Sonication-Assisted Method for Decellularization of Human Umbilical Artery for Small-Caliber Vascular Tissue Engineering.
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ABSTRACT:
SUBMITTER: Lin CH
PROVIDER: S-EPMC8196986 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Decellularization to produce bioscaffolds composed of the extracellular matrix (ECM) uses enzymatic, chemical and physical methods to remove antigens and cellular components from tissues. Effective decellularization methods depend on the characteristics of tissues, and in particular, tissues with dense, complex structure and abundant lipid content are difficult to completely decellularize. Our study enables future research on the development of methods and treatments for fabricating bioscaffolds via decellularization of complex and rigid skin tissues, which are not commonly considered for decellularization to date as their structural and functional characteristics could not be preserved after severe decellularization. In this study, decellularization of human dermal tissue was done by a combination of both chemical (0.05% trypsin-EDTA, 2% SDS and 1% Triton X-100) and physical methods (electroporation and sonication). After decellularization, the content of DNA remaining in the tissue was quantitatively confirmed, and the structural change of the tissue and the retention and distribution of ECM components were evaluated through histological and histochemical analysis, respectively. Conditions of the chemical pretreatment that increase the efficiency of physical stimulation as well as decellularization, and conditions for electroporation and sonication without the use of detergents, unlike the methods performed in previous studies, were established to enable the complete decellularization of the skin tissue. The combinatorial decellularization treatment formed micropores in the lipid bilayers of the skin tissues while removing all cell and cellular residues without affecting the ECM properties. Therefore, this procedure can be widely used to fabricate bioscaffolds by decellularizing biological tissues with dense and complex structures.
| S-EPMC9017362 | biostudies-literature
Project description:Preservation of tissue structure is often a primary goal when optimizing tissue and organ decellularization methods. Many current protocols nonetheless rely on detergents that aid extraction of cellular components but also damage tissue architecture. It may be more beneficial to leverage an innate cellular process such as apoptosis and promote cell removal without the use of damaging reagents. During apoptosis, a cell detaches from the extracellular matrix, degrades its internal components, and fragments its contents for easier clearance. We have developed a method that leverages this process to achieve tissue decellularization using only mild wash buffers. We have demonstrated that treating peripheral nerve tissue with camptothecin induced both an early marker of apoptosis, cleaved caspase-3 expression, as well as a late stage marker, TUNEL+ DNA fragmentation. Clearance of the cellular components was then achieved in an apoptosis-dependent manner using a gentle wash in hypertonic phosphate buffered saline followed by DNase treatment. This wash paradigm did not significantly affect collagen or glycosaminoglycan content, but it was sufficient to remove any trace of the cytotoxic compound based on conditioned media experiments. The resulting acellular tissue graft was immunogenically tolerated in vivo and exhibited an intact basal lamina microarchitecture mimicking that of native, unprocessed nerve. Hence, ex vivo induction of apoptosis is a promising method to decellularize tissue without the use of harsh reagents while better preserving the benefits of native tissue such as tissue-specific composition and microarchitecture.Statement of significanceTissue decellularization has expanded the ability to generate non-immunogenic organ replacements for a broad range of health applications. Current technologies typically rely on the use of harsh agents for clearing cellular debris, altering the tissue structure and potentially diminishing the pro-regenerative effects. We have developed a method for effectively, yet gently, removing cellular components from peripheral nerve tissue while preserving the native tissue architecture. The novelty of this process is in the induction of programmed cell death - or apoptosis - via a general cytotoxin, thereby enabling antigen clearance using only hypertonic wash buffers. The resulting acellular nerve scaffolds are nearly identical to unprocessed tissue on a microscopic level and elicit low immune responses comparable to an isograft negative control in a model of subcutaneous implantation.
| S-EPMC6126930 | biostudies-literature
Project description:Vascular disease is a leading cause of death worldwide, but surgical options are restricted by the limited availability of autologous vessels, and the suboptimal performance of prosthetic vascular grafts. This is especially evident for coronary artery by-pass grafts, whose small caliber is associated with a high occlusion propensity. Despite the potential of tissue-engineered grafts, compliance mismatch, dilatation, thrombus formation, and the lack of functional elastin are still major limitations leading to graft failure. This calls for advanced materials and fabrication schemes to achieve improved control on the grafts' properties and performance. Here, bioinspired materials and technical textile components are combined to create biohybrid cell-free implants for endogenous tissue regeneration. Clickable elastin-like recombinamers are processed to form an open macroporous 3D architecture to favor cell ingrowth, while being endowed with the non-thrombogenicity and the elastic behavior of the native elastin. The textile components (i.e., warp-knitted and electrospun meshes) are designed to confer suture retention, long-term structural stability, burst strength, and compliance. Notably, by controlling the electrospun layer's thickness, the compliance can be modulated over a wide range of values encompassing those of native vessels. The grafts support cell ingrowth, extracellular matrix deposition and endothelium development in vitro. Overall, the fabrication strategy results in promising off-the-shelf hemocompatible vascular implants for in situ tissue engineering by addressing the known limitations of bioartificial vessel substitutes.
| S-EPMC6877483 | biostudies-literature
Project description:Decellularization procedures have been developed and optimized for the entire organ or tissue blocks, by either perfusion of decellularizing agents through the tissue's vasculature or submerging large sections in decellularizing solutions. However, some research aims require the analysis of native as well as decellularized tissue slices side by side, but an optimal protocol has not yet been established to address this need. Thus, the main goal of this work was to develop a fast and efficient decellularization method for tissue slices-with an emphasis on lung-while attached to a glass slide. To this end, different decellularizing agents were compared for their effectiveness in cellular removal while preserving the extracellular matrix. The intensity of DNA staining was taken as an indicator of remaining cells and compared to untreated sections. The presence of collagen, elastin and laminin were quantified using immunostaining and signal quantification. Scaffolds resulting from the optimized protocol were mechanically characterized using atomic force microscopy. Lung scaffolds were recellularized with mesenchymal stromal cells to assess their biocompatibility. Some decellularization agents (CHAPS, triton, and ammonia hydroxide) did not achieve sufficient cell removal. Sodium dodecyl sulfate (SDS) was effective in cell removal (1% remaining DNA signal), but its sharp reduction of elastin signal (only 6% remained) plus lower attachment ratio (32%) singled out sodium deoxycholate (SD) as the optimal treatment for this application (6.5% remaining DNA signal), due to its higher elastin retention (34%) and higher attachment ratio (60%). Laminin and collagen were fully preserved in all treatments. The SD decellularization protocol was also successful for porcine and murine (mice and rat) lungs as well as for other tissues such as the heart, kidney, and bladder. No significant mechanical differences were found before and after sample decellularization. The resulting acellular lung scaffolds were shown to be biocompatible (98% cell survival after 72 h of culture). This novel method to decellularize tissue slices opens up new methodological possibilities to better understand the role of the extracellular matrix in the context of several diseases as well as tissue engineering research and can be easily adapted for scarce samples like clinical biopsies.
| S-EPMC8959585 | biostudies-literature
Project description:BackgroundThe development of a biological based small diameter vascular graft (d?<?6 mm), that can be properly stored over a long time period at -?196 °C, in order to directly be used to the patients, still remains a challenge. In this study the decellularized umbilical arteries (UAs) where vitrified, evaluated their composition and implanted to a porcine model, thus serving as vascular graft.MethodsHuman UAs were decellularized using 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and sodium dodecyl sulfate (SDS) detergents. Then, vitrified with vitrification solution 55 (VS55) solution, remained for 6 months in liquid nitrogen and their extracellular matrix composition was compared to conventionally cryopreserved UAs. Additionally, total hydroxyproline, sulphated glycosaminoglycan and DNA content were quantified in all samples. Finally, the vitrified umbilical arteries implanted as common carotid artery interposition graft to a porcine animal model.ResultsDecellularized and vitrified UAs characterized by proper preservation of extracellular matrix proteins and tissue architecture, whereas conventionally cryopreserved samples exhibited a disorganized structure. Total hydroxyproline content was preserved, although sulphated glycosaminoglycan and DNA contents presented significantly alterations in all samples. Implanted UAs successfully recellularized and remodeled as indicated by the histological analysis.ConclusionDecellularized and vitrified UAs retained their structure function properties and can be possible used as an alternative source for readily accessible small diameter vascular grafts.
| S-EPMC7260347 | biostudies-literature
Project description:We developed an automated system that can be used to decellularize whole human-sized organs and have shown lung as an example. Lungs from 20 to 30 kg pigs were excised en bloc with the trachea and decellularized with our established protocol of deionized water, detergents, sodium chloride, and porcine pancreatic DNase. A software program was written to control a valve manifold assembly that we built for selection and timing of decellularization fluid perfusion through the airway and the vasculature. This system was interfaced with a prototypic bioreactor chamber that was connected to another program, from a commercial source, which controlled the volume and flow pressure of fluids. Lung matrix that was decellularized by the automated method was compared to a manual method previously used by us and others. Automation resulted in more consistent acellular matrix preparations as demonstrated by measuring levels of DNA, hydroxyproline (collagen), elastin, laminin, and glycosaminoglycans. It also proved highly beneficial in saving time as the decellularization procedure was reduced from days down to just 24 h. Developing a rapid, controllable, automated system for production of reproducible matrices in a closed system is a major step forward in whole-organ tissue engineering.
| S-EPMC4290793 | biostudies-literature
Project description:Small incision lenticule extraction (SMILE) becomes a procedure to correct myopia. The extracted lenticule can be used for other clinical scenarios. To prepare for allogeneic implantation, lenticule decellularization with preserved optical property, stromal architecture and chemistry would be necessary. We evaluated different methods to decellularize thin human corneal stromal lenticules created by femtosecond laser. Treatment with 0.1% sodium dodecylsulfate (SDS) followed by extensive washes was the most efficient protocol to remove cellular and nuclear materials. Empty cell space was found inside the stroma, which displayed aligned collagen fibril architecture similar to native stroma. The SDS-based method was superior to other treatments with hyperosmotic 1.5 M sodium chloride, 0.1% Triton X-100 and nucleases (from 2 to 10 U/ml DNase and RNase) in preserving extracellular matrix content (collagens, glycoproteins and glycosaminoglycans). The stromal transparency and light transmittance was indifferent to untreated lenticules. In vitro recellularization showed that the SDS-treated lenticules supported corneal stromal fibroblast growth. In vivo re-implantation into a rabbit stromal pocket further revealed the safety and biocompatibility of SDS-decellularized lenticules without short- and long-term rejection risk. Our results concluded that femtosecond laser-derived human stromal lenticules decellularized by 0.1% SDS could generate a transplantable bioscaffold with native-like stromal architecture and chemistry.
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Project description:Endothelial progenitor cells (EPCs) play a significant role in multiple biological processes such as vascular homeostasis, regeneration, and tumor angiogenesis. This makes them a promising cell of choice for studying a variety of biological processes, toxicity assays, biomaterial-cell interaction studies, as well as in tissue-engineering applications. In this study, we report the generation of two clones of SV40-immortalized EPCs from umbilical cord blood. These cells retained most of the functional features of mature endothelial cells and showed no indication of senescence after repeated culture for more than 240 days. Extensive functional characterization of the immortalized cells by western blot, flow cytometry, and immunofluorescence studies substantiated that these cells retained their ability to synthesize nitric oxide, von Willebrand factor, P-Selectin etc. These cells achieved unlimited proliferation potential subsequent to inactivation of the cyclin-dependent kinase inhibitor p21, but failed to form colonies on soft agar. We also show their enhanced growth and survival on vascular biomaterials compared to parental cultures in late population doubling. These immortalized EPCs can be used as a cellular model system for studying the biology of these cells, gene manipulation experiments, cell-biomaterial interactions, as well as a variety of tissue-engineering applications.
| S-EPMC3483052 | biostudies-literature
Project description:Advances in the development of cornea substitutes by tissue engineering techniques have focused on the use of decellularized tissue scaffolds. In this work, we evaluated different chemical and physical decellularization methods on small intestine tissues to determine the most appropriate decellularization protocols for corneal applications. Our results revealed that the most efficient decellularization agents were the SDS and triton X-100 detergents, which were able to efficiently remove most cell nuclei and residual DNA. Histological and histochemical analyses revealed that collagen fibers were preserved upon decellularization with triton X-100, NaCl and sonication, whereas reticular fibers were properly preserved by decellularization with UV exposure. Extracellular matrix glycoproteins were preserved after decellularization with SDS, triton X-100 and sonication, whereas proteoglycans were not affected by any of the decellularization protocols. Tissue transparency was significantly higher than control non-decellularized tissues for all protocols, although the best light transmittance results were found in tissues decellularized with SDS and triton X-100. In conclusion, our results suggest that decellularized intestinal grafts could be used as biological scaffolds for cornea tissue engineering. Decellularization with triton X-100 was able to efficiently remove all cells from the tissues while preserving tissue structure and most fibrillar and non-fibrillar extracellular matrix components, suggesting that this specific decellularization agent could be safely used for efficient decellularization of SI tissues for cornea TE applications.
| S-EPMC3682956 | biostudies-literature
Project description:Patients with stress urinary incontinence mainly suffer from malfunction of the urethra closure mechanism. We established the decellularization of porcine urethras to produce acellular urethra bioscaffolds for future tissue engineering applications, using bioscaffolds or bioscaffold-derived soluble products. Cellular removal was evaluated by H&E, DAPI and DNA quantification. The presence of specific ECM proteins was assessed through immunofluorescence staining and colorimetric assay kits. Human skeletal muscle myoblasts, muscle progenitor cells and adipose-derived stromal vascular fractions were used to evaluate the recellularization of the acellular urethra bioscaffolds. The mechanochemical decellularization system removed ~93% of tissue's DNA, generally preserving ECM's components and microarchitecture. Recellularization was achieved, though methodological advances are required regarding cell seeding strategies and functional assessment. Through microdissection and partial digestion, different urethra ECM-derived coating substrates were formulated (i.e. containing smooth or skeletal muscle ECM) and used to culture MPCs in vitro. The skeletal muscle ECM substrates enhanced fiber formation leading to the expression of the main skeletal muscle-related proteins and genes, as confirmed by immunofluorescence and RT-qPCR. The described methodology produced a urethra bioscaffold that retained vital ECM proteins and was liable to cell repopulation, a crucial first step towards the generation of urethra bioscaffold-based Tissue Engineering products.
| S-EPMC5292742 | biostudies-literature