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Utilizing natural scaffold production derived from extracellular matrix components presents a promising strategy for advancing in vitro spermatogenesis. In this study, we employed decellularized human placental tissue as a scaffold, upon which neonatal mouse spermatogonial cells (SCs) were cultured three‐dimensional (3D) configuration. To assess cellular proliferation, we examined the expression of key markers (Id4 and Gfrα1) at both 1 and 14 days into the culture. Our quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR) analysis revealed a notable increase in Gfrα1 gene expression, with the 3D culture group exhibiting the highest levels. Furthermore, the relative frequency of Gfrα1‐positive cells significantly rose from 38.1% in isolated SCs to 46.13% and 76.93% in the two‐dimensional (2D) and 3D culture systems, respectively. Moving forward to days 14 and 35 of the culture period, we evaluated the expression of differentiating markers (Sycp3, acrosin, and Protamine 1). Sycp3 and Prm1 gene expression levels were upregulated in both 2D and 3D cultures, with the 3D group displaying the highest expression. Additionally, acrosin gene expression increased notably within the 3D culture. Notably, at the 35‐day mark, the percentage of Prm1‐positive cells in the 3D group (36.4%) significantly surpassed that in the 2D group (10.96%). This study suggests that the utilization of placental scaffolds holds significant promise as a bio‐scaffold for enhancing mouse in vitro spermatogenesis. [ABSTRACT FROM AUTHOR]

Background: Decellularized extracellular matrix (dECM) from several tissue sources has been proposed as a promising alternative to conventional scaffolds used in regenerative endodontic procedures (REPs). This systematic review aimed to evaluate the histological outcomes of studies utilizing dECM-derived scaffolds for REPs and to analyse the contributing factors that might influence the nature of regenerated tissues. Methods: The PRISMA 2020 guidelines were used. A search of articles published until April 2024 was conducted in Google Scholar, Scopus, PubMed and Web of Science databases. Additional records were manually searched in major endodontic journals. Original articles including histological results of dECM in REPs and in-vivo studies were included while reviews, in-vitro studies and clinical trials were excluded. The quality assessment of the included studies was analysed using the ARRIVE guidelines. Risk of Bias assessment was done using the (SYRCLE) risk of bias tool. Results: Out of the 387 studies obtained, 17 studies were included for analysis. In most studies, when used as scaffolds with or without exogenous cells, dECM showed the potential to enhance angiogenesis, dentinogenesis and to regenerate pulp-like and dentin-like tissues. However, the included studies showed heterogeneity of decellularization methods, animal models, scaffold source, form and delivery, as well as high risk of bias and average quality of evidence. Discussion: Decellularized ECM-derived scaffolds could offer a potential off-the-shelf scaffold for dentin-pulp regeneration in REPs. However, due to the methodological heterogeneity and the average quality of the studies included in this review, the overall effectiveness of decellularized ECM-derived scaffolds is still unclear. More standardized preclinical research is needed as well as well-constructed clinical trials to prove the efficacy of these scaffolds for clinical translation. Other: The protocol was registered in PROSPERO database #CRD42023433026. This review was funded by the Science, Technology and Innovation Funding Authority (STDF) under grant number (44426). [ABSTRACT FROM AUTHOR]

Whole lung engineering and the transplantation of its products is an ambitious goal and ultimately a viable solution for alleviating the donor-shortage crisis for lung transplants. There are several limitations currently impeding progress in the field with a major obstacle being efficient revascularization of decellularized scaffolds, which requires an extremely large number of cells when using larger pre-clinical animal models. Here, we developed a simple but effective experimental pulmonary bioengineering platform by utilizing the lung as a scaffold. Revascularization of pulmonary vasculature using human umbilical cord vein endothelial cells was feasible using a novel in-house developed perfusion-based bioreactor. The endothelial lumens formed in the peripheral alveolar area were confirmed using a transmission electron microscope. The quality of engineered lung vasculature was evaluated using box-counting analysis of histological images. The engineered mouse lungs were successfully transplanted into the orthotopic thoracic cavity. The engineered vasculature in the lung scaffold showed blood perfusion after transplantation without significant hemorrhage. The mouse-based lung bioengineering system can be utilized as an efficient ex-vivo screening platform for lung tissue engineering. [ABSTRACT FROM AUTHOR]

The objective of this study was to prepare Ficus benjamina leaves scaffolds for tissue engineering applications. Fresh leaves were decutinized with mixed solution of n-hexanes and diethyl ether in ratio of 9:1 and decellularized using 2.5% SDS (P1 and P2) and 5% SDS (P3 and P4) followed by incubation in 0.5% (P1, P3) and 1% (P2, P4) TnBP solution and finaly soaking in 4% NaOCl for 12 to 36h. The control and decellularized leaves scaffolds samples were characterized on the basis of macroscopic observations, histological/microscopic observations, DAPI staining, DNA quantification, density and porosity, swelling property, water vapor transmission rate, FTIR spectroscopic examination, and biocompatibility assessment. Cuticular wax was completely removed by nhexane and diethyl ether treatment and the treatment leaves were completely decellularized at 12 and 24h interval after bleaching as revealed by H&E, DAPI stained micrographs and SEM ultrastructure. DNA (ng/µl) quantity, density and of the decellularized Ficus benjamina leaves tissue also reduced significantly (P<0.05). However, porosity, swelling percent and water vapor transmission rate of decellularized leaf scaffolds increased significantly (P<0.05). Decellularized leaf scaffolds were found hemocompatible. FTIR analysis of both control and decellularized Ficus benjamina leaf samples showed well defined similar peaks in spectra. The subcutaneously implanted decellularized leaf scaffolds were infiltrated with host cells and found biocompatible as showed by histopathological, DAPI and SEM images. The results demonstrated that the use of SDS and TnBP by immersion technique is a suitable procedure for decellularization of Ficus benjamina plants leaves to produce biocompatible cellulose scaffolds. [ABSTRACT FROM AUTHOR]

Objective: Tracheal transplantation could be a better option for patients with long segmental laryngotracheal stenosis or defects, but the need for immunosuppressants limits its widespread use due to the antigenicity of the tracheal epithelium. Chemically treated or cryopreserved nonviable tracheal allografts have no immunogenicity but lead to necrosis and stenosis in long-term outcomes. The present report describes the 5-year outcomes of de-epithelialized viable tracheal allotransplantation without immunosuppressants in a patient with severe laryngotracheal stenosis. Methods: The recipient was a 47-year-old female with relapsing polychondritis affecting the larynx and cervical trachea and producing a 5 cm long stenosis that could not be repaired using resection and anastomosis. A tracheal allograft was obtained from a 45-year-old male donor and treated with a combination of 3% sodium dodecyl sulfate (SDS) and organ preservation solution for 138 hours. The allograft was revascularized by heterotopical implantation in the infrahyoid muscles of the recipient for 3 months and then transplantation to the laryngotracheal defect with a split-thickness skin graft sutured to the lumen and a silicon T-tube. No immunosuppressants were used postoperatively. Results: The allograft was de-epithelialized, and most of the cartilage rings remained viable after the treatment. The allograft was revascularized, viable, and mechanically stable after 3 months of heterotopic implantation. No apparent signs of rejection or destruction were observed. The T-tube was removed, and the internal lining of the allograft was repopulated 4 months after orthotopic transplantation, despite the skin graft necrotizing at 2 weeks. Endoscopy and computed tomography showed a patent airway 5 years after orthotopic transplantation. The patient was able to resume her usual quality of life. Conclusion: The present study demonstrates that transplantation of the de-epithelialized viable tracheal allograft without immunosuppressants is safe and promising for patients with long laryngotracheal stenosis or defects, especially for those with malignant tumor resections. [ABSTRACT FROM AUTHOR]

Reconstructive techniques to repair severe tissue defects include the use of autologous fasciocutaneous flaps, which may be limited due to donor site availability or lead to complications such as donor site morbidity. A number of synthetic or natural dermal substitutes are in use clinically, but none have the architectural complexity needed to reconstruct deep tissue defects. The perfusion decellularization of fasciocutaneous flaps is an emerging technique that yields a scaffold with the necessary composition and vascular microarchitecture and serves as an alternative to autologous flaps. In this study, we show the perfusion decellularization of porcine fasciocutaneous flaps using sodium dodecyl sulfate (SDS) at three different concentrations, and identify that 0.2% SDS results in a decellularized flap that is efficiently cleared of its cellular material at 86%, has maintained its collagen and glycosaminoglycan content, and preserved its microvasculature architecture. We further demonstrate that the decellularized graft has the porous structure and growth factors that would facilitate repopulation with cells. Finally, we show the biocompatibility of the decellularized flap using human dermal fibroblasts, with cells migrating as deep as 150 µm into the tissue over a 7-day culture period. Overall, our results demonstrate the promise of decellularized porcine flaps as an interesting alternative for reconstructing complex soft tissue defects, circumventing the limitations of autologous skin flaps. [ABSTRACT FROM AUTHOR]

Open in new tab Download slide OBJECTIVES Decellularized aortic homografts (DAH) were introduced as a new option for aortic valve replacement for young patients. METHODS A prospective, EU-funded, single-arm, multicentre study in 8 centres evaluating non-cryopreserved DAH for aortic valve replacement. RESULTS A total of 144 patients (99 male) were prospectively enrolled in the ARISE Trial between October 2015 and October 2018 with a median age of 30.4 years [interquartile range (IQR) 15.9–55.1]; 45% had undergone previous cardiac operations, with 19% having 2 or more previous procedures. The mean implanted DAH diameter was 22.6 mm (standard deviation 2.4). The median operation duration was 312 min (IQR 234–417), the median cardiopulmonary bypass time was 154 min (IQR 118–212) and the median cross-clamp time 121 min (IQR 93–150). No postoperative bypass grafting or renal replacement therapy were required. Two early deaths occurred, 1 due to a LCA thrombus on day 3 and 1 due ventricular arrhythmia 5 h postoperation. There were 3 late deaths, 1 death due to endocarditis 4 months postoperatively and 2 unrelated deaths after 5 and 7 years due to cancer and Morbus Wegener resulting in a total mortality of 3.47%. After a median follow-up of 5.9 years [IQR 5.1–6.4, mean 5.5 years. (standard deviation 1.3) max. 7.6 years], the primary efficacy end-points peak gradient with median 11.0 mmHg (IQR 7.8–17.6) and regurgitation of median 0.5 (IQR 0–0.5) of grade 0–3 were excellent. At 5 years, freedom from death/reoperation/endocarditis/bleeding/thromboembolism were 97.9%/93.5%/96.4%/99.2%/99.3%, respectively. CONCLUSIONS The 5-year results of the prospective multicentre ARISE trial continue to show DAH to be safe for aortic valve replacement with excellent haemodynamics. [ABSTRACT FROM AUTHOR]

Open in new tab Download slide OBJECTIVES Decellularized aortic homografts (DAH) were introduced in 2008 as a further option for paediatric aortic valve replacement (AVR). METHODS Prospective, multicentre follow-up of all paediatric patients receiving DAH for AVR in 8 European centres. RESULTS A total of 143 DAH were implanted between February 2008 and February 2023 in 137 children (106 male, 74%) with a median age of 10.8 years (interquartile range 6.6–14.6). Eighty-four (59%) had undergone previous cardiac operations and 24 (17%) had undergone previous AVR. The median implanted DAH diameter was 21 mm (interquartile range 19–23). The median operation duration was 348 min (227–439) with a median cardiopulmonary bypass time of 212 min (171–257) and a median cross-clamp time of 135 min (113–164). After a median follow-up of 5.3 years (3.3–7.2, max. 15.2 years), the primary efficacy end-points peak gradient (median 14 mmHg, 9–28) and regurgitation (median 0.5, interquartile range 0–1, grade 0–3) showed good results but an increase over time. Freedom from death/explantation/endocarditis/bleeding/thromboembolism at 5 years were 97.8 ± 1.2/88.7 ± 3.3/99.1 ± 0.9/100 and 99.2 ± 0.8%, respectively. Freedom from death/explantation/endocarditis/bleeding/thromboembolism at 10 years were 96.3 ± 1.9/67.1 ± 8.0/93.6 ± 3.9/98.6 ± 1.4 and 86.9 ± 11.6%, respectively. In total, 21 DAH were explanted. Seven were replaced by a mechanical AVR, 1 Ross operation was performed and a re-do DAH was implanted in 13 patients with no redo mortality. The calculated expected adverse events were lower for DAH compared to cryopreserved homograft patients (mean age 8.4 years), and in the same range as for Ross patients (9.2 years) and mechanical AVR (13.0 years). CONCLUSIONS This large-scale prospective analysis demonstrates excellent mid-term survival using DAH with adverse event rates comparable to paediatric Ross procedures. [ABSTRACT FROM AUTHOR]

One of the most important medical interventions for individuals with heart valvular disease is heart valve replacement, which is not without substantial challenges, particularly for pediatric patients. Due to their biological properties and biocompatibility, natural tissue-originated scaffolds derived from human or animal sources are one type of scaffold that is widely used in tissue engineering. However, they are known for their high potential for immunogenicity. Being free of cells and genetic material, decellularized xenografts, consequently, have low immunogenicity and, thus, are expected to be tolerated by the recipient's immune system. The scaffold ultrastructure and ECM composition can be affected by cell removal agents. Therefore, applying an appropriate method that preserves intact the structure of the ECM plays a critical role in the final result. So far, there has not been an effective decellularization technique that preserves the integrity of the heart valve's ultrastructure while securing the least amount of genetic material left. This study demonstrates a new protocol with untraceable cells and residual DNA, thereby maximally reducing any chance of immunogenicity. The mechanical and biochemical properties of the ECM resemble those of native heart valves. Results from this study strongly indicate that different critical factors, such as ionic detergent omission, the substitution of Triton X-100 with Tergitol, and using a lower concentration of trypsin and a higher concentration of DNase and RNase, play a significant role in maintaining intact the ultrastructure and function of the ECM. [ABSTRACT FROM AUTHOR]

Biomaterials play an important role in the development of advancing three dimensional (3D) in vitro skin models, providing valuable insights for drug testing and tissue-specific modeling. Commercial materials, such as collagen, fibrin or alginate, have been widely used in skin modeling. However, they do not adequately represent the molecular complexity of skin components. On this regard, the development of novel biomaterials that represent the complexity of tissues is becoming more important in the design of advanced models. In this study, we have obtained aged human decellularized dermal extracellular matrix (dECM) hydrogels extracted from cadaveric human skin and demonstrated their potential as scaffold for advanced skin models. These dECM hydrogels effectively reproduce the complex fibrillar structure of other common scaffolds, exhibiting similar mechanical properties, while preserving the molecular composition of the native dermis. It is worth noting that fibroblasts embedded within human dECM hydrogels exhibit a behavior more representative of natural skin compared to commercial collagen hydrogels, where uncontrolled cell proliferation leads to material shrinkage. The described human dECM hydrogel is able to be used as scaffold for dermal fibroblasts in a skin aging-on-a-chip model. These results demonstrate that dECM hydrogels preserve essential components of the native human dermis making them a suitable option for the development of 3D skin aging models that accurately represent the cellular microenvironment, improving existing in vitro skin models and allowing for more reliable results in dermatopathological studies. [ABSTRACT FROM AUTHOR]

Background: Epothilone B (EpoB) is a microtubule‐stabilizing agent with neuroprotective properties. Objectives: This study examines the regenerative properties of ANA supplemented with EpoB on a sciatic nerve deficit in male Wistar rats. Methods: For this purpose, the 10 mm nerve gap was filled with acellular nerve allografts (ANAs) containing EpoB at 0.1, 1, and 10 nM concentrations. The sensorimotor recovery was evaluated up to 16 weeks after the operation. Real‐time PCR, histomorphometry analysis, and electrophysiological evaluation were also used to evaluate the process of nerve regeneration. Results: ANA/EpoB (0.1 nM) significantly improved sensorimotor recovery in rats compared to ANA, ANA/EpoB (1 nM), and ANA/EpoB (10 nM) groups. This led to reduced muscle atrophy, improved sciatic functional index, and thermal paw withdrawal reflex latency, indicating nerve regeneration and target organ reinnervation. The electrophysiological and histomorphometry findings also confirmed the ANA/EpoB regenerative properties (0.1 nM). EpoB only enhanced ANA regenerative properties at 0.1 nM, with no therapeutic effects at higher concentrations. Conclusion: Totally, we concluded that ANA loaded with 0.1 nM EpoB can effectively reconstruct the transected sciatic nerve in rats, likely by enhancing axonal sprouting and extension. [ABSTRACT FROM AUTHOR]

The extracellular matrix (ECM), the main component of the extracellular space, mediates signal transmission between cells and controls their key functions: proliferation, differentiation, and migration. The relevance of studying ECM research is stipulated to the wide range of its biological properties, which can be used in regenerative medicine and bioengineering. Particular interest is presented the study of the regulatory activity on various cellular functions of cell-derived decellularized ECM (dECM). In this work, we tested the hypothesis about the modulating effect of dECM deposited by young MSC from Wharton's jelly on the aging phenotype of endometrial human multipotent mesenchymal stromal cells (eMSCs), which the cells acquired in response to oxidative stress. This aspect of ECM functioning in the context of eMSCs has not yet been considered. A comparative study of H2O2-induced senescence of eMSCs cultured on dECM and on plastic for a long time showed a significant change in the hallmarks of aging in the cell population maintained on dECM. Taken together, the results obtained suggest that the dECM is able to partially reverse (retard) premature senescence of eMSCs in response to oxidative stress, as well as expanding the understanding of the ECM as a regulator of the functional activity of cells. [ABSTRACT FROM AUTHOR]

The healthy skeletal muscle extracellular matrix (ECM) has several functions including providing structural integrity to myofibers, enabling lateral force transmission, and contributing to overall passive mechanical properties. In diseases such as Duchenne Muscular dystrophy, there is accumulation of ECM materials, primarily collagen, which results in fibrosis. Previous studies have shown that fibrotic muscle is often stiffer than healthy muscle, in part due to the increased number and altered architecture of collagen fibers within the ECM. This would imply that the fibrotic matrix is stiffer than the healthy matrix. However, while previous studies have attempted to quantify the extracellular contribution to passive stiffness in muscle, the outcomes are dependent on the type of method used. Thus, the goals of this study were to compare the stiffness of healthy and fibrotic muscle ECM and to demonstrate the efficacy of two methods for quantifying extracellular-based stiffness in muscle, namely decellularization and collagenase digestion. These methods have been demonstrated to remove the muscle fibers or ablate collagen fiber integrity, respectively, while maintaining the contents of the extracellular matrix. Using these methods in conjunction with mechanical testing on wildtype and D2.mdx mice, we found that a majority of passive stiffness in the diaphragm is dependent on the ECM, and the D2.mdx diaphragm ECM is resistant to digestion by bacterial collagenase. We propose that this resistance is due to the increased collagen cross-links and collagen packing density in the ECM of the D2.mdx diaphragm. Taken altogether, while we did not find increased stiffness of the fibrotic ECM, we did observe that the D2.mdx diaphragm conveyed resistance against collagenase digestion. These findings demonstrate how different methods for measuring ECM-based stiffness each have their own limitations and can produce different results.

The excessive deposition and cross-linking of core matrisome components typically result in abnormal remodeling of the extracellular matrix (ECM), leading to increased liver stiffness and worsening liver fibrosis. Exploring the biochemical properties of the ECM scaffold can deepen our understanding of the pathological mechanisms driving liver fibrosis and potentially facilitate the identification of therapeutic targets. While traditional sodium dodecyl sulfate (SDS)-based liver decellularization followed by proteomics can uncover the matrisome components within the ECM scaffold, it lacks the ability to reveal physicochemical characteristics like solubility. In our present study, using adult mouse liver as an example, we introduced a novel two-step workflow that combines our previously enhanced SDS (ESDS) decellularization with the conventional SDS method, enabling the identification of matrisome members with mild and/or high solubilities. Through this approach, we visualized the atlas of the mildly and highly insoluble matrisome contents in the adult mouse liver, as well as the regulatory network of highly insoluble matrisome that largely governs liver stiffness. Given the strong correlation between increased matrisome insolubility and heightened ECM stiffness, we believe that this methodology holds promise for future research focused on liver stiffness.

Host remodeling of decellularized extracellular matrix (dECM) material through the appropriate involvement of immune cells is essential for achieving functional organ/tissue regeneration. As many studies have focused on the role of macrophages, only few have evaluated the role of regulatory T cells (Tregs) in dECM remodeling. In this study, we used a mouse model of traumatic muscle injury to determine the role of Tregs in the constructive remodeling of vascular-derived dECM. According to the results, a certain number of Tregs could be recruited after dECM implantation. Notably, using anti-CD25 to reduce the number of Tregs recruited by the dECM was significantly detrimental to material remodeling based on a significant reduction in the number of M2 macrophages. In addition, collagen and elastic fibers, which maintain the integrity and mechanical properties of the material, rapidly degraded during the early stages of implantation. In contrast, the use of CD28-SA antibodies to increase the number of Tregs recruited by dECM promoted constructive remodeling, resulting in a decreased inflammatory response at the material edge, thinning of the surrounding fibrous connective tissue, uniform infiltration of host cells, and significantly improved tissue remodeling scores. The number of M2 macrophages increased whereas that of M1 macrophages decreased. Moreover, Treg-conditioned medium further enhanced material-induced M2 macrophage polarization in vitro. Overall, Treg is an important cell type that influences constructive remodeling of the dECM. Such findings contribute to the design of next-generation biomaterials to optimize the remodeling and regeneration of dECM materials.

Recombinant growth factors are used in tissue engineering to stimulate cell proliferation, migration, and differentiation. Conventional methods of growth factor delivery for therapeutic applications employ large amounts of these bioactive cues. Effective, localized growth factor release is essential to reduce the required dose and potential deleterious effects. The endogenous extracellular matrix (ECM) sequesters native growth factors through its negatively charged sulfated glycosaminoglycans. Mesenchymal stromal cells secrete an instructive extracellular matrix that can be tuned by varying culture and decellularization methods. In this study, mesenchymal stromal cell-secreted extracellular matrix was modified using λ-carrageenan as a macromolecular crowding (MMC) agent and decellularized with DNase as an alternative to previous decellularized extracellular matrices (dECM) to improve growth factor retention. Macromolecular crowding decellularized extracellular matrix contained 7.7-fold more sulfated glycosaminoglycans and 11.7-fold more total protein than decellularized extracellular matrix, with no significant difference in residual DNA. Endogenous BMP-2 was retained in macromolecular crowding decellularized extracellular matrix, whereas BMP-2 was not detected in other extracellular matrices. When implanted in a murine muscle pouch, we observed increased mineralized tissue formation with BMP-2-adsorbed macromolecular crowding decellularized extracellular matrix in vivo compared to conventional decellularized extracellular matrix. This study demonstrates the importance of decellularization method to retain endogenous sulfated glycosaminoglycans in decellularized extracellular matrix and highlights the utility of macromolecular crowding to upregulate sulfated glycosaminoglycan content. This platform has the potential to aid in the delivery of lower doses of BMP-2 or other heparin-binding growth factors in a tunable manner.

Age-related macular degeneration, a multifactorial inflammatory degenerative retinal disease, ranks as the leading cause of blindness in the elderly. Strikingly, there is a scarcity of curative therapies, especially for the atrophic advanced form of age-related macular degeneration, likely due to the lack of models able to fully recapitulate the native structure of the outer blood retinal barrier, the prime target tissue of age-related macular degeneration. Standard in vitro systems rely on 2D monocultures unable to adequately reproduce the structure and function of the outer blood retinal barrier, integrated by the dynamic interaction of the retinal pigment epithelium, the Bruch’s membrane, and the underlying choriocapillaris. The Bruch’s membrane provides structural and mechanical support and regulates the molecular trafficking in the outer blood retinal barrier, and therefore adequate Bruch’s membrane-mimics are key for the development of physiologically relevant models of the outer blood retinal barrier. In the last years, advances in the field of biomaterial engineering have provided novel approaches to mimic the Bruch’s membrane from a variety of materials. This review provides a discussion of the integrated properties and function of outer blood retinal barrier components in healthy and age-related macular degeneration status to understand the requirements to adequately fabricate Bruch’s membrane biomimetic systems. Then, we discuss novel materials and techniques to fabricate Bruch’s membrane-like scaffolds for age-related macular degeneration in vitro modeling, discussing their advantages and challenges with a special focus on the potential of Bruch’s membrane-like mimics based on decellularized tissue.

Background: Men’s infertility and lack of production of healthy and active sperm are concerns of recent years in mostcountries. Studies on the preparation of extracellular matrix (ECM) from decellularization of testis tissue and spermatogenesiscould provide proper results to solve some of the men’s infertility problems. This study aims to decellularize calftestis by different methods to reach a suitable scaffold and introduce it in spermatogenesis studies.Materials and Methods: In this experimental study, calf testis were decellularized by a freeze-de freeze, 1% sodiumdeoxycholate (SD), 0.1% sodium dodecyl sulfate (SDS), 0.1% SDS-vacuum, 1% SDS, 1% SDS-vacuum, and Triton-X100 methods. The content of DNA, collagen, and glycosaminoglycan (GAG) was analyzed using the kit and stainingwith Hematoxylin-Eosin, Masson’s trichrome, Alcian blue, and Orcein methods. The morphology of the scaffolds wasanalyzed with a scanning electron microscope (SEM).Results: Methods of 1% SDS, 1% SDS-vacuum, and 1% SD completely removed the cells. The preservation of collagenand GAG was confirmed using the staining kit and methods. The use of a vacuum showed greater porosity inthe SEM images. Toxicity and hemolysis were not observed in the scaffolds.Conclusion: Testis decellularization with 1% SDS and 1% SD, in addition to cell removal, could maintain the ECMstructure to a large extent without having cytotoxic and hemolysis effects.

Decellularized extracellular matrix (dECM), with its distinct biological properties, has gained significant attention as a natural biomaterial. Leveraging its potentials, we successfully developed a three-dimensional matrix-based oriented nerve graft by encapsulating a fibrous scaffold with multilayered conformationally intact and biologically active human bone marrow mesenchymal stem cell-derived decellularized extracellular matrix (hBMSC-dECM). Convincingly, the hBMSC-dECM group exhibited comparable functional recoveries to the autograft group by postoperative week 12. In the comprehensive analysis, the molecular regulations in the hBMSC-dECM group were more intricate and nuanced compared to the autograft group. Nevertheless, both groups displayed similar molecular regulatory processes in terms of vascularization and extracellular matrix. Notably, the hBMSC-dECM group demonstrated sustained high levels of regulation in axon and myelin regeneration at week 12, while the immunomodulation returned to the normal levels after peaking at week 2. Collectively, our findings illustrated the satisfactory construction of a cell-matrixed nerve graft that established a microenvironment conducive to nerve regeneration, and elucidated the distinct molecular regulation patterns and characteristics associated with different repair modes.

Chronic respiratory diseases often necessitate lung transplantation due to irreversible damage. Organ engineering offers hope through stem cell-based organ generation. However, the crucial sterilization step in scaffold preparation poses challenges. This study conducted a systematic review of studies that analysed the extracellular matrix (ECM) conditions of decellularised lungs subjected to different sterilisation processes. A search was performed for articles published in the PubMed, Web of Sciences, Scopus, and SciELO databases according to the PRISMA guidelines. Overall, five articles that presented positive results regarding the effectiveness of the sterilisation process were selected, some of which identified functional damage in the ECM. Was possible concluded that regardless of the type of agent used, physical or chemical, all of them demonstrated that sterilisation somehow harms the ECM. An ideal protocol has not been found to be fully effective in the sterilisation of pulmonary scaffolds for use in tissue and/or organ engineering.

This study aims to present an easily scalable, cost-efficient process of dECM extraction from porcine meniscus, dedicated to bioink preparation and 3D bioprinting. Due to its cartilage like structure and mechanical robustness, the meniscus is an exceptionally demanding tissue for extraction and decellularisation of its ECM. Its processing poses a great difficulty and renders the methods previously developed for soft tissues useless. A process, combining homogenisation, hydrolysis, supercritical CO2 (scCO2) extraction and lyophilisation, was developed to meet this challenge. This protocol allows for retaining its native compounds and biocompatibility while offering good printability and providing a stimulatory environment for cell proliferation and differentiation towards a meniscus-like phenotype. Also, this process is economically and ecologically friendly since it doesn't require the use of high amounts of solvents, detergents or expensive enzymes (DNase). The decellularisation process has been meticulously studied, demonstrating a substantial reduction in DNA content but still exceeding accepted thresholds. The study further explores the biocompatibility of the dECM, demonstrating no detrimental effects of remnant DNA on cell survival during extended in vitro culture, indicating excellent biocompatibility. These findings challenge the current definition of decellularisation effectiveness based solely on DNA content, proposing a broader assessment of biological effects.

Full osteochondral regeneration remains a major clinical challenge. Among other experimental cartilage regenerative approaches, decellularized cartilage (DCC) is considered a promising material for generating potentially implantable scaffolds useful as cartilage repair strategy. In this work, we focus on screening and comparing different decellularization methods, aiming to generate DCC potentially useful in biomedical context, and therefore, with biological activity and functional properties in terms of induction of differentiation and regeneration. Data indicates that enzymatic and detergents-based decellularization methods differentially affect ECM components, and that it has consequences in further biological behavior. SDS-treated DCC powder is not useful to be further processed in 2D or 3D structures, because these structures tend to rapidly solubilize, or disaggregate, in physiologic media conditions. Conversely, Trypsin-treated DCC powders can be processed to mechanically stable 2D films and 3D solid-foam scaffolds, presumably due to partial digestion of collagens during decellularization, which would ease crosslinking at DCC during solubilization and processing. In vitro cell culture studies indicate that these structures are biocompatible and induce and potentiate chondrogenic differentiation. In vivo implantation of DCC derived 3D porous scaffolds in rabbit osteochondral defects induce subchondral bone regeneration and fibrocartilage tissue formation after implantation. Therefore, this work defines an optimal cartilage tissue decellularization protocol able to generate DCC powders processable to biocompatible and bioactive 2D and 3D structures. These structures are useful for in vitro cartilage research and in vivo subchondral bone regeneration, while hyaline cartilage regeneration with DCC alone as implantable material remains elusive.

The bioengineering of corneal endothelial grafts consists of seeding in vitro cultured corneal endothelial cells onto a thin, transparent, biocompatible, and sufficiently robust carrier which can withstand surgical manipulations. This is one of the most realistic alternatives to donor corneas, which are in chronic global shortage. The anterior capsule of the crystalline lens has already been identified as one of the best possible carriers, but its challenging manual preparation has limited its use. In this study, we describe a femtosecond laser cutting process of the anterior capsule of whole lenses in order to obtain capsule discs of 8 mm diameter, similar to conventional endothelial grafts. Circular marks made on the periphery of the disc indicate its orientation. Immersion in water for 3 days is sufficient to completely remove the lens epithelial cells and to enable the seeding of corneal endothelial cells, which remain viable after 27 days of culture. Therefore, this method provides a transparent, decellularized disc ready to form viable tissue engineered endothelial grafts. [ABSTRACT FROM AUTHOR]

• Creation and characterization of muscle-derived extracellular matrix hydrogels from TSP2 KO mice shows altered composition, assembly, and biomechanical properties when compared to WT. • TSP2 KO hydrogels allow greater myocyte spreading and invasion as well increased tube formation by endothelial cells in vitro. • In vivo study showed excellent foreign body response of TSP2 KO hydrogels associated with enhanced cell invasion, innervation, and angiogenesis. • TSP2 KO hydrogels were used to treat VML injury in mice and were found to induce greater recruitment of repair cells and enhanced tissue remodeling. Volumetric muscle loss (VML) represents a clinical challenge due to the limited regenerative capacity of skeletal muscle. Most often, it results in scar tissue formation and loss of function, which cannot be prevented by current therapies. Decellularized extracellular matrix (DEM) has emerged as a native biomaterial for the enhancement of tissue repair. Here, we report the generation and characterization of hydrogels derived from DEM prepared from WT or thrombospondin (TSP)-2 null muscle tissue. TSP2-null hydrogels, when compared to WT, displayed altered architecture, protein composition, and biomechanical properties and allowed enhanced invasion of C2C12 myocytes and chord formation by endothelial cells. They also displayed enhanced cell invasion, innervation, and angiogenesis following subcutaneous implantation. To evaluate their regenerative capacity, WT or TSP2 null hydrogels were used to treat VML injury to tibialis anterior muscles and the latter induced greater recruitment of repair cells, innervation, and blood vessel formation and reduced inflammation. Taken together, these observations indicate that TSP2-null hydrogels enhance angiogenesis and promote muscle repair in a VML model. [ABSTRACT FROM AUTHOR]

One of the key objectives of regenerative medicine is the design of skin tissue engineering scaffolds to promote wound healing. These scaffolds provide a fresh viewpoint on skin injury repair by emulating body tissues in their structure. A suitable platform for cellular processes can be provided by natural scaffolds made from decellularized tissues while retaining the primary components. Resveratrol (RES), which has qualities like angiogenesis, antioxidant, antibacterial, and anti-inflammatory, is also useful in the healing of wounds. In this investigation, RES-loaded decellularized sheep pericardium scaffolds were created and tested on full-thickness wounds in a mouse model. According to the in vivo findings, the groups in which the wound was treated with decellularized pericardium (DP) had better wound healing than the control group and showed more production of angiogenic and anti-inflammatory substances. The secretion of these factors was greater in RES‐loaded decellularized pericardium (DP-RES) than in the scaffold without RES, and the macroscopic and histological data supported this. Therefore, the use of decellularization scaffolds with substances like RES for the regeneration of skin wounds can be further researched and evaluated in the preclinical stages. [ABSTRACT FROM AUTHOR]

Utilization of acellular scaffolds, extracellular matrix (ECM) without cell content, is growing in tissue engineering, due to their high biocompatibility, bioactivity ad mechanical support. Hence, the purpose of this research was to study the characteristics and biocompatibility of decellularized rat skin scaffolds using the osmotic shock method. First, the skin of male Wistar rats was harvested and cut into 1 × 1 cm2 pieces. Then, some of the harvested parts were subjected to the decellularization process by applying osmotic shock. Comparison of control and scaffold samples was conducted in order to assure cell elimination and ECM conservation by means of histological evaluations, quantification of biochemical factors, measurement of DNA amount, and photographing the ultrastructure of the samples by scanning electron microscopy (SEM). In order to evaluate stem cell viability and adhesion to the scaffold, adipose-derived mesenchymal stem cells (AD-MSCs) were seeded on the acellular scaffolds. Subsequently, MTT test and SEM imaging of the scaffolds containing cultured cells were applied. The findings indicated that in the decellularized scaffolds prepared by osmotic shock method, not only the cell content was removed, but also the ECM components and its ultrastructure were preserved. Also, the 99% viability and adhesion of AD-MSCs cultured on the scaffolds indicate the biocompatibility of the decellularized skin scaffold. In conclusion, decellularized rat skin scaffolds are biocompatible and appropriate scaffolds for future investigations of tissue engineering applications. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Tendon injuries repair is a significant burden for orthopaedic surgeons. Finding a proper graft material to repair tendon is one of the main challenges in orthopaedics, for which the requirement of substitute for tendon repair would be different for each clinical application. Among biological scaffolds, the use of decellularized tendon increasingly represents an interesting approach to treat tendon injuries and several articles have investigated the approaches of tendon decellularization. To understand the outcomes of the the approaches of tendon decellularization on effect of tendon transplantation, a literature review was performed. This review was conducted by searching in Pubmed and Embase and 64 studies were included in this study. The findings revealed that the common approaches to decellularize tendon include chemical, physical, and enzymatic decellularization methods or their combination. With the development of tissue engineering, researchers also put forward new theories such as automatic acellular machine, 3D printing technology to manufacture acellular scaffold. [ABSTRACT FROM AUTHOR]

More than 1000 donated aortic and pulmonary valves from predominantly European tissue banks were centrally decellularized and delivered to hospitals in Europe and Japan. Here, we report on the processing and quality controls before, during and after the decellularization of these allografts. Our experiences show that all tissue establishments, which provide native cardiovascular allografts for decellularization, meet comparably high-quality standards, regardless of their national origin. A total of 84% of all received allografts could be released as cell-free allografts. By far the most frequent reasons for rejection were non-release of the donor by the tissue establishment or severe contaminations of the native tissue donation. Only in 2% of all cases the specification for freedom from cells was not fulfilled, indicating that decellularization of human heart valves is a safe process with a very low discard ratio. In clinical use, cell-free cardiovascular allografts have been shown to be advantageous over conventional heart valve replacements, at least in young adults. These results open the discussion on the future gold standard and funding of this innovative therapeutic option for heart valve replacement. [ABSTRACT FROM AUTHOR]

For decades, bovine jugular vein conduits (BJV) and classic cryopreserved homografts have been the two most widely used options for pulmonary valve replacement (PVR) in congenital heart disease. More recently, decellularized pulmonary homografts (DPH) have provided an alternative avenue for PVR. Matched comparison of patients who received DPH for PVR with patients who received bovine jugular vein conduits (BJV) considering patient age group, type of heart defect, and previous procedures. 319 DPH patients were matched to 319 BJV patients; the mean age of BJV patients was 15.3 (SD 9.5) years versus 19.1 (12.4) years in DPH patients (p = 0.001). The mean conduit diameter was 24.5 (3.5) mm for DPH and 20.3 (2.5) mm for BJV (p < 0.001). There was no difference in survival rates between the two groups after 10 years (97.0 vs. 98.1%, p = 0.45). The rate of freedom from endocarditis was significantly lower for BJV patients (87.1 vs. 96.5%, p = 0.006). Freedom from explantation was significantly lower for BJV at 10 years (81.7 vs. 95.5%, p = 0.001) as well as freedom from any significant degeneration at 10 years (39.6 vs. 65.4%, p < 0.001). 140 Patients, matched for age, heart defect type, prior procedures, and conduit sizes of 20–22 mm (± 2 mm), were compared separately; mean age BJV 8.7 (4.9) and DPH 9.5 (7.3) years (p = n.s.). DPH showed 20% higher freedom from explantation and degeneration in this subgroup (p = 0.232). Decellularized pulmonary homografts exhibit superior 10-year results to bovine jugular vein conduits in PVR. [ABSTRACT FROM AUTHOR]

Focal chondral defects (FCDs) significantly impede quality of life for patients and impose severe economic costs on society. One of the most promising treatment options—autologous matrix‐induced chondrogenesis (AMIC)—could benefit from a scaffold that contains both of the primary cartilage matrix components—sulfated glycosaminoglycans (sGAGs) and collagen type II. Here, 17 different protocols were evaluated to determine the most optimum strategy for decellularizing (decelling) the bovine nucleus pulposus (bNP) to yield a natural biomaterial with a cartilaginous constituency. The resulting scaffold was then characterized with respect to its biochemistry, biomechanics and cytocompatibility. Results indicated that the optimal decell protocol involved pre‐crosslinking the tissue prior to undergoing decell with trypsin and Triton X‐100. The residual DNA content of the scaffold was found to be 32.64 ± 9.26 ng/mg dry wt. of tissue with sGAG and hydroxyproline (HYP) contents of 72.53 ± 16.43. and 78.38 ± 8.46 μg/mg dry wt. respectively. The dynamic viscoelastic properties were found to be preserved (complex modulus: 17.92–16.62 kPa across a range of frequencies) while the equilibrium properties were found to have significantly decreased (aggregate modulus: 11.51 ± 9.19 kPa) compared to the non‐decelled fresh bNP tissue. Furthermore, the construct was also found to be cytocompatible with bone marrow stem cells (BMSCs). While it was not permissive of cellular infiltration, the BMSCs were still found to have lined the laser drilled channels in the scaffold. Taken together, the biomaterial developed herein could be a valuable addition to the AMIC family of scaffolds or serve as an off‐the‐shelf standalone option for cartilage repair. [ABSTRACT FROM AUTHOR]

Cellular agriculture holds the potential to address sustainability, food security, and agricultural resilience. Within the cell-based meat supply chain, one of the key steps is developing sustainable scaffolding. In this study, we evaluated the impact of decellularized banana leaves, various coating materials including soy protein and gelatine, and different cell seeding strategies on cell viability, cell growth, cell alignment, and the response of the materials to thermal processing. Kinetics of the quality degradation of the scaffolds with and without cells were determined through kinetics equations. The efficiency of decellularization was verified through DNA quantification, which decreased from 445 ng/mg in fresh banana leaves to non-detectable levels in the decellularized samples. The alignment of cells on gelatin-coated samples was the highest among the samples, with a dominant orientation of 65.8°, compared to soy-coated and uncoated samples, with dominant orientations of 9.2° and -6.3°, respectively. The kinetics of shrinkage indicated that coating with soy and the presence of cells increased the activation energy due to the higher energy required for protein denaturation. The kinetics of area changes in plain scaffolds without cells followed a first-order pattern, while with seeded cells a second-order pattern was followed. Overall, the results showed that decellularized banana leaves provide sustainable scaffoldings for cellular agriculture applications. In addition, soy coating providedmany benefits for decellularized samples by supporting cell adhesion and cell proliferation. [ABSTRACT FROM AUTHOR]

Cell-derived matrices (CDMs) are scaffolds constructed by decellularization of cellular matrices from different tissues and organs. Since CDMs mimic the extracellular matrices (ECMs) of native tissues, it plays an essential role in the preparation of bioscaffolds. CDM scaffolds from mesenchymal stem cells (MSCs) have been reported to support cell adhesion and proliferation of its own cells. Therefore, in this study we aimed to test if growth of human Wharton's jelly-derived MSCs may be enhanced when cultured on their own CDMs. To do this, MSCs were induced to generate ECM using ascorbic acid. Thus, obtained matrices were decellularized and characterized quantitatively for changes in their biochemical components (total protein, collagen, glycosaminoglycans) and qualitatively for fibronectin, laminin, and collagen (I & IV) by immunostaining. Our results show the retention of essential ECM components in the decellularized WJ-MSC-derived matrix (WJ-CDM). The influence of WJ-CDM on proliferation and differentiation of WJ-MSCs were evaluated by comparing their growth on collagen and fibronectin-only coated plates. A non-coated tissue culture polystyrene plate (TCPS) served as control. Our cell proliferation results show that no significant changes were observed in the proliferation of MSCs when cultured on WJ-CDM as compared to the bio-coated and non-coated cultures. However, gene expression analysis of the differentiation process showed that osteogenic and adipogenic differentiation potential of the WJ-MSCs was significantly increased upon culturing them on WJ-CDM. In conclusion, the present study reveals that the WJ-MSCs cultured on WJ-CDM may augment osteogenic and adipogenic differentiation. [ABSTRACT FROM AUTHOR]

Advancements in regenerative medicine have highlighted the potential of decellularized extracellular matrix (ECM) as a scaffold for organ bioengineering. Although the potential of ECM in major organ systems is well-recognized, studies focusing on the angiogenic effects of pancreatic ECM are limited. This study investigates the capabilities of pancreatic ECM, particularly its role in promoting angiogenesis. Using a Triton-X-100 solution, porcine pancreas was successfully decellularized, resulting in a significant reduction in DNA content (97.1% removal) while preserving key pancreatic ECM components. A three-dimensional ECM hydrogel was then created from this decellularized tissue and used for cell culture. Biocompatibility tests demonstrated enhanced adhesion and proliferation of mouse embryonic stem cell-derived endothelial cells (mES-ECs) and human umbilical vein endothelial cells (HUVECs) in this hydrogel compared to conventional scaffolds. The angiogenic potential was evaluated through tube formation assays, wherein the cells showed superior tube formation capabilities in ECM hydrogel compared to rat tail collagen. The RT-PCR analysis further confirmed the upregulation of pro-angiogenic genes in HUVECs cultured within the ECM hydrogel. Specifically, HUVECs cultured in the ECM hydrogel exhibited a significant upregulation in the expression of MMP2, VEGF and PAR-1, compared to those cultured in collagen hydrogel or in a monolayer condition. The identification of ECM proteins, specifically PRSS2 and Decorin, further supports the efficacy of pancreatic ECM hydrogel as an angiogenic scaffold. These findings highlight the therapeutic promise of pancreatic ECM hydrogel as a candidate for vascularized tissue engineering application. [ABSTRACT FROM AUTHOR]

Decellularized dermal matrices (dDMs) have emerged as effective biomaterials that can revolutionize regenerative medicine, particularly in the field of wound healing and tissue regeneration. Derived from animal or human skin, dDMs offer great biocompatibility, remarkable biochemistry, and a macromolecular architecture equivalent to the native tissue. Notably, among the biomimetic extracellular matrix (ECM)-based scaffolds, dDMs stand out due to their inherent dermal microenvironment, holding high value for skin regeneration and reconstructive surgery. The integration of dDMs as a biomaterial base for bioinks in advanced manufacturing technologies opens promising avenues for crafting precise, biomimetic tissue engineering (TE) constructs with optimized recellularization properties. This mini review outlines the main sources, differential decellularization techniques applied to dDMs, and their significance intissue engineering and regenerative medicine. It subsequently delves into the different categories of decellularized materials obtained, their unique physical and biochemical attributes, as well as their applications to promote wound healing and regenerating skin and soft tissues. Additionally, the currently available market products based on dDMs are examined and the main outcomes are compared. Finally, the article highlights current barriers in the field and anticipates the future challenges and applications of dDMs-based therapies. [ABSTRACT FROM AUTHOR]

Background: Men's infertility and lack of production of healthy and active sperm are concerns of recent years in most countries. Studies on the preparation of extracellular matrix (ECM) from decellularization of testis tissue and spermatogenesis could provide proper results to solve some of the men's infertility problems. This study aims to decellularize calf testis by different methods to reach a suitable scaffold and introduce it in spermatogenesis studies. Materials and Methods: In this experimental study, calf testis were decellularized by a freeze-de freeze, 1% sodium deoxycholate (SD), 0.1% sodium dodecyl sulfate (SDS), 0.1% SDS-vacuum, 1% SDS, 1% SDS-vacuum, and Triton- X100 methods. The content of DNA, collagen, and glycosaminoglycan (GAG) was analyzed using the kit and staining with Hematoxylin-Eosin, Masson's trichrome, Alcian blue, and Orcein methods. The morphology of the scaffolds was analyzed with a scanning electron microscope (SEM). Results: Methods of 1% SDS, 1% SDS-vacuum, and 1% SD completely removed the cells. The preservation of collagen and GAG was confirmed using the staining kit and methods. The use of a vacuum showed greater porosity in the SEM images. Toxicity and hemolysis were not observed in the scaffolds. Conclusion: Testis decellularization with 1% SDS and 1% SD, in addition to cell removal, could maintain the ECM structure to a large extent without having cytotoxic and hemolysis effects. [ABSTRACT FROM AUTHOR]

The heart valve is crucial for the human body, which directly affects the efficiency of blood transport and the normal functioning of all organs. Generally, decellularization is one method of tissue-engineered heart valve (TEHV), which can deteriorate the mechanical properties and eliminate allograft immunogenicity. In this study, removable polyvinyl alcohol (PVA) is used to encapsulate decellularized porcine heart valves (DHVs) as a dynamic template to improve the processability of DHVs, such as suturing. Mechanical tests show that the strength and elastic modulus of DHVs treated with different concentrations of PVA significantly improve. Without the PVA layer, the valve would shift during suture puncture and not achieve the desired suture result. The in vitro results indicate that decellularized valves treated with PVA can sustain the adhesion and growth of human umbilical vein endothelial cells (HUVECs). All results above show that the DHVs treated with water-soluble PVA have good mechanical properties and cytocompatibility to ensure post-treatment. On this basis, the improved processability of DHV treated with PVA enables a new paradigm for the manufacturing of scaffolds, making it easy to apply. [ABSTRACT FROM AUTHOR]

The selection of an appropriate scaffold is imperative for the successful development of alternative animal protein in the form of cultured meat or lab-grown meat. Decellularized tissues have been suggested as a potential scaffold for cultured meat production owing to their capacity to support an optimal environment and niche conducive to cell proliferation and growth. This approach facilitates the systematic development of 3D tissues in the laboratory. Decellularized scaffold biomaterials have characteristics of high biocompatibility, biodegradation, and various bioactivities, which could potentially address the limitations associated with synthetic bio-scaffold materials. The present study involved the derivation and characterization of a decellularized scaffold from mushroom tissue following subsequent assessment of the scaffold's capacity to support myogenic differentiation. Mushroom sections were soaked in nuclease and detergent solution for 4 days. Furthermore, decellularization was confirmed by histology and DAPI staining, which showed the removal of cellular components and nuclei. Myoblast cells were seeded onto decellularized tissue, which exhibited excellent cytocompatibility and promoted myogenic growth and differentiation. The study's findings can serve as a foreground for the generation of an edible and natural scaffold for producing a safe and disease-free source of alternative animal protein, potentially reducing the burden on the health sector caused by conventional animal protein production and consumption. [ABSTRACT FROM AUTHOR]

Solubilizing extracellular matrix (ECM) materials and transforming them into hydrogels has expanded their potential applications both in vitro and in vivo. In this study, hydrogels are prepared by decellularization of human placental tissue using detergent and enzymes and by the subsequent creation of a homogenized acellular placental tissue powder (P‐ECM). A perfusion‐based decellularization approach is employed using detergent and enzymes. The P‐ECM with and without gamma irradiation is then utilized to prepare P‐ECM hydrogels. Physical and biological evaluations are conducted to assess the suitability of the P‐ECM hydrogels for biocompatibility. The decellularized tissue has significantly reduced cellular content and retains the major ECM proteins. Increasing the concentration of P‐ECM leads to improved mechanical properties of the P‐ECM hydrogels. The biocompatibility of the P‐ECM hydrogel is demonstrated through cell proliferation and viability assays. Notably, gamma‐sterilized P‐ECM does not support the formation of a stable hydrogel. Nonetheless, the use of HCl during the digestion process effectively decreases spore growth and bacterial bioburden. The study demonstrates that P‐ECM hydrogels exhibit physical and biological attributes conducive to soft tissue reconstruction. These hydrogels establish a favorable microenvironment for cell growth and the need for investigating innovative sterilization methods. [ABSTRACT FROM AUTHOR]

Over the past few decades, there has been a remarkable advancement in the field of transplantation. But the shortage of donors is still an urgent problem that requires immediate attention. As with xenotransplantation, bioengineered organs are promising solutions to the current shortage situation. And decellularization is a unique technology in organ-bioengineering. However, at present, there is no unified decellularization method for different tissues, and there is no gold-standard for evaluating decellularization efficiency. Meanwhile, recellularization, re-endothelialization and modification are needed to form transplantable organs. With this mind, we can start with decellularization and re-endothelialization or modification of small blood vessels, which would serve to address the shortage of small-diameter vessels while simultaneously gathering the requisite data and inspiration for further recellularization of the whole organ-scale vascular network. In this review, we collect the related experiments of decellularization and post-decellularization approaches of small vessels in recent years. Subsequently, we summarize the experience in relation to the decellularization and post-decellularization combinations, and put forward obstacle we face and possible solutions.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive lethal malignancy that accounts for more than 90% of pancreatic cancer diagnoses. Our research is focused on the physico-chemical properties of the tumour microenvironment (TME), including its tumoural extracellular matrix (tECM), as they may have an important impact on the success of cancer therapies. PDAC xenografts and their decellularized tECM offer a great material source for research in terms of biomimicry with the original human tumour. Our aim was to evaluate and quantify the physico-chemical properties of the PDAC TME. Both cellularized (native TME) and decellularized (tECM) patient-derived PDAC xenografts were analyzed. A factorial design of experiments identified an optimal combination of factors for effective xenograft decellularization. Our results provide a complete advance in our understanding of the PDAC TME and its corresponding stroma, showing that it presents an interconnected porous architecture with very low permeability and small pores due to the contractility of the cellular components. This fact provides a potential therapeutic strategy based on the therapeutic agent size.

Chronic respiratory diseases often necessitate lung transplantation due to irreversible damage. Organ engineering offers hope through stem cell-based organ generation. However, the crucial sterilization step in scaffold preparation poses challenges. This study conducted a systematic review of studies that analysed the extracellular matrix (ECM) conditions of decellularised lungs subjected to different sterilisation processes. A search was performed for articles published in the PubMed, Web of Sciences, Scopus, and SciELO databases according to the PRISMA guidelines. Overall, five articles that presented positive results regarding the effectiveness of the sterilisation process were selected, some of which identified functional damage in the ECM. Was possible concluded that regardless of the type of agent used, physical or chemical, all of them demonstrated that sterilisation somehow harms the ECM. An ideal protocol has not been found to be fully effective in the sterilisation of pulmonary scaffolds for use in tissue and/or organ engineering.

Large bone defect regeneration remains a major challenge for orthopedic surgeons. Tissue engineering approaches are therefore emerging in order to overcome this limitation. However, these processes can alter some of essential native tissue properties such as intermolecular crosslinks of collagen triple helices, which are known for their essential role in tissue structure and function. We assessed the persistence of extracellular matrix (ECM) properties in human fascia lata (HFL) and periosteum (HP) after tissue engineering processes such as decellularization and sterilization. Harvested from cadaveric donors (N = 3), samples from each HFL and HP were decellularized following five different chemical protocols with and without detergents (D1-D4 and D5, respectively). D1 to D4 consisted of different combinations of Triton, Sodium dodecyl sulfate and Deoxyribonuclease, while D5 is routinely used in the institutional tissue bank. Decellularized HFL tissues were further gamma-irradiated (minimum 25 kGy) in order to study the impact of sterilization on the ECM. Polarized light microscopy (PLM) was used to estimate the thickness and density of collagen fibers. Tissue hydration and content of hydroxyproline, enzymatic crosslinks, and non-enzymatic crosslinks (pentosidine) were semi-quantified with Raman spectroscopy. ELISA was also used to analyze the maintenance of the decorin (DCN), an important small leucine rich proteoglycan for fibrillogenesis. Among the decellularization protocols, detergent-free treatments tended to further disorganize HFL samples, as more thin fibers (+53.7%) and less thick ones (−32.6%) were recorded, as well as less collagen enzymatic crosslinks (−25.2%, p = 0.19) and a significant decrease of DCN (p = 0.036). GAG content was significantly reduced in both tissue types after all decellularization protocols. On the other hand, HP samples were more sensitive to the D1 detergent-based treatments, with more disrupted collagen organization and greater, though not significant loss of enzymatic crosslinks (−37.4%, p = 0.137). Irradiation of D5 HFL samples, led to a further and significant loss in the content of enzymatic crosslinks (−29.4%, p = 0.037) than what was observed with the decellularization process. Overall, the results suggest that the decellularization processes did not significantly alter the matrix. However, the addition of a gamma-irradiation is deleterious to the collagen structural integrity of the tissue.

Introduction: The decellularization and recellularization is a promising approach for tissue engineering and regenerative medicine. However, the decellularization process depletes important components like glycosaminoglycans (GAGs), affecting cell attachment and causing immunogenicity. Studies have explored various surface modification strategies to enhance recellularization. Methods: To optimize the decellularization method, we employed whole kidney perfusion and slice kidney immersion/agitation techniques. The decellularized extracellular matrix (dECM) was then analyzed using hematoxylin and eosin (H&E) staining, scanning electron microscope (SEM), and DNA quantification. To enhance cell proliferation efficiency, albumin coating and rotating culture were applied. Also, we evaluated in vitro blood clot formation on the albumin-coated dECM by immersing it in blood. Results: After decellularization, the unique structures of the kidney were preserved whether cellular components were removed. Subsequently, we utilized albumin coating and rotating culture for recellularization, and observed that albumin-coated dECM not only promoted high cell proliferation rates but also prevented blood clot formation. Conclusion: The albumin-coated dECM promoted cell proliferation and reduced blood clot formation in vitro. Also, dynamic culture condition using rotating culture allowed for improved cellular penetration into the dECM, leading to a conductive environment for enhanced tissue infiltration. This new approach suggests that the combined utilization of albumin coating and rotating culture conditions can improve the efficiency of recellularization.

The skin is an important organ of the human body that resists external threats but lacks sufficient self-regeneration ability when severe damage occurs. However, most of the available skin substitutes cannot achieve ideal restoration of complex structures and multiple functions of native skin tissues. Fortunately, the advent of decellularized extracellular matrix (dECM) offers a promising approach to overcome these obstacles. The dECM, derived from the natural extracellular matrix (ECM), possesses a similar structure and composition, which constructs an environment favorable for cell performance in regeneration. Moreover, dECM retains good bioactivity, low immunogenicity, and high availability, making it a suitable biomaterial for skin repair and regeneration. In this review, various decellularization methods and subsequent evaluations of dECM are introduced first, and the main sources of dECM are then presented. Furthermore, the recent progress of dECM-based biomaterials applied in skin regeneration and future perspectives are summarized.