Decellularized Extracellular Matrix Research Articles

Deer antler stem cell niche: An interesting perspective

In recent years, there has been considerable exploration into methods aimed at enhancing the regenerative capacity of transplanted and/or tissue-resident cells. Biomaterials, in particular, have garnered significant interest for their potential to serve as natural scaffolds for cells. In this editorial, we provide commentary on the study by Wang et al , in a recently published issue of World J Stem Cells , which investigates the use of a decellularized xenogeneic extracellular matrix (ECM) derived from antler stem cells for repairing osteochondral defects in rat knee joints. Our focus lies specifically on the crucial role of biological scaffolds as a strategy for augmenting stem cell potential and regenerative capabilities, thanks to the establishment of a favorable microenvironment (niche). Stem cell differentiation heavily depends on exposure to intrinsic properties of the ECM, including its chemical and protein composition, as well as the mechanical forces it can generate. Collectively, these physicochemical cues contribute to a bio-instructive signaling environment that offers tissue-specific guidance for achieving effective repair and regeneration. The interest in mechanobiology, often conceptualized as a form of “structural memory”, is steadily gaining more validation and momentum, especially in light of findings such as these.

Decellularized extracellular matrix enriched with GDNF enhances neurogenesis and remyelination for improved motor recovery after spinal cord injury

Motor functional improvement represents a paramount treatment objective in the post-spinal cord injury (SCI) recovery process. However, neuronal cell death and axonal degeneration following SCI disrupt neural signaling, impeding the motor functional recovery. In this study, we developed a multifunctional decellularized spinal cord-derived extracellular matrix (dSECM), crosslinked with glial cell-derived neurotrophic factor (GDNF), to promote differentiation of stem cells into neural-like cells and facilitate axonogenesis and remyelination. After decellularization, the immunogenic cellular components were effectively removed in dSECM, while the crucial protein components were retained which supports stem cells proliferation and differentiation. Furthermore, sustained release of GDNF from the dSECM facilitated axonogenesis and remyelination by activating the PI3K/Akt and MEK/Erk pathways. Our findings demonstrate that the dSECM-GDNF platform promotes neurogenesis, axonogenesis, and remyelination to enhance neural signaling, thereby yielding promising therapeutic effects for motor functional improvement after SCI. Statement of significanceThe dSECM promotes the proliferation and differentiation of MSCs or NSCs by retaining proteins associated with positive regulation of neurogenesis and neuronal differentiation, while eliminating proteins related to negative regulation of neurogenesis.After crosslinking, GDNF can be gradually released from the platform, thereby promoting neural differentiation, axonogenesis, and remyelination to enhance neural signaling through activation of the PI3K/Akt and MEK/Erk pathways.In vivo experiments demonstrated that dSECM-GDNF/MSC@GelMA hydrogel exhibited the ability to facilitate neuronal regeneration at 4 weeks post-surgery, while promoting axonogenesis and remyelination at 8 weeks post-surgery, ultimately leading to enhanced motor functional recovery.This study elucidates the ability of neural regeneration strategy to promote motor functional recovery and provides a promising approach for designing multifunctional tissue for SCI treatment.

Emerging Strategies for Optimizing Cell‐Derived Decellularized Extracellular Matrix Scaffolds in Tissue Engineering

As an ideal biological scaffold, cell‐derived decellularized extracellular matrix (dECM) attracts increasing attention and is prepared and studied for tissue engineering. However, a few barriers prevent its extensive clinical usage, indicating that it requires further optimization. This review starts with a brief introduction to the benefits and limitations of dECM as a biomaterial. Then, it provides a novel perspective summarizing and describing methods for dECM optimization, delineating strategies to optimize regular cell‐derived dECM sheet preparation and additional biochemical or mechanophysical procedures to further modify the scaffold. These innovative approaches equip engineered biomaterials based on cell‐derived dECMs with key properties and functionalities, thus providing a great opportunity for their broader future therapeutic applications. Finally, several ideas and directions for future development are also prospected.

Magnetic biocomposite scaffold based on decellularized tendon ECM and MNP-deposited halloysite nanotubes: physicochemical, thermal, rheological, mechanical and in vitro biological evaluations

The development of new three-dimensional biomaterials with advanced versatile properties is critical to the success of tissue engineering (TE) applications. Here, (a) bioactive decellularized tendon extracellular matrix (dECM) with a sol-gel transition feature at physiological temperature, (b) halloysite nanotubes (HNT) with known mechanical properties and bioactivity, and (c) magnetic nanoparticles (MNP) with superparamagnetic and osteogenic properties were combined to develop a new scaffold that could be used in prospective bone TE applications. Deposition of MNPs on HNTs resulted in magnetic nanostructures without agglomeration of MNPs. A completely cell-free, collagen- and glycosaminoglycan- rich dECM was obtained and characterized. dECM-based scaffolds incorporated with 1%, 2% and 4% MNP-HNT were analysed for their physical, chemical, and in vitro biological properties. Fourier-transform infrared spectroscopy, x-ray powder diffractometry and vibrating sample magnetometry analyses confirmed the presence of dECM, HNT and MNP in all scaffold types. The capacity to form apatite layer upon incubation in simulated body fluid revealed that dECM-MNP-HNT is a bioactive material. Combining dECM with MNP-HNT improved the thermal stability and compressive strength of the macroporous scaffolds upto 2% MNP-HNT. In vitro cytotoxicity and hemolysis experiments showed that the scaffolds were essentially biocompatible. Human bone marrow mesenchymal stem cells adhered and proliferated well on the macroporous constructs containing 1% and 2% MNP-HNT; and remained metabolically active for at least 21 d in vitro. Collectively, the findings support the idea that magnetic nanocomposite dECM scaffolds containing MNP-HNT could be a potential template for TE applications.

The extracellular matrix differentially directs myoblast motility and differentiation in distinct forms of muscular dystrophy: Dystrophic matrices alter myoblast motility

Extracellular matrix (ECM) pathologic remodeling underlies many disorders, including muscular dystrophy. Tissue decellularization removes cellular components while leaving behind ECM components. We generated “on-slide” decellularized tissue slices from genetically distinct dystrophic mouse models. The ECM of dystrophin- and sarcoglycan-deficient muscles had marked thrombospondin 4 deposition, while dysferlin-deficient muscle had excess decorin. Annexins A2 and A6 were present on all dystrophic decellularized ECMs, but annexin matrix deposition was excessive in dysferlin-deficient muscular dystrophy. Muscle-directed viral expression of annexin A6 resulted in annexin A6 in the ECM. C2C12 myoblasts seeded onto decellularized matrices displayed differential myoblast mobility and fusion. Dystrophin-deficient decellularized matrices inhibited myoblast mobility, while dysferlin-deficient decellularized matrices enhanced myoblast movement and differentiation. Myoblasts treated with recombinant annexin A6 increased mobility and fusion like that seen on dysferlin-deficient decellularized matrix and demonstrated upregulation of ECM and muscle cell differentiation genes. These findings demonstrate specific fibrotic signatures elicit effects on myoblast activity.

Novel muscle-derived extracellular matrix hydrogel promotes angiogenesis and neurogenesis in volumetric muscle loss

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.

Development and Characterization of Decellularized Lung Extracellular Matrix Hydrogels.

The use of extracellular matrix (ECM)-derived hydrogels in tissue engineering has become increasingly popular, as they can mimic cells' natural environment in vitro. However, maintaining the native biochemical content of the ECM, achieving mechanical stability, and comprehending the impact of the decellularization process on the mechanical properties of the ECM hydrogels are challenging. Here, a pipeline for decellularization of bovine lung tissue using two different protocols, downstream characterization of the effectiveness of decellularization, fabrication of reconstituted decellularized lung ECM hydrogels and assessment of their mechanical and cytocompatibility properties were described. Decellularization of the bovine lung was pursued using a physical (freeze-thaw cycles) or chemical (detergent-based) method. Hematoxylin and Eosin staining was performed to validate the decellularization and retention of major ECM components. For the evaluation of residual collagen and sulfated glycosaminoglycan (sGAG) content within the decellularized samples, Sirius red and Alcian blue staining techniques were employed, respectively. Mechanical properties of the decellularized lung ECM hydrogels were characterized by oscillatory rheology. The results suggest that decellularized bovine lung hydrogels can provide a reliable organotypicalternative to commercial ECM products by retaining most native ECM components. Furthermore, these findings reveal that the decellularization method of choice significantly affects gelation kinetics as well as the stiffness and viscoelastic properties of resulting hydrogels.

Hierarchically patterned triple-layered gelatin-based electrospun membrane functionalized by cell-specific extracellular matrix for periodontal regeneration

ObjectivesRegenerating the periodontium poses a critical challenge in oral medicine. To repair various periodontal defects, it is necessary to adopt a bio-scaffold that provides both the architecture and bioactive cues for local stem cells to migrate, reside, proliferate, and differentiate. The objective of this study is to combine a cell-specific decellularized extracellular matrix (ECM) and a biomimetic electrospinning scaffold to regenerate severely destructed periodontium. MethodsSEM, water contact angle (WCA), live/dead staining, swelling ratio, tensile test and immune-fluorescent staining were used to define the suitable topography for certain dental stem cells seeding and culturing. Transwell assay, CCK-8, Alizarin Red staining and PCR immune-fluorescent staining were used to determine ideal cell-specific ECM for PDLSCs/BMSCs migration, viability, and oriented differentiation. A biodegradable triple-layered electrospun scaffold (TLS) was fabricated by electrospinning with aligned fibers on both surfaces and a polyporous structure in the middle. The morphology and inter-porous structure of the TLS were characterized by SEM and mercury intrusion porosimetry (MIP). The surface of the TLS was functionalized with cell-specific ECM (Bi-ECM-TLS) through decellularization of the cell sheets cultured on the scaffold. The regenerative outcome of Bi-ECM-TLS was assessed by an in-situ rat periodontal defect model. Micro-CT, HE-staining, Masson’s trichome staining, Sirius Red staining and Immunofluorescent staining were used for histological analysis. ResultsAligned Gelatin/PCL fibrous membrane (GPA) was most effective for both PDLSCs and BMSCs in culture with WCA around 50 degrees and better mechanical strength than the rest. MSCs favored the same type of ECM (cell-specific ECM), and their regenerative properties were effectively induced with better chemotaxis, proliferative and differentiating behaviors. TLS characterization showed that TLS possessed aligned-random-aligned structure and inter-porous structure. In a rat model of periodontal defects, the TLS functionalized by BMSC-specific ECM for bone regeneration and PDLSC-specific ECM demonstrated highest BV/TV ratio, best bone structure and ligament fiber orientation and blood vessel formation, suggesting optimal performance in regenerating both alveolar bone and periodontal ligaments over TLS, single-ECM loaded TLS and r-Bi-ECM-TLS. SignificanceThis study highlights the importance of combining a cell-specific decellularized ECM and a biomimetic electrospinning scaffold for targeted periodontal tissue regeneration, with potential implications for periodontal tissue engineering and improved patient outcomes.

Human cornea-derived extracellular matrix hydrogel for prevention of post-traumatic corneal scarring: A translational approach.

Corneal scarring and opacification are a significant cause of blindness affecting millions worldwide. The current standard of care for corneal blindness is corneal transplantation, which suffers from several drawbacks. One alternative approach that has shown promise is the use of xenogeneic corneal extracellular matrix (ECM), but its clinical applicability is challenging due to safety concerns. This study reports the innovative use of human cornea-derived ECM to prevent post-traumatic corneal scarring. About 30 - 40% of corneas donated to the eye banks do not meet the standards defined for clinical use and are generally discarded, although they are completely screened for their safety. In this study, human cornea-derived decellularized ECM hydrogel was prepared from the non-transplantation grade human cadaveric corneas obtained from an accredited eye-bank. The prepared hydrogel was screened for its efficacy against corneal opacification following an injury in an animal model. Our in vivo study revealed that, the control collagen-treated group developed corneal opacification, while the prophylactic application of human cornea-derived hydrogel effectively prevented corneal scarring and opacification. The human hydrogel-treated corneas were indistinguishable from healthy corneas and comparable to those treated with the xenogeneic bovine corneal hydrogel. We also demonstrated that the application of the hydrogel retained the biological milieu including cell behavior, protein components, optical properties, curvature, and nerve regeneration by remodeling the corneal wound after injury. The hydrogel application is also sutureless, resulting in faster corneal healing. We envision that this human cornea-derived ECM-based hydrogel has potential clinical application in preventing scarring from corneal wounding. STATEMENT OF SIGNIFICANCE: There are significant challenges surrounding corneal regeneration after injury due to extensive scarring. Although there is substantial research on corneal regeneration, much of it uses synthetic materials with chemical cross-linking methods or xenogeneic tissue-based material devices which have to undergo exhaustive safety analysis before clinical trials. Herein, we demonstrate the potential application of a human corneal extracellular matrix hydrogel without any additional materials for scarless corneal tissue regeneration, and a method to reduce the wasting of donated allogenic corneal tissue from eye banks. We found no difference in efficacy between the usage of human tissues compared to xenogeneic sources. This may help ease clinical translation and can be used topically without sutures as an outpatient procedure.

An application of decellularized membrane as guided tissue regeneration

Decellularized Extracellular Matrix (DECM) has been established as a biomaterial that retains the natural properties of a tissue, promotes cell proliferation and cell differentiation. Periodontal regeneration requires the neovascularization niches and the proliferation and differentiation of the involved cells. DECM have various advantages and qualities in terms of stimulating periodontal tissue regeneration. Several methods for improving mechanical strength of the scaffolds have been identified like, crosslinking which is to enhance regenerative potential. This review focuses on the ability of DECM to repair damaged tissue in periodontal tissue engineering and addresses the future direction of periodontal regeneration in particular area.

Improvement of Myocardial Structure after Developed Fibrous Degeneration Under the Use of Allogenic Biomaterial

Introduction. The question of the possibility of recovery of postischemic myocardium remains relevant.Aim. The aim of the study was to study the effect of dispersed decellularized allogeneic extracellular matrix (allogeneic biomaterial, DAB) on the developed fibrous degeneration of the myocardium, as well as to reveal the possible mechanisms of cellular regeneration.Materials and methods. The muscular wall of the heart of rats was subjected to cryodestruction. After 45 days, the rats of the main group were intramyocardially injected with a suspension of allogeneic biomaterialinto the area of the affected myocardium, and the rats of the control group were injected with saline.Results and discussions. In the experimental group, there was a regression of the formed fibrous connective tissue, chemoattraction of progenitor cells, their differentiation and integration into the myocardium. The thickness of the muscular part of the wall of the left ventricle was three orders of magnitude higher than in the control group.Conclusion. Analysis of the results of the study indicates that the heart in adult mammals has a powerful regenerative reserve. It is likely that, based on the use of DAB, a protocol can be developed that allows the restoration of the heart muscle even in conditions of already developed fibrous degeneration.

Distinguished biomimetic dECM system facilitates early detection of metastatic breast cancer cells.

Breast cancer is the most prevalent malignant tumor affecting women's health. Bone is the most common distant metastatic organ, worsening the quality of life and increasing the mortality of patients. Early detection of breast cancer bone metastasis is urgent for halting disease progression and improving tumor prognosis. Recently, extracellular matrix (ECM) with biomimetic tissue niches opened a new avenue for tumor models in vitro. Here, we developed a biomimetic decellularized ECM (dECM) system to recapitulate bone niches at different situations, bone mimetic dECM from osteoblasts (BM-ECM) and bone tumor mimetic dECM from osteosarcoma cells (OS-ECM). The two kinds of dECMs exhibited distinct morphology, protein composition, and distribution. Interestingly, highly metastatic breast cancer cells tended to adhere and migrate on BM-ECM, while lowly metastatic breast cancer cells preferred the OS-ECM niche. Epithelial-to-mesenchymal transition was a potential mechanism to initiate the breast cancer cell migration on different biomimetic dECMs. Importantly, in the nude mice model, the dECM system captured metastatic breast cancer cells as early as 10 days after orthotopic transplantation in mammary gland pads, with higher signal on BM-ECM than that on OS-ECM. Collectively, the biomimetic dECM system might be a promising tumor model to distinguish the metastatic ability of breast cancer cells in vitro and to facilitate early detection of metastatic breast cancer cells in vivo, contributing to the diagnosis of breast cancer bone metastasis.

Growth factors and mechano-regulated reciprocal crosstalk with extracellular matrix tune the keratocyte–fibroblast/myofibroblast transition

Improper healing of the cornea after injury, infections or surgery can lead to corneal scar formation, which is associated with the transition of resident corneal keratocytes into activated fibroblasts and myofibroblasts (K–F/M). Myofibroblasts can create an extracellular matrix (ECM) niche in which fibrosis is promoted and perpetuated, resulting in progressive tissue opacification and vision loss. As a reversion back to quiescent keratocytes is essential to restore corneal transparency after injury, we characterized how growth factors with demonstrated profibrotic effects (PDGF, FGF, FBS, TGFβ1) induce the K–F/M transition, and whether their withdrawal can revert it. Indeed, the upregulated expression of αSMA and the associated changes in cytoskeletal architecture correlated with increases in cell contractility, fibronectin (Fn) and collagen matrix density and Fn fiber strain, as revealed by 2D cell culture, nanopillar cellular force mapping and a FRET-labeled Fn tension probe. Substrate mechanosensing drove a more complete K–F/M transition reversal following growth factor withdrawal on nanopillar arrays than on planar glass substrates. Using decellularized ECM scaffolds, we demonstrated that the K–F/M transition was inhibited in keratocytes reseeded onto myofibroblast-assembled, and/or collagen-1-rich ECM. This supports the presence of a myofibroblast-derived ECM niche that contains cues favoring tissue homeostasis rather than fibrosis.

Chondrogenic Differentiation of Adipose-Derived Stromal Cells Induced by Decellularized Cartilage Matrix/Silk Fibroin Secondary Crosslinking Hydrogel Scaffolds with a Three-Dimensional Microstructure.

Finding an ideal scaffold is always an important issue in the field of cartilage tissue engineering. Both decellularized extracellular matrix and silk fibroin have been used as natural biomaterials for tissue regeneration. In this study, a secondary crosslinking method of γ irradiation and ethanol induction was used to prepare decellularized cartilage extracellular matrix and silk fibroin (dECM-SF) hydrogels with biological activity. Furthermore, the dECM-SF hydrogels were cast in custom-designed molds to produce a three-dimensional multi-channeled structure to improve internal connectivity. The adipose-derived stromal cells (ADSC) were seeded on the scaffolds, cultured in vitro for 2 weeks, and implanted in vivo for another 4 and 12 weeks. The double crosslinked dECM-SF hydrogels exhibited an excellent pore structure after lyophilization. The multi-channeled hydrogel scaffold presents higher water absorption ability, surface wettability, and no cytotoxicity. The addition of dECM and a channeled structure could promote chondrogenic differentiation of ADSC and engineered cartilage formation, confirmed by H&E, safranin O staining, type II collagen immunostaining, and qPCR assay. In conclusion, the hydrogel scaffold fabricated by the secondary crosslinking method has good plasticity and can be used as a scaffold for cartilage tissue engineering. The multi-channeled dECM-SF hydrogel scaffolds possess a chondrogenic induction activity that promotes engineered cartilage regeneration of ADSC in vivo.

Injectable decellularzied extracellular matrix hydrogel derived from human umbilical cord: A novel perspective to deal with refractory wound via medical wastes

Refractory wounds are frequent and protracted diabetes or postoperative complications that greatly affect the quality of life and mental health of patients. As a result of repeated dressing replacements and local debridement of the existing therapeutic therapies, freshly formed granulation is once more infiltrated by inflammation and eliminated, which delays the healing process. Herein, the improved Fryetes' method, which retained most of the active factors in the extracellular matrix, was developed to prepare injectable decellularized extracellular matrix hydrogels (dECMH) from human umbilical cord tissue. The physical, biochemical, in vivo and in vitro biological properties were analysed in detail. The results showed that the pre-gel exhibited good injectability and could form the hydrogel within 30 min. Biochemical analysis indicated that the prepared decellularized extracellular matrix retained abundant structural proteins and wound healing-related growth factors. In vitro and in vivo experiments confirmed the excellent biocompatibility of dECMH and the extremely high migration and proliferation capacities of human umbilical vein endothelial cells in the hydrogels. Thus, the injectability, gel-forming ability, and excellent biological characteristics of the hydrogel are highly compatible with refractory wounds with various irregular shapes, showing great potential for clinical application in wound repair.

Construction and properties detection of 3D micro-structure scaffolds base on decellularized sheep kidney before and after crosslinking.

Decellularized extracellular matrix is one form of natural material in tissue engineering. The process of dECM retains the tissue microstructure, provides good cell adhesion sites, maintains most of biological signals that promotes the survival and differentiation ability of cells. In this study, sheep kidney was decellularized followed by histochemical staining, elemental analysis and scanning electron microscopy characterizations. The dECM scaffold was prepared with different sequences of freeze drying technology, crosslinking and the water absorption, porosity, mechanical strength with subsequent thermogravimetric analysis, Infrared spectroscopy and biocompatibility tests. Our results indicated that these decellularized treatments of sheep kidney can effectively remove DNA and retain uniform pore size distribution. After crosslinking the scaffold's water absorption decreased from 987.56 ± 40.21% to 934.39 ± 39.61%, the porosity decreased from 89.64 ± 3.2% to 85.09 ± 17.63%, and the compression modulus increased from 304.32 ± 25.43kPa to 459.53 ± 38.92kPa, with thermal process the percentage of weight loss decreased from 66.57% to 44.731%, in addition, the composition didn't change significantly, crosslinking could also promote the stability. In terms of biocompatibility, the number of viable cells increased significantly with the days. In conclusion, the crosslinked decellularized sheep kidney extracellular matrix scaffold reduced water absorption and porosity slightly, but has a significant increase in mechanical properties, and presented excellent biocompatibility which are beneficial to cell adhesion, growth and differentiation.

A cervical cancer tissue-derived decellularized extracellular matrix scaffold for cervical cancer tissue reconstruction in vitro

The prepare decellularized extracellular matrix (ECM) scaffold materials derived from human cervical carcinoma tissues for 3D culture of cervical carcinoma cells. Fresh human cervical carcinoma tissues were treated with sodium lauryl ether sulfate (SLES) solution to prepare decellularized ECM scaffolds. The scaffolds were examined for ECM microstructure and residual contents of key ECM components (collagen, glycosaminoglycan, and elastin) and genetic materials by pathological staining and biochemical content analysis. In vitro 3D culture models were established by injecting cultured cervical cancer cells into the prepared ECM scaffolds. The cells in the recellularized scaffolds were compared with those in a conventional 2D culture system for cell behaviors including migration, proliferation and epithelial-mesenchymal transition (EMT) wsing HE staining, immunohistochemical staining and molecular biological technology analysis. Resistance to 5-fluorouracil (5-Fu) of the cells in the two culture systems was tested by analyzing the cell apoptosis rates via flow cytometry. SLES treatment effectively removed cells and genetic materials from human cervical carcinoma tissues but well preserved the microenvironment structure and biological activity of ECM. Compared with the 2D culture system, the 3D culture models significantly promoted proliferation, migration, EMT and 5-Fu resistance of human cervical cancer cells. The decellularized ECM scaffolds prepared using human cervical carcinoma tissues provide the basis for construction of in vitro 3D culture models for human cervical cancer cells.

Different Decellularization Methods in Bovine Lung Tissue Reveals Distinct Biochemical Composition, Stiffness, and Viscoelasticity in Reconstituted Hydrogels.

Extracellular matrix (ECM)-derived hydrogels are in demand for use in lung tissue engineering to mimic the native microenvironment of cells in vitro. Decellularization of native tissues has been pursued for preserving organotypic ECM while eliminating cellular content and reconstitution into scaffolds which allows re-cellularization for modeling homeostasis, regeneration, or diseases. Achieving mechanical stability and understanding the effects of the decellularization process on mechanical parameters of the reconstituted ECM hydrogels present a challenge in the field. Stiffness and viscoelasticity are important characteristics of tissue mechanics that regulate crucial cellular processes and their in vitro representation in engineered models is a current aspiration. The effect of decellularization on viscoelastic properties of resulting ECM hydrogels has not yet been addressed. The aim of this study was to establish bovine lung tissue decellularization for the first time via pursuing four different protocols and characterization of reconstituted decellularized lung ECM hydrogels for biochemical and mechanical properties. Our data reveal that bovine lungs provide a reproducible alternative to human lungs for disease modeling with optimal retention of ECM components upon decellularization. We demonstrate that the decellularization method significantly affects ECM content, stiffness, and viscoelastic properties of resulting hydrogels. Lastly, we examined the impact of these aspects on viability, morphology, and growth of lung cancer cells, healthy bronchial epithelial cells, and patient-derived lung organoids.

Decellularized ECM hydrogels: prior use considerations, applications, and opportunities in tissue engineering and biofabrication.

Tissue development, wound healing, pathogenesis, regeneration, and homeostasis rely upon coordinated and dynamic spatial and temporal remodeling of extracellular matrix (ECM) molecules. ECM reorganization and normal physiological tissue function, require the establishment and maintenance of biological, chemical, and mechanical feedback mechanisms directed by cell-matrix interactions. To replicate the physical and biological environment provided by the ECM in vivo, methods have been developed to decellularize and solubilize tissues which yield organ and tissue-specific bioactive hydrogels. While these biomaterials retain several important traits of the native ECM, the decellularizing process, and subsequent sterilization, and solubilization result in fragmented, cleaved, or partially denatured macromolecules. The final product has decreased viscosity, moduli, and yield strength, when compared to the source tissue, limiting the compatibility of isolated decellularized ECM (dECM) hydrogels with fabrication methods such as extrusion bioprinting. This review describes the physical and bioactive characteristics of dECM hydrogels and their role as biomaterials for biofabrication. In this work, critical variables when selecting the appropriate tissue source and extraction methods are identified. Common manual and automated fabrication techniques compatible with dECM hydrogels are described and compared. Fabrication and post-manufacturing challenges presented by the dECM hydrogels decreased mechanical and structural stability are discussed as well as circumvention strategies. We further highlight and provide examples of the use of dECM hydrogels in tissue engineering and their role in fabricating complex in vitro 3D microenvironments.

Towards a standardized multi-tissue decellularization protocol for the derivation of extracellular matrix materials.

The goal of tissue decellularization is to efficiently remove unwanted cellular components, such as DNA and cellular debris, while retaining the complex structural and molecular milieu within the extracellular matrix (ECM). Decellularization protocols to date are centered on customized tissue-specific and lab-specific protocols that involve consecutive manual steps which results in variable and protocol-specific ECM material. The differences that result from the inconsistent protocols between decellularized ECMs affect consistency across batches, limit comparisons between results obtained from different laboratories, and could limit the transferability of the material for consistent laboratory or clinical use. The present study is the first proof-of-concept towards the development of a standardized protocol that can be used to derive multiple ECM biomaterials (powders and hydrogels) via a previously established automated system. The automated decellularization method developed by our group was used due to its short decellularization time (4 hours) and its ability to reduce batch-to-batch variability. The ECM obtained using this first iteration of a unified protocol was able to produce ECM hydrogels from skin, lung, muscle, tendons, cartilage, and laryngeal tissues. All hydrogels formed in this study were cytocompatible and showed gelation and rheological properties consistent with previous ECM hydrogels. The ECMs also showed unique proteomic composition. The present study represents the first step towards developing standardized protocols that can be used on multiple tissues in a fast, scalable, and reproducible manner.