脱细胞技术在组织工程血管中的应用进展

将天然血管经过脱细胞处理得到的脱细胞血管,被认为是一种具有广阔应用前景的组织工程血管支架材料.截至目前,细胞外基质(ECM)支架的制备方法仍缺乏统一标准.脱细胞方法的选择取决于组织来源和基质支架的用途,尤其对于脱细胞血管等需要长期承受血流冲击的基质支架材料来说,脱细胞方案的选择至关重要.细胞清除效率和细胞外基质支架的性...     

An overview of tissue and whole organ decellularization processes

Biologic scaffold materials composed of extracellular matrix (ECM) are typically derived by processes that involve decellularization of tissues or organs. Preservation of the complex composition and three-dimensional ultrastructure of the ECM is highly desirable but it is recognized that all methods of decellularization result in disruption of the architecture and potential loss of surface structure and composition. Physical methods and chemical and biologic agents are used in combination to lyse cells, followed by rinsing to remove cell remnants. Effective decellularization methodology is dictated by factors such as tissue density and organization, geometric and biologic properties desired for the end product, and the targeted clinical application. Tissue decellularization with preservation of ECM integrity and bioactivity can be optimized by making educated decisions regarding the agents and techniques utilized during processing. An overview of decellularization methods, their effect upon resulting ECM structure and composition, and recently described perfusion techniques for whole organ decellularization techniques are presented herein.     

On the Decellularization of Fresh or Frozen Human Umbilical Arteries: Implications for Small-Diameter Tissue Engineered Vascular Grafts

Most tissues, including those to be decellularized for tissue engineering applications, are frozen for long term preservation. Such conventional cryopreservation has been shown to alter the structure and mechanical properties of tissues. Little is known, however, how freezing affects decellularization of tissues. The purpose of this study was two-fold: to examine the effects of freezing on decellularization of human umbilical arteries (HUAs), which represent a potential scaffolding material for small-diameter tissue-engineered vascular grafts, and to examine how decellularization affects the mechanical properties of frozen HUAs. Among many decellularization methods, hypotonic sodium dodecyl sulfate solution was selected as the decellularizing agent and tested on fresh HUAs to optimize decellularization conditions. The efficiency of decellularization was evaluated by DNA assay and histology every 12 up to 48 h. The optimized decellularization protocol was then performed on frozen HUAs. The stiffness, burst pressure, and suture retention strength of fresh HUAs and frozen HUAs before and after decellularization were also examined. It appeared that freezing decreased the efficiency of decellularization, which may be attributed to the condensed extracellular matrix caused by freezing. While the stiffness of fresh HUAs did not change significantly after decellularization, decellularization reduced the compliance of frozen HUAs. Interestingly, the stiffness of decellularized frozen HUAs was similar to that of decellularized fresh HUAs. Although little difference in stiffness was observed, we suggest avoiding freezing if more efficient and complete decellularization is desired.     

组织脱细胞方法的研究进展

脱细胞组织和器官作为一种生物材料,已经在组织工程和再生医学中成功应用,各种组织的脱细胞方法也受到关注.组织脱细胞的方法可以分为物理、化学和酶学方法,各组织脱细胞的效率和结果与组织的来源、结构和组成、脱细胞的方法等都有关,不同的方法对不同的组织不仅清除细胞的效果不同,对细胞外基质(ECM)的影响也不同,这反过来又会使宿主对移植的脱细胞材料产生不同的反应.因此,我们要根据组织特点和不同脱细胞方法的特点来选择最优的脱细胞方法,以期达到理想的效果.本文介绍了最常用的一些脱细胞方法,包括物理、化学和酶学方法,并简单描述了它们对组织支架的影响.     

Comparison of decellularization techniques for preparation of extracellular matrix scaffolds derived from three-dimensional cell culture

Extracellular matrix (ECM) scaffolds derived from cultured cells have drawn increasing attention for use in tissue engineering. We have developed a method to prepare cultured cell-derived ECM scaffolds by combining three-dimensional cell culture, decellularization, and selective template removal. Cell-ECM-template complexes were first formed by culture of cells in a poly(lactic-co-glycolic acid) (PLGA) mesh template to deposit their own ECM. The complexes were subsequently decellularized to remove cellular components. Finally, the PLGA template was selectively removed to obtain the ECM scaffolds. Seven decellularization methods were compared for their decellularization effects during scaffold preparation. They were: freeze-thaw cycling (-80°C, six times) with ammonia water (25 mM); 0.1% Triton? X-100 (TX100) with 1.5M KCl aqueous solution; freeze-thaw cycling alone; ammonia water alone; TX100 extraction; osmotic shock with 1.5M KCl; and freeze-thaw cycling with 3M NaCl. Among these methods, the methods of freeze-thaw cycling with NH(4) OH and TX100 with 1.5M KCl showed the best effect on the removal of cellular components from the complexes, while the other five methods could only partially remove cellular components. The ECM scaffolds prepared by these two methods had similar gross appearances and microstructures. In vivo implantation of the ECM scaffolds prepared by these two methods induced mild host responses. The two decellularization methods were demonstrated to be effective for preparation of cultured cell-derived ECM scaffolds.     

Decellularization of tissues and organs

Decellularized tissues and organs have been successfully used in a variety of tissue engineering/regenerative medicine applications, and the decellularization methods used vary as widely as the tissues and organs of interest. The efficiency of cell removal from a tissue is dependent on the origin of the tissue and the specific physical, chemical, and enzymatic methods that are used. Each of these treatments affect the biochemical composition, tissue ultrastructure, and mechanical behavior of the remaining extracellular matrix (ECM) scaffold, which in turn, affect the host response to the material. Herein, the most commonly used decellularization methods are described, and consideration give to the effects of these methods upon the biologic scaffold material.     

The role of protein solubilization in antigen removal from xenogeneic tissue for heart valve tissue engineering

Decellularization techniques have been developed in an attempt to reduce the antigenicity of xenogeneic biomaterials, a critical barrier in their use as tissue engineering scaffolds. However, numerous studies have demonstrated inadequate removal and subsequent persistence of antigens in the biomaterial following decellularization, resulting in an immune response upon implantation. Thus, methods to enhance antigen removal (AR) are critical for the use of xenogeneic biomaterials in tissue engineering and regenerative medicine. In the present study, AR methods incorporating protein solubilization principles were investigated for their ability to reduce antigenicity of bovine pericardium (BP) for heart valve tissue engineering. Bovine pericardium following AR (BP-AR) was assessed for residual antigenicity, tensile properties, and extracellular matrix composition. Increasing protein solubility during AR significantly decreased the residual antigenicity of BP-AR-by an additional 80% compared to hypotonic solution or 60% compared to 0.1% (w/v) SDS decellularization methods. Moreover, solubilizing agents have a dominant effect on reducing the level of residual antigenicity of BP-AR beyond that achieved by AR additives alone. Tested AR methods did not compromise the tensile properties of BP-AR compared to native BP. Furthermore, residual cell nuclei did not correlate to residual antigenicity, demonstrating that residual nuclei counts may not be an appropriate indicator of successful AR. In conclusion, AR strategies promoting protein solubilization significantly reduced residual antigens compared to decellularization methods without compromising biomaterial functional properties. This study demonstrates the importance of solubilizing protein antigens for their removal in the generation of xenogeneic scaffolds.     

Preservation of aortic root architecture and properties using a detergent-enzymatic perfusion protocol

Aortic valve degeneration and dysfunction is one of the leading causes for morbidity and mortality. The conventional heart-valve prostheses have significant limitations with either life-long anticoagulation therapeutic associated bleeding complications (mechanical valves) or limited durability (biological valves). Tissue engineered valve replacement recently showed encouraging results, but the unpredictable outcome of tissue degeneration is likely associated to the extensive tissue processing methods. We believe that optimized decellularization procedures may provide aortic valve/root grafts improved durability. We present an improved/innovative decellularization approach using a detergent-enzymatic perfusion method, which is both quicker and has less exposure of matrix degenerating detergents, compared to previous protocols. The obtained graft was characterized for its architecture, extracellular matrix proteins, mechanical and immunological properties. We further analyzed the engineered aortic root for biocompatibility by cell adhesion and viability in vitro and heterotopic implantation in vivo. The developed decellularization protocol was substantially reduced in processing time whilst maintaining tissue integrity. Furthermore, the decellularized aortic root remained bioactive without eliciting any adverse immunological reaction. Cell adhesion and viability demonstrated the scaffold's biocompatibility. Our optimized decellularization protocol may be useful to develop the next generation of clinical valve prosthesis with a focus on improved mechanical properties and durability.     

Comparison of aortic valve allograft decellularization techniques in the rat

Rodent models have been essential to understanding the immune-mediated failure of aortic valve allografts (AVAs). Decellularization has been proposed to reduce the immunogenicity of AVAs. The objective of this study was to determine the most effective method to decellularize AVAs for use in a rat model. Three different decellularization techniques were compared in Lewis aortic valves. Detergent decellularization involved a series of hypotonic and hypertonic Tris buffers at 4 degrees C for 48 h/buffer containing 0.5% Triton X-100 followed by a 72 h washout in phosphate-buffered saline. Osmotic decellularization was performed in similar manner to the detergent-based technique except without the addition of Triton X-100. Enzymatic decellularization consisted of trypsin/EDTA at 37 degrees C for 48 h. Assessment was performed with light microscopy (H&E, Movat's pentachrome), immunohistochemistry for residual cellular elements, and hydroxyproline assays. Detergent-based methodology effected near-complete decellularization of both the leaflets and aortic wall in addition to preservation of the extracellular matrix (ECM). Osmotic lysis was associated with preservation of ECM and moderate decellularization. Enzymatic decellularization resulted in complete decellularization but extensive degeneration and fragmentation of the ECM. When implanted into the infrarenal aorta of allogeneic rats for 1 week, valves decellularized with detergent-based and osmotic methodology failed to stimulate an allogeneic immune response as evidenced by an absence of T cell infiltrates. Osmotic lysis protocols with low dose detergent appear to be most effective at both removing antigenic cellular elements and preserving ECM.     

脱细胞基质制备方法及其在涎腺组织工程中的应用

背景:由脱细胞基质组成的生物支架被广泛应用于动物及临床研究,以修复和重建组织与器官,但所有的脱细胞方法都会在一定程度上破坏基质结构与功能。目的:综述脱细胞基质制备方法、优缺点及其在涎腺组织工程研究中的应用。方法:应用计算机检索CNKI数据库、中国生物医学文献数据库、PubMed数据库及Elsevier数据库2008至2019年发表的相关文献,检索词为“脱细胞基质,制备方法,涎腺,组织工程,再生;decellularization,preparation method,parotid gland tissue engineering”,共纳入74篇文献。结果与结论:大部分组织与器官脱细胞基质制备方法需要化学、生物(酶)、物理及以上几种方法联合使用,具体方法取决于组织与器官的厚度、组成和性质。虽然不是所有脱细胞方法均可去除组织与器官中的细胞成分,但完全去除细胞的组织与器官具有重塑组织特异性的优势,为接种细胞的增殖和分化提供有利的微环境。由于涎腺结构复杂和组织工程化在临床应用中的挑战,应用于患者的临床移植进展有限,该领域的体内研究仅限于动物,而基于颌下腺脱细胞基质生物支架材料方面的应用有望为涎腺组织工程提供有利的来源。     

不同保护剂对大鼠肾脏冻融法脱细胞的影响

目的:利用不同保护剂对大鼠肾脏进行预处理,辅助冻融法脱细胞获得脱细胞支架,优化冻融脱细胞工艺。方法:采用不同保护剂预处理大鼠肾脏,放入-20℃环境下进行冷冻,12 h后进行37℃水浴复温,然后利用Triton X-100灌注24 h、PBS灌注2 h。最后通过CT三维重构、染色切片、蛋白质定量以及力学性能分析等手段评估所得脱细胞支架。结果:未添加保护剂组血管网络损伤较大,洗脱效果一般。添加不同保护剂组的血管网络也均存在一定的损伤,但其中10%DMSO和5%海藻糖组血管网络保留得较为完整。但10%DMSO组DNA等物质残留过多,5%海藻糖组洗脱细胞效果良好。结论:加载保护剂能够对冻融法脱细胞起到一定的促进作用,且能保护大鼠肾脏内部血管网络等结构,但不同保护剂对冻融法脱细胞的作用效果不同。     

Comparison of three methods for the derivation of a biologic scaffold composed of adipose tissue extracellular matrix

Extracellular matrix (ECM)-based scaffold materials have been used successfully in both preclinical and clinical tissue engineering and regenerative medicine approaches to tissue reconstruction. Results of numerous studies have shown that ECM scaffolds are capable of supporting the growth and differentiation of multiple cell types in vitro and of acting as inductive templates for constructive tissue remodeling after implantation in vivo. Adipose tissue represents a potentially abundant source of ECM and may represent an ideal substrate for the growth and adipogenic differentiation of stem cells harvested from this tissue. Numerous studies have shown that the methods by which ECM scaffold materials are prepared have a dramatic effect upon both the biochemical and structural properties of the resultant ECM scaffold material as well as the ability of the material to support a positive tissue remodeling outcome after implantation. The objective of the present study was to characterize the adipose ECM material resulting from three methods of decellularization to determine the most effective method for the derivation of an adipose tissue ECM scaffold that was largely free of potentially immunogenic cellular content while retaining tissue-specific structural and functional components as well as the ability to support the growth and adipogenic differentiation of adipose-derived stem cells. The results show that each of the decellularization methods produced an adipose ECM scaffold that was distinct from both a structural and biochemical perspective, emphasizing the importance of the decellularization protocol used to produce adipose ECM scaffolds. Further, the results suggest that the adipose ECM scaffolds produced using the methods described herein are capable of supporting the maintenance and adipogenic differentiation of adipose-derived stem cells and may represent effective substrates for use in tissue engineering and regenerative medicine approaches to soft tissue reconstruction.     

Mass transfer trends occurring in engineered ex vivo tissue scaffolds

In vivo the vasculature provides an effective delivery system for cellular nutrients; however, artificial scaffolds have no such mechanism, and the ensuing limitations in mass transfer result in limited regeneration. In these investigations, the regional mass transfer properties that occur through a model scaffold derived from the human umbilical vein (HUV) were assessed. Our aim was to define the heterogeneous behavior associated with these regional variations, and to establish if different decellularization technologies can modulate transport conditions to improve microenvironmental conditions that enhance cell integration. The effect of three decellularization methods [Triton X-100 (TX100), sodium dodecyl sulfate (SDS), and acetone/ethanol (ACE/EtOH)] on mass transfer, cellular migration, proliferation, and metabolic activity were assessed. Results show that regional variation in tissue structure and composition significantly affects both mass transfer and cell function. ACE/EtOH decellularization was shown to increase albumin mass flux through the intima and proximate-medial region (0-250 μm) when compared with sections decellularized with TX100 or SDS; although, mass flux remained constant over all regions of the full tissue thickness when using TX100. Scaffolds decellularized with TX100 were shown to promote cell migration up to 146% further relative to SDS decellularized samples. These results show that depending on scaffold derivation and expectations for cellular integration, specificities of the decellularization chemistry affect the scaffold molecular architecture resulting in variable effects on mass transfer and cellular response.     

无细胞胶原基质的研究进展

天然胶原类组织,经过脱细胞处理后,降低免疫原性,可作为组织工程的支架材料,本文就脱细胞的方法以及无细胞胶原基质的研究进展作一综述。     

Matrix composition and mechanics of decellularized lung scaffolds

The utility of decellularized native tissues for tissue engineering has been widely demonstrated. Here, we examine the production of decellularized lung scaffolds from native rodent lung using two different techniques, principally defined by use of either the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) or sodium dodecyl sulfate (SDS). All viable cellular material is removed, including at least 99% of DNA. Histochemical staining and mechanical testing indicate that collagen and elastin are retained in the decellularized matrices with CHAPS-based decellularization, while SDS-based decellularization leads to loss of collagen and decline in mechanical strength. Quantitative assays confirm that most collagen is retained with CHAPS treatment but that about 80% of collagen is lost with SDS treatment. In contrast, for both detergent methods, at least 60% of elastin content is lost along with about 95% of native proteoglycan content. Mechanical testing of the decellularized scaffolds indicates that they are mechanically similar to native lung using CHAPS decellularization, including retained tensile strength and elastic behavior, demonstrating the importance of collagen and elastin in lung mechanics. With SDS decellularization, the mechanical integrity of scaffolds is significantly diminished with some loss of elastic function as well. Finally, a simple theoretical model of peripheral lung matrix mechanics is consonant with our experimental findings. This work demonstrates the feasibility of producing a decellularized lung scaffold that can be used to study lung matrix biology and mechanics, independent of the effects of cellular components.     

Altered structural and mechanical properties in decellularized rabbit carotid arteries

Recently, major achievements in creating decellularized whole tissue scaffolds have drawn considerable attention to decellularization as a promising approach for tissue engineering. Decellularized tissues are expected to have mechanical strength and structure similar to the native tissues from which they are derived. However, numerous studies have shown that mechanical properties change after decellularization. Often, tissue structure is observed by histology and electron microscopy, but the structural alterations that may have occurred are not always evident. Here, a variety of techniques were used to investigate changes in tissue structure and relate them to altered mechanical behavior in decellularized rabbit carotid arteries. Histology and scanning electron microscopy revealed that major extracellular matrix components were preserved and fibers appeared intact, although collagen appeared looser and less crimped after decellularization. Transmission electron microscopy confirmed the presence of proteoglycans (PG), but there was decreased PG density and increased spacing between collagen fibrils. Mechanical testing and opening angle measurements showed that decellularized arteries had significantly increased stiffness, decreased extensibility and decreased residual stress compared with native arteries. Small-angle light scattering revealed that fibers had increased mobility and that structural integrity was compromised in decellularized arteries. Taken together, these studies revealed structural alterations that could be related to changes in mechanical properties. Further studies are warranted to determine the specific effects of different decellularization methods on the structure and performance of decellularized arteries used as vascular grafts.     

Qualitative and Quantitative Evaluation of a Novel Detergent-Based Method for Decellularization of Peripheral Nerves

Tissue engineering is an emerging strategy for the development of nerve substitutes for peripheral nerve repair. Especially decellularized peripheral nerve allografts are interesting alternatives to replace the gold standard autografts. In this study, a novel decellularization protocol was qualitatively and quantitatively evaluated by histological, biochemical, ultrastructural and mechanical methods and compared to the protocol described by Sondell et al. and a modified version of the protocol described by Hudson et al. Decellularization by the method described by Sondell et al. resulted in a reduction of the cell content, but was accompanied by a loss of essential extracellular matrix (ECM) molecules such as laminin and glycosaminoglycans. This decellularization also caused disruption of the endoneurial tubes and an increased stiffness of the nerves. Decellularization by the adapted method of Hudson et al. did not alter the ECM composition of the nerves, but an efficient cell removal could not be obtained. Finally, decellularization by the method developed in our lab by Roosens et al. led to a successful removal of nuclear material, while maintaining the nerve ultrastructure and ECM composition. In addition, the resulting ECM scaffold was found to be cytocompatible, allowing attachment and proliferation of adipose-derived stem cells. These results show that our decellularization combining Triton X-100, DNase, RNase and trypsin created a promising scaffold for peripheral nerve regeneration.     

Comparison of different decellularization procedures of porcine heart valves

BACKGROUND: Tissue engineering of heart valves should avoid the disadvantages of conventional prostheses. In this study we tested different decellularization procedures for their potential of cell removal and their ability to preserve the matrix. METHODS: Specimens of porcine aortic and pulmonary roots were treated with either trypsin or sodium-dodecyl-sulfate (SDS) or Triton-X 100 and sodium-deoxycholate with a range of concentrations. Tissue samples were then processed for scanning electron microscopy and laser scanning microscopy. RESULTS: Trypsin achieved only incomplete decellularization and caused severe structural alterations of the matrix. In contrast SDS removed cells completely but caused strong structural alterations. Treatment with Triton-X100 and sodium-deoxycholate achieved both complete decellularization and preservation of the matrix structure. CONCLUSION: Techniques of decellularization are highly variable in efficiency and matrix preservation and was best achieved in our study with Triton-X100 and sodium deoxycholate.     

脑去细胞生物支架的制备与鉴定

通过化学萃取加震荡法制备脑去细胞外基质,并对其进行形态学分析和成分鉴定。健康成年SD大鼠20只,分成正常对照组和去细胞组。去细胞组大鼠经心脏灌流后取脑,通过化学萃取加震荡法,依次用3%的TritionX-100、1%的SDS、4%的脱氧胆酸钠震荡萃取,无菌蒸馏水漂洗制备脑去细胞外基质(dBECM)。通过扫描电镜观察dBECM微观形态,用HE染色和荧光DAPI染色分析其去细胞化的效果,进一步通过Masson染色和免疫荧光染色进行成分的鉴定。通过HE染色及荧光DAPI染色可以发现,该方法去细胞化程度完全(去细胞程度大于99%),仅保留大量细胞外基质,未见明显的细胞及细胞核成分残留;Masson染色和免疫荧光染色发现,dBECM保留弹性蛋白(4.0%±1.1%)、层粘连蛋白(19.0%±1.6%)、纤维连接蛋白(9.0%±2.1%)、IV型胶原蛋白(16.0%±1.9%)等成分。化学萃取加震荡法可有效地去除大鼠脑组织内的细胞成分,制备得到的脑去细胞外基质较好地维持宏观和微观的三维结构,保留细胞外基质支架的蛋白成分,是一种简便且理想的脑去细胞外基质制备技术。