Decellularization Protocol Research Articles

Decellularized Extracellular Matrix for Modeling Cardiac Extracellular Microenvironment.

The extracellular matrix (ECM) forms most of the tissue microenvironment and is in a constant and dynamic equilibrium with cells. The decellularization process employs physical or chemical methods, or a combination of them, to remove the cellular components of tissues and organs while preserving the architecture and composition of the ECM. Depending on the methodology used, the decellularized ECM (dECM) is then suitable for research or clinical applications. Here, we describe an optimized protocol for the efficient decellularization of the human myocardium to generate 3D scaffolds of well-preserved cardiac extracellular matrix that can be used for in vitro or in vivo studies.

Decellularization of Pig Lung to Yield Three-Dimensional Scaffold for Lung Tissue Engineering.

Lately, the need for three-dimensional (3D) cell culture has been recognized in order to closely mimic the organization of native tissues. Thus, 3D scaffolds started to be employed to facilitate the 3D cell organization and enable the artificial tissue formation for the emerging tissue engineering applications. 3D scaffolds can be prepared by various techniques, each with certain advantages and disadvantages. Decellularization is an easy method based on removal of cells from native tissue sample, yielding extracellular matrix (ECM) scaffold with preserved architecture and bioactivity. This chapter provides a detailed protocol for decellularization of pig lung and also some basic assays for evaluation of its effectivity, such as determination of DNA content and histological verification of the selected ECM components. Such decellularized scaffold can subsequently be used for various tissue engineering applications, for example, for recellularization with cells of interest, for natural ECM hydrogel preparation, or as a bioink for 3D bioprinting.

Decellularisation and Characterisation of Porcine Pleura as Bioscaffolds in Tissue Engineering

Persistent air leaks caused by thoracic surgery, physical trauma, or spontaneous pneumothoraces are a cause of patient morbidity with need for extended chest tube durations and surgical interventions. Current treatment measures involve mechanical closure of air leaks in the compromised pleura. Organ and membrane decellularisation offers a broad range of biomimetic scaffolds of allogeneic and xenogeneic origins, exhibiting innate tissue‐specific characteristics. We explored a physicochemical method for decellularising porcine pleural membranes (PPM) as potential tissue‐engineered surrogates for lung tissue repair. Decellularised PPM (dPPM) was characterised with histology, quantitative assays, mechanical testing, and sterility evaluation. Cytotoxicity and recellularisation assays assessed biocompatibility of decellularised PPM (dPPM). Haematoxylin and Eosin (H&E) staining showed an evident reduction in stained nuclei in the dPPM, confirmed with nuclear staining and analysis ( ∗∗∗∗p < 0.0001). Sulphated glycosaminoglycans (sGAG) and collagen histology demonstrated minimal disruption to the gross structural assembly of core extracellular matrix (ECM) in dPPM. Confocal imaging demonstrated realignment of ECM fibres in dPPM against native control. Quantitative analysis defined a significant change in the angular distribution ( ∗∗∗∗p < 0.0001) and coherence ( ∗∗∗p < 0.001) of fibre orientations in dPPM versus native ECM. DNA quantification indicated ≥85% reduction in native nuclear dsDNA in dPPM ( ∗∗p < 0.01). Collagen and sGAG quantification indicated reductions of both ( ∗∗p < 0.01). dPPM displayed increased membrane thickness ( ∗∗∗p < 0.001). However, Young’s modulus (459.67 ± 10.36 kPa) and ultimate tensile strength (4036.22 ± 155.1 kPa) of dPPM were comparable with those of native controls at (465.82 ± 10.51 kPa) and (3912.9 ± 247.42 kPa), respectively. In vitro cytotoxicity and scaffold biocompatibility assays demonstrated robust human mesothelial cell line (MeT‐5A) attachment and viability. DNA quantification in reseeded dPPM with MeT‐5A cells exhibited significant increase in DNA content at day 7 ( ∗∗p < 0.01) and day 15 ( ∗∗∗∗p < 0.0001) against unseeded dPPM. Here, we define a decellularisation protocol for porcine pleura that represents a step forward in their potential tissue engineering applications as bioscaffolds.

Cell-Derived Matrix: Production, Decellularization, and Application of Wound Repair.

Chronic nonhealing wounds significantly reduce patients' quality of life and are a major burden on healthcare systems. Over the past few decades, tissue engineering materials have emerged as a viable option for wound healing, with cell-derived extracellular matrix (CDM) showing remarkable results. The CDM's compatibility and resemblance to the natural tissue microenvironment confer distinct advantages to tissue-engineered scaffolds in wound repair. This review summarizes the current processes for CDM preparation, various cell decellularization protocols, and common characterization methods. Furthermore, it discusses the applications of CDM in wound healing, including skin defect and wound repair, angiogenesis, and engineered vessels, and offers perspectives on future developments.

Which detergent is most suitable for the generation of an acellular pancreas bioscaffold?

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.

Preparation of Corneal Tissue Matrix Bioink for 3D Bioprinting.

There are many protocols available to decellularize tissues for the preparation of bioink for 3D bioprinting purposes. Almost all the methods comprise multiple chemicals and enzymes in different combinations. Here we describe the usage of sodium chloride that enables the decellularization of corneal tissues from human and animal sources, which is a simple, rapid, and detergent-free method, unlike conventional decellularization protocols. The method described here is for cornea tissue decellularization and its digestion and bioink preparation for 3D bioprinting applications. We demonstrate the efficient decellularization of tissues by retaining the extracellular matrix.

Injectable Xenogeneic Dental Pulp Decellularized Extracellular Matrix Hydrogel Promotes Functional Dental Pulp Regeneration.

The present challenge in dental pulp tissue engineering scaffold materials lies in the development of tissue-specific scaffolds that are conducive to an optimal regenerative microenvironment and capable of accommodating intricate root canal systems. This study utilized porcine dental pulp to derive the decellularized extracellular matrix (dECM) via appropriate decellularization protocols. The resultant dECM was dissolved in an acid pepsin solution to form dECM hydrogels. The analysis encompassed evaluating the microstructure and rheological properties of dECM hydrogels and evaluated their biological properties, including in vitro cell viability, proliferation, migration, tube formation, odontogenic, and neurogenic differentiation. Gelatin methacrylate (GelMA) hydrogel served as the control. Subsequently, hydrogels were injected into treated dentin matrix tubes and transplanted subcutaneously into nude mice to regenerate dental pulp tissue in vivo. The results showed that dECM hydrogels exhibited exceptional injectability and responsiveness to physiological temperature. It supported the survival, odontogenic, and neurogenic differentiation of dental pulp stem cells in a 3D culture setting. Moreover, it exhibited a superior ability to promote cell migration and angiogenesis compared to GelMA hydrogel in vitro. Additionally, the dECM hydrogel demonstrated the capability to regenerate pulp-like tissue with abundant blood vessels and a fully formed odontoblast-like cell layer in vivo. These findings highlight the potential of porcine dental pulp dECM hydrogel as a specialized scaffold material for dental pulp regeneration.

Bioengineered Small-Diameter Vascular Xenografts as an Alternative to Autologous Vascular Grafting for Emergency Revascularization – a Preliminary Study

Abstract Background Autologous vascular arterial or venous graft are not available in 10-40% of patients, due to vascular pathologies, and the utility of decellularized biological scaffolds would be a solution for those cases. The purpose of this research was to obtain a functional acellular xenograft, prior to in-vivo testing as a vascular graft in an experimental animal. Materials and method Two batches of carotid vasculo-nervous bundles were collected from porcine models from a local slaughterhouse. The arterial grafts were dissected and isolated, obtaining carotid arteries with a caliber of 5–6 mm and a length of approximately 10–12 cm. Two decellularization protocols were used, immersion (n = 10) and perfusion (n = 9). The resulting grafts underwent histological examination, DNA analysis, electrophoresis and spectrophotometry. Results Due to severe tissue damage and friability, the batch that was decellularized using perfusion was not examined. The histological examination of grafts stained with hematoxylin-eosin and DAPI highlighted the absence of nuclei. Spectrophotometry revealed a 90% decellularization, and electrophoresis of revealed the migration band of the material extracted from the fresh tissue, as well as the absence of migration bands in the case of the material extracted from decellularized tissues. Conclusion We successfully used the immersion protocol to obtain a functional acellular vascular graft, in contrast to perfusion decellularization, where intraluminal high pressures damage the extracellular matrix.

Stomach engineering: region-specific characterization of the decellularized porcine stomach

PurposePatients affected by microgastria, severe gastroesophageal reflux, or those who have undergone subtotal gastrectomy, have commonly described reporting dumping syndromes or other symptoms that seriously impair the quality of their life. Gastric tissue engineering may offer an alternative approach to treating these pathologies. Decellularization protocols have great potential to generate novel biomaterials for large gastric defect repair. There is an urgency to define more reliable protocols to foster clinical applications of tissue-engineered decellularized gastric grafts.MethodsIn this work, we investigated the biochemical and mechanical properties of decellularized porcine stomach tissue compared to its native counterpart. Histological and immunofluorescence analyses were performed to screen the quality of decellularized samples. Quantitative analysis was also performed to assess extracellular matrix composition. At last, we investigated the mechanical properties and cytocompatibility of the decellularized tissue compared to the native.ResultsThe optimized decellularization protocol produced efficient cell removal, highlighted in the absence of native cellular nuclei. Decellularized scaffolds preserved collagen and elastin contents, with partial loss of sulfated glycosaminoglycans. Decellularized gastric tissue revealed increased elastic modulus and strain at break during mechanical tensile tests, while ultimate tensile strength was significantly reduced. HepG2 cells were seeded on the ECM, revealing matrix cytocompatibility and the ability to support cell proliferation.ConclusionOur work reports the successful generation of acellular porcine gastric tissue able to support cell viability and proliferation of human cells.

Development of Methods to Obtain Grafts for the Anterior Abdominal Wall in Surgical Treatment of External Abdominal Hernias

INTRODUCTION: Currently, tissue engineering is one of the most promising fields of medicine dealing with search for solutions to the problems of creation of biocompatible structures capable of partial or complete replacement of organs or tissues of an organism. An advanced method of tissue engineering implies using an extracellular matrix (ECM) cleared from the cellular material. The main problem of obtaining an ECM consists in the difficulty of selecting a suitable decellularization method and exposure time.
 AIM: Development of a method for obtaining grafts for the anterior abdominal wall (AAW) and evaluation of the histological properties of the ECM to determine the optimal protocol for donor tissue decellularization providing a complete removal of cellular material without any significant damage to the scaffold.
 MATERIALS AND METHODS: The experiments were conducted on 10 White Flemish Giant male rabbits. All the animals were withdrawn from the experiment, after which, on autopsy, AAW grafts were obtained for evaluation of the decellularization properties of three detergents (alkaline solutions): 2% SDS (sodium dodecyl sulfate) solution, 2% CHAPS (zwitterionic surfactant, 3-[(3-cholamidopropyl)-dimethylammonio]-2-hydroxy-1-propansulfonate) solution, and 1% Triton-X 100 solution. The decellularization quality was controlled in histological examination.
 RESULTS: Morphological evaluation of tissue samples revealed that after decellularization of the material with 2% SDS solution and 2% CHAPS solution, the number of cells remaining in the preparations was comparable and significantly less (Me = 10 in a microscope field) than after treatment with 1% Triton-X 100 solution (Me = 45 in a microscope field). However, decellularization with 2% CHAPS solution led to fiber ruptures and tissue edema manifested by an increase in the distance between the fibers (Me = 163 µm).
 CONCLUSIONS: Decellularization of the anterior abdominal wall tissues with 2% SDS solution provides a sufficient cell elimination while preserving the fiber integrity and ECM structure. 2% CHAPS solution was comparable to 2% SDS solution in terms of the quality of cell elements removal, but its more aggressive effect on collagen fibers was noted (their multiple ruptures and a more pronounced edema). 1% Triton-X 100 solution appeared to be of low efficiency in elimination of cellular material, although it had a minimal effect on the integrity of ECM network.

Decellularization of Massive Bone Allografts By Perfusion: A New Protocol for Tissue Engineering.

In terms of large bone defect reconstructions, massive bone allografts may sometimes be the only solution. However, they are still burdened with a high postoperative complication rate. Our hypothesis is that the immunogenicity of residual cells in the graft is involved in this issue. Decellularization by perfusion might therefore be the answer to process and create more biologically effective massive bone allografts. Seventy-two porcine bones were used to characterize the efficiency of our sodium hydroxide-based decellularization protocol. A sequence of solvent perfusion through each nutrient artery was set up to ensure the complete decellularization of whole long bones. Qualitative (histology and immunohistochemistry [IHC]) and quantitative (fluoroscopic absorbance and enzyme-linked immunosorbent assay) evaluations were performed to assess the decellularization and the preservation of the extracellular matrix in the bone grafts. Cytotoxicity and compatibility were also tested. Comparatively to nontreated bones, our experiments showed a very high decellularization quality, demonstrating that perfusion is mandatory to achieve an entire decellularization. Moreover, results showed a good preservation of the bone composition and microarchitecture, Haversian systems and vascular network included. This protocol reduces the human leukocyte antigen antigenic load of the graft by >50%. The majority of measured growth factors is still present in the same amount in the decellularized bones compared to the nontreated bones. Histology and IHC show that the bones were cell compatible, noncytotoxic, and capable of inducing osteoblastic differentiation of mesenchymal stem cells. Our decellularization/perfusion protocol allowed to create decellularized long bone graft models, thanks to their inner vascular network, ready for in vivo implantation or to be further used as seeding matrices.

Developing Bovine Brain-Derived Extracellular Matrix Hydrogels: a Screen of Decellularization Methods for Their Impact on Biochemical and Mechanical Properties.

Tissue models that recapitulate the key biochemical and physical aspects of the brain have been highly pursued in neural tissue engineering. Decellularization of native organs offers the advantage of preserving the composition of native extracellular matrix (ECM). Brain ECM has distinct features which play a major role in neural cell behavior. Cell instructive ligands and mechanical properties take part in the regulation of cellular processes in homeostasis and diseases. One of the main challenges in decellularization is maintaining mechanical integrity in reconstituted hydrogels and achieving physiologically relevant stiffness. The effect of the decellularization process on different mechanical aspects, particularly the viscoelasticity of brain-derived hydrogels, has not been addressed. In this study, we developed bovine brain-derived hydrogels for the first time. We pursued seven protocols for decellularization and screened their effect on biochemical content, hydrogel formation, and mechanical characteristics. We show that bovine brain offers an easily accessible alternative for in vitro brain tissue modeling. Our data demonstrate that the choice of decellularization method strongly alters gelation as well as the stiffness and viscoelasticity of the resulting hydrogels. Lastly, we investigated the cytocompatibility of brain ECM hydrogels and the effect of modulated mechanical properties on the growth and morphological features of neuroblastoma cells.

Creation of a decellularized vaginal matrix from healthy human vaginal tissue for potential vagina reconstruction: experimental studies.

When a disorder causes the absence of a healthy, full-size vagina, various neovaginal creation methods are available. Sometimes dilation or stretching of the vaginal cavity is sufficient, but intestinal or dermal flap tissue is generally required. However, different inherent tissue properties cause complications. Therefore, a lost body part should be replaced with a similar material. The use of organ-specific acellular vaginal tissue carries great potential, as its similar architecture and matrix composition make it suitable for vaginal regeneration. The authors developed an optimized protocol for decellularization of healthy human vaginal tissue. Resected colpectomy tissue from 12 healthy transgender patients was used. Successful decellularization was confirmed by applying acellular criteria from in-vivo remodeling reports. Suitability as a tissue-mimicking scaffold for vaginal reconstruction was determined by visible structural features, biocompatibility during stretching, and the presence of visible collagen, elastin, laminin, and fibronectin. Histological examination confirmed the preservation of structural features, and minimal cellular residue was seen during fluorescence microscopy, DNA and RNA quantification, and fragment length examination. Biomechanical testing showed decreased peak load (55%, P <0.05), strain at rupture (23%, P <0.01), and ultimate tensile stress (55%, P <0.05) after decellularization, while the elastic modulus (68%) did not decrease significantly. Fluorescence microscopy revealed preserved Fibronectin-I/II/III and Laminin-I/II, while Collagen-I and Ficolin-2B were decreased but mostly retained. The absence of cellular residue, moderately altered biomechanical extracellular matrix properties, and mostly preserved structural proteins appear to make our decellularized human vaginal matrix a suitable tissue-mimicking scaffold for vagina transplantation when tissue survival through vascularization and innervation are accomplished in the future.

Freeze-thaw sheep pericardium decellularization without detergents: A pilot study

Decellularization in tissue engineering preserves the extracellular matrix for the development of bioequivalent tissues. Detergent-based decellularization protocols can adversely affect the extracellular matrix (ECM) by destroying structural proteins. Furthermore, some of them are associated with residual cytotoxicity. The freeze-thaw method is not toxic, but the influence of its parameters on the results of the treatment has not been studied. This study explores the influence of a wide range of parameters of freeze-thaw protocols on the efficacy of decellularization and structural and mechanical properties of the ECM. The samples of sheep pericardium were decellularized in liquid nitrogen and phosphate-buffered saline for 5–50 cycles and subjected to histological and immunofluorescence analysis and tensile testing. A local decellularization was prominent for protocols with 20 and 30 cycles with 2-minute freezing and 2 or 5-minute thawing. Increasing the freeze-thaw duration to 5 or 10 min did not improve the results, and all the protocols with a low number of cycles did not result in effective decellularization. The ECM was well preserved and its mechanical properties were not significantly altered. One of the protocols resulted in sufficient decellularization which demonstrates the ability to successfully decellularize tissue and highlights the further need to investigate the influencing factors.

Impact of various detergent-based immersion and perfusion decellularization strategies on the novel caprine pancreas derived extracellular matrix scaffold.

Limited availability of the organs donors has facilitated the establishment of xenogeneic organ sources for transplantation. Numerous studies have decellularized several organs and assessed their implantability in order to provide such organs. Among all the decellularized organs studies for xenotransplantation, the pancreas has garnered very limited amount of research. The presently offered alternatives for pancreas transplantation are unable to liberate patients from donor dependence. The rat and mice pancreas are not of an accurate size for transplantation but can only be used for in-vitro studies mimicking in-vivo immune response in humans, while the porcine pancreas can cause zoonotic diseases as it carries porcine endogenous retrovirus (PERV- A/B/C). Therefore, we propose caprine pancreas as a substitute for these organs, which not only reduces donor dependence but also poses no risk of zoonosis. Upon decellularization the extracellular matrix (ECM) of different tissues responds differently to the detergents used for decellularization at physical and physiological level; this necessitates a comprehensive analysis of each tissue independently. This study investigates the impact of decellularization by ionic (SDS and SDC), non-ionic (Triton X-100 and Tween-20), and zwitterionic detergents (CHAPS). All these five detergents have been used to decellularize caprine pancreas via immersion (ID) and perfusion (PD) set-up. In this study, an extensive comparison of these two configurations (ID and PD) with regard to each detergent has been conducted. The final obtained scaffold with each set-up has been evaluated for the left-over cytosolic content, ECM components like sGAG, collagen, and fibronectin were estimated via Prussian blue and Immunohistochemical staining respectively, and finally for the tensile strength and antimicrobial activity. All the detergents performed consistently superior in PD than in ID. Conclusively, PD with SDS, SDC, and TX-100 successfully decellularizes caprine pancreatic tissue while retaining ECM architecture and mechanical properties. This research demonstrates the viability of caprine pancreatic tissue as a substitute scaffold for porcine organs and provides optimal decellularization protocol for this xenogeneic tissue. This research aims to establish a foundation for further investigations into potential regenerative strategies using this ECM in combination with other factors.

Utilization of a Decellularized Skin Scaffold for Repair of a Cleft Palate in a Dog: A Case Report.

Cleft palates are oral deformities that mostly affect puppies. They are frequently extensive and characterized by bone and palatal mucosa malformation. This deformity is a serious condition that may result in the death of the dog, therefore surgical treatment is recommended. Tissue bioengineering has emerged as a valuable option to treat cleft palates by applying acellular biological scaffolds as grafts. This case report proposed a new approach for surgical correction of canine cleft palate through a grafting technique using a decellularized scaffold. A decellularized portion of skin was implanted to correct a large cleft palate in a 3-month-old female Pug dog. The skin fragment was obtained from a dog cadaver and a decellularization protocol was performed. Under general anesthesia, a bilateral mucoperiosteal separation of the entire length of cleft margins was performed, and the scaffold was then positioned between the tissue and the bone palate. The interaction of the grafted scaffold with the oral mucosa and palatine layers resulted in total cleft closure, without postsurgical rejection or infection, indicating the applicability of this technique in dog's cleft palate correction. This is the first reported case demonstrating this new technique, which resulted in full cleft closure and healing.

Breast Cancer Cell Type and Biomechanical Properties of Decellularized Mouse Organs Drives Tumor Cell Colonization.

Tissue engineering has emerged as an indispensable tool for the reconstruction of organ-specific environments. Organ-derived extracellular matrices (ECM) and, especially, decellularized tissues (DCL) are recognized as the most successful biomaterials in regenerative medicine, as DCL preserves the most essential organ-specific ECM properties such as composition alongside biomechanics characterized by stiffness and porosity. Expansion of the DCL technology to cancer biology research, drug development, and nanomedicine is pending refinement of the existing DCL protocols whose reproducibility remains sub-optimal varying from organ to organ. We introduce a facile decellularization protocol universally applicable to murine organs, including liver, lungs, spleen, kidneys, and ovaries, with demonstrated robustness, reproducibility, high purification from cell debris, and architecture preservation, as confirmed by the histological and SEM analysis. The biomechanical properties of as-produced DCL organs expressed in terms of the local and total stiffness were measured using our facile methodology and were found well preserved in comparison with the intact organs. To demonstrate the utility of the developed DCL model to cancer research, we engineered three-dimensional tissue constructs by recellularization representative decellularized organs and collagenous hydrogel with human breast cancer cells of pronounced mesenchymal (MDA-MB-231) or epithelial (SKBR-3) phenotypes. The biomechanical properties of the DCL organs were found pivotal to determining the cancer cell fate and progression. Our histological and scanning electron microscopy (SEM) study revealed that the larger the ECM mean pore size and the smaller the total stiffness (as in lung and ovary), the more proliferative and invasive the mesenchymal cells became. At the same time, the low local stiffness ECMs (ranged 2.8-3.6 kPa) did support the epithelial-like SKBR-3 cells' viability (as in lung and spleen), while stiff ECMs did not. The total and local stiffness of the collagenous hydrogel was measured too low to sustain the proliferative potential of both cell lines. The observed cell proliferation patterns were easily interpretable in terms of the ECM biomechanical properties, such as binding sites, embedment facilities, and migration space. As such, our three-dimensional tissue engineering model is scalable and adaptable for pharmacological testing and cancer biology research of metastatic and primary tumors, including early metastatic colonization in native organ-specific ECM.

Semi-synthetic degradable notochordal cell-derived matrix hydrogel for use in degenerated intervertebral discs: Initial in vitro characterization.

Low back pain is the leading cause of disability worldwide, but current therapeutic interventions are palliative or surgical in nature. Loss of notochordal cells (NCs) and degradation of the healthy matrix in the nucleus pulposus (NP), the central tissue of intervertebral discs (IVDs), has been associated with onset of degenerative disc changes. Recently, we established a protocol for decellularization of notochordal cell derived matrix (NCM) and found that it can provide regenerative cues to nucleus pulposus cells of the IVD. Here, we combined the biologically regenerative properties of decellularized NCM with the mechanical tunability of a poly(ethylene glycol) hydrogel to additionally address biomechanics in the degenerate IVD. We further introduced a hydrolysable PEG-diurethane crosslinker for slow degradation of the gels in vivo. The resulting hydrogels were tunable over a broad range of stiffness's (0.2 to 4.5 kPa), matching that of NC-rich and -poor NP tissues, respectively. Gels formed within 30 min, giving ample time for handling, and remained shear-thinning post-polymerization. Gels also slowly released dNCM over 28 days as measured by GAG effusion. Viability of encapsulated bone marrow stromal cells after extrusion through a needle remained high. Although encapsulated NCs stayed viable over two weeks, their metabolic activity decreased, and their phenotype was lost in physiological medium conditions in vitro. Overall, the obtained gels hold promise for application in degenerated IVDs but require further tuning for combined use with NCs.

Efficient Decellularization of the Full-Thickness Rat-Derived Abdominal Wall to Produce Acellular Biologic Scaffolds for Tissue Reconstruction: Promising Evidence Acquired from In Vitro Results.

Functional restoration of abdominal wall defects represents one of the fundamental challenges of reconstructive surgery. Synthetic grafts or crosslinked animal-derived biological grafts are characterized by significant adverse reactions, which are mostly observed after their implantation. The aim of this study was to evaluate the efficacy of the decellularization protocol to produce a completely acellular full-thickness abdominal wall scaffold. Full-thickness abdominal wall samples were harvested from Wistar rats and submitted to a three-cycle decellularization process. Histological, biochemical, and DNA quantification analyses were applied to evaluate the effect of the decellularization protocol. Mechanical testing and immunogenicity assessment were also performed. Histological, biochemical, and DNA analysis results showed efficient decellularization of the abdominal wall samples after the third cycle. Decellularized abdominal wall scaffolds were characterized by good biochemical and mechanical properties. The data presented herein confirm the effective production of a rat-derived full-thickness abdominal wall scaffold. Expanding this approach will allow the exploitation of the capacity of the proposed decellularization protocol in producing acellular abdominal wall scaffolds from larger animal models or human cadaveric donors. In this way, the utility of biological scaffolds with preserved in vivo remodeling properties may be one step closer to its application in clinical studies.

Perfusion and Ultrasonication Produce a Decellularized Porcine Whole-Ovary Scaffold with a Preserved Microarchitecture.

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