Optimization of TX-100/SDS-based decellularized vascular material using ultrasound and chemical treatment: evaluation of structure and biosafety

Hydrogels derived from decellularized extracellular matrices (ECM) of animal origin show immense potential for regenerative applications due to their excellent cytocompatibility and biomimetic properties. Despite these benefits, the impact of decellularization protocols on the properties and immunogenicity of these hydrogels remains relatively unexplored. In this study, porcine skeletal muscle ECM (smECM) underwent decellularization using mechanical disruption (MD) and two commonly employed decellularization detergents, sodium deoxycholate (SDC) or Triton X-100. To mitigate immunogenicity associated with animal-derived ECM, all decellularized tissues were enzymatically treated with α-galactosidase to cleave the primary xenoantigen-the α-Gal antigen. Subsequently, the impact of the different decellularization protocols on the resultant hydrogels was thoroughly investigated. All methods significantly reduced total DNA content in hydrogels. Moreover, α-galactosidase treatment was crucial for cleaving α-Gal antigens, suggesting that conventional decellularization methods alone are insufficient. MD preserved total protein, collagen, sulfated glycosaminoglycan, laminin, fibronectin, and growth factors more efficiently than other protocols. The decellularization method impacted hydrogel gelation kinetics and ultrastructure, as confirmed by turbidimetric and scanning electron microscopy analyses. MD hydrogels demonstrated high cytocompatibility, supporting satellite stem cell recruitment, growth, and differentiation into multinucleated myofibers. In contrast, the SDC and Triton X-100 protocols exhibited cytotoxicity. Comprehensive in vivo immunogenicity assessments in a subcutaneous xenotransplantation model revealed MD hydrogels' biocompatibility and low immunogenicity. These findings highlight the significant influence of the decellularization protocol on hydrogel properties. Our results suggest that combining MD with α-galactosidase treatment is an efficient method for preparing low-immunogenic smECM-derived hydrogels with enhanced properties for skeletal muscle regenerative engineering and clinical applications.

Different decellularization methods can affect the integrity and the biomechanical and biocompatible properties of the tracheal matrix. Natural cross-linking with genipin can be applied to improve those properties. The goals of this study were to evaluate the effects of different decellularization methods on the properties of genipin-cross-linked decellularized tracheal matrices in rabbits. The tracheas of New Zealand rabbits were decellularized by the Triton-X 100-processed method (TPM) and the detergent-enzymatic method (DEM) and were then cross-linked with genipin. Mechanical tests, haematoxylin-eosin staining, Masson trichrome staining, Safranin O staining, DAPI staining, scanning electronic microscopy (SEM), and biocompatibility tests were used to evaluate the treatment. The bioengineered trachea and control trachea were then implanted into allogeneic rabbits for 30days. The structural and functional analyses were performed after transplantation. The biomechanical tests demonstrated that the biomechanical properties of the decellularized tracheas decreased and that genipin improved them (p < 0.05). The histological staining results revealed that most of the mucosal epithelial cells were removed and that the decellularized trachea had lower immunogenicity than the control group. The analysis of SEM revealed that the decellularized trachea retained the micro- and ultra-structural architectures of the trachea and that the matrices cross-linked with genipin were denser. The biocompatibility evaluation and in vivo implantation experiments showed that the decellularized trachea treated with the DEM had better biocompatibility than that treated with the TPM and that immunogenicity in the cross-linked tissues was lower than that in the uncross-linked tissues (p < 0.05). Compared with the trachea treated with the TPM, the rabbit trachea processed by the DEM had better biocompatibility and lower immunogenicity, and its structural and mechanical characteristics were effectively improved after the genipin treatment, which is suitable for engineering replacement tracheal tissue.

The decellularized extracellular matrix (ECM) of cartilage is a widely used natural bioscaffold for constructing tissue-engineered cartilage due to its good biocompatibility and regeneration properties. However, current decellularization methods for accessing decellularized cartilaginous tissues require multiple steps and a relatively long duration to produce decellularized cartilage. In addition, most decellularization strategies lead to damage of the microstructure and loss of functional components of the cartilaginous matrix. In this study, a novel decellularization strategy based on a hydrostatic pressure (HP) bioreactor was introduced, which aimed to improve the efficiency of producing integral decellularized cartilage pieces by combining physical and chemical decellularization methods in a perfusing manner. Two types of cartilaginous tissues, auricular cartilage (AC) and nucleus pulposus (NP) fibrocartilage, were selected for comparison of the effects of ordinary, positive, and negative HP-based decellularization according to the cell clearance ratio, microstructural changes, ECM components, and mechanical properties. The results indicated that applying positive HP improved the efficiency of producing decellularized AC, but no significant differences in decellularization efficiency were found between the ordinary and negative HP-treated groups. However, compared with the ordinary HP treatment, the application of the positive or negative HP did not affect the efficiency of decellularized NP productions. Moreover, neither positive nor negative HP influenced the preservation of the microstructure and components of the AC matrix. However, applying negative HP disarranged the fibril distribution of the NP matrix and reduced glycosaminoglycans and collagen type II contents, two essential ECM components. In addition, the positive HP was beneficial for maintaining the mechanical properties of decellularized cartilage. The recellularization experiments also verified the good biocompatibility of the decellularized cartilage produced by the present bioreactor-based decellularization method under positive HP. Overall, applying positive HP-based decellularization resulted in a superior effect on the production of close-to-natural scaffolds for cartilage tissue engineering. Impact statement In this study, we successfully constructed a novel hydrostatic pressure (HP) bioreactor and used this equipment to produce decellularized cartilage by combining physical and chemical decellularization methods in a perfusing manner. We found that positive HP-based decellularization could improve the production efficiency of integral decellularized cartilage pieces and promote the maintenance of matrix components and mechanical properties. This new decellularization strategy exhibited a superior effect in the production of close-to-natural scaffolds and positively impacts cartilage tissue engineering.

Long segment trachea defects are repaired by tracheal substitution, while decellularized technology has been effectively employed to prepare tissue engineering trachea (TET). However, its clinical application is restricted by the long preparation cycle, while poor vascularization is associated with the transplantation failure. In the present study, we used sodium lauryl ether sulfate (SLES) to develop a novel rapid decellularized tracheal preparation method, then constructed a TET with revascularization functions. Summarily, we decellularized rabbit trachea using various SLES concentrations. Results from histological analysis, immunohistochemical and DAPI staining, as well as DNA quantitative assay, revealed that 1-0.1% (v/v) SLES treatment not only entirely removed cellular components to reduce its immunogenicity, but also retained the tracheal matrix's gross structure. SEM images, safranine O-fast green staining, total collagen content assay and collagen II immunofluorescence revealed that low SLES concentrations preserved the bioactive components of the decellularized tracheal matrix. Next, we performed cytobiocompatible and cytotoxin assays to verify biocompatibility of the decellularized tracheal matrix, and is confirmed by the omentum transplantation of rats. Results from omentum transplantation revealed that the decellularized tracheal matrix had low immunogenicity and excellent biocompatibility. Its revascularization capacity was confirmed by histologic appearance and CD31 immunofluorescence. Based on these findings, we selected 0.1% (v/v) as the optimal SLES concentration for preparing a decellularized tracheal matrix. Next, we seeded allogeneic bone marrow stem cells (BMSC) onto the matrix to construct TET patches. In vivo tracheal defect reconstruction confirmed the biocompatibility and revascularization capacity of this novel TET, and the formation of a vascular network around the patch promoted submucosa and mucosa regeneration without significant stenosis, 4 weeks post-surgery. In conclusion, we used SLES to successfully develop a novel decellularized approach for the preparation of TET, which has low immunogenic and inflammatory responses, as well as excellent biocompatibility, and revascularization ability in vivo without additional exogenous cytokines.

Decellularization treatment has been widely used to decrease the potential immunogenicity and improve the anticalcification properties of bio-derived materials, which may be utilized as an alternative method for the preparation of bioprosthetic heart valves. However, the excessive decellularization treatments will deteriarate the properties of heart valves. Among the decellularizaton parameters, detergent concentration and processing time are considered as those of the most key factors. Therefore, it should be meaningful to balance the decellularization efficiency and properties of bioprosthetic heart valves by optimizing the detergent concentration and processing time. In this study, three groups of the decellularized heart valves treated by sodium deoxycholate (SD) with different concentration and processing time were investigated through histological, biochemical, and mechanical analysis. Similar decellularization efficiency can be concluded through histological staining, DNA and α-Gal quantification results. Extracellular matrix contents quantification and tensile test results revealed that there is no obvious difference among the three decellularized heart valves. in vitro cytotoxicity assay showed that the remnant detergent is not enough to cause cell death, which indicated that the decellularized porcine aortic heart valves may be suitable for further in vivo research. In conclusion, Triton X-100/SD may be a suitable protocol used for heart valves decellularization. And it is feasible to vary the detergent processing time by changing the detergent concentration without compromising the decellularization efficiency.

Ultrasound modified polysaccharides: A review of structure, physicochemical properties, biological activities and food applications

Xenogeneic menisci offer a promising biomaterial with high biocompatibility, closely resembling the native meniscus. However, to enable clinical application in humans, decellularization is essential to remove cellular components that may trigger graft rejection. We developed a combined decellularization protocol for bovine-derived meniscus tissue, integrating physical stimulation (sonication and vacuum), chemical treatments (hypotonic and hypertonic solutions, and sodium dodecyl sulfate [SDS]), and enzymatic digestion (trypsin and nucleases). The decellularization efficiency was confirmed not only by DNA reduction rate, but also by residual DNA, DNA fragmentation and histology. Subsequently, cytotoxicity, biocompatibility, and mechanical properties were assessed. This combined decellularization achieved a DNA removal efficiency of up to 94.94%, enabled rapid decellularization within 5days, and preserved collagen content, resulting in a high-quality xeno meniscal implant (XMI). The XMI produced through this combined decellularization process demonstrates strong potential as a scaffold for the treatment of meniscal injuries.

The lack of a definitive strategy for soft tissue regeneration highlights the need to evaluate new approaches for soft tissue replacement that is of clinical relevance. As tissue engineering presents a promising strategy for soft tissue regeneration, the focus of this thesis is mainly on the development of tissue-inspired scaffolds for adipose tissue engineering applications. In order to achieve this objective, studies were carried out to evaluate the efficacy of two novel materials, namely adipose tissue-derived extracellular matrix (ECM) material and bullfrog skin-derived collagen. Taking inspiration from the native tissue itself, decellularized tissue-derived ECM is regarded as a promising material for tissue engineering applications. However, it has been shown that different decellularization methods play an influential role over the property of the final decellularized ECM material. Two novel decellularization methods were established during the course of this thesis for the isolation of ECM material from adipose tissue without the need of any harsh chemicals. The ECM material isolated using these two methods led to the preservation of more biological components including growth factors, matricellular proteins and structural proteins, as compared to previously established decellularization methods. The extracted ECM material was subsequently used as a bioactive material to enrich other biomaterials for tissue engineering applications for the first time. It was found that the ECM material was an effective enrichment due to the well preservation of various valuable components within ECM material. This ECM enrichment improved the cell-material interaction for both synthetic (i.e. Polycaprolactone) and natural (i.e. Ovalbumin) materials. In addition, the ECM coating was observed to have minimal pro-inflammatory response and improved in vitro wound healing, endothelialization, and adipogenesis. In parallel, in vitro and in vivo studies were also carried out to demonstrate how bullfrog skin-derived collagen could be a promising alternative source of collagen for the fabrication of tunable acid-soluble collagen (AC) scaffolds. The material properties and cell proliferation capabilities of the AC scaffolds could be further modulated using different concentrations of crosslinker to suit various tissue engineering applications. In addition, a decrease in both in vitro and in vivo degradation rate was observed with increasing crosslinker concentration. Overall, by combining engineering tools and techniques with biological assays and animal studies, adipose tissue-derived ECM material and bullfrog skin-derived collagen were shown to be promising materials for soft tissue engineering applications. The novel concept of using ECM material to improve cell–material interactions demonstrates the potential exploitation of adipose tissue as useful bioactive coating material. Overall, an ECM-containing hybrid scaffold is an acellular scaffold with cellular benefits and is therefore a great stepping-stone towards the development of engineered adipose tissue replacement for clinical applications.

Human acellular amniotic membrane (HAAM) has emerged as a promising tool in the field of regenerative medicine, particularly for wound healing and tissue regeneration. HAAM provides a natural biological scaffold with low immunogenicity and good anti-infective and anti-scarring results. Despite its potential, the clinic application of HAAM faces challenges, particularly with respect to the preparation methods and its low mechanical strength. This review provides a comprehensive overview of HAAM, covering its preparation, sterilization, preclinical research, and clinical applications. This review also discusses promising decellularization and sterilization methods, such as Supercritical Carbon Dioxide (SC-CO2), and the need for further research into the regenerative mechanisms of HAAM. In addition, we discuss the potential of HAAM as a skin dressing and cell delivery system in preclinical research and clinical applications. Both the safety and effectiveness of HAAM have been validated by extensive research, which provides a robust foundation for its clinical application.

Decellularization removes cellular antigens while preserving the ultrastructure and composition of extracellular matrix (ECM). Decellularized ECM (DECM) scaffolds have been widely used in various tissue engineering applications with varying levels of success. The mechanical, architectural and bioactive properties of a DECM scaffold depend largely on the method of decellularization and dictate its clinical efficacy. This article highlights the advantages and challenges associated with the clinical use of DECM scaffolds. Poor mechanical strength is a significant disadvantage of some DECM scaffolds in the repair of load-bearing tissues as well as critical-size defects, where long-term mechanical support is required for the regenerating tissue. Combining DECM scaffolds with synthetic biocompatible polymers could provide a useful strategy to circumvent the issues of poor mechanical stability. This article reviews studies that have combined DECM scaffolds from various tissues with synthetic polymers to create hybrid scaffolds using electrospinning. These hybrid scaffolds provide a mechanical backbone while retaining the bioactive properties of DECM, thus offering a significant advantage for tissue engineering and regenerative medicine applications.

The application of decellularized scaffolds for artificial tissue reconstruction has been an approach with great therapeutic potential in regenerative medicine. Recently, biomimetic ovarian tissue reconstruction was proposed to reestablish ovarian endocrine functions. Despite many decellularization methods proposed, there is no established protocol for whole ovaries by detergent perfusion that is able to preserve tissue macro and microstructure with higher efficiency. This generated biomaterial may have the potential to be applied for other purposes beyond reproduction and be translated to other areas in the tissue engineering field. Therefore, this study aimed to establish and standardize a protocol for porcine ovaries' decellularization based on detergent perfusion and ultrasonication to obtain functional whole-ovary scaffolds. For that, porcine ovaries (n = 5) were perfused with detergents (0.5% SDS and 1% Triton X-100) and submitted to an ultrasonication bath to produce acellular scaffolds. The decellularization efficiency was evaluated by DAPI staining and total genomic DNA quantification. ECM morphological evaluation was performed by histological, immunohistochemistry, and ultrastructural analyses. ECM physico-chemical composition was evaluated using FTIR and Raman spectroscopy. A cytocompatibility and cell adhesion assay using murine fibroblasts was performed. Results showed that the proposed method was able to remove cellular components efficiently. There was no significant ECM component loss in relation to native tissue, and the scaffolds were cytocompatible and allowed cell attachment. In conclusion, the proposed decellularization protocol produced whole-ovaries scaffolds with preserved ECM composition and great potential for application in tissue engineering.

Various decellularization methods based on physical, chemical, or biological treatments have been proposed to date. Each of them has advantages and disadvantages. To remove cells completely and to inhibit extracellular matrix (ECM) denaturation at the same time seems to be impossible and may not be the ideal decellularization method. Residual substrates and some changes of the ECM resulted in good recellularization or tissue regeneration in some cases. In this chapter, various decellularization methods are summarized and each decellularization method is divided into three modes of action, (1) cell disruption, (2) removal of cellular components, and (3) making large molecules smaller by digestion or degradation. Efficacy of each action and change (denaturation) in ECM components are defined by the decellularization method and its conditions, which lead to successful or unsuccessful results of recellularization and tissue or organ regeneration.

Aims/Purpose: The purpose of this research was to evaluate the decellularization efficiency of various individual protocols and their combinations for bovine cornea intended for ocular surface biomaterial applications.Methods: Two fundamental decellularization protocols, namely sodium chloride (NaCl) and Ammonium &amp; Triton X‐100 were taken as benchmark and their combinations with freeze–thaw and lyophilization, were assessed whether, the decellularization efficiency improved (n = 3). Overall, protocols that were investigated were as follows: (1) Lyophilization followed by NaCl, (2) Freeze–Thaw followed by NaCl, (3) Lyophilization, Ammonium and Triton X‐100 (1%), (4) Ammonium and Triton X‐100 (5%), (5) Freeze–thaw, Ammonium and Triton X‐100 (1%), (6) Lyophilization, Ammonium and Triton X‐100 (5%), (7) Freeze–thaw and (8) NaCl. These methods were assessed using gel electrophoresis for qualitative DNA analysis and NanoDrop for quantitative DNA content. Efficient decellularization was achieved in the presence of DNA content below 50 ng/mg ECM dry weight and &lt;200 bp DNA fragment length.Results: Gel electrophoresis revealed DNA content over 200 bp in groups 1, 2,7 and 8 while other groups fulfilled the criterion for efficient decellularization. DNA content as measured by NanoDrop indicated efficient decellularization in all groups, except group 7.Conclusions: Our study provides valuable insights into the effectiveness of combined and individual decellularization protocols for bovine cornea. Our findings suggest, that NaCl and its combinations with lyophilization and freeze–thaw yield in ineffective decellularization. On the other hand, Ammonium and Triton X‐100, individually or in combinations, result in favourable decellularization.

Decellularization is the process of obtaining acellular tissues with low immunogenic cellular components from animals or plants while maximizing the retention of the native extracellular matrix structure, mechanical integrity and bioactivity. The decellularized tissue obtained through the tissue decellularization technique retains the structure and bioactive components of its native tissue; it not only exhibits comparatively strong mechanical properties, low immunogenicity and good biocompatibility but also stimulates in situ neovascularization at the implantation site and regulates the polarization process of recruited macrophages, thereby promoting the regeneration of damaged tissue. Consequently, many commercial products have been developed as promising therapeutic strategies for the treatment of different tissue defects and lesions, such as wounds, dura, bone and cartilage defects, nerve injuries, myocardial infarction, urethral strictures, corneal blindness and other orthopedic applications. Recently, there has been a growing interest in the decellularization of fish tissues because of the abundance of sources, less religious constraints and risks of zoonosis transmission between mammals. In this review, we provide a complete overview of the state-of-the-art decellularization of fish tissues, including the organs and methods used to prepare acellular tissues. We enumerated common decellularized fish tissues from various fish organs, such as skin, scale, bladder, cartilage, heart and brain, and elaborated their different processing methods and tissue engineering applications. Furthermore, we presented the perspectives of (i) the future development direction of fish tissue decellularization technology, (ii) expanding the sources of decellularized tissue and (iii) innovating decellularized tissue bio-inks for 3D bioprinting to unleash the great potential of decellularized tissue in tissue engineering and regenerative medicine applications.

Scaffolds play a key role in the process of regeneration and morphogenesis of tissue or organ. We have studied a novel application of ultrasound irradiation to prepare decellularized tissue for tissue-engineered scaffolds. The aim of the present study is to investigate the possibility of ultrasound energy on decellularization of cartilage tissue. The samples were decellularized using 20 kHz of ultrasonic irradiation in circulated 0.3% sodium chloride (NaCl) constituent of 2% sodium dodecyl sulfate (SDS) solution. To evaluate tissue integrity and cell removal, we used hematoxylin-eosin staining. To estimate the degree of irradiated ultrasound on the sample, we used computer simulation to calculate the irradiated power distribution in solution, corresponding to the irradiation energy against to the sample. From histological results, it shows that the ultrasonic power could assist the decellularization on meniscal tissue. The decellularization efficiency was better than the other preparation methods. Because of distributions of ultrasonic irradiation to samples were non-uniform, the efficiency of cell removal could be improved by using 3 dimensional scanning system which uniformly irradiate to the sample. In conclusion, ultrasonic chemical process has a potential to improve the efficiency of decellularization for menisci.