Decellularization Agents Research Articles

Decellularization Agents on Porosity and Pore Size in Bovine Pericardium Scaffolds for Pediatric Heart Surgery

Background: Tissue engineering in pediatric and congenital heart surgery relies on developing biomaterials with extracellular matrix (ECM)-like properties. The acellular bovine pericardium membrane (BPM) is a promising scaffold due to its strength, low infection rates, and cost-effectiveness. Decellularization is essential to optimize BPM's properties for tissue regeneration. This study investigates the effects of different decellularization methods on BPM's porosity and pore size. Methods: A true experimental design was used to evaluate BPM scaffolds treated with sodium dodecyl sulfate (SDS), hydrogen peroxide (H2O2), or ASB-16, compared to a control group. Porosity and pore size were measured using image analysis software after 4 weeks of incubation. Statistical analysis was performed using the Kruskal-Wallis test, Mann-Whitney U test, and one-way ANOVA. Results: ASB-16 decellularization significantly increased BPM porosity (50.14 ± 3.71%) compared to the control (3.06 ± 0.99%) and other treatments (SDS: 6.23 ± 2.94%, H2O2: 4.47 ± 1.34%). Pore size was also significantly larger in the ASB-16 group (26.9 ± 5.93 µm) compared to SDS (8.99 ± 2.77 µm), H2O2 (3.13 ± 1.00 µm), and control (2.00 ± 0.29 µm). Conclusion: ASB-16 decellularization effectively enhances BPM porosity and pore size, making it a promising method for optimizing scaffolds in tissue engineering applications. Further research should focus on its impact on cell proliferation and tissue regeneration.

Current Strategies for Tracheal Decellularization: A Systematic Review.

The process of decellularization is crucial for producing a substitute for the absent tracheal segment, and the choice of agents and methods significantly influences the outcomes. This paper aims to systematically review the efficacy of diverse tracheal decellularization agents and methods using the PRISMA flowchart. Inclusion criteria encompassed experimental studies published between 2018 and 2023, written in English, and detailing outcomes related to histopathological anatomy, DNA quantification, ECM evaluation, and biomechanical characteristics. Exclusion criteria involved studies related to 3D printing, biomaterials, and partial decellularization. A comprehensive search on PubMed, NCBI, and ScienceDirect yielded 17 relevant literatures. The integration of various agents and methods has proven effective in the process of tracheal decellularization, highlighting the distinct advantages and drawbacks associated with each agent and method.

An overview of the production of tissue extracellular matrix and decellularization process.

Thousands of patients need an organ transplant yearly, while only a tiny percentage have this chance to receive a tissue/organ transplant. Nowadays, decellularized animal tissue is one of the most widely used methods to produce engineered scaffolds for transplantation. Decellularization is defined as physically or chemically removing cellular components from tissues while retaining structural and functional extracellular matrix (ECM) components and creating an ECM-derived scaffold. Then, decellularized scaffolds could be reseeded with different cells to fabricate an autologous graft. Effective decellularization methods preserve ECM structure and bioactivity through the application of the agents and techniques used throughout the process. The most valuable agents for the decellularization process depend on biological properties, cellular density, and the thickness of the desired tissue. ECM-derived scaffolds from various mammalian tissues have been recently used in research and preclinical applications in tissue engineering. Many studies have shown that decellularized ECM-derived scaffolds could be obtained from tissues and organs such as the liver, cartilage, bone, kidney, lung, and skin. This review addresses the significance of ECM in organisms and various decellularization agents utilized to prepare the ECM. Also, we describe the current knowledge of the decellularization of different tissues and their applications.

Optimization Techniques of Single-Detergent Based Protocols for Heart Tissue Decellularization.

The extracellular matrix (ECM) scaffolds are considered a gold standard for the engineering of appropriate grafts used in regenerative medicine for tissue repair, and decellularization of myocardial tissue is one of the most studied processes for obtaining natural ECM to date. Decellularization methods, agents used, or treatment durations can be varied to optimize cardiac tissue decellularization parameters. In this work we performed a morphological and morphometric analysis of cardiac tissue subjected to decellularization protocols based on Sodium Deoxycholate (SD) or Sodium Lauryl Sulfate (SLS) to identify factors that allow optimization of single-detergent based protocols for cardiac ECM manufacturing. For this, Wistar rat hearts (n=10) were subjected to 5 different decellularization protocols (n=2) and then histologically processed to achieve H&E or Azan trichrome stained sections for the morphological and morphometric analysis of the obtained ECM. The results of this study showed that SLS alters the spatial distribution of cardiac ECM collagen fibers, and SD can be successfully used in tailoring single-based detergent decellularization protocols by appropriately adjusting the application times of hypo/hyperosmotic shocks, which increases the lytic action of the detergent, and the washing times for the efficient elimination of cellular residues.

Decellularization of Articular Cartilage: A Hydrochloric Acid-Based Strategy.

Removing cellular material from a tissue, a process called decellularization, reduces the risk of adverse host reactions, allows for efficient decontamination, and extends the shelf-life of the matrix. It facilitates the use of cartilage tissue as human-derived allograft, thus providing the field of cartilage regeneration with a biomaterial unmatched in its similarity to native cartilage in terms of structure, composition, and mechanical properties.The dense extracellular matrix of articular cartilage requires a particularly thorough process to achieve the removal of cells, cell debris, and reagents used in the process. In our studies (Nürnberger et al., EBioMedicine 64:103196, 2021; Schneider et al., Tissue Eng Part C Methods 22(12):1095-1107, 2016), we have successfully developed a protocol for achieving decellularization via physical, chemical, and enzymatic steps. Combining freeze-thaw cycles for devitalization, hydrochloric acid as decellularization agent and the enzymatic removal of glycosaminoglycans, results in an acellular scaffold that is fully biocompatible and promotes cellular attachment. The structure and sophisticated architecture of collagen type II is left intact.This chapter provides a comprehensive guide to the steps and reagents needed to decellularize articular cartilage. In addition to the standard decell-deGAG protocol, a fast option is given which is suitable for thin specimen. Histological evaluation is presented to illustrate treatment success.

Exhale and Decell: Environmentally Sustainable Sterilization and Decellularization of Cartilage Grafts via Supercritical Carbon Dioxide

PURPOSE: Current decellularization techniques have important limitations, including obligatory use of harsh ionized substances or detergents that deplete bioactive cytokines and compromise the physical integrity of the resulting extracellular matrix (ECM) scaffold. Furthermore, the detergents used are increasingly recognized as harmful to the environment and are poised to be restricted from commercial use. Supercritical carbon dioxide (scCO2) is an appealing alternative, applying the principles behind supercritical fluid extraction and sterilization to the removal of immunogenic cellular components. We endeavor to evaluate the efficacy of scCO2 in both sterilization and decellularization of ovine costal cartilage xenograft and human costal cartilage allograft to develop a highly efficacious, environmentally sustainable, and biocompatible alternative to currently available decellularization strategies. METHODS AND MATERIALS: Xenograft preparation: Racks of lamb were purchased from a local butcher. Floating ribs were minced into ∼8 mm3 cubes or zested into flakes (1 mm × 1 mm). Allograft harvest: Human costal cartilage was obtained from patients undergoing microsurgical breast reconstruction necessitating partial rib resection and processed via mincing or zested as above. All samples were subjected to: 1) sterilization; 2) sterilization and decellularization; or 3) sterilization, decellularization, and a pretreatment wash. Sterilization: Samples were placed in a NovaSterilis ™ Nova2200 unit and subject to standard NovaSterilis ™ sterilization parameters. At least 3 samples with biologic indicators were placed within the packaging and processed with each run. Decellularization: Samples were subject to the standard parameters of the Nova2200 system as previously described by NovaSterilis ™ with an ethanol to scCO2 volume ratio of 1:3.3. Wash: Samples were serially soaked in saline and agitated with exposure to 16 mL 3% H2O2 and a 30-minute scCO2 run at 35° C and 1,436 psi. Histology: After treatment, H&E, DAPI, and safranin-O staining were performed. DNA Quantification: DNA content was quantified in unprocessed and decellularized graft samples using the DNeasy Blood & Tissue kit (Qiagen Inc.). RESULTS: Sterilization conditions were sufficient for a 6 log10 sterilization of B. atrophaeus. H&E staining revealed preservation of tissue architecture after both sterilization and decellularization in both allograft and xenograft cartilage. DAPI staining demonstrated depletion of nuclei in the decellularized zested samples, but persistence of visible nuclei in the minced samples. Safranin-O staining revealed immunogenic GAG depletion after decellularization. DNA content in minced and zested samples was 192.2 ng DNA/mg tissue and 321.6 ng DNA/mg tissue, respectively, while DNA content in sterilized and decellularized minced and zested samples were 24.8 ng DNA/mg tissue and 11.6 ng DNA/mg tissue, respectively (industry standard requirement for decellularization <50 ng DNA/mg tissue); after sterilization, decellularization, and the chemical wash, minced and zested samples contained 17.6 ng DNA/mg tissue and 4 ng DNA/mg tissue respectively. CONCLUSION: These preliminary data suggest that scCO2 sufficiently sterilizes and decellularizes cartilage derived from both human and animal sources, thereby supporting scCO2 as an efficacious, commercially appealing, and ecologically responsible alternative to current decellularization strategies. Biomechanical testing on treated samples, as well as the evaluation of scCO2 as a decellularization agent for human nasal septal cartilage are ongoing.

Comparative Assessment of Various Concentration and Exposure Time of Sodium Dodecyl Sulfate as Decellularization Agents for Small-Vessels Vascular Tissue Engineering

BACKGROUND: Finding the optimum vascular grafts (VG) to replace damaged blood arteries in cardiac surgery is still a work in progress. To be employed, a tissue-engineered VG (TEVG) must have the appropriate biological and mechanical qualities. Decellularized arteries may be a better TEVG than synthetic grafts because of their natural three-dimensional architecture. AIM: The goal of this study was to compare different concentrations and times of sodium dodecyl sulfate (SDS) to decellularize tissue to find the best decellularized VG. METHODS: In all decellularized scaffolds, which are 1% SDS-2 weeks group, hematoxylin and eosin and Masson’s trichrome staining exhibited looser collagen networks and fewer nuclei. RESULTS: The orientation of collagen fibers was identical to native vascular scaffolds. Collagen I deposition was seen in the immunohistochemistry assay. A tensile strength test revealed that the decellularized scaffold (0.5% SDS for 4 weeks and 0.5% SDS for 2 weeks) had exceeded the native arteries’ maximal strength. In comparison to 1% SDS in 4 weeks treated groups, scanning electron microscopy following decellularization revealed no endothelial cells on the inner side of 1% SDS in 2 weeks group with minimum extracellular matrix damage. The endothelial cells remained marginally visible on the inner side of all 0.5% SDS treated groups. The 3-(4,5-dimethylthiazol-2yl)2,5-diphenyltetrazolium bromide test was used to determine the cytotoxicity of the decellularized scaffolds. CONCLUSION: This study reveals that exposing a bovine mesenteric artery to 1% SDS for 2 weeks is an excellent procedure for extracting the most acellular VG, potentially serving as a biological scaffold for TEVGs.

Novel Decellularization Method for Tissue Slices.

Decellularization procedures have been developed and optimized for the entire organ or tissue blocks, by either perfusion of decellularizing agents through the tissue’s vasculature or submerging large sections in decellularizing solutions. However, some research aims require the analysis of native as well as decellularized tissue slices side by side, but an optimal protocol has not yet been established to address this need. Thus, the main goal of this work was to develop a fast and efficient decellularization method for tissue slices—with an emphasis on lung—while attached to a glass slide. To this end, different decellularizing agents were compared for their effectiveness in cellular removal while preserving the extracellular matrix. The intensity of DNA staining was taken as an indicator of remaining cells and compared to untreated sections. The presence of collagen, elastin and laminin were quantified using immunostaining and signal quantification. Scaffolds resulting from the optimized protocol were mechanically characterized using atomic force microscopy. Lung scaffolds were recellularized with mesenchymal stromal cells to assess their biocompatibility. Some decellularization agents (CHAPS, triton, and ammonia hydroxide) did not achieve sufficient cell removal. Sodium dodecyl sulfate (SDS) was effective in cell removal (1% remaining DNA signal), but its sharp reduction of elastin signal (only 6% remained) plus lower attachment ratio (32%) singled out sodium deoxycholate (SD) as the optimal treatment for this application (6.5% remaining DNA signal), due to its higher elastin retention (34%) and higher attachment ratio (60%). Laminin and collagen were fully preserved in all treatments. The SD decellularization protocol was also successful for porcine and murine (mice and rat) lungs as well as for other tissues such as the heart, kidney, and bladder. No significant mechanical differences were found before and after sample decellularization. The resulting acellular lung scaffolds were shown to be biocompatible (98% cell survival after 72 h of culture). This novel method to decellularize tissue slices opens up new methodological possibilities to better understand the role of the extracellular matrix in the context of several diseases as well as tissue engineering research and can be easily adapted for scarce samples like clinical biopsies.

Interindividual heterogeneity affects the outcome of human cardiac tissue decellularization

The extracellular matrix (ECM) of engineered human cardiac tissues corresponds to simplistic biomaterials that allow tissue assembly, or animal derived off-the-shelf non-cardiac specific matrices. Decellularized ECM from human cardiac tissue could provide a means to improve the mimicry of engineered human cardiac tissues. Decellularization of cardiac tissue samples using immersion-based methods can produce acceptable cardiac ECM scaffolds; however, these protocols are mostly described for animal tissue preparations. We have tested four methods to decellularize human cardiac tissue and evaluated their efficiency in terms of cell removal and preservation of key ECM components, such as collagens and sulfated glycosaminoglycans. Extended exposure to decellularization agents, namely sodium dodecyl sulfate and Triton-X-100, was needed to significantly remove DNA content by approximately 93% in all human donors. However, the biochemical composition of decellularized tissue is affected, and the preservation of ECM architecture is donor dependent. Our results indicate that standardization of decellularization protocols for human tissue is likely unfeasible, and a compromise between cell removal and ECM preservation must be established in accordance with the scaffold’s intended application. Notwithstanding, decellularized human cardiac ECM supported human induced pluripotent-derived cardiomyocyte (hiPSC-CM) attachment and retention for up to 2 weeks of culture, and promoted cell alignment and contraction, providing evidence it could be a valuable tool for cardiac tissue engineering.

Development of decellularized aortic scaffold for regenerative medicine using Sapindus mukorossi fruit pericarp extract

The aim of this study is to develop a novel decellularization method using aqueous extract of soap nut pericarp (SPE) and its evaluation using hematoxylin–eosin staining, scanning electron microscopy, diamidino-2-phenylindol (DAPI) staining, mechanical testing, sodium dodecyl sulfate polyacrylamide gel electrophoresis and DNA quantification. The presently available decellularization agent raises some concerns due to the potential for presence of residual cytotoxic agents in the extracellular matrix. Histological analysis of hematoxylin and eosin and masson’s trichrome stained processed aortic samples shows complete decellularization with preservation of extracellular matrix microarchitecture at 120 h. Further, staining of tissue samples with DAPI demonstrates complete removal of DNA fragments. Quantitative evaluation of DNA in the decellularized aorta tissues demonstrated a significant (P < 0.01) decrease in DNA content as compared to native tissues. Collagen quantification assay indicate no significant (P> 0.05) difference in its content between native and decellularized caprine aorta. Tensile strength of the decellularized scaffolds decreased non-significantly (P > 0.05) when compared to native tissues. There was no significant (P > 0.05) difference in young’s modulus of elasticity, stiffness and stretch ratio between native aortic tissues and decellularized aortic scaffolds. Histological and scanning electron microscopic examination of in vitro cultured scaffold demonstrated the cell viability and proliferation of primary chicken embryo fibroblasts. SPE treatment is thus capable of producing cytocompatible decellularized caprine aorta scaffold with preservation of extracellular matrix architecture for vascular tissue engineering and could be applied widely as one of the decellularization agent.

Optimizing the Decellularized Porcine Liver Scaffold Protocol

There are few existing methods for shortening the decellularization period for a human-sized whole-liver scaffold. Here, we describe a protocol that enables effective decellularization of the liver obtained from pigs weigh 120 ± 4.2 kg within 72 h. Porcine livers (approx. 1.5 kg) were decellularized for 3 days using a combination of chemical and enzymatic decellularization agents. After trypsin, sodium deoxycholate, and Triton X-100 perfusion, the porcine livers were completely translucent. Our protocol was efficient to promote cell removal, the preservation of extracellular matrix (ECM) components, and vascular tree integrity. In conclusion, our protocol is efficient to promote human-sized whole-liver scaffold decellularization and thus useful to generate bioengineered livers to overcome the shortage of organs.

Decellularized ECM-derived bioinks: Prospects for the future.

Decellularization aims to remove cells from tissue ultrastructure while preserving the mechanical and biological properties, which makes the decellularized extracellular matrix (dECM) an appropriate scaffold for tissue engineering applications. Three-dimensional (3D) bioprinting technology as a reproducible and accurate method can print the combination of ECM and autologous cells layer by layer to fabricate patient based cell-laden structures representing the intrinsic cues of natural ECM. This review defines ECM, classifies decellularization agents and techniques, and explains different sources of ECM. Then, bioprinting techniques, bioink concept, applications of dECM bioinks, and finally the future perspectives of 3d bioprinting technology are discussed.

Development of perfusion bioreactor for whole organ engineering — a culture system that enhances cellular engraftment, survival and phenotype of repopulated pancreas

Whole organ engineering has emerged as a promising alternative avenue to fill the gap of donor organ shortage in organ transplantation. Recent breakthroughs in the decellularization of solid organs and repopulation with desired cell populations have generated neo-organ constructs with promising functional outcomes. The realization of this goal requires engineering advancement in the perfusion-based bioreactors to (i) efficiently deliver decellularization agents, followed by (ii) its reconstruction with relevant cell types and (iii) maintenance of viability and function of the repopulated organ. In this study, we report the development and assembly of a perfusion bioreactor with the potential to enable regenerative reconstruction of pancreas. The assembled bioreactor is versatile to efficiently decellularize multiple organs, as demonstrated by complete decellularization of pancreas, liver and heart in the same set-up. Further, the same system is amenable to support organ repopulation with diverse cell types. Using our in-house bioreactor system, we demonstrate pancreas repopulation with both immortalized MIN-6 beta cells and differentiating human pluripotent stem cells. Importantly, we show the significant advantage of perfusion culture over static culture in enhancing cell engraftment, viability and phenotypic maintenance of the repopulated pancreas. In addition, this study is a significant step forward for whole organ engineering as it will facilitate cost-effective and easy assembly of perfusion bioreactors to enable rapid advancement in regenerative organ reconstruction.

Chemical Decellularization Methods and Its Effects on Extracellular Matrix

Background: Extracellular matrix (ECM) produced by tissue decellularization processes as a biological scaffold due to its unique properties compared to other scaffolds for migration and implantation of stem cells have been used successfully in the field of tissue engineering and regenerative medicine in the last years. The objective of this manuscript was to provide an overview of the chemical decellularization methods, evaluation of decellularized ECM and the potential effect of the chemical decellularization agents on the biochemical composition.Methods: We searched in Google Scholar, PubMed, Scopus, and Science Direct. The literature search was done by using the following keywords: “ECM, biologic scaffold, decellularization, chemical methods, tissue engineering.” We selected articles have been published from 2000 to 2016, and 15 full texts and 97 abstracts were reviewed.Results:Employing an optimization method to minimize damage to the ECM ultrastructure as for a result of the lack of reduction in mechanical properties and also the preservation of essential proteins such as laminin, fibronectin, Glycosaminoglycans (GAGs), growth factor is required. Various methods include chemical, physical and enzymatic technics were studied. However, on each of these methods can have undesirable effects on ECM.Conclusion: It is suggested that instead of the Sodium dodecyl sulfate (SDS) which have high strength degradation, we can use zwitterionic separately or in combination with SDS. Tributyl phosphate (TBP) due to its unique properties can be used in decellularization process.

Efficient decellularization for tissue engineering of the tendon-bone interface with preservation of biomechanics.

Interfaces between tendon/ligament and bone (“entheses”) are highly specialized tissues that allow for stress transfer between mechanically dissimilar materials. Entheses show very low regenerative capacity resulting in high incidences of failure after surgical repair. Tissue engineering is a promising approach to recover functionality of entheses. Here, we established a protocol to decellularize porcine entheses as scaffolds for enthesis tissue engineering. Chemical detergents as well as physical treatments were investigated with regard to their efficiency to decellularize 2 mm thick porcine Achilles tendon entheses. A two-phase approach was employed: study 1 investigated the effect of various concentrations of sodium dodecyl sulfate (SDS) and t-octylphenoxypolyethoxy-ethanol (Triton X-100) as decellularization agents. The most efficient combination of SDS and Triton was then carried forward into study 2, where different physical methods, including freeze-thaw cycles, ultrasound, perfusion, and hydrostatic washing were used to enhance the decellularization effect. Cell counts, DNA quantification, and histology showed that washing with 0.5% SDS + 1% Triton X-100 for 72 h at room temperature could remove ~ 98% cells from the interface. Further investigation of physical methods proved that washing under 200 mmHg hydrostatic pressure shortened the detergent exposing time from 72 h to 48 h. Biomechanical tensile testing showed that the biomechanical features of treated samples were preserved. Washing under 200 mmHg hydrostatic pressure with 0.5% SDS + 1% Triton X-100 for 48 h efficiently decellularized entheses with preservation of matrix structure and biomechanical features. This protocol can be used to efficiently decellularize entheses as scaffolds for tissue engineering.

The impact of decellularization agents on renal tissue extracellular matrix.

The combination of patient-specific cells with scaffolds obtained from natural sources may result in improved regeneration of human tissues. Decellularization of the native tissue is the first step in this technology. Effective decellularization uses agents that lyse cells and remove all cellular materials, leaving intact collagenous extracellular matrices (ECMs). Removing cellular remnants prevents an immune response while preserving the underlying structure. In this study, the impact of five decellularization agents (0.1 N NaOH, 1% peracetic acid, 3% Triton X-100, 1% sodium dodecyl sulfate (SDS), and 0.05% trypsin/EDTA) on renal tissue was examined using slices of porcine kidneys. The NaOH solution induced the most efficient cell removal, and resulted in the highest amount of cell viability and proliferation after recellularization, although it also produced the most significant damage to collagenous fiber networks, glycosaminoglycans (GAGs) and fibroblast growth factor (FGF). The SDS solution led to less severe damage to the ECM structure but it resulted in lower metabolic activity and less proliferation. Peracetic acid and Triton X-100 resulted in minimum disruption of ECMs and the most preserved GAGs and FGF. However, these last two agents were not as efficient in removing cellular materials as NaOH and SDS, especially peracetic acid, which left more than 80% of cellular material within the ECM. As a proof of principle, after completing the comparison studies using slices of renal ECM, the NaOH process was used to decellularize a whole kidney, with good results. The overall results demonstrate the significant effect of cell lysing agents and the importance of developing an optimized protocol to avoid extensive damage to the ECM while retaining the ability to support cell growth.

Impact of decellularization on porcine myocardium as scaffold for tissue engineered heart tissue.

Decellularized myocardium has been proposed to construct tissue engineered heart tissue, providing the advantage of natural extracellular architecture. Various decellularization protocols have been developed, but the impact of individual decellularization reagent in the protocol remains unclear. The aim of this study is to evaluate the structural impact of three commonly used decellularization reagents on the porcine myocardium. We decellularized porcine heart tissue with trypsin, Triton X-100 or SDS, and analyzed the morphological characteristics of the remaining tissue by SEM, AFM and two-photon LSM. We further recellularized the scaffold with rat myocardial fibroblasts and cardiomyocytes separately. According to the H&E staining and DNA quantification, SDS decellularized more efficiently in comparison to the other two reagents. Moreover, we found distinct surface microarchitecture differences among groups. The changed structure of tissue might result in varied proliferation myocardial fibroblasts and biophysical performance of the engineered heart tissue. This study demonstrated that the microstructure of decellularized porcine heart tissue vary with decellularization agents. Compared to trypsin and Triton X-100, SDS not only decellularized more efficiently but also preserved the biocompatible microstructure of ECM for recellularization.

Impact of Detergent-Based Decellularization Methods on Porcine Tissues for Heart Valve Engineering

To date an optimal decellularization protocol of heart valve leaflets (HVL) and pericardia (PER) with an adequate preservation of the extracellular matrix (ECM) is still lacking. This study compares a 4 day Triton X-100-based protocol with faster SDC-based protocols for the decellularization of cardiac tissues. Decellularized and non-treated HVL and PER were processed for histological, biochemical and mechanical analysis to determine the effect of these agents on the structure, ECM components, and biomechanical properties. Tissues treated with SDC-based protocols still showed nuclear material, whereas tissues treated with Triton X-100 1%+ENZ±TRYP were completely cell free. For both decellularized tissues, an almost complete washout of glycosaminoglycans, a reduction of soluble collagen and an alteration of the surface ultrastructure was observed. Interestingly, only the elastic fibers of pericardial tissue were affected and this tissue had a decreased maximum load. This study showed that both detergents had a similar impact on the ECM. However, Triton X-100 1% +DNase/RNase (ENZ)±Trypsin (TRYP) is the only protocol that generated completely cell free bioscaffolds. Also, our study clearly demonstrated that the decellularization agents have more impact on pericardial tissues than on heart valve leaflets. Thus, for the purpose of tissue engineering of heart valves, it is advisable to use valvular rather than pericardial matrices.

Histological Evaluation of Decellularized Skeletal Muscle Tissue Using Two Different Decellularization Agents.

The aim of the present study was to determine effect of two decellularized agents, sodium dodecyl sulphate (SDS) and Triton X-100, to the skeletal muscle tissue. Final scaffold was evaluated by several histological techniques to analyse preservation of essential structures including collagen and elastic fibres, basement membranes, glycosaminoglycans and also to confirm elimination of nuclear and cytoplasmic components which are redundant in effectively prepared decellularized scaffolds. Comparison of tissue scaffolds processed with different detergents proved that SDS is superior to Triton X-100 as it can effectively decellularize muscle tissue.

Trypsin as enhancement in cyclical tracheal decellularization: Morphological and biophysical characterization

There are different types of tracheal disorders (e.g. cancer, stenosis and fractures). These can cause respiratory failure and lead to death of patients. Several attempts have been made for trachea replacement in order to restore the airway, including anastomosis and implants made from synthetic or natural materials. Tracheal allotransplantation has shown high rejection rates, and decellularization has emerged as a possible solution. Decellularization involves the removal of antigens from cells in the organ or tissue, leaving a matrix that can be used as 3D cell-scaffold. Although this process has been used for tracheal replacement, it usually takes at least two months and time is critical for patients with tracheal disorders. Therefore, there is necessary to develop a tracheal replacement process, which is not only effective, but also quick to prepare. The aim of this research was to develop a faster trachea decellularization protocol using Trypsin enzyme and Ethylenediaminetetraacetic acid (EDTA) as decellularization agents. Three protocols of cyclic trachea decellularization (Protocols A, B, and C) were compared. Following Protocol A (previously described in the literature), 15consecutivecycles were performed over 32days. Protocol B (a variation of Protocol A) — EDTA being added — with 15consecutivecycles performed over 60days. Finally, Protocol C, with the addition of Trypsin as a decellularization agent, 5consecutivecycles being performed over 10days. For the three protocols, hematoxylin–eosin (H&E) staining and DNA residual content quantification were performed to establish the effectiveness of the decellularization process. Scanning Electron Microscopy (SEM) was used to observe the changes in porosity and microarrays. To evaluate the structural matrices integrity, Thermogravimetric Analysis (TGA) and biomechanical test were used. None of the protocols showed significant alteration or degradation in the components of the extracellular matrix (ECM). However, in Protocol C, more cellular components were removed in less time, making it the most efficient process. In addition, the cell tracking and viability was evaluated with chondrocytes seeding on the scaffold obtained by Protocol C, which showed an adequate cell scaffolding ability of this matrix.