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Optimization of Decellularization Procedure in Rat Esophagus for Possible Development of a Tissue Engineered Construct.

ABSTRACT: Background: Current esophageal treatment is associated with significant morbidity. The gold standard therapeutic strategies are stomach interposition or autografts derived from the jejunum and colon. However, severe adverse reactions, such as esophageal leakage, stenosis and infection, accompany the above treatments, which, most times, are life threating. The aim of this study was the optimization of a decellularization protocol in order to develop a proper esophageal tissue engineered construct. Methods: Rat esophagi were obtained from animals and were decellularized. The decellularization process involved the use of 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) and sodium dodecyl sulfate (SDS) buffers for 6 h each, followed by incubation in a serum medium. The whole process involved two decellularization cycles. Then, a histological analysis was performed. In addition, the amounts of collagen, sulphated glycosaminoglycans and DNA content were quantified. Results: The histological analysis revealed that only the first decellularization cycle was enough to produce a cellular and nuclei free esophageal scaffold with a proper extracellular matrix orientation. These results were further confirmed by biochemical quantification. Conclusions: Based on the above results, the current decellularization protocol can be applied successfully in order to produce an esophageal tissue engineered construct.

SUBMITTER: Mallis P

PROVIDER: S-EPMC6466343 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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Optimization of Decellularization Procedure in Rat Esophagus for Possible Development of a Tissue Engineered Construct.

Mallis Panagiotis P Chachlaki Panagiota P Katsimpoulas Michalis M Stavropoulos-Giokas Catherine C Michalopoulos Efstathios E

Bioengineering (Basel, Switzerland) 20181224 1

<b>Background:</b> Current esophageal treatment is associated with significant morbidity. The gold standard therapeutic strategies are stomach interposition or autografts derived from the jejunum and colon. However, severe adverse reactions, such as esophageal leakage, stenosis and infection, accompany the above treatments, which, most times, are life threating. The aim of this study was the optimization of a decellularization protocol in order to develop a proper esophageal tissue engineered co ...[more]

PMID: 30586900

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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 SIGNIFICANCE:Tissue 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.

Project description:BackgroundIrreversible electroporation (IRE) is a non-thermal cell ablation approach that induces selective damage to cell membranes only. The purpose of the current study was to evaluate and optimize its use for in-vivo myocardial decellularization.MethodsForty-two Sprague-Dawley rats were used to compare myocardial damage of seven different IRE protocols with anterior myocardial infarction damage. An in-vivo open thoracotomy model was used, with two-needle electrodes in the anterior ventricular wall. IRE protocols included different combinations of pulse lengths (70 vs. 100 ?seconds), frequency (1, 2, 4 Hz), and number (10 vs. 20 pulses), as well as voltage intensity (50, 250 and 500 Volts). All animals underwent baseline echocardiographic evaluation. Degree of myocardial ablation was determined using repeated echocardiography measurements (days 7 and 28) as well as histologic and morphometric analysis at 28 days.ResultsAll animals survived 28 days of follow-up. Compared with 50V and 250V, electroporation with 500V was associated with significantly increased myocardial scar and reduction in ejection fraction (67.4%±4% at baseline vs. 34.6%±20% at 28 days; p <0.01). Also, compared with pulse duration of 70 ?sec, pulses of 100 ?sec were associated with markedly reduced left ventricular function and markedly increased relative scar area ratio (28%±9% vs. 16%±3%, p = 0.02). Decreasing electroporation pulse frequency (1Hz vs. 2Hz, 2Hz vs. 4Hz) was associated with a significant increase in myocardial damage. Electroporation protocols with a greater number of pulses (20 vs. 10) correlated with more profound tissue damage (p<0.05). When compared with myocardial infarction damage, electroporation demonstrated a considerable likeness regarding the extent of the inflammatory process, but with relatively higher levels of extra-cellular preservation.ConclusionsIRE has a graded effect on the myocardium. The extent of ablation can be controlled by changing pulse length, frequency and number, as well as by changing electric field intensity.

Project description:BackgroundDevelopment of a pre-vascularized tissue-engineered construct with intrinsic vascular system for cell growth and tissue formation still faces many difficulties due to the complexity of the vascular network of natural bone tissue. The present study was to design and form a new vascularized tissue-engineered construct using pre-differentiated rADSCs, arteriovenous vascular bundle and porous nHA-PA 66 scaffold.MethodsrADSCs were pre-differentiated to endothelial cells (rADSCs-Endo) and then incorporated in nHA-PA 66 scaffolds in vitro. Subsequently, in vivo experiments were carried out according to the following groups: Group A (rADSCs-Endo/nHA-PA 66 scaffold with arteriovenous vascular bundle), Group B (rADSCs/nHA-PA 66 scaffold with arteriovenous vascular bundle); Group C (nHA-PA66 scaffold with arteriovenous vascular bundle), Group D (nHA-PA 66 scaffold only). The vessel density and vessel diameter were measured based on histological and immunohistochemical evaluation, furthermore, the VEGF-C, FGF-2 and BMP-2 protein expressions were also evaluated by western blot analysis.ResultsThe results of in vivo experiments showed that the vessel density and vessel diameter in group A were significantly higher than the other three groups. Between Group B and C, no statistical difference was observed at each time point. In accordance with the results, there were dramatically higher expressions of VEGF-C and FGF-2 protein in Group A than that of Group B, C and D at 2 or 4 weeks. Statistical differences were not observed in VEGF-C and FGF-2 expression between Group B and C. BMP-2 was not expressed in any group at each time point.ConclusionsCompared with muscular wrapping method, arteriovenous vascular bundle implantation could promote vascularization of the scaffold; and the angiogenesis of the scaffold was significantly accelerated when pre-differentiated rADSCs (endothelial differentiation) were added. These positive results implicate the combination of pre-differentiated rADSCs (endothelial differentiation) and arteriovenous vascular bundle may achieve rapidly angiogenesis of biomaterial scaffold.

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Optimization of Decellularization Procedure in Rat Esophagus for Possible Development of a Tissue Engineered Construct.

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Optimization of Decellularization Procedure in Rat Esophagus for Possible Development of a Tissue Engineered Construct.

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