Haemocompatibility testing allows selective adaption of GA-free SULEEI-preparation strategy for bovine pericardium
Authors: Dittfeld C , Koenig U , Welzel C , Jannasch A , Matschke K , Sperling C , Tugtekin S M , Maitz M 
Published: October 2021

Abstract

Background/Introduction
Bovine pericardium is the main natural source for patches or valve substitutes in cardiac surgery, but still, long-term durability is limited. An alternative, glutaraldehyde (GA)-free preparation combining decellularization, riboflavin/UVA crosslinking, and low-energy electron irradiation (SULEEI) procedure was established to avoid this. Protocol optimization is ongoing to further improve the final material. Haemocompatibility in vitro testing with fresh, human whole blood has advantages for material analysis, as it provides essential information on the activation of the complement system and immune cells therefore substantiating and possibly reducing the amount of animal testing.

Purpose
The haemocompatibility testing system was applied to reveal advantageous protocol adaptation of GA-free SULEEI preparation of pericardia.

Methods
Decellularized bovine pericardia were UVA-crosslinked in riboflavin/dextran solution. Protocol SULEEI B adaption includes the addition of trypsin in decellularization buffer and a 10x reduced riboflavin/dextran concentration during prolonged UVA irradiation with reduced intensity. Electron irradiation with 36 kGy was retained. Pericardia were incubated in blood chambers with heparinized human blood (2h, 37°C, GA-fixed/native controls). Inflammation (granulocyte loss and activation [CD11b]), complement activation (C5a) and haemostasis parameters (F1+2, PF4, granulocyte/platelet conjugates, platelet loss) were analyzed via flow cytometry or ELISA. Surface cell adhesion was investigated immunohistologically and via REM-analyses. Nuclear fragments were quantified in HE-stained sections.

Results
Haemostasis parameters F1+2 and PF4 were significantly higher in SULEEI A-pericardium (133±25 nmol/l and 1507±677 U/ml) vs. GA-fixation (2.9±2.9 nmol/l and 320±160 U/ml). Also, granulocyte/platelet conjugates (76.3±18.8%) and platelet loss (40.4±29.7%) were significantly higher after SULEEI A preparation in comparison to GA-fixation (39.3±9.9% and 20.5±6.4%). SULEEI B treated pericardia did not differ significantly. C5a complement activation was significantly lower in SULEEI A samples but comparable in SULEEI B to GA-fixed material. This is in contrast to a higher rate of nuclear fragments in SULEEI A tissue HE stained sections. Granulocyte activation of SULEEI pericardium (A: 89.8±29.7%; B: 106.2±18.8%) was significantly higher compared to native (46.1±22.6%) and for SULEEI B higher than in GA-fixed samples (67.2±9%). Granulocyte loss was comparable to GA-fixed pericardium after SULEEI B protocol. REM-analyses and histological evaluation visualized a dense blood cell and fibrin covering particular of SULEEI A-pericardium.

Conclusions
Haemocompatibility testing reveals advantageous properties of SULEEI B-pericardium. The impact of treatment substances such as dextran on high inflammatory response and dense surface covering in SULEEI A protocol is focus in ongoing experiments.

Funding Acknowledgement
Type of funding sources: Public grant(s) – EU funding. Main funding source(s): EU/EFRE