tissue engineering vascular scaffold fabrication by investigating the effect of ultrahigh pressure and freezing thawing treatment on natural vessels and to observe the decellularization results of different concentration nuclease solutions after the natural vessels being treated by repeated freeze thawing and ultrahigh pressure.
2.To observe the effects that 0.1% Peracetic Acid had on the biological features of the acellular vascular scaffold by analyzing the biocompatibility and biomechanics of the dealed scaffolds treated by 0.1% Peracetic Acid.
3.Various intensities of ultrasonication was tested in order to observe the effects that ultrasonication had on the biological features of the acellular vascular scaffold by analyzing the biocompatibility and biomechanics of the dealed scaffolds.
4.To investigate the effects of a Freeze-Drying technique preservation protocol on the biocompatibility and biomechanical properties of acellular vascular scaffold.
Results and conclusions:
1 . The resulting scaffold has been shown to be biocompatible with biochemical and mechanical properties similar to those of natural vessels following ultrahigh pressure and repeated freeze thawing treatment, the vessels were completely cell free when they were treated by nuclease solutions in a fairly low concentration with short treatment time. This tissue processing of decellularization by ultrahigh pressure and repeated freeze thawing treatment can decrease both the treatment time and concentration of nuclease solutions remarkably; it may provide a new idea and method for fabrication of bioscaffolds.
2.From the histological and ultrastructural analysis, we can see that treated by 0.1% Peracetic Acid, the main performance indexes of the acellular scaffold changed unremarkably in compared with normal blood vessels. This tissue processing of disinfection by 0.1% Peracetic Acid treatment has not obvious effect on the biocompatibility and biomechanics of the acellular scaffold, and it can be an alternative of disinfector for the acellular bioscaffold preparation.
3.Ultrasonication treatment with the intensity of 300W and a pulse time of 1 s for a total of 1 min was found to be the optimal treatment. This process did not have significant effect upon the biochemical constituents, nor did it denature the collagen. Moreover, the acellular sonicated scaffold retained the essential biomechanical characteristics of the native tissue. These findings show us that ultrasonication can provide a novel method to enhance the recellularization of decellularized natural tissues.
4.From the vitro investigation we find that treated by Freeze-Drying technique preservation protocol, the main performance indexes of the acellular scaffold changed unremarkably in compared with normal blood vessels. This tissue processing of Freeze-Drying technique preservation protocol has no obvious effects on the biocompatibility and biomechanics of the acellular scaffolds, and it can be a good preservation protocol for the acellular bioscaffold preparation.
Summary: In our studies, a novel solution for the fast fbrication, simple sterilization and long preservation of the tissue engineered acelluluar vascular scaffolds is found out. Meanwhile, we also investigate a way to initially solve the problem of opening up the collagenous matrix and subsequently allowing cells to enter and migrate into the scaffold of their own accord without altering the biological or mechanical properties of the acellular vascular scaffold.
目前国内外常用的主要脱细胞方法包括:机械法,酶消化法,化学去垢剂法等。以上几种方法各有利弊,物理方法对支架结构的破坏较小但脱细胞效果较差,而化学去垢剂方法脱细胞效果较好却容易破坏血管的胶原结构。另外,在利用化学去垢剂制备生物材料时,去垢剂的残留会导致脱细胞生物材料具有一定程度的细胞毒性。酶消化法制备脱细胞基质材料简单易行,但是高浓度的酶可引起支架胶原结构的破坏,而低浓度酶又无法完全去除动脉壁深部的细胞成份,脱细胞效果并不理想。因此寻找一种更高效和安全的脱细胞方法一直是脱细胞组织工程血管支架研究的一个热点。
同其它异体移植材料一样,脱细胞血管支架的研究应用也面临着传播供体潜在疾病以及在移植物处理应用过程中二次污染的问题。传统的生物材料灭菌方法主要有以环氧乙烷为代表的化学法和以钴60照射为代表的电离辐射法两种。但是这些方法都存在操作要求条件较高,过程较复杂以及可能降低材料的机械强度等缺点。因此,我们希望找到一种操作简便、灭菌效果确实、费用低廉、残留低且对材料的生物特性影响较小的脱细胞血管支架消毒方法。
目前仍没有足够的证据表明细胞能够长入脱细胞血管基质支架材料内部。因此,如何在保留脱细胞血管支架足够机械强度的前提下,提高脱细胞血管基质材料的疏松性和孔隙率,这也是目前组织工程脱细胞血管支架研究中一个亟待解决的难点。
最后,脱细胞血管材料制备后如何长期保存也是一个需要解决的问题,我们希望找到一种简单可行的储存技术来长期保存制备的脱细胞血管支架以达到临床和科研上“随取随用”的目的。
本实验采用天然血管作为组织工程血管支架的来源:
1.通过反复冻融及超高压处理,摸索上述处理与完全去除血管材料固有细胞所需核酸酶酶浓度的关系,以及对血管材料生物力学特性的影响,从而得到一种体外快速高效构建组织工程化小血管支架材料的方法。
2.在上述试验的基础上,我们将0.1%的过氧乙酸作为脱细胞血管支架的消毒剂,对经过氧乙酸处理的脱细胞血管支架进行组织学评估、超微结构观察、生物力学以及细胞毒性测定,以观察过氧乙酸对脱细胞血管支架材料生物相容性和生物力学特性的影响。
3.利用超声处理的空化效应,对制备的脱细胞血管支架进行不同强度及不同时间的超声处理。对经超声处理后支架材料的孔隙大小、生物相容性、细胞毒性、生物力学效应及与种子细胞亲和性进行评估,从而得出脱细胞血管材料基质疏松的最佳超声强度参数以及作用时间。
4.利用冷冻干燥技术,对制备好的脱细胞血管支架进行真空冷冻干燥处理。通过对经冻干处理并保存的脱细胞血管支架材料的各种生物学特性的分析,以判断冷冻干燥技术是否可以作为脱细胞生物支架材料长期保存的一种方法。
本研究结果表明:
1.采用反复冻融加超高压结合低浓度核酸酶消化、缓冲液冲洗的脱细胞方法可以在较短的时间内完全除去支架内细胞成份,且对材料的主要生物力学指标以及胶原结构无明显影响。相对于单一的超高压处理或反复冻融处理,反复冻融及超高压两种方法结合不仅能明显提高制备脱细胞血管支架的效率,其对于超高压设备参数的要求也明显降低,使得超高压生物处理这一过程在国产设备上即能完成。因此,其为今后脱细胞血管支架的快速高效制备提供一种新的思路和方法。
2.体外试验表明经过0.1%浓度过氧乙酸处理过的脱细胞血管材料,其细胞毒性、生物力学及胶原含量与未经过氧乙酸处理前相比没有明显的差异。因此应用0.1%浓度过氧乙酸作为组织工程血管支架制备过程中的消毒灭菌剂是可行的。
3.超声处理参数为强度300瓦,处理间隔1秒,处理时长1分钟时,脱细胞兔股动脉支架材料的疏松程度和材料的生物力学强度处于一个较均衡的水平。即材料疏松程度较未处理前明显提高,而生物力学强度无明显降低。因此,针对不同长度及厚度的血管材料,应用不同参数的超声处理,可以达到在不明显降低材料生物力学特性的前提下,使脱细胞血管基质材料结构明显疏松的目的。
4.经过冻干保存处理的脱细胞血管材料,其细胞毒性、生物力学以及其自身胶原结构与未经处理前相比没有明显的差异,冻干保存后的材料在体内依然具有良好的生物相容性。因此我们认为:冷冻干燥处理作为组织工程脱细胞血管支架此类管状生物材料的长期储存方法是可行的。
综上所述,通过系列的试验,我们找到了一种组织工程脱细胞血管支架材料快速制备、简便高效消毒及长时间储存的方法。同时我们也初步解决了脱细胞血管基质材料结构致密,种子细胞无法长入的问题,在组织工程脱细胞血管支架的研究上又前进了一步。
Cardiovascular diseases are prevalent and debilitating lesions that affect the quality of life among populations worldwide. Large numbers of patients suffers from diseases of the vascular system, resulting in a clear clinical need for developing functional arterial replacements. As a novel solution, tissue engineering has made significant progress toward the creation of vascular grafts for the repair of damaged or malformed vessels. It aims to address these lesions by integrating engineered, living substitutes with their native counterparts in vivo. For such a purpose, competent vascular scaffolding materials are essential. To date, two major categories of vascular scaffolding materials have been employed: synthetic polymers and natural collagen derivatives. Compared with synthetic polymers, natural acellular vascular scaffolds have the advantages of preserving ECM proteins important for cell attachment and the desired mechanical properties which made it the most intriguing materials used to create scaffolds for tissue engineering vascular applications.
Different decellularization methods have been developed to fabricate acellular vascular scaffolds for the purpose of tissue engineering blood vessel, mostly comprising physical methods、detergents and zymatic extraction methods. Recent studies revealed that vascular grafts that had been decellularized using detergents were more resistant to cellular in-growth than those treated with enzymatic extraction. Otherwise, there are also flaws of the enzymatic extraction method; long digestion time may cause a biomechanical damage of the biomaterials and short time treatment results in a residue of the original cells. Then finding an ideal way to produce acellular vascular scaffolds becomes an interesting subject.
As with any form of allografts, the risks of disease transmission from either the donor to recipient or from environmental contamination acquired during retrieval and processing of the graft must be considered. Historically, two methods have been commonly applied for the sterilisation of allograft biomaterials; chemical sterilisation utilising ethylene oxide gas and sterilisation with ionising radiation. Whilst both of these techniques have been demonstrated to be effective sterilisation procedures concerns have been raised about their potentially deleterious effects on important properties of the grafts. For these reasons, it is desirable to apply a high-level disinfection or sterilisation procedure to such grafts following retrieval and processing.
On the same time, it is also suggested that following acellular treatment, the structure of the acellular scaffold is still compact. How to open up the collagenous matrix and subsequently allow cells to enter and migrate into the scaffold of their own accord without altering the biological or mechanical properties of the scaffold, this will also be the subject for our works. Finally, we want to find out a preservation protocol after fabrication of the acellular vascullar scaffolds to meet the emergency request of the clinical and scientific research.
Object:
1.This study was to find a new way for tissue engineering vascular scaffold fabrication by investigating the effect of ultrahigh pressure and freezing thawing treatment on natural vessels and to observe the decellularization results of different concentration nuclease solutions after the natural vessels being treated by repeated freeze thawing and ultrahigh pressure.
2.To observe the effects that 0.1% Peracetic Acid had on the biological features of the acellular vascular scaffold by analyzing the biocompatibility and biomechanics of the dealed scaffolds treated by 0.1% Peracetic Acid.
3.Various intensities of ultrasonication was tested in order to observe the effects that ultrasonication had on the biological features of the acellular vascular scaffold by analyzing the biocompatibility and biomechanics of the dealed scaffolds.
4.To investigate the effects of a Freeze-Drying technique preservation protocol on the biocompatibility and biomechanical properties of acellular vascular scaffold.
Results and conclusions:
1 . The resulting scaffold has been shown to be biocompatible with biochemical and mechanical properties similar to those of natural vessels following ultrahigh pressure and repeated freeze thawing treatment, the vessels were completely cell free when they were treated by nuclease solutions in a fairly low concentration with short treatment time. This tissue processing of decellularization by ultrahigh pressure and repeated freeze thawing treatment can decrease both the treatment time and concentration of nuclease solutions remarkably; it may provide a new idea and method for fabrication of bioscaffolds.
2.From the histological and ultrastructural analysis, we can see that treated by 0.1% Peracetic Acid, the main performance indexes of the acellular scaffold changed unremarkably in compared with normal blood vessels. This tissue processing of disinfection by 0.1% Peracetic Acid treatment has not obvious effect on the biocompatibility and biomechanics of the acellular scaffold, and it can be an alternative of disinfector for the acellular bioscaffold preparation.
3.Ultrasonication treatment with the intensity of 300W and a pulse time of 1 s for a total of 1 min was found to be the optimal treatment. This process did not have significant effect upon the biochemical constituents, nor did it denature the collagen. Moreover, the acellular sonicated scaffold retained the essential biomechanical characteristics of the native tissue. These findings show us that ultrasonication can provide a novel method to enhance the recellularization of decellularized natural tissues.
4.From the vitro investigation we find that treated by Freeze-Drying technique preservation protocol, the main performance indexes of the acellular scaffold changed unremarkably in compared with normal blood vessels. This tissue processing of Freeze-Drying technique preservation protocol has no obvious effects on the biocompatibility and biomechanics of the acellular scaffolds, and it can be a good preservation protocol for the acellular bioscaffold preparation.
Summary: In our studies, a novel solution for the fast fbrication, simple sterilization and long preservation of the tissue engineered acelluluar vascular scaffolds is found out. Meanwhile, we also investigate a way to initially solve the problem of opening up the collagenous matrix and subsequently allowing cells to enter and migrate into the scaffold of their own accord without altering the biological or mechanical properties of the acellular vascular scaffold.
引文
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5. Conklin BS, Richter ER, Kreutziger KL, et al. Development and evaluation of a novel decellularized vascular xenograft.J Med Eng Phys. 2002; 24 (3): 173-183.
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15.柴岗,张艳,刘伟等.组织工程骨在颅颌面骨缺损临床修复中的应用.中华医学杂志.2003; 83(19):1676-1681.
16. Atala A, Bauer SB, Soker S, et al. Tissue-engineered autologous bladders for patients needing cystoplasty. Lancet.2006; 367(9518):1241-1246.
17. Pokrywczyńska M, Drewa T. Treatment of liver failure using tissue engineering techniques. Pol Merkur Lekarski.2007; 23(133):66-69.
18. Kofidis T, Müller-Stahl K, Haverich A. Myocardial restoration and tissue engineering of heart structures.Methods Mol Med.2007;140:273-290.
19. Schmidt D, Mol A, Kelm JM, et al. In vitro heart valve tissue engineering. Methods Mol Med.2007; 140:319-330.
20. Kagami H, Wang S, Hai B. Restoring the function of salivary glands. Oral Dis. 2008; 14 (1):15 -24.
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