In vivo Determination of Biocompatibility of Bladder Acellular Matrix in a Rabbit Model

The present study was carried out for in-vivo biocompatibility testing of cross-linked bladder acellular matrix graft (BAMG) with of 0.6% Glutaraldehyde(GA), 1% Hexamethylene diisocyanate (HMD), 1% 1,4-butanediol diglycidyl ether (BDDGE) and 1% 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) for 12, 24, 48 and 72 h duration at room temperature. The uncross-linked acellular bladder was used as control. In-vivo biocompatibility testing of above cross-linked bladder acellular matrix grafts was determined by subcutaneous implantation of graft on either side of spine of rabbits. The native bladder, acellular and cross-linked BAMG were implanted in separate rabbit under general anesthesia. These grafts were retrieved back at 15, 30, 60 and 90 post-operative implantation days and were evaluated using parameters like macroscopic and microscopic examination, immunoblotting, lymphocyte proliferation assay and ELISA. Macroscopically, all the retrieved biomaterials were found covered with fibrous connective tissue. However, it was thin at 15 days and later on its density increased with maximum at day 90. By day 90, EDC treated grafts for 24 h and 72 h were completely resorbed while EDC treated 12 and 48 h grafts were partially resorbed; whereas GA, HMD and BDDGE cross-linked biomaterials were partially resorbed. The histopathological examination revealed that the bladder acellular matrix graft treated with GA underwent minimum degradation at day 90 post-implantation, which indicated that degradation was least as compared to other cross-linking agents. The native bladder was found to induce more host inflammatory reaction as compared to its acellular graft. All the cross-linked bladder acellular matrix graft showed similar type of inflammatory reaction as compared to acellular uncross-linked bladder matrix (control), which was confined to the periphery of the graft without infiltration within the graft. The reaction became more intense at day 90 in GA and BDDGE treated BAMG samples. The HMD-12 h and HMD-48 h treated BAMG showed good healing of tissue appeared within the graft which was having less inflammatory exudates by day 90. The HMD-24 h and HMD-72 h treated graft showed heavy infiltration of cellular debris and disintegrated collagen scaffold in time dependent manner. The EDC-24 h and EDC-72 h treated BAMG showed host reaction, confined to the periphery around graft up to 60 days post-implantation, but at day 90 the graft was completely reabsorbed. At different cross-linking time intervals the host graft reaction of BDDGE treated graft was similar. The immunoblotting revealed that HMD and BDDGE cross-linked graft showed humoral response. In lymphocyte proliferation assay, the minimum SI was recorded in the tissues cross-linked with GA-48 h which indicated that GA cross-linked tissue had least ability to trigger CMI response in host and the maximum SI was seen in HMD-48 h treated samples. The GA and EDC cross-linked BAMG showed minimum SI value as compared to other cross-linked samples. ELISA revealed less immune response towards the EDC cross-linked grafts.