The Anti-calcification Effect of Dithiobispropionimidate, Carbodiimide and Ultraviolet Irradiation Cross-linking Compared to Glutaraldehyde in Rabbit Implantation Models

BACKGROUND: Glutaraldehyde (GA) is a widely used cross-linking agent for improving mechanical properties and resistance to enzymatic degradation of collagenous tissue, but it has several drawbacks such as calcification and cytotoxicity. The aim of this study was to find the alternative effective cross-linking methods to GA. MATERIALS AND METHODS: Bovine pericardium was processed with GA with ethanol+octanol and glycine detoxification, and polyethylene glycol (PG) space filler, dimethyl 3,3'-dithiobispropionimidate (DTBP), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) treatment, and the physical fixation of ultraviolet irradiation were done. The biologic material properties of variously treated pericardial tissues were assessed by biochemical, mechanical and histological tests. Treated pericardial tissues were also implanted subcutaneously or intramuscularly into the rabbit for 10 weeks to assess the xenoreactive antibody response of immunoglobulin G and M, their anti-calcification effect. RESULTS: The biochemical and mechanical properties of EDC fixed pericardial tissues were comparable to the GA fixed tissue. The cytotoxicity was lowest in space filler treated GA fixed group. In rabbit subcutaneous or intramuscular implantation models, decellularization, space filler, EDC treatment group showed significantly lower calcium content than GA only and DTBP treatment group (p<0.05, analysis of variance). The titer of anti Galalpha1-3Galbeta1-4GlcNAc-R antibodies did not change in the postimplantation serial enzyme-linked immunosorbent assay. Hematoxylin and eosin and von Kossa staining showed that decellularization, space filler, EDC, and ultraviolet treatment had less inflammatory cell infiltration and calcium deposits. CONCLUSION: The decellularization process, PG filler, and EDC treatments are good alternative cross-linking methods compared to GA only fixation and primary amine of DTBP treatment for cardiovascular xenograft preservation in terms of the collagen cross-linking stability and in vivo anti-calcification effects.

Effects of Cross-Linking Agents on the Stability of Human Acellular Dermal Matrix

PURPOSE: Human acellular dermal matrix(ADM) is widely used in the treatment of congenital anomalies and soft tissue deficiencies. But it is rapidly degraded in the body and does not provide satisfactory results. There is a need to improve collagen fiber stability through various methods and ultimately regulate the speed of degradation. METHODS: The ADMs were added with various cross- linking agents called glutaraldehyde, dimethyl 3,3'-dithiobispropionimidate to produce cross-linked acellular dermal matrices. 1,4-butanediol diglycidyl ether solution was applied with a pH of 4.5 and 9.0, respectively. The stability of cross-linked dermal matrix was observed by measuring the shrinkage temperature and the degradation rates. The cross- and non-cross linked dermis were placed in the rat abdomen and obtained after 8, 12 and 16 weeks. RESULTS: The shrinkage temperature significantly increased and the degradation rate significantly decreased, compared to the control(p<0.05). All of cross- linked dermises were observed grossly in 16 weeks, but most of non-cross linked dermis were absorbed in 8 weeks. Histologically, the control group ADM was found to have been infiltrated with fibroblasts and most of dermal stroma were transformed into the host collagen fibers. However, infiltration of fibroblasts in the experiment was insignificant and the original collagen structure was intact. CONCLUSION: Collagen cross-linking increases the structural stability and decreases degradation of acellular dermis. Therefore, decrease in body absorption and increase in duration can be expected.

Role of positive charge of lysine residue on ribosome-inactivating property of gelonin.

The report that gelonin cross-linked with monoclonal antibodies with the use of 2-iminothiolane (2-IT) exhibited higher cytotoxicity than the conjugates prepared with the use of N-succinimidyl-3-(2-pyridylthio) propionate (SPDP) alone, has prompted us to investigate the effect of epsilon-NH2 group modification with 2-IT on the ribosome-inactivating property (RIP) of gelonin. The purified gelonin was modified with 2-IT at a different molar ratio and their effects on immunoreactivity and ribosome-inactivating property were compared with those of N-succinimidyl 6-[3-(2-pyridyldithio) propionamido] hexanoate (long chain-SPDP) and SPDP modified gelonin derivatives. Modification of single amino group with 2-IT results in about 25-50% inhibition of immunoreactivity and 60-70% loss of protein synthesis inhibition activity. Modification of 2-3 amino groups further hampers both immunoreactivity and protein synthesis inhibition property of gelonin. Both the long chain-SPDP with SPDP modifications showed more pronounced effects on immunoreactivity and RIP activity as compared to the similar ratio of 2-IT modification(s). It may, therefore, be concluded that the positive charge plays an important role in the immunological as well as the protein synthesis inhibitory effect of gelonin.

Chemically attenuated larvae of S. mansoni as a novel tool in schistosomiasis research: 2. Chemical attenuation of cercariae by diimidoesters

A novel approach to chemicallu attenuate cercariae fo S. mansoni is presented. The method utilizes the biologically active surface proteins/glycoproteins which are essential for the survival of the organism as a target for inactivation. The inactivation was achieved by reaction with 0.01 M dimethil adipimidate, dimethyl pimelimidate or dimethyl suberimidate at pH 8.5. The cercariae lost their viability, but retained the ability to exclude trypan blue for up to 2 years when stored at 4-C in a manner similar to live cecariae and in contrast to dead cercariae witch took up the dye immediately. In addition, the attenuated cercariae reacted with monoclonal and policlonal antischistosome antibodies in an indirect immunofluorescence assay indacting the retention and preservation of surface antigens after attenuation. The immunochemical reactivity of the attenuated cercariae was preserved after storage for 2 years at 4-C as shown by reaction with antisera from infected mice and rats in IIF assay. Attenuated cercariae revealed the presence of antischistosome antibodies as early as one week after infection in mice and rats. The presence of receptors for the Fc portion of human IgG on the attenuated cercariae interfered in their use as an immunodiagnostic reagent for human schistosomiasis. The attenuated cercariae were also used to screen cultures for monoclonal antischistosome antibodies. preliminary results indicated that immunozation with attenuated cercariae was capable of imparting protective immunity in mice