Effect of intracellular uptake of nanoparticle-encapsulated trehalose on the hemocompatibility of allogeneic valves in the VS83 vitrification protocol

Abstract

Trehalose is a disaccharide molecule consisting of two molecules of glucose. Industrially, trehalose is derived from corn starch and utilized as a drug. This study aims to examine whether the integration of nanoparticle-encapsulated trehalose to the Ice-Free Cryopreservation (IFC) method for preserving heart valves has better cell viability, benefits to protect the extracellular matrix (ECM), and reduce immune response after storage. For the experiment to be carried out, we obtained materials, and the procedures were carried out in the following manner. The initial step was the preparation of hydroxyapatite nanoparticles, followed by precipitation to acquire Apatite colloidal suspensions. Animals were obtained, and their tissue isolation and grouping were done ethically. All samples were then divided into four groups, Control group, Conventional Frozen Cryopreservation (CFC) group, IFC group, and IFC + T (IFC with the addition of 0.2 M nanoparticle-encapsulated Trehalose) group. Histological analysis was carried out via H&E staining, ECM components were stained with Modified Weigert staining, and the Gomori Ammonia method was used to stain reticular fibers. Alamar Blue assay was utilized to assess cell viability. Hemocompatibility was evaluated, and samples were processed for immunohistochemistry (TNFα and IL-10). Hemocompatibility was quantified using Terminal Complement Complex (TCC) and Neutrophil elastase (NE) as an indicator. The results of the H&E staining revealed less formation of extracellular ice crystals and intracellular vacuoles in the IFC + T group compared with all other groups. The CFC group’s cell viability showed better viability than the IFC group, but the highest viability was exhibited in the IFC + T group (70.96 ± 2.53, P < 0.0001, n = 6). In immunohistochemistry, TNFα levels were lowest in both IFC and IFC + T group, and IL-10 expression had significantly reduced in IFC and IFC + T group. The results suggested that the nanoparticle encapsulated trehalose did not show significant hemocompatibility issues on the cryopreserved heart valves.

Cryopreservation of Animals and Cryonics: Current Technical Progress, Difficulties and Possible Research Directions

“…Vitrification is the preferred cooling method in cryonics as the body is preserved in a stable glassy state ( 8 , 67 ) but achieving this requires high CPA concentrations which can be damaging and toxic. As the future of cryonics is reanimation, a significant amount of research is currently focused on developing safer and effective CPA options including antifreeze proteins (AFPs) ( 68 ), trehalose ( 69 ), DP6 ( 70 ), nanotechnology engineered cryoprotectants ( 71 ), neutral amino acids ( 6 ). A major milestone was recorded in 2007 when major cryonics organizations claimed to have successfully vitrified the brain without ice formation ( 9 ), still post-thaw recovery not being promised drove the award-winning research of G. Fahy and R. McIntyre in 2015 ( 72 ).…”