RESUMO
Last twenties, tissue engineering has rapidly advanced to address the shortage of organ donors. Decellularization techniques have been developed to mitigate immune rejection and alloresponse in transplantation. However, a clear definition of effective decellularization remains elusive. This study compares various decellularization protocols using the human fascia lata model. Morphological, structural and cytotoxicity/viability analyses indicated that all the five tested protocols were equivalent and met Crapo's criteria for successful decellularization. Interestingly, only the in vivo immunization test on rats revealed differences. Only one protocol exhibited Human Leucocyte Antigen (HLA) content below 1% residual threshold, the only criterion preventing rat immunization with an absence of rat anti-human IgG switch after one month (N=4 donors for each of the 7 groups, added by negative and positive controls, n=28). By respecting a refined set of criteria, i.e. lack of visible nuclear material, <50ng DNA/mg dry weight of extracellular matrix, and <1% residual HLA content, the potential for adverse host reactions can be drastically reduced. In conclusion, this study emphasizes the importance of considering not only nuclear components but also major histocompatibility complex in decellularization protocols and proposes new guidelines to promote safer clinical development and use of bioengineered scaffolds.
Assuntos
Fascia Lata , Antígenos HLA , Engenharia Tecidual , Humanos , Animais , Engenharia Tecidual/métodos , Antígenos HLA/imunologia , Ratos , Alicerces Teciduais/química , Materiais Biocompatíveis/química , Masculino , Matriz Extracelular Descelularizada/química , Matriz Extracelular/química , Matriz Extracelular/metabolismoRESUMO
Over the past decade, organoids have emerged as a prevalent and promising research tool, mirroring the physiological architecture of the human body. However, as the field advances, the traditional use of animal or tumor-derived extracellular matrix (ECM) as scaffolds has become increasingly inadequate. This shift has led to a focus on developing synthetic scaffolds, particularly hydrogels, that more accurately mimic three-dimensional (3D) tissue structures and dynamics in vitro. The ECM-cell interaction is crucial for organoid growth, necessitating hydrogels that meet organoid-specific requirements through modifiable physical and compositional properties. Advanced composite hydrogels have been engineered to more effectively replicate in vivo conditions, offering a more accurate representation of human organs compared to traditional matrices. This review explores the evolution and current uses of decellularized ECM scaffolds, emphasizing the application of decellularized ECM hydrogels in organoid culture. It also explores the fabrication of composite hydrogels and the prospects for their future use in organoid systems.
Assuntos
Matriz Extracelular Descelularizada , Hidrogéis , Organoides , Engenharia Tecidual , Hidrogéis/química , Humanos , Matriz Extracelular Descelularizada/química , Engenharia Tecidual/métodos , Animais , Alicerces Teciduais/química , Matriz Extracelular/químicaRESUMO
The field of bone tissue engineering (BTE) has witnessed a revolutionary breakthrough with the advent of three-dimensional (3D) bioprinting technology, which is considered an ideal choice for constructing scaffolds for bone regeneration. The key to realizing scaffold biofunctions is the selection and design of an appropriate bioink, and existing bioinks have significant limitations. In this study, a composite bioink based on natural polymers (gelatin and alginate) and liver decellularized extracellular matrix (LdECM) was developed and used to fabricate scaffolds for BTE using 3D bioprinting. Through in vitro studies, the concentration of LdECM incorporated into the bioink was optimized to achieve printability and stability and to improve the proliferation and osteogenic differentiation of loaded rat bone mesenchymal stem cells (rBMSCs). Furthermore, in vivo experiments were conducted using a Sprague Dawley rat model of critical-sized calvarial defects. The proposed rBMSC-laden LdECM-gelatin-alginate scaffold, bioprinted layer-by-layer, was implanted in the rat calvarial defect and the development of new bone growth was studied for four weeks. The findings showed that the proposed bioactive scaffolds facilitated angiogenesis and osteogenesis at the defect site. The findings of this study suggest that the developed rBMSC-laden LdECM-gelatin-alginate bioink has great potential for clinical translation and application in solving bone regeneration problems.
Assuntos
Bioimpressão , Fígado , Células-Tronco Mesenquimais , Osteogênese , Ratos Sprague-Dawley , Engenharia Tecidual , Alicerces Teciduais , Animais , Alicerces Teciduais/química , Engenharia Tecidual/métodos , Bioimpressão/métodos , Ratos , Células-Tronco Mesenquimais/citologia , Osteogênese/fisiologia , Fígado/citologia , Impressão Tridimensional , Matriz Extracelular Descelularizada/química , Regeneração Óssea/fisiologia , Gelatina/química , Diferenciação Celular , Alginatos/química , Proliferação de Células , Matriz Extracelular/química , Osso e Ossos/fisiologia , TintaRESUMO
Cancer stem cells (CSCs) are most likely the main cause of lung cancer formation, metastasis, drug resistance, and genetic heterogeneity. Three-dimensional (3D) ex vivo cell culture models can facilitate stemness improvement and CSC enrichment. Considering the critical role of extracellular matrix (ECM) on CSC properties, the present study developed a thermo-responsive hydrogel using the porcine decellularized lung for 3D cell culture, and the cell-laden hydrogel culturing model was used to explore the CSC characteristics and potential utilization in CSC-specific drug evaluation. Results showed that the lung dECM hydrogel (LEH) was composed of the main ECM components and displayed excellent cellular compatibility. In addition, lung cancer cells 3D cultured in LEH displayed the overexpression of metastasis-related genes and enhanced migration properties, as compared with those in two-dimensional (2D) conditions. Notably, the CSC features, including the expression level of stemness-associated genes, colony formation capability, drug resistance, and the proportion of cancer stem-like cells (CD133+), were also enhanced in 3D cells. Furthermore, the attenuation effect of epigallocatechin gallate (EGCG) on CSC properties in the 3D model was observed, confirming the potential practicability of the 3D culture on CSC-targeted drug screening. Overall, our results suggest that the fabricated LEH is an effective and facile platform for 3D cell culture and CSC-specific drug evaluation.
Assuntos
Técnicas de Cultura de Células em Três Dimensões , Hidrogéis , Neoplasias Pulmonares , Células-Tronco Neoplásicas , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Humanos , Hidrogéis/química , Hidrogéis/farmacologia , Técnicas de Cultura de Células em Três Dimensões/métodos , Animais , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Matriz Extracelular Descelularizada/farmacologia , Matriz Extracelular Descelularizada/química , Pulmão/metabolismo , Pulmão/patologia , Pulmão/citologia , Suínos , Catequina/análogos & derivados , Catequina/farmacologia , Catequina/química , Matriz Extracelular/metabolismo , Matriz Extracelular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Técnicas de Cultura de Células/métodosRESUMO
The transplantation of islet beta cells offers an alternative to heterotopic islet transplantation for treating type 1 diabetes mellitus (T1DM). However, the use of systemic immunosuppressive drugs in islet transplantation poses significant risks to the body. To address this issue, we constructed an encapsulated hybrid scaffold loaded with islet beta cells. This article focuses on the preparation of the encapsulated structure using 3D printing, which incorporates porcine pancreas decellularized extracellular matrix (dECM) to the core scaffold. The improved decellularization method successfully preserved a substantial proportion of protein (such as Collagen I and Laminins) architecture and glycosaminoglycans in the dECM hydrogel, while effectively removing most of the DNA. The inclusion of dECM enhanced the physical and chemical properties of the scaffold, resulting in a porosity of 83.62% ± 1.09% and a tensile stress of 1.85 ± 0.16 MPa. In teams of biological activity, dECM demonstrated enhanced proliferation, differentiation, and expression of transcription factors such as Ki67, PDX1, and NKX6.1, leading to improved insulin secretion function in MIN-6 pancreatic beta cells. In the glucose-stimulated insulin secretion experiment on day 21, the maximum insulin secretion from the encapsulated structure reached 1.96 ± 0.08 mIU ml-1, representing a 44% increase compared to the control group. Furthermore, conventional capsule scaffolds leaverage the compatibility of natural biomaterials with macrophages to mitigate immune rejection. Here, incorporating curcumin into the capsule scaffold significantly reduced the secretion of pro-inflammatory cytokine (IL-1ß, IL-6, TNF-α, IFN-γ) secretion by RAW264.7 macrophages and T cells in T1DM mice. This approach protected pancreatic islet cells against immune cell infiltration mediated by inflammatory factors and prevented insulitis. Overall, the encapsulated scaffold developed in this study shows promise as a natural platform for clinical treatment of T1DM.
Assuntos
Curcumina , Matriz Extracelular Descelularizada , Diabetes Mellitus Tipo 1 , Células Secretoras de Insulina , Alicerces Teciduais , Animais , Diabetes Mellitus Tipo 1/terapia , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/citologia , Alicerces Teciduais/química , Curcumina/farmacologia , Curcumina/química , Camundongos , Matriz Extracelular Descelularizada/química , Matriz Extracelular Descelularizada/farmacologia , Suínos , Transplante das Ilhotas Pancreáticas , Cápsulas/química , Insulina/metabolismo , Diabetes Mellitus Experimental/terapia , Linhagem Celular , Matriz Extracelular/metabolismo , Matriz Extracelular/químicaRESUMO
Tissue engineering presents a promising approach for the treatment of meniscal injuries, yet the development of meniscal scaffolds that exhibit both superior biomechanical properties and biocompatibility remains a considerable challenge. In this study, decellularized skin matrix (DSM) scaffolds were first prepared using porcine skin through decellularization and freeze-drying techniques. The DSM scaffold has favorable porosity, hydrophilicity, and biocompatibility. Importantly, the collagen content and tensile modulus of the scaffold are comparable to those of native meniscus (44.13 ± 2.396 mg/g vs. 42.41 ± 2.40 mg/g and 103.30 ± 2.98 MPa vs. 128.80 ± 9.115 MPa). Subsequently, the peptide PFSSTKT (PFS) with mesenchymal stem cells (MSCs) recruitment capability was used to modify DSM to construct DSM-PFS scaffolds. Compared to the DSM scaffold, the optimized DSM-PFS scaffold enhanced in vitro collagen and glycosaminoglycan (GAG) production and upregulated the expression of cartilage-specific genes. Furthermore, the DSM-PFS scaffold was more effective in recruiting MSCs in vitro. In vivo studies in rabbit models showed that the DSM-PFS scaffold successfully promoted meniscus tissue regeneration. Three months post-implantation, meniscus tissue formation can be observable, and after six months, the neo-meniscus exhibited tissue structure and tensile properties similar to the native meniscus. Notably, the DSM-PFS scaffold exhibited significant chondroprotective effects, slowing osteoarthritis (OA) progression. In conclusion, the DSM-PFS scaffold may represent a promising candidate for future applications in meniscus tissue engineering. STATEMENT OF SIGNIFICANCE: We developed a decellularized skin matrix (DSM) meniscus scaffold using whole-layer porcine skin, demonstrating superior biomechanical strength and biocompatibility. Following modification with the stem cell-recruiting peptide PFS, the optimized DSM-PFS scaffold outperformed the DSM scaffold in cell attraction, collagen and glycosaminoglycan production, and cartilage-specific gene expression. Implanted into rabbit knee joints, the cell-free DSM-PFS scaffold induced meniscal tissue formation within three months, achieving the histological structure and tensile strength of the native meniscus by six months. Moreover, it significantly protected the cartilage. Our findings provide new insights into the fabrication of scaffolds for meniscal tissue engineering, with the DSM-PFS scaffold emerging as an ideal candidate for future applications.
Assuntos
Meniscectomia , Menisco , Regeneração , Alicerces Teciduais , Animais , Coelhos , Alicerces Teciduais/química , Pele/patologia , Matriz Extracelular Descelularizada/química , Suínos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Sistema Livre de Células/química , Engenharia Tecidual/métodosRESUMO
Extracellular matrix sponge plays a positive role in the wound healing process, but requires proper structural strength and biological properties. In order to solve the problem of lyophilized dissolution of placenta-derived sponge, glutaraldehyde was selected for use in the lyophilized crosslinking process to improve the necessary mechanical properties of the placental decellularization matrix sponge. In this work, the effects of three cross-linking methods of glutaraldehyde (Fumigation/Slurry/Soak) on the physical and biological characteristics of lyophilised sponges derived from placental acellular matrix was investigated. The results revealed that the sponges prepared by all three cross-linking methods exhibited excellent blood coagulation ability and stability. The fumigation cross-linked sponges had good mechanical properties of soft and elastic, and safe cytotoxicity, which were more compatible with the requirements of wound dressing. The slurry cross-linking process was uneven due to the stacked matrix materials, resulting in obvious cracks and easy to break when stretching. The soak crosslinking can obtain a higher degree of crosslinking, which leads to the poor antibacterial performance and the harder sponge scaffold with larger elastic modulus and smaller tensile ratio. In general, fumigation cross-linking is more suitable for the preparation of acellular sponge derived from placenta materials which can maintain basic mechanical properties and biological validity.
Assuntos
Reagentes de Ligações Cruzadas , Glutaral , Placenta , Glutaral/química , Placenta/citologia , Feminino , Reagentes de Ligações Cruzadas/química , Gravidez , Animais , Matriz Extracelular Descelularizada/química , Matriz Extracelular Descelularizada/farmacologia , Humanos , Alicerces Teciduais/química , Liofilização/métodos , Coagulação Sanguínea/efeitos dos fármacos , Resistência à Tração , Matriz Extracelular/química , Teste de MateriaisRESUMO
Decellularized extracellular matrix (dECM) hydrogels are engineered constructs that are widely-used in the field of regenerative medicine. However, the development of ECM-based hydrogels for bone tissue engineering requires enhancement in its osteogenic properties. For this purpose, we initially employed bone-derived dECM hydrogel (dECM-Hy) in combination with calcium phosphate cement (CPC) paste to improve the biological and structural properties of the dECM hydrogel. A decellularization protocol for bovine bone was developed to prepare dECM-Hy, and the mechanically-tuned dECM/CPC-Hy was built based on both rheological and mechanical characteristics. The dECM/CPC-Hy displayed a double swelling ratio and compressive strength. An interconnected structure with distinct hydroxyapatite crystals was evident in dECM/CPC-Hy. The expression levels of Alp, Runx2 and Ocn genes were upregulated in dECM/CPC-Hy compared to the dECM-Hy. A 14-day follow-up of the rats receiving subcutaneous implanted dECM-Hy, dECM/CPC-Hy and mesenchymal stem cells (MSCs)-embedded (dECM/CPC/MSCs-Hy) showed no toxicity, inflammatory factor expression or pathological changes. Radiography and computed tomography (CT) of the calvarial defects revealed new bone formation and elevated number of osteoblasts-osteocytes and osteons in dECM/CPC-Hy and dECM/CPC/MSCs-Hy compared to the control groups. These findings indicate that the dECM/CPC-Hy has substantial potential for bone tissue engineering.
Assuntos
Cimentos Ósseos , Regeneração Óssea , Fosfatos de Cálcio , Células-Tronco Mesenquimais , Animais , Fosfatos de Cálcio/química , Regeneração Óssea/efeitos dos fármacos , Bovinos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Ratos , Cimentos Ósseos/química , Cimentos Ósseos/farmacologia , Matriz Extracelular Descelularizada/química , Matriz Extracelular Descelularizada/farmacologia , Hidrogéis/química , Hidrogéis/farmacologia , Osteogênese/efeitos dos fármacos , Ratos Sprague-Dawley , Engenharia Tecidual , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Matriz Extracelular/química , Matriz Extracelular/metabolismoRESUMO
Breast cancer (BC) is the second deadliest cancer after lung cancer. Similar to all cancers, it is also driven by a 3D microenvironment. The extracellular matrix (ECM) is an essential component of the 3D tumor micro-environment, wherein it functions as a scaffold for cells and provides metabolic support. BC is characterized by alterations in the ECM. Various studies have attempted to mimic BC-specific ECMs using artificial materials, such as Matrigel. Nevertheless, research has proven that naturally derived decellularized extracellular matrices (dECMs) are superior in providing the essential in vivo-like cues needed to mimic a cancer-like environment. Developing in vitro 3-D BC models is not straightforward and requires extensive analysis of the data established by researchers. For the benefit of researchers, in this review, we have tried to highlight all developmental studies that have been conducted by various scientists so far. The analysis of the conclusions drawn from these studies is also discussed. The advantages and drawbacks of the decellularization methods employed for generating BC scaffolds will be covered, and the review will shed light on how dECM scaffolds help develop a BC environment. The later stages of the article will also focus on immunogenicity issues arising from decellularization and the origin of the tissue. Finally, this review will also discuss the biofabrication of matrices, which is the core part of the bioengineering process.
Assuntos
Neoplasias da Mama , Matriz Extracelular Descelularizada , Alicerces Teciduais , Humanos , Neoplasias da Mama/patologia , Alicerces Teciduais/química , Matriz Extracelular Descelularizada/química , Feminino , Medicina de Precisão , Antineoplásicos/química , Antineoplásicos/farmacologia , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Animais , Engenharia Tecidual , Avaliação Pré-Clínica de Medicamentos , Microambiente TumoralRESUMO
Due to the limited supply of autologous bone grafts, there is a need to develop more bone matrix materials to repair bone defects. Xenograft bone is expected to be used for clinical treatment due to its exact structural similarity to natural bone and its high biocompatibility. In this study, decellularized antler cancellous bone matrix (DACB) was first prepared, and then the extent of decellularization of DACB was verified by histological staining, which demonstrated that it retained the extracellular matrix (ECM). The bioactivity of DACB was assessed using C3H10T1/2 cells, revealing that DACB enhanced cell proliferation and facilitated cell adhesion and osteogenic differentiation. When evaluated by implanting DACB into nude mice, there were no signs of necrosis or inflammation in the epidermal tissues. The bone repair effect of DACB was verified in vivo using sika deer during the antler growth period as an animal model, and the molecular mechanisms of bone repair were further evaluated by transcriptomic analysis of the regenerated tissues. Our findings suggest that the low immunogenicity of DACB enhances the production of bone extracellular matrix components, leading to effective osseointegration between bone and DACB. This study provides a new reference for solving bone defects.
Assuntos
Chifres de Veado , Osso Esponjoso , Cervos , Camundongos Nus , Osteogênese , Alicerces Teciduais , Animais , Chifres de Veado/química , Alicerces Teciduais/química , Camundongos , Proliferação de Células , Diferenciação Celular , Matriz Extracelular Descelularizada/química , Engenharia Tecidual/métodos , Matriz Extracelular/metabolismo , Regeneração Óssea , Linhagem Celular , Adesão CelularRESUMO
The incidence of liver diseases is high worldwide. Many factors can cause liver fibrosis, which in turn can lead to liver cirrhosis and even liver cancer. Due to the shortage of donor organs, immunosuppression, and other factors, only a few patients are able to undergo liver transplantation. Therefore, how to construct a bioartificial liver that can be transplanted has become a global research hotspot. With the rapid development of three-dimensional (3D) bioprinting in the field of tissue engineering and regenerative medicine, researchers have tried to use various 3D bioprinting technologies to construct bioartificial livers in vitro. In terms of the choice of bioinks, liver decellularized extracellular matrix (dECM) has many advantages over other materials for cell-laden hydrogel in 3D bioprinting. This review mainly summarizes the acquisition of liver dECM and its application in liver 3D bioprinting as a bioink with respect to availability, printability, and biocompatibility in many aspects and puts forward the current challenges and prospects.
Assuntos
Bioimpressão , Matriz Extracelular Descelularizada , Fígado , Impressão Tridimensional , Engenharia Tecidual , Humanos , Bioimpressão/métodos , Fígado/metabolismo , Fígado/citologia , Engenharia Tecidual/métodos , Animais , Matriz Extracelular Descelularizada/química , Matriz Extracelular Descelularizada/metabolismo , Alicerces Teciduais/química , Hidrogéis/química , Matriz Extracelular/metabolismo , Matriz Extracelular/química , Materiais Biocompatíveis/químicaRESUMO
Bone extracellular matrix (ECM) has been shown to mimic aspects of the tissue's complex microenvironment, suggesting its potential role in promoting bone repair. However, current ECM-based therapies suffer from limitations such as inefficient scale-up, lack of mechanical integrity, and sub-optimal efficacy. Here, we fabricated hydrogels from decellularized ECM (dECM) from wild type (WT) and thrombospondin-2 knock-out (TSP2KO) mouse bones. TSP2KO bone ECM hydrogel was found to have distinct mechanical properties and collagen fibril assembly from WT. Furthermore, TSP2KO hydrogel promoted mesenchymal stem cell (MSC) attachment, spreading, and invasion in vitro. Similarly, it promoted formation of tube-like structures by human umbilical vein endothelial cells (HUVECs). When applied to a murine calvarial defect model, TSP2KO hydrogel enhanced repair, in part, due to increased angiogenesis. Our study suggests the pro-angiogenic therapeutic potential of TSP2KO bone ECM hydrogel in bone repair. STATEMENT OF SIGNIFICANCE: The study describes the first successful preparation of a novel hydrogel made from decellularized bones from wild-type mice and mice lacking thrombospondin-2 (TSP2). Hydrogels from TSP2 knock-out (TSP2KO) bones have unique characteristics in structure and biomechanics. These gels interacted well with cells in vitro and helped repair damaged bone in a mouse model. Therefore, TSP2KO bone-derived hydrogel has translational potential for accelerating repair of bone defects that are otherwise difficult to heal. This study not only creates a new material with promise for accelerated healing, but also validates tunability of native biomaterials by genetic engineering.
Assuntos
Matriz Extracelular , Células Endoteliais da Veia Umbilical Humana , Hidrogéis , Trombospondinas , Animais , Humanos , Camundongos , Osso e Ossos/efeitos dos fármacos , Regeneração Óssea/efeitos dos fármacos , Matriz Extracelular Descelularizada/química , Matriz Extracelular Descelularizada/farmacologia , Matriz Extracelular/metabolismo , Matriz Extracelular/química , Células Endoteliais da Veia Umbilical Humana/metabolismo , Hidrogéis/química , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Camundongos Knockout , Neovascularização Fisiológica/efeitos dos fármacos , Trombospondinas/metabolismo , Trombospondinas/genéticaRESUMO
Decellularized extracellular matrix (dECM) hydrogels loaded with adipose-derived stromal cells (ASC) or their conditioned medium (ASC CM) present a promising and versatile treatment approach for tissue vascularization and regeneration. These hydrogels are easy to produce, store, personalize, manipulate, and deliver to the target tissue. This literature review aimed to investigate the applications of dECM hydrogels with ASC or ASC CM for in vivo tissue vascularization. Fourteen experimental studies have been reviewed using vessel density as the primary outcome parameter for in vivo vascularization. The studies consistently reported an increased efficacy in augmenting angiogenesis by the ASC or ASC CM-loaded hydrogels compared to untreated controls. However, this systematic review shows the need to standardize procedures and characterization, particularly of the final administered product(s). The findings from these experimental studies highlight the potential of dECM hydrogel with ASC or ASC CM in novel tissue regeneration and regenerative medicine applications.
Assuntos
Tecido Adiposo , Matriz Extracelular Descelularizada , Hidrogéis , Neovascularização Fisiológica , Células Estromais , Animais , Humanos , Tecido Adiposo/citologia , Meios de Cultivo Condicionados , Matriz Extracelular Descelularizada/química , Hidrogéis/química , Medicina Regenerativa/métodos , Células Estromais/transplante , Células Estromais/citologia , Engenharia Tecidual/métodosRESUMO
Pancreatic bioengineering is a potential therapeutic alternative for type 1 diabetes (T1D) in which the pancreas is decellularized, generating an acellular extracellular matrix (ECM) scaffold, which may be reconstituted by recellularization with several cell types to generate a bioartificial pancreas. No consensus for an ideal pancreatic decellularization protocol exists. Therefore, we aimed to determine the best-suited detergent by comparing sodium dodecyl sulfate (SDS), sodium deoxycholate (SDC), and Triton X-100 at different concentrations. Murine (n=12) and human pancreatic tissue from adult brain-dead donors (n=06) was harvested in accordance with Institutional Ethical Committee of the University of São Paulo Medical School (CEP-FMUSP) and decellularized under different detergent conditions. DNA content, histological analysis, and transmission and scanning electron microscopy were assessed. The most adequate condition for pancreatic decellularization was found to be 4% SDC, displaying: a) effective cell removal; b) maintenance of extracellular matrix architecture; c) proteoglycans, glycosaminoglycans (GAGs), and collagen fibers preservation. This protocol was extrapolated and successfully applied to human pancreas decellularization. The acellular ECM scaffold generated was recelullarized using human pancreatic islets primary clusters. 3D clusters were generated using 0.5×104 cells and then placed on top of acellular pancreatic slices (25 and 50 µm thickness). These clusters tended to connect to the acellular matrix, with visible cells located in the periphery of the clusters interacting with the ECM network of the bioscaffold slices and continued to produce insulin. This study provided evidence on how to improve and accelerate the pancreas decellularization process, while maintaining its architecture and extracellular structure, aiming at pancreatic bioengineering.
Assuntos
Ácido Desoxicólico , Detergentes , Pâncreas , Dodecilsulfato de Sódio , Engenharia Tecidual , Alicerces Teciduais , Animais , Detergentes/química , Detergentes/farmacologia , Humanos , Pâncreas/citologia , Camundongos , Dodecilsulfato de Sódio/farmacologia , Ácido Desoxicólico/farmacologia , Ácido Desoxicólico/química , Alicerces Teciduais/química , Engenharia Tecidual/métodos , Octoxinol/química , Matriz Extracelular , Diabetes Mellitus Tipo 1 , Microscopia Eletrônica de Varredura , Matriz Extracelular Descelularizada/químicaRESUMO
Decellularized scaffolds retain the main bioactive substances of the extracellular matrix, which can better promote cell proliferation and matrix reconstruction at the defect site, and have great potential for morphological and functional restoration in patients with tissue defects. Due to the safety of the material source of allogeneic decellularized scaffolds, there is a great limitation in their clinical application, so the preparation and evaluation of xenodermal acellular scaffolds have attracted much attention. In terms of skin tissue structure and function, porcine skin has a high degree of similarity to human skin and has the advantages of sufficient quantity and no ethical issues. However, there is a risk of immune rejection after xenodermal acellular scaffold transplantation. To address the above problems, this paper focuses on porcine dermal decellularized scaffolds prepared using two common decellularization preparation methods and compares the decellularization efficiency, retention of active components of the extracellular matrix, structural characterization of the decellularized scaffolds, and the effect of porcine dermal decellularized scaffolds on mouse Raw264.7 macrophages, so as to make a functional evaluation of the active components and immune effects of porcine dermal decellularized scaffolds, and to provide a reference for filling trauma-induced defects in humans.
Assuntos
Matriz Extracelular Descelularizada , Alicerces Teciduais , Animais , Camundongos , Suínos , Alicerces Teciduais/química , Células RAW 264.7 , Matriz Extracelular Descelularizada/química , Matriz Extracelular/química , Materiais Biocompatíveis/química , Humanos , Proliferação de Células , Engenharia TecidualRESUMO
This study evaluates the efficacy of a decellularized intestine tissue-derived extracellular matrix (Intestine ECM) as a scaffold for culturing colorectal cancer (CRC) organoids and establishing cell-derived xenograft (CDX) models, comparing its performance to traditional Matrigel. Intestine ECM demonstrates comparable support for organoid formation and cellular function, highlighting its potential as a more physiologically relevant and reproducible platform. Our findings suggest that Intestine ECM enhances the mimetic environment for colon epithelium, supporting comparable growth and improved differentiation compared to Matrigel. Moreover, when used as a delivery carrier, Intestine ECM significantly increases the growth rate of CDX models using patient-derived primary colorectal cancer cells. This enhancement demonstrates Intestine ECM's role not only as a scaffold but also as a vital component of the tumor microenvironment, facilitating more robust tumorigenesis. These findings advocate for the broader application of Intestine ECM in cancer model systems, potentially leading to more accurate preclinical evaluations and the development of targeted cancer therapies.
Assuntos
Neoplasias Colorretais , Organoides , Microambiente Tumoral , Neoplasias Colorretais/patologia , Neoplasias Colorretais/terapia , Animais , Humanos , Camundongos , Matriz Extracelular Descelularizada/química , Alicerces Teciduais/química , Laminina , Matriz Extracelular , Xenoenxertos , Linhagem Celular Tumoral , Mucosa Intestinal/citologia , Combinação de Medicamentos , Proteoglicanas , Colágeno , Ensaios Antitumorais Modelo de Xenoenxerto , Diferenciação CelularRESUMO
Current engineered synthetic scaffolds fail to functionally repair and regenerate ruptured native tendon tissues, partly because they cannot satisfy both the unique biological and biomechanical properties of these tissues. Ideal scaffolds for tendon repair and regeneration need to provide porous topographic structures and biological cues necessary for the efficient infiltration and tenogenic differentiation of embedded stem cells. To obtain crimped and porous scaffolds, highly aligned poly(l-lactide) fibers were prepared by electrospinning followed by postprocessing. Through a mild and controlled hydrogen gas foaming technique, we successfully transformed the crimped fibrous mats into three-dimensional porous scaffolds without sacrificing the crimped microstructure. Porcine derived decellularized tendon matrix was then grafted onto this porous scaffold through fiber surface modification and carbodiimide chemistry. These biofunctionalized, crimped, and porous scaffolds supported the proliferation, migration, and tenogenic induction of tendon derived stem/progenitor cells, while enabling adhesion to native tendons. Together, our data suggest that these biofunctionalized scaffolds can be exploited as promising engineered scaffolds for the treatment of acute tendon rupture.
Assuntos
Materiais Biocompatíveis , Teste de Materiais , Regeneração , Tendões , Alicerces Teciduais , Alicerces Teciduais/química , Tendões/citologia , Animais , Suínos , Porosidade , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Engenharia Tecidual , Proliferação de Células/efeitos dos fármacos , Tamanho da Partícula , Matriz Extracelular Descelularizada/química , Matriz Extracelular Descelularizada/farmacologia , Poliésteres/químicaRESUMO
Limited treatments and a lack of appropriate animal models have spurred the study of scaffolds to mimic lung disease in vitro. Decellularized human lung and its application in extracellular matrix (ECM) hydrogels has advanced the development of these lung ECM models. Controlling the biochemical and mechanical properties of decellularized ECM hydrogels continues to be of interest due to inherent discrepancies of hydrogels when compared to their source tissue. To optimize the physiologic relevance of ECM hydrogel lung models without sacrificing the native composition we engineered a binary fabrication system to produce a Hybridgel composed of an ECM hydrogel reinforced with an ECM cryogel. Further, we compared the effect of ECM-altering disease on the properties of the gels using elastin poor Chronic Obstructive Pulmonary Disease (COPD) vs non-diseased (ND) human lung source tissue. Nanoindentation confirmed the significant loss of elasticity in hydrogels compared to that of ND human lung and further demonstrated the recovery of elastic moduli in ECM cryogels and Hybridgels. These findings were supported by similar observations in diseased tissue and gels. Successful cell encapsulation, distribution, cytotoxicity, and infiltration were observed and characterized via confocal microscopy. Cells were uniformly distributed throughout the Hybridgel and capable of survival for 7 days. Cell-laden ECM hybridgels were found to have elasticity similar to that of ND human lung. Compositional investigation into diseased and ND gels indicated the conservation of disease-specific elastin to collagen ratios. In brief, we have engineered a composited ECM hybridgel for the 3D study of cell-matrix interactions of varying lung disease states that optimizes the application of decellularized lung ECM materials to more closely mimic the human lung while conserving the compositional bioactivity of the native ECM. STATEMENT OF SIGNIFICANCE: The lack of an appropriate disease model for the study of chronic lung diseases continues to severely inhibit the advancement of treatments and preventions of these otherwise fatal illnesses due to the inability to recapture the biocomplexity of pathologic cell-ECM interactions. Engineering biomaterials that utilize decellularized lungs offers an opportunity to deconstruct, understand, and rebuild models that highlight and investigate how disease specific characteristics of the extracellular environment are involved in driving disease progression. We have advanced this space by designing a binary fabrication system for a ECM Hybridgel that retains properties from its source material required to observe native matrix interactions. This design simulates a 3D lung environment that is both mechanically elastic and compositionally relevant when derived from non-diseased tissue and pathologically diminished both mechanically and compositionally when derived from COPD tissue. Here we describe the ECM hybridgel as a model for the study of cell-ECM interactions involved in COPD.
Assuntos
Pulmão , Doença Pulmonar Obstrutiva Crônica , Humanos , Doença Pulmonar Obstrutiva Crônica/patologia , Pulmão/patologia , Matriz Extracelular Descelularizada/química , Matriz Extracelular Descelularizada/farmacologia , Hidrogéis/química , Hidrogéis/farmacologia , Matriz Extracelular/química , Modelos Biológicos , Criogéis/química , AnimaisRESUMO
Hypothyroidism is caused by insufficient stimulation or disruption of the thyroid. However, the drawbacks of thyroid transplantation have led to the search for new treatments. Decellularization allows tissue transplants to maintain their biomimetic structures while preserving cell adhesion, proliferation, and differentiation. This study aimed to decellularize human thyroid tissues using a structure-preserving optimization strategy and present preliminary data on recellularization. Nine methods were used for physical and chemical decellularization. Quantitative and immunohistochemical analyses were performed to investigate the DNA and extracellular matrix components of the tissues. Biomechanical properties were determined by compression test, and cell viability was examined after seeding MDA-T32 papillary thyroid cancer (PTC) cells onto the decellularized tissues. Decellularized tissues exhibited a notable decrease (<50 ng mg-1DNA, except for Groups 2 and 7) compared to the native thyroid tissue. Nonetheless, collagen and glycosaminoglycans were shown to be conserved in all decellularized tissues. Laminin and fibronectin were preserved at comparatively higher levels, and Young's modulus was elevated when decellularization included SDS. It was observed that the strain value in Group 1 (1.63 ± 0.14 MPa) was significantly greater than that in the decellularized tissues between Groups 2-9, ranging from 0.13 ± 0.03-0.72 ± 0.29 MPa. Finally, viability assessment demonstrated that PTC cells within the recellularized tissue groups successfully attached to the 3D scaffolds and sustained metabolic activity throughout the incubation period. We successfully established a decellularization optimization for human thyroid tissues, which has potential applications in tissue engineering and transplantation research. Our next goal is to conduct recellularization using the methods utilized in Group 1 and transplant the primary thyroid follicular cell-seeded tissues into anin vivoanimal model, particularly due to their remarkable 3D structural preservation and cell adhesion-promoting properties.
Assuntos
Sobrevivência Celular , Matriz Extracelular , Glândula Tireoide , Engenharia Tecidual , Alicerces Teciduais , Engenharia Tecidual/métodos , Humanos , Glândula Tireoide/citologia , Matriz Extracelular/metabolismo , Matriz Extracelular/química , Alicerces Teciduais/química , Colágeno/química , Adesão Celular , Glicosaminoglicanos/metabolismo , Glicosaminoglicanos/química , Linhagem Celular Tumoral , DNA , Módulo de Elasticidade , Proliferação de Células , Neoplasias da Glândula Tireoide/patologia , Matriz Extracelular Descelularizada/química , Laminina/química , Fenômenos Biomecânicos , Diferenciação Celular , Câncer Papilífero da Tireoide/patologia , Fibronectinas/química , Fibronectinas/metabolismoRESUMO
The use of decellularized extracellular matrix products in tissue regeneration is quite alluring yet practically challenging due to the limitations of its availability, harsh processing techniques, and host rejection. Scaffolds obtained by either incorporating extracellular matrix (ECM) material or coating the surface can resolve these challenges to some extent. However, these scaffolds lack the complex 3D network formed by proteins and growth factors observed in natural ECM. This study introduces an approach utilizing 3D nanofiber scaffolds decorated with dECM to enhance cellular responses and promote tissue regeneration. Notably, the dECM can be customized according to specific cellular requirements, offering a tailored environment for enhanced therapeutic outcomes. Two types of 3D expanded scaffolds, namely radially aligned scaffolds (RAS) and laterally expanded scaffolds (LES) fabricated by the gas-foaming expansion were utilized. To demonstrate the proof-of-concept, human dermal fibroblasts (HDFs) seeded on these scaffolds for up to 8 weeks, resulted in uniform and highly aligned cells which deposited ECM on the scaffolds. These cellular components were then removed from the scaffolds through decellularization (e.g., SDS treatment and freeze-thaw cycles). The dECM-decorated 3D expanded nanofiber scaffolds can direct and support cell alignment and proliferation along the underlying fibers upon recellularization. An in vitro inflammation assay indicates that dECM-decorated LES induces a lower immune response than dECM-decorated RAS. Further, subcutaneous implantation of dECM-decorated RAS and LES shows higher cell infiltration and angiogenesis within 7 and 14 days than RAS and LES without dECM decoration. Taken together, dECM-decorated 3D expanded nanofiber scaffolds hold great potential in tissue regeneration and tissue modeling. STATEMENT OF SIGNIFICANCE: Decellularized ECM scaffolds have attained widespread attention in biomedical applications due to their intricate 3D framework of proteins and growth factors. Mimicking such a complicated architecture is a clinical challenge. In this study, we developed natural ECM-decorated 3D electrospun nanofiber scaffolds with controlled alignments to mimic human tissue. Fibroblasts were cultured on these scaffolds for 8 weeks to deposit natural ECM and decellularized by either freeze-thawing or detergent to obtain decellularized ECM scaffolds. These scaffolds were tested in both in-vitro and in-vivo conditions. They displayed higher cellular attributes with lower immune response making them a good grafting tool in tissue regeneration.