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Background: Decellularized allograft tissues have been identified as a potential extracellular matrix (ECM) scaffold on which to base recellularized tissue-engineered vascular and valvular substitutes. Decreased antigenicity and the capacity to recellularize suggest that such constructs may have favorable durability. Detergent/enzyme decellularization methods remove cells and cellular debris while leaving intact structural protein "scaffolds." Allograft pulmonary artery tissues decellularized with an anionic detergent/enzyme methodology were tested in a long-term implantation model that used arterial wall repairs in the great vessels of juvenile sheep., Methods: Twelve test sheep were implanted (n = 4) for each of three different scaffold protocols that compared traditional dimethylsulfoxide cryopreservation, cryopreservation followed by decellularization, and decellularization of fresh tissue. Four additional sheep served as controls (n = 2 sham, n = 2 fresh tissue). Patches were fashioned and implanted into pulmonary artery and aortic defects. Panel reactive antibodies (PRA) were measured over time (10 to 20 weeks). Explant histopathology determined recellularization morphology as well as calcium, collagen, and elastin distribution within explanted tissue., Results: Unlike traditionally cryopreserved tissues, the decellularized tissues contained no residual cells or cellular debris before implantation, which correlated with measurable reductions in PRA. Decellularized explants demonstrated time-dependent migration of recipient cells through matrix, typically staining positive for alpha-smooth muscle actin with no calcification., Conclusions: The properties demonstrated seem consistent with characteristics necessary for implantable tissue-engineered scaffolds. The decellularization method described appears to create a biologically suitable ECM scaffold for in vivo migration of phenotypically appropriate cells while avoiding antigenicity and calcification.

The time-dependent penetration of the cryoprotectant dimethylsulfoxide (DMSO) into porcine aortic conduit and the effectiveness of dilutional removal of cryoprotectant from these tissues were evaluated using high performance liquid chromatography. Incubation of intact conduit tissues in 1.4 mol/liter (10%) DMSO at 4 degrees C revealed that the cryoprotectant penetrated the tissues in a nonlinear time-dependent manner requiring 80 to 120 min to achieve a tissue concentration of approximately 0.7 mol/liter tissue water volume. This concentration approximated 54% of the theoretical media concentration (1.3 mol/liter) occurring through dilution effects of addition of the tissue to the medium. The osmolality of the medium decreased linearly in a time-dependent manner from 2975 to 2860 mOsmol/kg water over 80 min for a negative slope of 1.28 mOsmol/kg water/min. Analysis of the concentrations of DMSO in tissues undergoing dilutional removal at 4 degrees C revealed that dilutional removal at low temperatures may not be an effective means of removing DMSO from cryopreserved cardiovascular tissues. It requires approximately 40 to 50 min at 4 degrees C for tissue concentrations of DMSO to be reduced to 0.24 mol/liter tissue water volume, which is far in excess of the concentration expected to be present in the tissue and suspending solution (< 0.03 mol/liter media) at equilibrium. It is suggested that protocols for removal of DMSO from thawed allograft heart valves which do not allow the temperature to increase above 4 degrees C during the removal process may not provide sufficient incubation time to permit the cryoprotectant (DMSO) to diffuse from the tissues during a clinical post-thawing preparation for clinical transplantation.

Quantitative analysis of proteoglycans (PGs) revealed that the content of PG material from cryopreserved aorta, measured as uronate-positive material, was similar to that from fresh tissue (440 +/- 30 versus 430 +/- 7 micrograms/g wet tissue). Gel permeation column chromatography studies suggested that three PG fractions from cryopreserved tissue had molecular weights similar to PG fractions from fresh tissue; K(av) = 0.13, 0.47 (I), 0.20 (II), and 0.43 (III) from cryopreserved tissue and K(av) = 0.13, 0.50 (I), 0.23 (II), and 0.40 (III) from fresh tissue. Sequential extraction of tissue with guanidine-HCl (Gdn-HCl) followed by digestions with collagenase, elastase, and papain also demonstrated that there was no difference between fresh and cryopreserved tissues in the distribution of PGs in the extracts. Transmission electron microscopy analysis revealed less densely packed collagen fibers in cryopreserved tissues compared to fresh tissues. These studies indicate that there is no significant alteration in the content, molecular size, or distribution of PGs in properly cryopreserved tissue.

Cryopreservation and storage of human heart valves have become an accepted means of maintaining a usable supply of heart valves for outflow track reconstructive surgery. Valves are typically stored at the vapor phase temperature of liquid nitrogen, -130 degrees C and below, to reduce the chance of recrystallization within the tissues. Concern over the effects of submersion of the valves in liquid nitrogen, i.e., plunging to -196 degrees C, prompted this study. Cryopreserved valves were plunged into liquid nitrogen, held for 5 min, and then processed (thawed) by standardized protocols. The thawed valves were then assessed using scanning electron microscopy and the more traditional histology at the light microscope level. Cuspal tissues plunged into liquid nitrogen appear to have numerous microfractures over both surfaces of the tissue, penetrating into the collagen/proteoglycan matrix. Control cryopreserved valves do not exhibit these microfractures. Histologically, the submerged valves appear normal. The clinical use of valves which have been submerged in liquid nitrogen is discussed.