Ying C. Song

Vitreous cryopreservation maintains the function of vascular grafts

Avoidance of ice formation during cooling can be achieved by vitrification, which is defined as solidification in an amorphous glassy state that obviates ice nucleation and growth. We show that a vitrification approach to storing vascular tissue results in markedly improved tissue function compared with a standard method involving freezing. The maximum contractions achieved in vitrified vessels were >80% of fresh matched controls with similar drug sensitivities, whereas frozen vessels exhibited maximal contractions below 30% of controls and concomitant decreases in drug sensitivity. In vivo studies of vitrified vessel segments in an autologous transplant model showed no adverse effects of vitreous cryopreservation compared with fresh tissue grafts.

Mechanical Properties and Compositions of Tissue Engineered and Native Arteries

With the goal of mimicking the mechanical properties of a given native tissue, tissue engineers seek to culture replacement tissues with compositions similar to those of native tissues. In this report, differences between the mechanical properties of engineered arteries and native arteries were correlated with differences in tissue composition. Engineered arteries failed to match the strengths or compliances of native tissues. Lower strengths of engineered arteries resulted partially from inferior organization of collagen, but not from differences in collagen density. Furthermore, ultimate strengths of engineered vessels were significantly reduced by the presence of residual polyglycolic acid polymer fragments, which caused stress concentrations in the vessel wall. Lower compliances of engineered vessels resulted from minimal smooth muscle cell contractility and a lack of organized extracellular elastin. Organization of elastin and collagen in engineered arteries may have been partially hindered by high concentrations of sulfated glycosaminoglycans. Tissue engineers should continue to regulate cell phenotype and promote synthesis of proteins that are known to dominate the mechanical properties of the associated native tissue. However, we should also be aware of the potential negative impacts of polymer fragments and glycosaminoglycans on the mechanical properties of engineered tissues.

In vivo evaluation of the effects of a new ice-free cryopreservation process on autologous vascular grafts.

Conventionally cryopreserved vascular grafts have performed poorly as arterial grafts. One possible mechanism that causes the poor function is the extracellular ice damage in tissue. We used a novel new ice-free cryopreservation (namely, vitrification) method for prevention of ice formation in cryopreserved venous grafts. This study was designed to evaluate the in vivo effects of the vitrification process on autologous vascular grafts using a short-term transplantation model and to examine the morphology and patency of vitrified grafts in correlation with control grafts. New Zealand White rabbits underwent a right common carotid interposition bypass graft. Fresh and vitrified reversed ipsilateral external jugular veins were used as autologous grafts. Animals were sacrificed at either 2 or 4 weeks after implantation, and fresh and vitrified vein grafts were harvested for histology studies. The results, comparing the patency of fresh and vitrified grafts, demonstrated similar short-term patency rates (approximately 90%). There were no signs of media disruption, aneurysm, or graft stenosis in vitrified vein grafts. Vitrification had not altered the pathophysiological cascade of events that occur when a vein graft is inserted into the arterial system. The vitrification process had no adverse effects locally or systemically in vivo. In addition, vitrification has preserved endothelial cell and smooth muscle cell integrity posttransplantation. In conclusion, this study, using an autologous animal model, clearly demonstrated a significant benefit of vitrification for preservation of graft function, and vitrification may be an acceptable approach for preservation of blood vessels or engineered tissue constructs.

Vitreous Preservation of Rabbit Articular Cartilage

The growing need for improved methods of viable tissue cryopreservation has stimulated debate regarding the relative merits of traditional freezing methods versus ice-free vitrification methods. Articular cartilage has proved refractory to satisfactory cryopreservation using conventional freezing methods. Therefore, full-thickness rabbit femoral head articular cartilage was used to compare a freezing method of cryopreservation and an ice-free, vitrification method of cryopreservation with fresh controls in vitro. Chondrocyte viability was determined in vitro using vital fluorescent staining with calcein-acetoxymethyl ester and an oxidation-reduction assay with alamarBlue™ (Accumed International, Westlake, Ohio). Cryosubstitution with 1% osmium tetroxide in 100% methanol was used to detect ice during cryopreserved storage. Cryosubstitution studies of frozen and vitrified articular cartilage pieces revealed negligible ice in the vitrified specimens and extensive ice formation in frozen specimens with the ex...

Vitrification of porcine articular cartilage

The limited availability of fresh osteochondral allograft tissues necessitates the use of banking for long-term storage. A vitrification solution containing a 55% cryoprotectant formulation, VS55, previously studied using rabbit articular cartilage, was evaluated using porcine articular cartilage. Specimens ranging from 2 to 6 mm in thickness were obtained from 6mm distal femoral cartilage cores and cryopreserved by vitrification or freezing. The results of post-rewarming viability assessments employing alamarBlue demonstrated a large decrease (p<0.001) in viability in all three sizes of cartilage specimen vitrified with VS55. This is in marked contrast with prior experience with full thickness, 0.6 mm rabbit cartilage. Microscopic examination following cryosubstitution confirmed ice formation in the chondrocytes of porcine cartilage vitrified using VS55. Experiments using a more concentrated vitrification formulation (83%), VS83, showed a significant treatment benefit for larger segments of articular cartilage. Differences between the VS55 and the VS83 treatment groups were significant at p<0.001 for 2 mm and 4 mm plugs, and at p<0.01 for full thickness, 6 mm plugs. The percentage viability in fresh controls, compared to VS55 and VS83, was 24.7% and 80.7% in the 2 mm size group, 18.2% and 55.5% in the 4 mm size group, and 5.2% and 43.6% in the 6 mm group, respectively. The results of this study continue to indicate that vitrification is superior to conventional cryopreservation with low concentrations of dimethyl sulfoxide by freezing for cartilage. The vitrification technology presented here may, with further process development, enable the long-term storage and transportation of living cartilage for repair of human articular surfaces.

Mechanisms of bioprosthetic heart valve calcification1

Background. Cryopreserved human heart valves are used in approximately 20% of the tissue heart valve procedures performed annually. The pathophysiology of allograft failure is not fully understood. The authors proposed the hypothesis that the rapid deterioration observed in some allograft heart valve recipients is caused by disruptive interstitial ice damage that occurs during cryopreservation and subsequently leads to accelerated valve degeneration on implantation. Methods. This hypothesis was tested by comparison of the standard commercial heart valve freezing method of cryopreservation and an ice-free, vitrification method of cryopreservation with fresh controls in a subcutaneous, juvenile rat implant model of calcification. Calcium concentration in explants was determined by atomic absorption spectroscopy. Results. Statistically significant calcification (P <0.05) was observed in both syngeneic and allogeneic cryopreserved valves relative to fresh valves. The ice-free cryopreservation method demonstrated significant reduction of allogeneic heart valve calcification (P <0.01). Comparison of fresh syngeneic and allogeneic grafts at the 3-week time point demonstrated significantly higher calcium content in allograft valve explants (P <0.005). Conclusions. These findings demonstrate that allogeneic valve calcification is influenced by two factors, the cryopreservation method used and immunogenicity. Alternative cryopreservation methods that avoid ice formation may improve the in vivo performance of cryopreserved allogeneic heart valves.