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Ann Thorac Surg 2004;77:186-190
© 2004 The Society of Thoracic Surgeons
a Cryobiology Unit, Complejo Hospitalario Universitario Juan Canalejo, La Coruña, Spain
b Transplant Coordination Office, Complejo Hospitalario Universitario Juan Canalejo, La Coruña, Spain
c Unit of Experimental Surgery, Complejo Hospitalario Universitario Juan Canalejo, La Coruña, Spain
d Department of Cardiac Surgery, Complejo Hospitalario Universitario Juan Canalejo, La Coruña, Spain
e Pathology Department, Complejo Hospitalario Universitario Juan Canalejo, La Coruña, Spain
f Statistics Department, Complejo Hospitalario Universitario Juan Canalejo, La Coruña, Spain
Accepted for publication July 29, 2003.
* Address reprint requests to Dr Vázquez, Unit of Cryobiology, Carretera del Pasaje s/n, Hospital Teresa Herrera, 15006 La Coruña, Spain.
e-mail: esther_rendal{at}canalejo.cesga.es
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Abstract |
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METHODS: The tissue sources were hearts from 40 pigs with 1 hour of warm ischemic time. The aortic and pulmonary valves were dissected after 2 or 24 hours of cold ischemic time. They were stored in antibiotic solution for 20 hours at 4°C with or without an antifungal agent. The samples were cryopreserved using a programed temperature decrease method. After 1 week of storage in a liquid nitrogen tank, either in a gas or a liquid phase, the cardiac valves were slowly thawed and examined.
RESULTS: Pulmonary valves showed greater viability than aortic valves. Decreased cellular viability was observed independent of cold ischemic time, treatment with amphotericin B, or the storage method used. Treatment with or without amphotericin B had no influence on cellular viability. Conversely it was observed that there was greater cellular viability among those valves stored in a liquid phase. As far as the histologic structure of the valve is concerned we did not observe any influence either in the treatment with amphotericin B or the storage method used although it was observed that reduction of the cold ischemic time minimized histologic injury.
CONCLUSIONS: Optimization of preservation methods may decrease the negative effects of cryopreservation on cell viability and histologic structure of the valve.
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Introduction |
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It has been proposed that the durability of allograft valves depends on fibroblast viability of the valves at the time of implantation [3, 4]. Nevertheless this issue remains controversial. Some authors [5] have suggested that several tissue processing variables such as ischemic time, antibiotic disinfection, cryopreservation, and thawing methods all affect viability.
In spite of improved cryopreservation techniques, significant morphologic and metabolic changes still occur with structural changes that may lead to implant failure. The presence of fractures in the thawed grafts is another significant problem. There is no clear consensus as to which of the two methods of storage, either the gas or liquid phase, is preferable.
The purpose of the present study was to determine the influence of cold ischemic time (CIT), sterilization with or without fungicides, and storage on the viability of the valve leaflets and on the histologic structure of the arterial wall, valve leaflets, and myocardium.
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Material and methods |
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Experimental design
Each valve was randomly assigned to one of the following groups. The control group consisted of fresh heart valves obtained after dissection with 1 hour of WIT. They were not placed in antibiotic solution and were not cryopreserved. The rest of the valves were cryopreserved for 1 week. Protocols for eight study groups were designed using the following variables: (1) CIT of 2 or 24 hours; (2) with or without the addition of amphotericin B (5 µg/mL) to the antibiotic solution; (3) cryopreservation in either liquid or gas phase.
Groups were labeled as follows: group 1, 2 hours of CIT with amphotericin, gas phase; group 2, 24 hours of CIT with amphotericin, gas phase; group 3, 2 hours of CIT with amphotericin, liquid phase; group 4, 24 hours of CIT with amphotericin, liquid phase; group 5, 2 hours of CIT without amphotericin, gas phase; group 6, 24 hours of CIT without amphotericin, gas phase; group 7, 2 hours of CIT without amphotericin, liquid phase; and group 8, 24 hours of CIT without amphotericin, liquid phase.
Cryopreservation
Samples were taken from the antibiotic solution and placed in a sterile Gambro DF 700 bag containing a solution composed of 100 mL RPMI 1640 medium plus 10% dimethylsulfoxide (DMSO), maintained at 4°C for 30 minutes before cryopreservation.
Cryopreservation was carried out in a biological refrigerator (CM25 Carburos Metalicos SA, Madrid, Spain) with a programed temperature decrease of -1°C/min until a temperature of -40°C was reached, and faster rates thereafter until -150°C was reached.
Samples were then transferred to a liquid nitrogen tank, either in gas or in liquid phase, and stored for 1 week.
Valves stored in a liquid phase were transferred to a gas phase for 24 hours, kept for 10 minutes at room temperature, and then in a water bath at 37°C until completely thawed. Valves stored in a gas phase were placed at room temperature for 10 minutes and then placed in a water bath at 37°C until thawing was complete. To minimize the toxic effects of DMSO, sequential washing and progressive dilution in saline solution at 4°C removed the cryoprotectant.
Controls
In the control group each valve was first visually examined for the presence of fractures. It was then inspected microscopically and anatomicopathologic and flow cytometry studies were carried out looking for the presence of fractures, presence of myocardial wall and valve leaflet defects, and viability of the valve leaflets.
The same studies were repeated after 1 week of storage in nitrogen tanks after thawing was carried out in the study groups.
Anatomicopathologic study
For the anatomicopathologic studies tissues were fixed in 10% formol, embedded in paraffin, and dyed with hematoxylin-eosin and orcein. The structures studied were the arterial walls, valve leaflets, and myocardium.
In the arterial walls the structures studied were the endothelial cells and the elastic lamellae. The endothelial cells were subdivided into three groups: attached; partially detached, which can be attached, detached less than 50%, or detached more than 50%; and completely detached (which can be slightly or greatly detached). In the elastic lamellae, circular fragmentation ranged from none to total with an intermediate degree.
In the valve leaflets the structures studied were the endothelial cells, subdivided into three groups of attached, partially detached (less than 50% or more than 50%); elastic lamellae where circular fragmentation ranged from none to total with an intermediate degree; fibroblasts, when analysis was possible, including number of fibroblasts (few, normal, or above normal) and distribution (regular or irregular); and inflammation (acute or chronic).
In the myocardium the variables analyzed were ischemia, subdivided into three groups (focal, multifocal, or confluent); and inflammation, subdivided into two groups (acute or chronic).
Cell viability
To analyze cellular viability we initially extracted valve fibroblasts. For this purpose leaflets were removed from the valve and washed in phosphate buffered saline solution (PBS) and then minced and transferred into a digestion buffer (M199 medium, 15% heat-inactivated bovine fetal serum, penicillin [100 U/mL], streptomycin [100 µg/mL), amphotericin B [5 µg/mL], and 0.08% collagenase) and incubated on a shaker at 37°C until the fragments were digested. Cells were then washed twice in culture medium (M199 medium, 15% heat-inactivated bovine fetal serum, penicillin [100 U/mL], streptomycin [100 µg/mL], amphotericin B [5 µg/mL] before viability was determined with cytometry using propidium iodure and fluorescent diacetate (FDA). The pellet was resuspended in phosphate-buffered saline. Fluorescent diacetate was added to obtain a final concentration of 10 µmol/L and reacted with cells at room temperature for 10 minutes. Next propidium iodure was added for a final concentration of 1 µmol/L. After 3 to 5 minutes samples were analyzed by flow cytometry. Cells were scanned using argon ion laser (wavelength 488 nm). Emitted light passed through a 635-nm band-pass filter for Pianalysis. Counts negative for fluorescent diacetate and propidium iodure were classified as debris and not considered cells. Conversely those counts positive for fluorescent diacetate and negative for propidium iodure were considered viable.
Statistical analysis
Statistical analysis was performed using the logistic regression model where overall significance (p < 0.05) was attained to analyze the effect of cryopreservation and thawing on the viability of the valve leaflets and on the histologic structure of the arterial wall, valve leaflet and myocardium (SPSS statistical package for the personal computer).
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Results |
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Histology
Treatment with fungicides and the storage method used had no influence on the histologic structure of the arterial walls, myocardium, or valve leaflets either in the control or in the sample groups. However an important effect of CIT could be observed in the cryopreserved samples. Valves with a 24-hour compared with those with a 2-hour CIT showed mainly multifocal or confluent ischemia (78% versus 33%). The valve walls showed more frequently a partial detachment of the endothelium (50% versus 15%), as it was the number of valves with circular fragmentation (40% versus 15%). Figure 1
demonstrates the absence of parallelism of elastic lamellae occurring predominantly in the valves with a 24-hour CIT (40% versus 15%). Valve leaflets with a 24-hour CIT showed a greater degree of partial (40% versus 20%) and complete endothelial detachment (30% versus 10%) and the fibroblasts appeared in smaller numbers (70% versus 90%) with an irregular distribution (50% versus 30%) and a greater degree of pyknosis (45% versus 30%) when compared with those with a 2-hour of CIT (Fig 2).
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Comment |
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Cryopreserved homografts have shown favorable clinical results after implantation that seem to be attributable to viable fibroblasts present in the valve [7, 8]. Viable fibroblasts synthesize the main constituents of extracellular matrix: collagen, elastin, reticulin, and mucopolysaccharides [9]; therefore longevity of the implantation is likely related to the viability of fibroblasts in the implanted valves [10].
Nevertheless it appears that cryopreservation can damage cells and affect cell viability. The process involved in cryopreservation is relatively complex and has many variables: harvesting (warm and cold ischemic time), sterilization (antibiotics, including antifungal media for 24 hours), freezing (fluid shifts and ice crystal formation), storage, and thawing [11]. At each step there is a potential for cellular injury [12].
Controversy surrounds the importance of viable fibroblasts within the allograft leaflet matrix at the time of implantation. There is evidence that cryopreserved valves viable at the time of cryopreservation have a much lower level of structural deterioration than nonviable valves [13, 14]. In addition Yacoub and coworkers [15] observed that valves with a larger degree of fibroblast viability had an improved long-term durability.
Several methods for determining valve viability have been proposed [13, 16]. Double staining with fluorescein diacetate-propidium iodure (FDA-PI) is reported to be a rapid method for assessing cell viability compared with the trypan blue dye exclusion method [17]. Verghese and colleagues [18] define viable valves as those in which at least 50% of the fibroblasts are preserved [18]. In our study after cryopreservation viability was reduced to 56% in all groups studied independently of cold ischemic time, treatment with amphotericin B, or storage method used.
Multiple combinations of antibiotics have been used with or without antifungal drugs, to obtain a sterile graft for implantation with different results as to cellular viability. Nevertheless it has been observed that cryopreservation after a period of antibiotic sterilization significantly reduces viability and with the use of antifungal drugs (amphotericin B) this viability is reduced even further [13, 19, 20]. Amphotericin B has been removed from some valve protocols because of its negative effect on viability. Most investigators use amphotericin B at a concentration of 25 to 100 µg/mL [18, 21, 22]. In our study however viability was reduced after cryopreservation independently of the treatment with amphotericin B. This could be due to the use of a lower concentration of amphotericin B (5 µg/mL), which was considered sufficient to cover the necessary antifungal level.
The ischemic time is divided into WIT, which is defined as the interval from heart function cessation until procurement of the heart, and CIT, which is the interval between procurement and dissection. Ischemic time has been shown to affect cell viability [20, 22, 23].
In most studies the isolated influence of WIT was analyzed but Suh and associates [20] also studied the influence of CIT. They showed that CIT for 24 hours with less than 12 hours of WIT did not have a significant effect on cellular metabolism. In our study fibroblasts were well preserved with 1 hour of WIT and either 2 or 24 hours of CIT. The amount of viable cells did not seem to change significantly between 2 and 24 hours of CIT.
In the different published studies cardiac valves have been stored either in a gas or liquid phase. Although there were no reasons to think that this could have an influence on cellular viability we found a greater cell viability of valves stored in a liquid phase that in a gas phase.
A comparison between pulmonary and aortic valves cell viability has not been previously assessed. Although there was no obvious reason to suspect that there would be a significant difference [13] we found that pulmonary valves had greater cell viability than the aortic valves. This findings suggest that these valves behave differently during cryopreservation.
Regarding the histologic structure of cardiac valves, different authors were able to observe changes after cryopreservation. Mitchell and associates [24] and Vogt and associates [25] showed that cryopreserved aortic valves presented loss of surface endothelium but near-complete preservation of the elastic lamina. Armiger and associates [21] in turn expressed that in most cryopreserved valve leaflets fibroblasts showed a diminished cellularity and irregular distribution.
The histologic structure of the valves analyzed in our study revealed a remarkable effect of CIT even with a short period of WIT. Valves with a 24-hour CIT showed a greater degree of both partial and complete detachment of the endothelium in the arterial wall and in the leaflets. The fibroblasts were present in smaller numbers and with an irregular distribution and they were also more pyknotic. Finally ischemia in the myocardium was mainly of a multifocal nature and no influence was observed with or without treatment with amphotericin. We conclude that refinement in the cryopreservation techniques may improve the viability of fibroblasts and minimize histologic structure damage of the cardiac valves. The impact of the reduced postcryopreservation viability of fibroblasts and the histologic changes observed should be assessed in terms of the success and longevity of the implanted valvular grafts.
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Acknowledgments |
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