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.

Functional improvement of infarcted heart by co-injection of embryonic stem cells with temperature-responsive chitosan hydrogel.

Transplantation of embryonic stem cells (ESCs) can improve cardiac function in treatment of myocardial infarction. The low rate of cell retention and survival within the ischemic tissues makes the application of cell transplantation techniques difficult. In this study, we used a temperature-responsive chitosan hydrogel (as scaffold) combined with ESCs to maintain viable cells in the infarcted tissue. Temperature-responsive chitosan hydrogel was prepared and injected into the infarcted heart wall of rat infarction models alone or together with mouse ESCs. The result showed that the 24-h cell retention and 4 week graft size of both groups was significantly greater than with a phosphate buffered saline control. After 4 weeks of implantation, heart function, wall thickness, and microvessel densities within the infarct area improved in the chitosan + ESC, chitosan, and ESC group more than the PBS control. Of the three groups, the chitosan + ESC performed best. Results of this study indicate that temperature-responsive chitosan hydrogel is an injectable scaffold that can be used to deliver stem cells to infarcted myocardium. It can also increase cell retention and graft size. Cardiac function is well preserved, too.

Cryoprotectant delivery and removal from murine insulinomas at vitrification-relevant concentrations.

Development of optimal cryopreservation protocols requires delivery and removal of cryoprotective agents (CPAs) in such a way that negative osmotic and cytotoxic effects on cells are minimized. This is especially true for vitrification, where high CPA concentrations are employed. In this study, we report on the determination of cell membrane permeability parameters for water (L(p)) and solute (P(s)), and on the design and experimental verification of CPA addition and removal protocols at vitrification-relevant concentrations for a murine insulinoma cell line, betaTC-tet cells. Using membrane permeability values and osmotic tolerance limits, mathematical modeling and computer simulations were used to design CPA addition and removal protocols at high concentrations. The cytotoxic effects of CPAs were also evaluated. Cells were able to tolerate the addition and removal of 2.5M dimethyl sulfoxide (DMSO) and 2.5M 1,2 propanediol (PD) in single steps, but required multi-step addition and removal with 3.0M DMSO, 3.0M PD, and a vitrification-relevant concentration of 3.0M DMSO+3.0M PD. Cytotoxicity studies revealed that betaTC-tet cells were able to tolerate the presence of single component 6.0M DMSO and 6.0M PD and to a lesser extent 3.0M DMSO+3.0M PD. These results determine the time and concentration domain of CPA exposure that cells can tolerate and are essential for designing cryopreservation protocols for free cells as well as cells in engineered tissues.

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.

Feasibility of vitrification as a storage method for tissue-engineered blood vessels.

It is well established that, in multicellular systems, conventional cryopreservation results in damaging ice formation, both in the cells and in the surrounding extracellular matrix. As an alternative to conventional cryopreservation, we performed a feasibility study using vitrification (ice-free cryopreservation) to cryopreserve tissue-engineered blood vessels. Fresh, frozen, and vitrified tissue-engineered blood vessels were compared using histological methods, cellular viability, and mechanical properties. Cryosubstitution methods were used to determine the location of ice in conventionally cryopreserved engineered vessels. Ice formation was negligible (0.0 +/- 0.0% of vessel area) in the vitrified specimens, and extensive (68.3 +/- 4.5% of vessel area) in the extracellular matrix of frozen specimens. The metabolic assay and TUNEL staining results indicated that vitrified tissue had similar viability to fresh controls. The contractility results for vitrified samples were >82.7% of fresh controls and, in marked contrast, the results for frozen samples were only 10.7% of fresh controls (p < 0.001). Passive mechanical testing revealed enhanced tissue strength after both freezing and vitrification. Vitrification is a feasible storage method for tissue-engineered blood vessel constructs, and their successful storage brings these constructs one step closer to clinical utility.

Vitreous preservation of articular cartilage grafts.

Articular cartilage has proved refractory to satisfactory cryopreservation using conventional freezing methods. Therefore, an ice-free cryopreservation method by vitrification was tested. Osteochondral plugs from New Zealand White rabbits were preserved using either a freezing method or an ice-free vitrification method of cryopreservation. Preserved and fresh control plugs were implanted in the tibial plateau of allogeneic recipients. A modified O'Driscoll grading scale, based on gross pathology, histopathology, and histochemistry, was used to evaluate the explants.The histology of fresh and vitrified explants was essentially the same, while the frozen cryopreserved explants were devoid of chondrocytes and only fibroblastlike cells were observed. The O'Driscoll grading indicated that both fresh and vitrified plugs performed significantly better than frozen plugs (p < or =.05). The results demonstrate the feasibility of vitrification as a storage method for cartilaginous tissues.

Morphological analyses of ice-free and frozen cryopreserved heart valve explants.

BACKGROUND AND AIM OF THE STUDY: The pathophysiology of allogeneic heart valve failure is not fully understood. It is hypothesized that the rapid deterioration seen in some allograft heart valve recipients is due to disruptive interstitial ice damage that occurs during cryopreservation by freezing. METHODS: The hypothesis was tested by comparing a standard commercial heart valve freezing and ice-free, vitrification cryopreservation methods with fresh controls in: (i) a subcutaneous, juvenile rat implant model of calcification; and (ii) a descending thoracic aorta implant rat model for histopathology. Calcium concentrations in one- to six-week explants were determined using atomic absorption spectroscopy; calcification rates were also determined. RESULTS: The calcification rate of frozen valves was significantly greater (p < 0.01) than that of vitrified valves in both syngeneic and allogeneic recipients, supporting prior observations that ice-free cryopreservation reduces allogeneic heart valve calcification. Cryopreservation by freezing and vitrification resulted in mild morphological changes in two- and four-week explants, a slight decrease in leaflet cellularity, and a more rapid onset of intimal hyperplasia than in fresh valve explants. The allograft explant groups exhibited similar changes, regardless of how the valves were processed. CONCLUSION: These findings provide only weak support for the tested hypothesis, and further studies in a large animal model are warranted.

Mechanisms of bioprosthetic heart valve calcification.

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.