sodium-borohydride and Calcinosis

Previous studies have shown that ethanol pretreatment of glutaraldehyde (GA)-fixed porcine aortic valve cusps (GPAV) significantly reduces bioprosthetic leaflet calcification. The anti-calcification mechanism is due to extraction of cholesterol and phospholipids, and a permanent alteration in collagen structure. It was noted in experimental implants that ethanol-pretreated GPAV occasionally show low levels of calcification. The study aim was to investigate whether this was due to unreacted aldehyde residues and other reducible compounds resulting from GA cross-linking.. GPAV were cross-linked in GA (0.6%) and stored at pH 7.4 in 0.2% GA. Cusps were pretreated with ethanol (80%, pH 7.4) for 24 h. Experimental groups included ethanol-pretreated cusps and GA-fixed controls that were pretreated with either sodium borohydride or sodium cyanoborohydride. Differential scanning calorimetry was used to measure shrink temperature as a measure of cross-linking. Subdermal implants of valve cusp tissue were carried out in 21-day-old Sprague-Dawley male rats. Implants were retrieved at 21 days and samples assessed for the extent of calcification using chemical analyses for Ca, and microscopy studies.. Ethanol pretreatment significantly inhibited calcification compared with controls (13.3 +/- 5.6 versus 119.2 +/- 6.6 micrograms Ca/mg tissue; p < 0.001). However, sodium borohydride reduction under optimized conditions combined with ethanol pretreatment optimally reduced calcification (1.16 +/- 0.1 microgram Ca/mg; p < 0.05), whereas levels after sodium cyanoborohydride treatment (23.6 +/- 10.4 micrograms Ca/mg) were not significantly different to those after ethanol alone. Neither reducing agent was effective in inhibiting calcification without ethanol pretreatment. Furthermore, the reducing agents had no significant effect on shrink temperature.. Inhibition of GPAV calcification with ethanol pretreatment can be enhanced through the optimized use of reducing agents. This indicates that reducible aldehyde-related moieties are likely responsible for breakthrough calcification, even after ethanol pretreatment.

Topics: Animals; Aortic Valve; Bioprosthesis; Borohydrides; Calcinosis; Cross-Linking Reagents; Ethanol; Glutaral; Heart Valve Prosthesis; Male; Rats; Rats, Sprague-Dawley; Reducing Agents; Skin; Solvents

A novel chemical modification of biological tissues was developed aimed at improving biocompatibility and calcification resistance. This method involved the additional grafting of sulfonated PEO (PEO-SO(3)) or heparin after conventional glutaraldehyde (GA) fixation of bovine pericardium (BP). The amino groups of PEO-SO(3) or heparin were utilized to react to the GA residues to block them. The PEO-SO(3) or heparin grafted tissues demonstrated a slightly higher shrinkage temperature and tensile strength, but greater resistance to collagenase digestion, than GA treated ones. These results suggest that modified tissues have improved durability due to the grafting and filling effect of PEO-SO(3) or heparin in addition to the GA cross-linking. At the direct contact cytotoxicity test in vitro, PEO-SO(3) or heparin grafted tissue was shown to be nontoxic, while relatively significant cytotoxicity was observed for the GA treated tissues, possibly due to the release of GA. From the in vivo calcification study, calcium contents deposited on the modified tissues were much less than those on GA treated tissues. Such a decreased calcification might be explained by the decrease of residual GA groups during the additional treatment, and the space-filling effect and the nonadhesive property and/or the blood compatibility of PEO-SO(3) or heparin grafted covalently. The newly modified tissue patch was observed to show improved pathological assessibility including less inflammation and tissue reactions. This simple modification method may be useful for calcification-resistant and blood-compatible tissue patches for cardiovascular implants.

Topics: Animals; Aorta, Thoracic; Biocompatible Materials; Bioprosthesis; Borohydrides; Calcinosis; Cattle; Collagenases; Cross-Linking Reagents; Dermatologic Surgical Procedures; Dogs; Fixatives; Glutaral; Heparin; L Cells; Male; Materials Testing; Mice; Oxidation-Reduction; Pericardium; Polyethylene Glycols; Pulmonary Artery; Rats; Rats, Sprague-Dawley; Skin; Specimen Handling; Sulfonic Acids; Temperature; Tensile Strength

Postimplant calcific degeneration is a frequent cause of clinical failures of glutaraldehyde cross-linked porcine bioprosthetic heart valves (BPHV). It was demonstrated previously that 2-amino oleic acid (AOA) used as a bioprosthesis treatment was highly effective in mitigating aortic valve cusp but not aortic wall calcification. Our main objective was to study the efficacy of various AOA exposure conditions for inhibiting calcification of both cusps and aortic wall tissues using rat subdermal implants. BPHV tissues were treated with a saturated AOA solution for different time intervals before experimental. Aortic wall AOA levels were consistently lower than that of the cusps after the same exposure times. The diffusion of calcium ion across both cusp and aortic wall tissues was evaluated, and the results demonstrated that there was an AOA exposure time-dependent retardation of calcium ion penetration for cusp but not aortic wall. An 8-month extraction study was performed to determine the stability of AOA binding. When Tween 80 was used as an extraction medium, cusp and aortic wall retained 12.9 and 48.7%, respectively, of their initial AOA levels. AOA inhibition of calcification in rat subdermal implants (60 days) was found to be exposure time-dependent with maximum treatment time (120 h), resulting in the lowest calcium levels (20.1 +/- 10.3 and 71.4 +/- 5.4 micrograms/mg of cusp and aortic wall, respectively) as compared with controls (219.1 +/- 6.8 and 104.9 +/- 8.5 micrograms/mg of cusp and aortic wall, respectively). The significance of AOA binding on BPHV tissue was determined by either blocking or reducing BPHV's (cusp and aortic wall) free aldehyde residues with lysine or NaBH4, respectively, before AOA treatment. For aortic cusps, the AOA contents after 72 h were 98.3 +/- 2.7, 34.2 +/- 3.6, and 54.1 +/- 3.0 nM/mg of tissue for AOA (control), lysine-pretreated (plus AOA) and NaBH4-pretreated (plus AOA) tissues, respectively. However, their calcium levels after 60 days of rat subdermal implant were all comparable (i.e., 48.1 +/- 6.2, 38.2 +/- 9.1, and 47.0 +/- 15.0 micrograms calcium per mg of tissue). Similar results were observed on BPHV aortic wall. It can thus be concluded that AOA inhibition of BPHV calcification is exposure time-dependent, but the efficacy of AOA for aortic wall is less than that noted for aortic cusps, perhaps because of lower AOA binding and differences in calcium diffusion kinetics.

Topics: Animals; Aortic Valve; Bioprosthesis; Borohydrides; Calcinosis; Calcium; Diffusion; Glutaral; Heart Valve Prosthesis; Lysine; Male; Oleic Acids; Rats; Rats, Sprague-Dawley; Swine; Time Factors