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.     

A mechanical evaluation of three decellularization methods in the design of a xenogeneic scaffold for tissue engineering the temporomandibular joint disc

Tissue-engineered temporomandibular joint (TMJ) discs offer a viable treatment option for patients with severe joint internal derangement. To date, only a handful of TMJ tissue engineering studies have been carried out and all have incorporated the use of synthetic scaffold materials. These current scaffolds have shown limited success in recapitulating morphological and functional aspects of the native disc tissue. The present study is the first to investigate the potential of a xenogeneic scaffold for use in tissue engineering the TMJ disc. The effects of decellularization agents on the disc's mechanical properties were assessed using three common decellularization protocols: Triton X-100, sodium dodecyl sulfate (SDS) and an acetone/ethanol solution. Decellularized scaffolds were subsequently characterized through cyclic mechanical testing at physiologically relevant frequencies to determine which chemical agent most accurately preserved the native tissue properties. Results have shown that porcine discs treated with SDS most closely matched the energy dissipation capabilities and resistance to deformation of the native tissue. Treatments using Triton X-100 caused the resultant tissue to become relatively softer with inferior energy dissipation capabilities, while treatment using acetone/ethanol led to a significantly stiffer and dehydrated material. These findings support the potential of a porcine-derived scaffold decellularized by SDS as a xenograft for TMJ disc reconstruction.     

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.     

Decellularization methods of porcine kidneys for whole organ engineering using a high-throughput system

End-stage renal failure is a devastating disease, with donor organ transplantation as the only functional restorative treatment. The current number of donor organs meets less than one-fifth of demand, so regenerative medicine approaches have been proposed as potential therapeutic alternatives. One such approach for whole large-organ bioengineering is to combine functional renal cells with a decellularized porcine kidney scaffold. The efficacy of cellular removal and biocompatibility of the preserved porcine matrices, as well as scaffold reproducibility, are critical to the success of this approach. We evaluated the effectiveness of 0.25 and 0.5% sodium dodecyl sulfate (SDS) and 1% Triton X-100 in the decellularization of adult porcine kidneys. To perform the decellularization, a high-throughput system was designed and constructed. In this study all three methods examined showed significant cellular removal, but 0.5% SDS was the most effective detergent (<50?ng DNA/mg dry tissue). Decellularized organs retained intact microarchitecture including the renal vasculature and essential extracellular matrix components. The SDS-treated decellularized scaffolds were non-cytotoxic to primary human renal cells. This method ensures clearance of porcine cellular material (which directly impacts immunoreactivity during transplantation) and preserves the extracellular matrix and cellular compatibility of these renal scaffolds. Thus, we have developed a rapid decellularization method that can be scaled up for use in other large organs, and this represents a step toward development of a transplantable organ using tissue engineering techniques.     

大鼠单叶肝去细胞生物支架的制备与鉴定

目的通过灌注法制备大鼠单叶肝去细胞生物支架,并对其进行鉴定。方法健康成年SD大鼠20只,随机分为去细胞组和正常对照组,每组10只。去细胞组经门静脉灌胃针插管,恒温37℃依次灌注肝素化PBS溶液,1%Triton X-100(p H 7.5~8.0)及PBS溶液。HE、Masson染色及扫描电子显微镜观察组织学及超微结构改变;免疫荧光结合4’,6-二脒基-2-苯基吲哚(DAPI)观察2组胶原蛋白Ⅳ和Ⅰ、层黏连蛋白和纤维连接蛋白观察细胞外基质的主要成分;DNA定性和定量分析组织中残留DNA浓度和片段长度;聚甲基丙烯酸甲酯铸型观察肝脏内血管分布情况。结果 Triton X-100灌注4h左右即可制备单叶肝脏去细胞生物支架。在灌注过程中,肝内细胞和细胞碎片逐渐被清洗,最终变成半透明状。HE、Masson、免疫荧光染色及扫描电子显微镜显示,去细胞组肝生物支架较完整地保留了细胞外支架的成分,未见明显细胞及细胞核成分残留;去细胞组支架DNA残留量较正常对照组下降了97.32%,琼脂糖凝胶电泳未见明显的DNA条带。血管铸型标本显示,去细胞组血管分布与正常对照组相仿,其分支完整、清晰。结论运用Triton X-100灌注法所制备的大鼠单叶肝生物支架去细胞彻底,较完整地保留细胞外基质和血管网络结构,是一种简单易行且较为理想的制备实验用单叶肝生物支架的方法。     

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。     

灌注法制备离体大鼠心脏脱细胞基质

目的 探索制备离体大鼠心脏脱细胞生物支架材料的新方法,为心脏组织工程研究提供三维立体天然支架.方法 取30只成年SD大鼠心脏,运用冻融加化学萃取的组织工程学方法 (胰蛋白酶、十二烷基硫酸钠和曲拉通X-100)处理离体大鼠心脏,同时观察心脏大体形态及颜色变化,并对脱细胞支架进行基因组DNA分析;HE染色,免疫荧光法,扫描和透射电镜进一步检测鉴定脱细胞支架的生物学特征.结果 心脏脱细胞支架外观透明,包膜完整,维持心脏三维立体结构,肉眼可见心脏内脉管系统;脱细胞支架DNA残留量不及对照组的3%;HE染色、扫描和透射电镜结果 显示,心脏脱细胞生物支架去细胞彻底,细胞外基质网状结构保留完整;免疫荧光结果 表明,胶原、弹性蛋白等细胞外支架成分保留较完整,未见明显细胞核成分残留.结论 运用冻融加化学萃取法所制备的离体心脏脱细胞生物支架去细胞彻底,细胞外基质保留较完整,是较为理想的心脏三维立体生物支架材料.     

Evaluation of decellularized esophagus as a scaffold for cultured esophageal epithelial cells

Recently, decellularized tissue has been reported to have the potential to regenerate a variety of tissues. However, the optimal protocol for a decellularized esophagus has not been studied. Here, we investigated the effect of different decellularization protocols on the histology and biocompatibility of decellularized esophagi in view of future applications to tissue engineering. The esophageal mucosal epithelium (EP) from 4-week-old Wistar rats was enzymatically dissociated and cultured with growth-arrested feeder cells. Two methods for decellularization using deoxycholic acid (DEOX) or Triton X-100 (TRITON) were compared on esophagi from adult Wistar rats. Those treated with DEOX showed superior mechanical properties, maintenance of extracellular matrix, and lower DNA content than those treated with TRITON. To evaluate the biocompatibility of the scaffold, cultured (passage 3) esophageal epithelial cells were seeded inside the decellularized esophagus and cultured for 7 days. The cells seeded onto the decellularized esophagus were examined histologically and immunocytochemically. Esophageal epithelial cells were stratified into three to four cellular layers in vitro inside the decellularized esophagus, to show polarity. The results from immunocytochemistry indicated that the seeded epithelial cells expressed characteristic marker proteins for native esophageal EP. Decellularized esophagus showed suitable compatibility as a scaffold material for esophageal tissue engineering.     

Matrix alteration and not residual sodium dodecyl sulfate cytotoxicity affects the cellular repopulation of a decellularized matrix

It has been suggested that residual cytotoxic sodium dodecyl sulfate (SDS) is responsible for the low levels of cell in-growth observed in SDS decellularized tissues. To determine whether this is the case, we used 2 washing methods to remove residual SDS and extensive biochemical, mechanical, and structural analyses to determine the effects of SDS-based decellularization on porcine anterior cruciate ligament (ACL) tissue and its propensity for cellular repopulation. The level of residual SDS in decellularized tissue was reduced using 2 different washing techniques (pH = 9 buffer, 75% ethanol). After washing in pH = 9 or 75% ethanol, residual SDS concentrations in decellularized tissues were found to be approximately 8 and 23 times less than reported SDS cytotoxic levels, respectively. It was found that SDS treatment significantly reduced glycosaminoglycan levels, increased collagen crimp amplitude and periodicity, and increased susceptibility of collagen to degradation by the gelatinase enzyme trypsin. The level of repopulation and viability of autologous ACL fibroblasts in the decellularized tissue after 28 days of culture were found to be the same regardless of the washing technique and resulting level of residual SDS in the tissue. This strongly indicates that alterations in tissue matrix biochemistry or structure from SDS treatment and not residual SDS cytotoxicity are responsible for the low cell re-population observed in SDS decellularized tissues.     

猪小肠黏膜下层脱细胞支架的制备及组织学评价

目的评价不同浓度的十二烷基磺酸钠(SDS)处理猪小肠黏膜下层(SIS)的脱细胞效果,筛选最佳脱细胞浓度,为组织工程化角膜上皮的制备提供支架材料。方法配制0.1%、0.2%、0.3%、0.5%SDS随机分成A、B、C、D 4组,分别脱细胞处理SIS 15min、30min、1h、2h(n=20),同时观察SIS的大体形态变化,HE和4,6-二脒基-2-苯基吲哚(DAPI)染色观察脱细胞前后SIS的生物学特性,并对脱细胞支架进行基因组DNA分析(n=5)。结果脱细胞SIS肉眼观察呈乳白色、半透明的膜状物,具有一定的弹性、韧性及透光性;HE和DAPI染色光学显微镜观察结果显示,A组及B组处理30min时SIS生物支架无细胞及DNA残留,组织结构保留完整,胶原纤维间孔隙率增加;A组及B组处理30min脱细胞率可达90%以上。结论 0.1%及0.2%SDS处理30min条件下脱细胞效果较好,能更好地降低SIS的免疫原性,是SDS脱细胞处理SIS比较理想的浓度时间条件,为组织工程化角膜上皮的构建奠定理论基础。     

去细胞全肾生物支架的制备与鉴定

目的 通过灌注加浸泡法制备全肾细胞外基质支架,并对该生物支架进行鉴定。方法 健康成年SD大鼠20只,取肾,行肾动脉留置针插管,灌注压3.6mmHg,恒温37℃依次浸泡灌注肝素化PBS溶液、0.05%胰蛋白酶溶液、1%十二烷基硫酸钠（SDS）溶液、1% Trition X-100溶液以及含青、链霉素的PBS溶液。处理后,行DNA定量与定性分析,透射电镜、HE染色及免疫荧光观察残留细胞核成分,进一步丙烯腈-丁二烯-苯乙烯树脂(ABS)铸型观察肾内血管分布情况。结果 去细胞组DNA含量较对照组下降了97%,琼脂糖凝胶电泳未见明显DNA条带;透射电镜、HE染色及免疫荧光观察去细胞组保留了大量细胞外基质,未见明显细胞核成分残留;铸型标本显示去细胞组血管较稀疏,分支完整、清晰。结论 联合酶消化法与去垢剂洗涤法,经灌注加浸泡法处理的全肾,可有效清除肾内所有细胞成分,较好地保留细胞外基质支架和血管网络结构,是一种简单易行且较为理想的制备组织工程肾生物支架的方法。     

去细胞化全肝生物支架循环灌注培养条件下体外细胞再植*

背景：通过去细胞化技术制备全肝生物支架成为缓解供体短缺的新技术,优化肝脏组织的去细胞化流程成为新的课题。 目的：通过去细胞化技术建立完整保留肝脏三维结构和脉管系统的全肝生物支架,利用自主设计的循环灌注培养装置实现生物支架体外细胞再植。 方法：通过门静脉路径循环灌注去垢剂Triton X-100,十二烷基硫酸钠,并用磷酸盐缓冲液洗脱残留去垢剂。通过动态循环灌注培养装置进行支架与HepG2细胞的共培养,观察植入细胞在全肝生物支架内的功能表达。 结果与结论：经去垢剂灌注后,支架呈现保持肝脏三维结构的透明结构,苏木精-伊红染色以及扫描电镜结果显示细胞成分被完全移除,Masson’s Trichrome染色可见大量胶原纤维,免疫组化结果证实纤维连接蛋白和层粘连蛋白保存完整。循环灌注培养下细胞白蛋白表达量及尿素合成量较平板培养明显提高。说明去细胞化肝脏生物支架可作为体外肝脏组织重建的基础材料,动态循环灌注方法可实现支架中细胞再植。      

Effect of extraction protocols and epidermal growth factor on the cellular repopulation of decellularized anterior cruciate ligament allografts

We are developing a decellularized bone-anterior cruciate ligament (ACL)-bone allograft for treatment of ACL disruption in young or active patients. This study demonstrates the feasibility of seeding decellularized ACL tissue with primary ligament fibroblasts. Porcine ACLs were decellularized by one of three protocols, each differing only by the detergent/solvent used during the second wash (SDS, Triton-X, or TnBP). Porcine ACL fibroblasts were obtained by explant and seeded onto tissue samples of decellularized ACL. Culture conditions were varied to compare the relative effect of three different decellularization protocols on cellular repopulation. Culture condition variables included (1) the number of cells used for seeding, (2) the addition of epidermal growth factor (EGF), and (3) culture duration. Cellular ingrowth was assessed by metabolic activity (MTT assay), DNA quantification (Hoescht dye), and histology (H&E staining). Cell counting on histological sections demonstrated that Triton-X-and TnBP-treated ligaments were more receptive to cellular ingrowth than SDS-treated samples. The addition of EGF to culture medium did not significantly increase cellular ingrowth. Both the Triton-X and TnBP decellularization treatments provide suitable, naturally derived scaffolds for the ingrowth of primary ACL fibroblasts, and should be further investigated in the development of an allograft-derived bone-ACL-bone graft.     

Decellularized human Schneiderian membrane: Electron microscopic study as a bioscaffold and preliminary cell seeding

BackgroundPerforation of maxillary sinus mucous membrane is one of the most prevalent complications during open sinus lift surgery. Moreover, such complication can usually be managed by an absorbable membrane. As far as absorbable membranes are concerned, decellularized maxillary sinus mucous membrane, which is an extracellular matrix, can be used as a biologic scaffold and an insulating membrane in sinus lifting surgery.Materials and methodsThe decellularization process of the maxillary sinus membrane was performed by means of physical and chemical procedures (liquid nitrogen and sodium dodecyl sulfate). Then this membrane was used as a bioscaffold for culturing with adult mesenchymal stem cells, which were derived from adipose tissue.ResultsHistologic evaluation of the decellularized scaffold revealed that cells of the Schneiderian membrane were compatibly removed via SDS 1%. Moreover, the scan with electron microscope (S6N – Leo vp1450, Germany) of the scaffold indicated that the collagen fibers of the decellularized maxillary sinus membrane were intact. Furthermore, the culture studies carried out showed that this scaffold supported cell seeding.ConclusionThe decellularized human maxillary Schneiderian membrane has a 3D structure similar to that of the extracellular matrix of human normal tissues. As a matter of fact, it can be used as a bioscaffold to support cell seeding.     

Development and characterization of an acellular human pericardial matrix for tissue engineering

This study aimed to produce an acellular human tissue scaffold with a view to recellularization with autologous cells to produce a tissue-engineered pericardium that can be used as a patch for cardiovascular repair. Human pericardia from cadaveric donors were treated sequentially with hypotonic buffer, SDS in hypotonic buffer, and a nuclease solution. Histological analysis of decellularized matrices showed that the human pericardial tissue retained its histioarchitecture and major structural proteins. There were no whole cells or cell fragments. There were no significant differences in the hydroxyproline (normal and denatured collagen) and glycosaminoglycan content of the tissue before and after decellularization (p > 0.05). There were no significant changes in the ultimate tensile strength after decellularization (p > 0.05). However, there was an increased extensibility when the tissue strips were cut parallel to the visualized collagen bundles (p = 0.005). No indication of contact or extract cytotoxicity was found when using human dermal fibroblasts and A549 cells. In summary, successful decellularization of the human pericardium was achieved producing a biocompatible matrix that retained the major structural components and strength of the native tissue.     

Effects of decellularization on the mechanical and structural properties of the porcine aortic valve leaflet

The potential for decellularized aortic heart valves (AVs) as heart valve replacements is based on the assumption that the major cellular immunogenic components have been removed, and that the remaining extracellular matrix (ECM) should retain the necessary mechanical properties and functional design. However, decellularization processes likely alter the ECM mechanical and structural properties, potentially affecting long-term durability. In the present study, we explored the effects of an anionic detergent (sodium dodecyl sulfate (SDS)), enzymatic agent (Trypsin), and a non-ionic detergent (Triton X-100) on the mechanical and structural properties of AV leaflets (AVLs) to provide greater insight into the initial functional state of the decellularized AVL. The overall extensibility represented by the areal strain under 60 N/m increased from 68.85% for the native AV to 139.95%, 137.51%, and 177.69% for SDS, Trypsin, and Triton X-100, respectively, after decellularization. In flexure, decellularized AVLs demonstrated a profound loss of stiffness overall, and also produced a nonlinear moment-curvature relation compared to the linear response of the native AVL. Effective flexural moduli decreased from 156.0+/-24.6 kPa for the native AV to 23.5+/-5.8, 15.6+/-4.8, and 19.4+/-8.9 kPa for SDS, Trypsin, and Triton X-100 treated leaflets, respectively. While the overall leaflet fiber architecture remained relatively unchanged, decellularization resulted in substantial microscopic disruption. In conclusion, changes in mechanical and structural properties of decellularized leaflets were likely associated with disruption of the ECM, which may impact the durability of the leaflets.     

Optimization of a natural collagen scaffold to aid cell–matrix penetration for urologic tissue engineering

The goal of this study was to fabricate a 3-dimensional (3-D) porous scaffold derived from bladder submucosa (BSM) and further recellularize the scaffold with human bladder cells for cell-based urethral tissue engineering. Fresh porcine BSM was soaked with peracetic acid (PAA) at different concentrations (0,1,3,5 and 10%) and then treated with Triton X-100 for decellularization. DNA content analysis showed that nuclear material was removed from the BSM scaffold. Treatment with 5% PAA led to high porosity on the surface of the matrix with retention of less cellular material and maintained about 75% of normal tensile strength. In 3-D dynamic culture, cells formed even multiple layers on the surface of matrix. Cells also penetrated deeper into the lamina propria of the matrix compared to untreated matrix. Immunocytochemical staining indicated that the grafted bladder cells expressed urothelial- and smooth muscle-specific markers both, in vitro and in vivo. This study demonstrates that decellularized/oxidized BSM possesses 3-D porosity for cell infiltration into the matrix. Further, cells seeded on decellularized/oxidized BSM and grown in dynamic culture, significantly promoted cell–matrix penetration in vitro and promoted cell growth in vivo. Scaffolds with such characteristics have potential applications in cell-based urological tissue engineering.     

Decellularized human cornea for reconstructing the corneal epithelium and anterior stroma

In this project, we strived to develop a decellularized human cornea to use as a scaffold for reconstructing the corneal epithelium and anterior stroma. Human cadaver corneas were decellularized by five different methods, including detergent- and nondetergent-based approaches. The success of each method on the removal of cells from the cornea was investigated. The structural integrity of decellularized corneas was compared with the native cornea by electron microscopy. The integrity of the basement membrane of the epithelium was analyzed by histology and by the expression of collagen type IV, laminin, and fibronectin. Finally, the ability of the decellularized corneas to support the growth of human corneal epithelial cells and fibroblasts was assessed in vitro. Corneas processed using Triton X-100, liquid nitrogen, and poly(ethylene glycol) resulted in incomplete removal of cellular material. Corneas processed with the use of sodium dodecyl sulfate (SDS) or with sodium chloride (NaCl) plus nucleases successfully removed all cellular material; however, only the NaCl plus nuclease treatment kept the epithelial basement membrane completely intact. Corneas processed with NaCl plus nuclease supported both fibroblast and epithelial cell growth in vitro, while corneas treated with SDS supported the growth of only fibroblasts and not epithelial cells. Decellularized human corneas provide a scaffold that can support the growth of corneal epithelial cells and stromal fibroblasts. This approach may be useful for reconstructing the anterior cornea and limbus using autologous cells.     

Evaluation of decellularization protocols for production of tubular small intestine submucosa scaffolds for use in oesophageal tissue engineering

Small intestine submucosa (SIS) has emerged as one of a number of naturally derived extracellular matrix (ECM) biomaterials currently in clinical use. In addition to clinical applications, ECM materials form the basis for a variety of approaches within tissue engineering research. In our preliminary work it was found that SIS can be consistently and reliably made into tubular scaffolds which confer certain potential advantages. Given that decellularization protocols for SIS are applied to sheet-form SIS, it was hypothesized that a tubular-form SIS would behave differently to pre-existing protocols. In this work, tubular SIS was produced and decellularized by the conventional peracetic acid–agitation method, peracetic acid under perfusion along with two commonly used detergent–perfusion protocols. The aim of this was to produce a tubular SIS that was both adequately decellularized and possessing the mechanical properties which would make it a suitable scaffold for oesophageal tissue engineering, which was one of the goals of this work. Analysis was carried out via mechanical tensile testing, DNA quantification, scanning electron and light microscopy, and a metabolic assay, which was used to give an indication of the biocompatibility of each decellularization method. Both peracetic acid protocols were shown to be unsuitable methods with the agitation-protocol-produced SIS, which was poorly decellularized, and the perfusion protocol resulted in poor mechanical properties. Both detergent-based protocols produced well-decellularized SIS, with no adverse mechanical effects; however, one protocol emerged, SDS/Triton X-100, which proved superior in both respects. However, this SIS showed reduced metabolic activity, and this cytotoxic effect was attributed to residual reagents. Consequently, the use of SIS produced using the detergent SD as the decellularization agent was deemed to be the most suitable, although the elimination of the DNase enzyme would give further improvement.