Extraction and Biological Evaluation of Matrix-Bound Nanovesicles (MBVs) from High-Hydrostatic Pressure-Decellularized Tissues

Abstract

Decellularized tissues are widely used as promising materials in tissue engineering and regenerative medicine. Research on the microstructure and components of the extracellular matrix (ECM) was conducted to improve the current understanding of decellularized tissue functionality. The presence of matrix-bound nanovesicles (MBVs) embedded within the ECM was recently reported. Results of a previous experimental investigation revealed that decellularized tissues prepared using high hydrostatic pressure (HHP) exhibited good in vivo performance. In the current study, according to the hypothesis that MBVs are one of the functional components in HHP-decellularized tissue, we investigated the extraction of MBVs and the associated effects on vascular endothelial cells. Using nanoparticle tracking assay (NTA), transmission electron microscopy (TEM), and RNA analysis, nanosized (100–300 nm) and membranous particles containing small RNA were detected in MBVs derived from HHP-decellularized small intestinal submucosa (SIS), urinary bladder matrix (UBM), and liver. To evaluate the effect on the growth of vascular endothelial cells, which are important in the tissue regeneration process, isolated SIS-derived MBVs were exposed to vascular endothelial cells to induce cell proliferation. These results indicate that MBVs can be extracted from HHP-decellularized tissues and may play a significant role in tissue remodeling.

Characterisation of Matrix-Bound Nanovesicles (MBVs) Isolated from Decellularised Bovine Pericardium: New Frontiers in Regenerative Medicine

“…These results confirm the presence of MBVs in dBP, a subclass of extracellular vesicles (EVs), distinguished as adhering to the ECM, while EVs were believed to be present solely in body fluids [24]. The size range and structure of MBVs from the TEM results herein reported correspond to MBVs from different tissues described in the literature [23,25,35]. Moreover, the nanovesicle structure also showed additional vesicular internal structures, as observed also for other EVs [45].…”

“…Thus, we can hypothesise that MVBs at adequate concentrations allow and enhance cell viability through a richer milieu, as proved by the bioactive molecules found in their cargo. The findings of MBV cytocompatibility were also confirmed by immunofluorescence analysis, as no cellular toxicity or significant changes in cellular morphology were seen according to the literature [28,30,35].…”

“…In different studies, MBVs, even when isolated from different tissue sources, have shown the ability to recapitulate the immunomodulatory and regenerative potential of dECM, and have been successfully used both in vitro and in vivo for different regenerative medicine applications, suggesting their vast potential for biomedical applications [27][28][29][30][31][32][33][34]. Even though MBVs seem to be a ubiquitous component of the ECM, maintained even after the tissue decellularisation process, to date, they have only been isolated from small intestine submucosa, urinary bladder matrix, dermis, brain, oesophagus, heart, muscle, liver, ovary, pancreas, and tendons [23][24][25]35]. The research characterising these MBVs has shown that there are slight differences in the cargo, surface markers, and, thus, biological potential, of each tissue-specific MBV.…”

Preparation and Use of Decellularized Extracellular Matrix for Tissue Engineering

“…This technique was successful in removing cells in SIS and UBM, but was not as effective in liver tissue due to the thickness of the pieces. The authors were able to successfully collect MBVs from the dECM [ 268 ]. Charoensombut N et al (2022) used hydrostatic pressure, increased by 65.3 MPa/min to 980 MPa, held for 10 min, and reduced by 65.3 MPa/min until atmospheric pressure was reached, to decellularize rat uterine tissue in PBS at 30 °C; this was followed by washing with 0.9% NaCl, 0.05 M magnesium chloride hexahydrate, 0.2 mg/mL DNase I, and 1% penicillin and streptomycin for seven days.…”