Porcine pulmonary valve decellularization with NaOH-based vs detergent process: preliminary in vitro and in vivo assessments

Abstract

BackgroundGlutaraldehyde fixed xenogeneic heart valve prosthesis are hindered by calcification and lack of growth potential. The aim of tissue decellularization is to remove tissue antigenicity, avoiding the use of glutaraldehyde and improve valve integration with low inflammation and host cell recolonization. In this preliminary study, we investigated the efficacy of a NaOH-based process for decellularization and biocompatibility improvement of porcine pulmonary heart valves in comparison to a detergent-based process (SDS-SDC0, 5%).MethodsNative cryopreserved porcine pulmonary heart valves were treated with detergent and NaOH-based processes.Decellularization was assessed by Hematoxylin and eosin/DAPI/alpha-gal/SLA-I staining and DNA quantification of native and processed leaflets, walls and muscles.Elongation stress test investigated mechanical integrity of leaflets and walls (n = 3 tests/valve component) of valves in the native and treated groups (n = 4/group).Biochemical integrity (collagen/elastin/glycosaminoglycans content) of leaflet-wall and muscle of the valves (n = 4/group) was assessed and compared between groups with trichrome staining (Sirius Red/Miller/Alcian blue).Secondly, a preliminary in vivo study assessed biocompatibility (CD3 and CD68 immunostaining) and remodeling (Hematoxylin and eosin/CD31 and ASMA immunofluorescent staining) of NaOH processed valves implanted in orthotopic position in young Landrace pigs, at 1 (n = 1) and 3 months (n = 2).ResultsDecellularization was better achieved with the NaOH-based process (92% vs 69% DNA reduction in the wall). Both treatments did not significantly alter mechanical properties. The detergent-based process induced a significant loss of glycosaminoglycans (p < 0,05).In vivo, explanted valves exhibited normal morphology without any sign of graft dilatation, degeneration or rejection. Low inflammation was noticed at one and three months follow-up (1,8 +/− 3,03 and 0,9836 +/− 1,3605 CD3 cells/0,12 mm2 in the leaflets). In one animal, at three months we documented minimal calcification in the area of sinus leaflet and in one, microthrombi formation on the leaflet surface at 1 month. The endoluminal side of the valves showed partial reendothelialization.ConclusionsNaOH-based process offers better porcine pulmonary valve decellularization than the detergent process. In vivo, the NaOH processed valves showed low inflammatory response at 3 months and partial recellularization. Regarding additional property of securing, this treatment should be considered for the new generation of heart valves prosthesis.Graphical abstractGraphical abstract of the study

Toward acellular xenogeneic heart valve prostheses: Histological and biomechanical characterization of decellularized and enzymatically deglycosylated porcine pulmonary heart valve matrices

“…The majority of recent studies related to antigens removal from xenogeneic heart valve grafts have been focusing on the development of decellularization protocols, which disregarded the importance of the removal of glycans 68 …”

“…xenogeneic heart valve grafts have been focusing on the development of decellularization protocols, which disregarded the importance of the removal of glycans. 68 To our knowledge, so far, no data have been published about in vitro tests with PNGaseF, Endoglycosidase H, or O-glycosidase treatments on whole tissue matrices. PNGaseF allows to achieve an efficient removal of carbohydrates, in particular N-glycans, while the mechanical stability of tissues is preserved, as revealed by our results.…”

Preclinical study of a self-expanding pulmonary valve for the treatment of pulmonary valve disease

“…Through the suture technique, pericardia were bound on the support frame so that the tension was dispersed on the support frame, thus reducing the tension borne on the pinhole. Calcification of the leaflet limits the durability of the valve product [ 18 , 19 ]. Calcification of valve material is an important factor affecting the service life of the biological valve.…”

Decellularized tissue-engineered heart valves calcification: what do animal and clinical studies tell us?

“…All of those categorized were tabulated and compared to each other's depending on the host animal (in the case of animal studies), the type of heart valves involved, the technique of decellularization, cell seeding or not and type of cells used, duration of the study follow-up, the size of the group involved and finally the techniques used to evaluate calcification occurrence). Also, so far nearly around five specific articles comparing different decellularization techniques effect on calcification have been published; in vivo studies [49][50][51] and in vitro studies [52,53].…”

“… unseeded Pig 3 m Pig Von Kossa staining Only one animal showed minimal calcification after 3 months. P [ 89 ] No mention Allogeneic Xenogeneic Sheep Pig Enzymatic 48-h enzyme incubation was done. (what enzyme?)…”