RESUMO
Solubilized, gel-forming decellularized extracellular matrix (dECM) is used in a wide range of basic and translational research and due to its inherent bioactivity can promote structural and functional tissue remodeling. The animal-derived protease pepsin has become the standard proteolytic enzyme for the solubilization of almost all types of collagen-based dECM. In this study, pepsin was compared with papain, α-amylase, and collagenase for their potential to solubilize porcine liver dECM. Maximum preservation of bioactive components and native dECM properties was used as a decisive criterion for further application of the enzymes, with emphasis on minimal destruction of the protein structure and maintained capacity for physical thermogelation at neutral pH. The solubilized dECM digests, and/or their physically gelled hydrogels were characterized for their rheological properties, gelation kinetics, GAG content, proteomic composition, and growth factor profile. This study highlights papain as a plant-derived enzyme that can serve as a cost-effective alternative to animal-derived pepsin for the efficient solubilization of dECM. The resulting homogeneous papain-digested dECM preserved its thermally triggered gelation properties similar to pepsin digests, and the corresponding dECM hydrogels demonstrated their enhanced bioadhesiveness in single-cell force spectroscopy experiments with fibroblasts. The viability and proliferation of human HepaRG cells on dECM gels were similar to those on pure rat tail collagen type I gels. Papain is not only highly effective and economically attractive for dECM solubilization but also particularly interesting when digesting human-tissue-derived dECM for regenerative applications, where animal-derived materials are to be avoided.
Assuntos
Matriz Extracelular , Papaína , Ratos , Suínos , Humanos , Animais , Matriz Extracelular/química , Papaína/metabolismo , Matriz Extracelular Descelularizada , Pepsina A/análise , Pepsina A/metabolismo , Pepsina A/farmacologia , Proteômica , Hidrogéis/química , Engenharia Tecidual/métodos , Alicerces Teciduais/químicaRESUMO
Three-dimensional tissue cultures are important models for the study of cell-cell and cell-matrix interactions, as well as, to investigate tissue repair and reconstruction pathways. Therefore, we designed a reproducible and easy to handle printable bioreactor system (Teburu), that is applicable for different approaches of pathway investigation and targeted tissue repair using human tissue slices as a three-dimensional cell culture model. Here, we definitively describe Teburu as a controlled environment to reseed a 500-µm thick decellularized human liver slice using human mesenchymal stroma cells. During a cultivation period of eight days, Teburu, as a semi-open and low consumption system, was capable to maintain steady pH and oxygenation levels. Its combination with additional modules delivers an applicability for a wide range of tissue engineering approaches under optimal culture conditions.
Assuntos
Bioimpressão , Reatores Biológicos , Impressão Tridimensional , Técnicas de Cultura de Tecidos/instrumentação , Desenho de Equipamento , Humanos , Fígado/química , Fígado/citologia , Fígado/ultraestrutura , Engenharia Tecidual/instrumentação , Alicerces Teciduais/químicaRESUMO
BACKGROUND: The extracellular matrix (ECM) is a three-dimensional network of proteins that encases and supports cells within a tissue and promotes physiological and pathological cellular differentiation and functionality. Understanding the complex composition of the ECM is essential to decrypt physiological processes as well as pathogenesis. In this context, the method of decellularization is a useful technique to eliminate cellular components from tissues while preserving the majority of the structural and functional integrity of the ECM. RESULTS: In this study, we employed a bottom-up proteomic approach to elucidate the intricate network of proteins in the decellularized extracellular matrices of murine liver and kidney tissues. This approach involved the use of a novel, perfusion-based decellularization protocol to generate acellular whole organ scaffolds. Proteomic analysis of decellularized mice liver and kidney ECM scaffolds revealed tissue-specific differences in matrisome composition, while we found a predominantly stable composition of the core matrisome, consisting of collagens, glycoproteins, and proteoglycans. Liver matrisome analysis revealed unique proteins such as collagen type VI alpha-6, fibrillin-2 or biglycan. In the kidney, specific ECM-regulators such as cathepsin z were detected. CONCLUSION: The identification of distinct proteomic signatures provides insights into how different matrisome compositions might influence the biological properties of distinct tissues. This experimental workflow will help to further elucidate the proteomic landscape of decellularized extracellular matrix scaffolds of mice in order to decipher complex cell-matrix interactions and their contribution to a tissue-specific microenvironment.
RESUMO
Vascular surgery faces a critical demand for novel vascular grafts that are biocompatible and thromboresistant. This urgency particularly applies to bypass operations involving small caliber vessels. In the realm of tissue engineering, the development of fully vascularized organs holds great promise as a solution to organ shortage for transplantation. To achieve this, it is imperative to (re-)construct a biocompatible and non-thrombogenic vascular network within these organs. In this systematic review, we identify, classify and discuss basic principles and methods used to perform in vitro/ex vivo dynamic thrombogenicity testing of perfusable tissue engineered organs and tissues. We conducted a pre-registered systematic review of studies published in the last 23 years according to PRISMA-P Guidelines, comprising a systematic data extraction, in-depth analysis and risk of bias assessment of 116 included studies. We identified shaking (n=28), flow loop (n=17), ex vivo (arterio-venous shunt, n=33) and dynamic in vitro models (n=38) as main approaches for thrombogenicity assessment. This comprehensive review unveils a prevalent lack of standardization and serves as a valuable guide in the design of standardized experimental setups.
RESUMO
BACKGROUND: Tissue engineered bioscaffolds based on decellularized composites have gained increasing interest for treatment of various diaphragmatic impairments, including muscular atrophies and diaphragmatic hernias. Detergent-enzymatic treatment (DET) constitutes a standard strategy for diaphragmatic decellularization. However, there is scarce data on comparing DET protocols with different substances in distinct application models in their ability to maximize cellular removal while minimizing extracellular matrix (ECM) damage. METHODS: We decellularized diaphragms of male Sprague Dawley rats with 1 % or 0.1 % sodium dodecyl sulfate (SDS) and 4 % sodium deoxycholate (SDC) by orbital shaking (OS) or retrograde perfusion (RP) through the vena cava. We evaluated decellularized diaphragmatic samples by (1) quantitative analysis including DNA quantification and biomechanical testing, (2) qualitative and semiquantitative analysis by proteomics, as well as (3) qualitative assessment with macroscopic and microscopic evaluation by histological staining, immunohistochemistry and scanning electron microscopy. RESULTS: All protocols produced decellularized matrices with micro- and ultramorphologically intact architecture and adequate biomechanical performance with gradual differences. The proteomic profile of decellularized matrices contained a broad range of primal core and ECM-associated proteins similar to native muscle. While no outstanding preference for one singular protocol was determinable, SDS-treated samples showed slightly beneficial properties in comparison to SDC-processed counterparts. Both application modalities proved suitable for DET. CONCLUSION: DET with SDS or SDC via orbital shaking or retrograde perfusion constitute suitable methods to produce adequately decellularized matrices with characteristically preserved proteomic composition. Exposing compositional and functional specifics of variously treated grafts may enable establishing an ideal processing strategy to sustain valuable tissue characteristics and optimize consecutive recellularization. This aims to design an optimal bioscaffold for future transplantation in quantitative and qualitative diaphragmatic defects.
Assuntos
Diafragma , Engenharia Tecidual , Ratos , Animais , Masculino , Engenharia Tecidual/métodos , Proteômica , Ratos Sprague-Dawley , Matriz Extracelular/química , Proteínas da Matriz Extracelular/análise , Proteínas da Matriz Extracelular/metabolismo , Ácido Desoxicólico/análise , Ácido Desoxicólico/metabolismoRESUMO
The role of extracellular matrix (ECM) composition and turnover in mechano-signaling and the metamorphic fate of cells seeded into decellularized tissue can be elucidated by recent developments in non-invasive imaging and biotechnological analysis methods. Because these methods allow accurate quantification of the composition and structural integrity of the ECM, they can be critical in establishing standardized decellularization protocols. This study proposes quantification of the solid fraction, the single-component fraction and the viscoelasticity of decellularized pancreatic tissues using compact multifrequency magnetic resonance elastography (MRE) to assess the efficiency and quality of decellularization protocols. MRE of native and decellularized pancreatic tissues showed that viscoelasticity parameters depend according to a power law on the solid fraction of the decellularized matrix. The parameters can thus be used as highly sensitive markers of the mechanical integrity of soft tissues. Compact MRE allows consistent and noninvasive quantification of the viscoelastic properties of decellularized tissue. Such a method is urgently needed for the standardized monitoring of decellularization processes, evaluation of mechanical ECM properties, and quantification of the integrity of solid structural elements remaining in the decellularized tissue matrix.
Assuntos
Matriz Extracelular , Pâncreas , Matriz Extracelular/química , Pâncreas/diagnóstico por imagem , Hormônios Pancreáticos/análise , ViscosidadeRESUMO
The production of biomaterials that endow significant morphogenic and microenvironmental cues for the constitution of cell integration and regeneration remains a key challenge in the successful implementation of functional organ replacements. Despite the vast development in the production of biological and architecturally native matrices, the complex compositions and pivotal figures by which the human matrisome mediates many of its essential functions are yet to be defined. Here we present a thorough analysis of the native human liver proteomic landscape using decellularization and defatting protocols to create extracellular matrix scaffolds of natural origin that can further be used in both bottom-up and top-down approaches in tissue engineering based organ replacements. Furthermore, by analyzing human liver extracellular matrices in different stages of fibrosis and cirrhosis, we have identified distinct attributes of these tissues that could potentially be exploited therapeutically and thus require further investigation. The general experimental pipeline presented in this study is applicable to any type of tissue and can be widely used for different approaches in regenerative medicine and in the construction of novel biomaterials for organ engineering approaches.
Assuntos
Proteômica , Alicerces Teciduais , Matriz Extracelular , Humanos , Fígado , Engenharia TecidualRESUMO
Although more than one million liver transplantations have been carried out worldwide, the literature on liver resections in transplanted livers is scarce. We herein report a total number of fourteen patients, who underwent liver resection after liver transplantation (LT) between September 2004 and 2017. Hepatocellular carcinomas and biliary tree pathologies were the predominant indications for liver resection (n = 5 each); other indications were abscesses (n = 2), post-transplant lymphoproliferative disease (n = 1) and one benign tumor. Liver resection was performed at a median of 120 months (interquartile range (IQR): 56.5-199.25) after LT with a preoperative Model for End-Stage Liver Disease (MELD) score of 11 (IQR: 6.75-21). Severe complications greater than Clavien-Dindo Grade III occurred in 5 out of 14 patients (36%). We compared liver resection patients, who had a treatment option of retransplantation (ReLT), with actual ReLTs (excluding early graft failure or rejection, n = 44). Bearing in mind that late ReLT was carried out at a median of 117 months after first transplantation and a median of MELD of 32 (IQR: 17.5-37); three-year survival following liver resection after LT was similar to late ReLT (50.0% vs. 59.1%; p = 0.733). Compared to ReLT, liver resection after LT is a rare surgical procedure with significantly shorter hospital (mean 25, IQR: 8.75-49; p = 0.034) and ICU stays (mean 2, IQR: 1-8; p < 0.001), acceptable complications and survival rates.