Albert E. Heacox

Retained structural integrity of collagen and elastin within cryopreserved human heart valve tissue as detected by two-photon laser scanning confocal microscopy ☆

Cryopreservation is commonly used for the long-term storage of heart valve allografts. Despite the excellent hemodynamic performance and durability of cryopreserved allografts, reports have questioned whether cryopreservation affects the valvular structural proteins, collagen and elastin. This study uses two-photon laser scanning confocal microscopy (LSCM) to evaluate the effect of cryopreservation on collagen and elastin integrity within the leaflet and conduit of aortic and pulmonary human heart valves. To permit pairwise comparisons of fresh and cryopreserved tissue, test valves were bisected longitudinally with one segment imaged fresh and the other imaged after cryopreservation and brief storage in liquid nitrogen. Collagen was detected by second harmonic generation (SHG) stimulation and elastin by autofluorescence excitation. Qualitative analysis of all resultant images indicated the maintenance of collagen and elastin structure within leaflet and conduit post-cryopreservation. Analysis of the optimized percent laser transmission (OPLT) required for full dynamic range imaging of collagen and elastin showed that OPLT observations were highly variable among both fresh and cryopreserved samples. Changes in donor-specific average OPLT in response to cryopreservation exhibited no consistent directional trend. The donor-aggregated results predominantly showed no statistically significant change in collagen and elastin average OPLT due to cryopreservation. Since OPLT has an inverse relationship with structural signal intensity, these results indicate that there was largely no statistical difference in collagen and elastin signal strength between fresh and cryopreserved tissue. Overall, this study indicates that the conventional cryopreservation of human heart valve allografts does not detrimentally affect their collagen and elastin structural integrity.

Structural integrity of collagen and elastin in SynerGraft® decellularized-cryopreserved human heart valves.

SynerGraft® (SG) decellularized-cryopreserved cardiac valve allografts have been developed to provide a valve replacement option that has reduced antigenicity, retained structural integrity, and the ability to be stored long-term until needed for implantation. However, it is critical to ensure that both the SG processing and cryopreservation of these allografts do not detrimentally affect the extracellular matrix architecture within the tissue. This study evaluates the effects of SG decellularization and subsequent cryopreservation on the extracellular matrix integrity of allograft heart valves. Human aortic and pulmonary valves were trisected, with one-third of each either left fresh (no further processing after dissection), decellularized, or decellularized and cryopreserved. Two-photon laser scanning confocal microscopy was used to visualize collagen and elastin in leaflets and conduits. The optimized percent laser transmission (OPLT) required for full dynamic range imaging of each site was determined, and changes in OPLT were used to infer changes in collagen and elastin signal intensity. Collagen fiber crimp period and collagen and elastin fiber diameter were measured in leaflet tissue. Statistically significant differences in OPLT and the dimensional characteristics of collagen and elastin in study groups were determined through single factor ANOVA. The majority of donor-aggregated average OPLT observations showed no statistically significant differences among all groups, indicating no difference in collagen or elastin signal strength. Morphometric analysis of collagen and elastin fibers revealed no significant alterations in treated leaflet tissues relative to fresh tissues. Collagen and elastin structural integrity within allograft heart valves are maintained through SynerGraft® decellularization and subsequent cryopreservation.

Factors affecting the viability of cryopreserved allograft heart valves

Allograft heart valves have been used for more than 25 years as replacements for diseased aortic valves and repair of congenital abnormalities. In many instances, particularly paediatric surgery, it is the valve of choice due to its non-obstructive flow, relative freedom from calcification and thromboembolism without anticoagulation therapy. In order to permit valve size matching for recipients, it is necessary to find methods of storage which will maintain cellular viability. The best method which allows infinite, convenient storage is cryopreservation.

Impact of γ-Irradiation on Extracellular Matrix of Porcine Pulmonary Valves

BACKGROUND The extracellular matrix plays an important role in heart valve function. To improve the processing of porcine pulmonary valves for clinical use, we have studied the influence of cryopreservation, decellularization, and irradiation on extracellular matrix components. METHODS Decellularization was carried out followed by DNAseI/RNAseA digestion and isotonic washout. Valves were cryopreserved in 10% DMSO/10% fetal bovine serum, and then subjected to 25-40 kGy γ-radiation. Extracellular matrix constituents were evaluated by histologic staining, immunohistochemistry, transmission electron microscopy, and liquid chromatography/mass spectrometry. RESULTS Histologic, immunohistochemical, ultrastructural, and biochemical analyses demonstrated a marked reduction in the expression of extracellular matrix components particularly in the valves that had been γ-irradiated following decellularization and cryopreservation. In this group, histology and immunohistochemistry showed an obvious reduction in staining for chondroitin sulphates, versican, hyaluronan, and collagens. Transmission electron microscopy revealed the smallest fibril diameter of collagen, shortest D-period, and loss of compactness of collagen fiber packaging and fragmentation of elastic fibers. Biochemical analysis showed loss of collagen and elastin crosslinks. Decellularization followed by cryopreservation showed some reduction in staining for collagens and versican, smaller diameter, shorter D-period in collagen fibers, and ridges in elastic fibers. Cryopreservation alone showed minimal changes in ECM staining intensity, collagen, and elastin ultrastructure and biochemistry. CONCLUSION γ-Irradiated valves that have been decellularized and cryopreserved produces significant changes in the expression of ECM components, thus providing useful information for improving valve preparation for clinical use and also some indication as to why irradiated human heart valves were not clinically successful.

Short-term follow-up of cryopreserved allograft valves and valved conduits from the CryoLife clinical registry

CryoLife, a laboratory which specializes in the cryopreservation of human tissues for transplant, first started preserving valves in Autumn 1984. The hearts which arrive at CryoLife are currently procured by 144 independent organ and tissue procurement agencies in the United States, Canada and West Germany. This procured tissue is preserved by CryoLife as a service to those agencies or doctors to whom the tissue has been assigned. In order to assure consistent quality control, all agencies follow the guidelines printed in the CryoLife (3) heart valve procurement protocol. The protocol provides information for screening for infectious diseases and diseases of the heart which would render the tissue less than ideal. For instance, when the required serum sample is returned by the procuring group, tests are performed for HIV (AIDS), hepatitis, syphilis and CMV (cytomegalovirus). These internal tests are compared with pretest data which accompanies the organ and are used as a double check to confirm that the possibility of transmission of these diseases is eliminated. The donor form also contains a detailed medical history of the donor, which helps eliminate donors of questionable standards or with a history of cardiac complications. The protocol also contains the precise details for the surgical extraction of the heart so that uniformity amongst participating centres is maintained.