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.     

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...     

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 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.     

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.     

Evaluation of the structural integrity and extracellular matrix components of tracheal allografts following cyclical decellularization techniques: comparison of three protocols

Tracheal reconstruction is indicated in cases of malignancy, traumatic injury, and subglottic or tracheal stenosis. Recent progress in airway transplantation has provided renewed optimism for potential solutions for defects involving more than half of the tracheal length in adults or one-third of the tracheal length in children. Biologic scaffolds derived from decellularized tissues and organs have shown great promise in tracheal allotransplantation, and cyclical decellularization techniques have been hypothesized as abrogating the need for immunosuppressive therapy. In this study, we performed a direct comparison of three decellularization protocols (Protocols A, B, and C) previously described in the literature, two of which were described in tracheal tissue (Protocols A and B). We concentrated on the immunogenicity within the epithelium and mucosa, quantified and qualified the extracellular matrix (ECM) components, and performed compliance measurements on large circumferential decellularized tracheal scaffolds following cyclical decellularization techniques using all three protocols. Quantitative measurements of glycosaminoglycans (GAGs) showed a significant decrease in the mucosal component following 17 cycles of all 3 protocols as well as a significant decrease of GAGs in the cartilaginous component following cycles 1, 9, and 17 of Protocol A and cycle 17 of Protocol C. Compliance measurements were also shown to be different between the protocols, with grafts becoming more compliant at physiologic pressures after cyclical decellularization with Protocols A and B and slightly less compliant but remaining similar to native trachea using Protocol C. Positive staining for anti-major histocompatibility complex Class I (anti-MHCI) and anti-MHCII remained within the submucosal glandular components despite multiple cycles of decellularization using all three protocols. This study illustrated that there are significant differences in ECM composition and resultant structural integrity of decellularized tracheal scaffolds depending on the decellularization protocol. Protocol B was shown to maintain the GAGs components despite an increase in tracheal compliance, while Protocol C decreases GAGs components following multiple cycles, despite showing a tracheal compliance resembling that of the native trachea at physiologic airway pressures.     

Biologic scaffold composed of skeletal muscle extracellular matrix

Biologic scaffolds prepared from the extracellular matrix (ECM) of decellularized mammalian tissues have been shown to facilitate constructive remodeling in injured tissues such as skeletal muscle, the esophagus, and lower urinary tract, among others. The ECM of every tissue has a unique composition and structure that likely has direct effects on the host response and it is plausible that ECM harvested from a given tissue would provide distinct advantages over ECM harvested from nonhomologous tissues. For example, a tissue specific muscle ECM scaffold may be more suitable for constructive remodeling of skeletal muscle than non-homologous ECM tissue sources. The present study describes an enzymatic and chemical decellularization process for isolating skeletal muscle ECM scaffolds using established decellularization criteria and characterized the structure and chemical composition of the resulting ECM. The results were compared to those from a non-muscle ECM derived from small intestine (SIS). Muscle ECM was shown to contain growth factors, glycosaminoglycans, and basement membrane structural proteins which differed from those present in SIS. Myogenic cells survived and proliferated on muscle ECM scaffolds in vitro, and when implanted in a rat abdominal wall injury model in vivo was shown to induce a constructive remodeling response associated with scaffold degradation and myogenesis in the implant area; however, the remodeling outcome did not differ from that induced by SIS by 35 days post surgery. These results suggest that superior tissue remodeling outcomes are not universally dependent upon homologous tissue derived ECM scaffold materials.     

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.     

Biologic scaffolds composed of central nervous system extracellular matrix

Acellular biologic scaffolds are commonly used to facilitate the constructive remodeling of three of the four traditional tissue types: connective, epithelial, and muscle tissues. However, the application of extracellular matrix (ECM) scaffolds to neural tissue has been limited, particularly in the central nervous system (CNS) where intrinsic regenerative potential is low. The ability of decellularized liver, lung, muscle, and other tissues to support tissue-specific cell phenotype and function suggests that CNS-derived biologic scaffolds may help to overcome barriers to mammalian CNS repair. A method was developed to create CNS ECM scaffolds from porcine optic nerve, spinal cord, and brain, with decellularization verified against established criteria. CNS ECM scaffolds retained neurosupportive proteins and growth factors and, when tested with the PC12 cell line in vitro, were cytocompatible and stimulated proliferation, migration, and differentiation. Urinary bladder ECM (a non-CNS ECM scaffold) was also cytocompatible and stimulated PC12 proliferation but inhibited migration rather than acting as a chemoattractant over the same concentration range while inducing greater rates of PC12 differentiation compared to CNS ECM. These results suggest that CNS ECM may provide tissue-specific advantages in CNS regenerative medicine applications and that ECM scaffolds in general may aid functional recovery after CNS injury.     

Method of preparing a decellularized porcine tendon using tributyl phosphate

Extracellular matrix (ECM) materials are currently utilized for soft tissue repair applications such as vascular grafts, tendon reconstruction, and hernia repair. These materials are derived from tissues such as human dermis and porcine small intestine submucosa, which must be rendered acellular to prevent disease transmission and decrease the risk of an immune response. The ideal decellularization technique removes cells and cellular remnants, but leaves the original collagen architecture intact. The tissue utilized in this study was the central tendon of the porcine diaphragm, which had not been previously investigated for soft tissue repair. Several treatments were investigated during this study including peracetic acid, TritonX-100, sodium dodecyl sulfate, and tri(n-butyl) phosphate (TnBP). Of the decellularization treatments investigated, only 1% TnBP was effective in removing cell nuclei while leaving the structure and composition of the tissue intact. Overall, the resulting acellular tissue scaffold retained the ECM composition, strength, resistance to enzymatic degradation, and biocompatibility of the original tissue, making 1% TnBP an acceptable decellularization treatment for porcine diaphragm tendon.     

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.     

The quest for an optimized protocol for whole-heart decellularization: a comparison of three popular and a novel decellularization technique and their diverse effects on crucial extracellular matrix qualities

Decellularized cardiac extracellular matrix (ECM) has been introduced as a template for cardiac tissue engineering, providing the advantages of a prevascularized scaffold that mimics native micro- and macroarchitecture to a degree difficult to achieve with synthetic materials. Nonetheless, the decellularization protocols used to create acellular myocardial scaffolds vary widely throughout the literature. In this study we performed a direct comparison of three previously described protocols while introducing and evaluating a novel, specifically developed fourth protocol, by decellularizing whole rat hearts through software-controlled automatic coronary perfusion. Although all protocols preserved the macroarchitecture of the hearts and all resulting scaffolds could successfully be reseeded with C2C12 myoblasts, assessing their biocompatibility for three-dimensional in vitro studies, we found striking differences concerning the microcomposition of the ECM scaffolds on a histological and biochemical level. While laminin could still be detected in all groups, other crucial ECM components, like elastin and collagen IV, were completely removed by at least one of the protocols. Further, only three protocols maintained a glycosaminoglycan content comparable to native tissue, whereas the remaining DNA content within the ECM varied highly throughout all four tested protocols. This study showed that the degree of acellularity and resulting ECM composition of decellularized myocardial scaffolds strongly differs depending on the decellularization protocol.     

In vivo degradation of 14C-labeled porcine dermis biologic scaffold

Biologic scaffold materials are used for repair and reconstruction of injured or missing tissues. Such materials are often composed of allogeneic or xenogeneic extracellular matrix (ECM) manufactured by decellularization of source tissue, such as dermis. Dermal ECM (D-ECM) has been observed to degrade and remodel in vivo more slowly than other biologic scaffold materials, such as small intestinal submucosa (SIS-ECM). Histologic examination is a common method for evaluating material degradation, but it lacks sensitivity and is subject to observer bias. Utilization of ¹⁴C-proline labeled ECM is a quantitative alternative for measuring degradation of ECM scaffolds. Using both methods, the amount of degradation of D-ECM and SIS-ECM was determined at 2, 4, and 24 weeks post-implantation in a rodent model. Results utilizing ¹⁴C liquid scintillation counting (LSC) analysis showed distinct differences in degradation at the three time points. D-ECM material in situ stayed the same at 76% remaining from 2 to 4 weeks post-implantation, and then decreased to 44% remaining at 24 weeks. In the same time period, implanted SIS-ECM material decreased from 72% to 13% to 0%. Visual examination of device degradation by histology overestimated degradation at 2 weeks and underestimated device degradation at 24 weeks, compared to the ¹⁴C method.     

The basement membrane component of biologic scaffolds derived from extracellular matrix

The extracellular matrix (ECM) has been successfully used as a scaffold for constructive remodeling of multiple tissues in both preclinical studies and in human clinical applications. The basement membrane is a specialized form of the ECM that supports and facilitates the growth of epithelial cell populations. The morphology and the molecular composition of the ECM, including the basement membrane, vary depending upon the organ from which the ECM is harvested and the methods by which it is processed for use as a medical device. Processing steps, such as decellularization, lyophilization, disinfection, and terminal sterilization, may affect the morphology and composition of an ECM scaffold, including, but not limited to, the integrity of a basement membrane complex. The present study evaluated the presence and integrity of a basement membrane complex in processed ECM derived from three different tissues: the urinary bladder, small intestine, and liver. Immunohistochemical determination of the presence and localization of three basement membrane molecules, collagen IV, laminin, and collagen VII, was conducted for each ECM scaffold. Scanning electron microscopy (SEM) was used to further explore the surface ultrastructure of selected ECM scaffolds. The effect of a surface basement membrane presence upon the pattern of in vitro growth of two separate cell types, NIH 3T3 fibroblasts and human microvascular endothelial cells (HMEC), was also evaluated for each ECM scaffold. Results showed that the only intact basement membrane complex was found on the luminal surface of the ECM derived from the urinary bladder and that the basement membrane was an effective barrier to penetration of the scaffold by the seeded cells. We conclude that the urinary bladder ECM but not the small intestine- or liver-derived ECM contains a surface with composition and morphology consistent with that of an intact basement membrane complex, that the basement membrane complex can survive processing, and that the basement membrane structure can modulate in vitro cell growth patterns.     

Consequences of ineffective decellularization of biologic scaffolds on the host response

Biologic scaffold materials composed of extracellular matrix (ECM) are routinely used for a variety of clinical applications. Despite known variations in tissue remodeling outcomes, quantitative criteria by which decellularization can be assessed were only recently described and as a result, the amount of retained cellular material varies widely among commercial products. The objective of this study was to evaluate the consequences of ineffective decellularization on the host response. Three different methods of decellularization were used to decellularize porcine small intestinal ECM (SIS-ECM). The amount of cell remnants was quantified by the amount and fragmentation of DNA within the scaffold materials. The M1/M2 phenotypic polarization profile of macrophages, activated in response to these ECM scaffolds, was assessed in vitro and in vivo using a rodent model of body wall repair. The results show that, in vitro, more aggressive decellularization is associated with a shift in macrophage phenotype predominance from M1 to M2. While this shift was not quantitatively apparent in vivo, notable differences were found in the distribution of M1 vs. M2 macrophages within the various scaffolds. A clear association between macrophage phenotype and remodeling outcome exists and effective decellularization remains an important component in the processing of ECM-based scaffolds.     

Regenerative medicine and developmental biology: the role of the extracellular matrix

The principles and ultimate goals of regenerative medicine and developmental biology involve a complex sequence of events, culminating in the formation of structurally and functionally normal tissues and organs. The molecular composition of the extracellular matrix (ECM) plays a critical role in cellular migration and differentiation events. Mammalian ECM, derived from various tissues and organs, has been used as a biologic scaffold for therapeutic regenerative applications. Hundreds of thousands of human patients have benefited from the use of biologic scaffolds composed of naturally occurring ECM. The mechanisms by which ECM induces constructive remodeling instead of scar tissue formation are only beginning to be understood. This article reviews composition of mammalian ECM, its poorly understood role in developmental biology, and the clinical applications that have resulted from the use of this naturally occurring scaffold.     

组织器官去细胞的研究进展

经去细胞处理的组织和器官已广泛应用于组织工程和再生医学.组织去细胞的功效与组织来源和去细胞方法密切相关.每种处理方法都会不同程度地影响去细胞后遗留支架的生化组分、超微结构以及机械性能.进而影响宿主对支架材料的反应.拟对最常用的去细胞方法及不同方法对生物支架的作用特点作一综述.     

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.     

Damage associated molecular patterns within xenogeneic biologic scaffolds and their effects on host remodeling

The immune response is an important determinant of the downstream remodeling of xenogeneic biologic scaffolds in vivo. Pro-inflammatory responses have been correlated with encapsulation and a foreign body reaction, while anti-inflammatory reactions are associated with constructive remodeling. However, the bioactive and bioinductive molecules within the extracellular matrix (ECM) that induce this polarization are unclear, although it is likely that cellular remnants such as damage associated molecular patterns (DAMPs) retained within the scaffold may play a role. The present study investigated the immunomodulatory effects of common ECM scaffolds. Results showed that tissue source, decellularization method and chemical crosslinking modifications affect the presence of the well characterized DAMP - HMGB1. In addition, these factors were correlated with differences in cell proliferation, death, secretion of the chemokines CCL2 and CCL4, and up regulation of the pro-inflammatory signaling receptor toll-like receptor 4 (TLR4). Inhibition of HMGB1 with glycyrrhizin increased the pro-inflammatory response, increasing cell death and up regulating chemokine and TLR4 mRNA expression. The present study suggests the importance of HMGB1 and other DAMPS as bioinductive molecules within the ECM scaffold. Identification and evaluation of other ECM bioactive molecules will be an area of future interest for new biomaterial development.