Evaluation of the degree of cross-linking in UV irradiated porcine valves

A porcine heart valve was irradiated by Ultraviolet (UV) rays (10 W, 254 nm) for 2, 4, 8 and 24 hours at 4 degrees C to cross-link the structural collagen matrix. The degree of cross-linking was evaluated by assaying the released amount of hydroxyproline (Hyp) from the matrix, and comparing it with the positive controls of valves treated by glutaraldehyde (GA) solution (0.625 wt%) and the negative controls of non-treated fresh valves. The undigested weight ratio of the specimens increased by increasing the UV irradiation time. The undigested weight of the leaflets, tunica interna and tunica externa of the fresh, GA-treated and UV-irradiated specimens after collagenase digestion was compared. As UV irradiation increased, the amount of released hydroxyproline was gradually decreased until 8 hours of irradiation, after which the released hydroxyproline-reduction occurred slightly until 24 hours of irradiation time in this system. A total 47.68% of the hydroxyproline in the valve was cross-linked by UV irradiation after 24 hours, while 73.74% of the hydroxyproline in the positive control was crossed-linked. Light microscopic observation revealed that the typical crimp pattern of collagen fibers decreased and was rearranged into a dense flattened pattern as the UV irradiation induced interfibrilar cross-linking. GA-treated valves demonstrated a denser matrix pattern than the UV-irradiated specimens. Cross-linked collagenous tissue prepared by UV irradiation would be useful for improving durability and reducing the disadvantages related to using a chemical cross-linking agent.

Viability and enzymatic activity of cryopreserved porcine heart valve

Fibroblast viability of a natural tissue valve for replacing a defective heart valve through allograft or xenograft has been suggested to affect its clinical durability. In this study, the cell viability and enzymatic activity of porcine heart valve leaflets were examined in regard to concerning to the preservation process [variable warm ischemic time (WIT), cold ischemic time (CIT), and cryopreservation]. Porcine heart enblocs were obtained and valve dissection was performed after 2, 12, 24, or 36 hours, in respective groups A, B, C, and D, as WIT. Each group was stored for 24 hours as CIT and cryopreserved. Leaflets were dissected from a valved conduit after each process, and cell viability and enzymatic activity in the leaflet were investigated using trypan blue staining and API ZYM kits. WIT extension significantly decreased fibroblast viability (p < 0.05, 92.25 +/- 2.7% at 2 hours, 84.9 +/- 6.7% at 12 hours, 57.0 +/- 10.2% at 24 hours, 55.9 +/- 7.9% at 36 hours), while CIT for 24 hours was also influenced significantly (p < 0.05), whereas cryopreservation demonstrated no effect on cellular viability. In enzyme activity observation, several enzymes related to lipid or nucleotide degradation (esterase, esterase lipase, particularly phosphatase, phosphohydrolase) were remarkably changed following the valve-fabrication process. After 24 hours CIT, these enzymatic activities in groups B, C and D significantly increased, but the activities decreased after cryopreservation. Particularly, both the viability and enzymatic activity showed remarkable changes after CIT in group B (WIT = 12 hours). These results suggest that WIT is more important than CIT in maintaining viability of the valve, and that completing all the cryopreservation process within 12 hours after acquisition is recommended.