Treatment of vascular graft infection by in situ replacement with cryopreserved aortic allografts: An experimental study

Purpose: The purposes of this study were to prove the efficacy of cryopreserved aortic allografts to treat an established vascular graft infection by in situ replacement in an animal model and to evaluate the role of the antibiotics normally used to decontaminate the allografts. Methods: Twenty-three dogs underwent infrarenal aortic replacement with a gelatin-sealed knitted polyester graft contaminated in vitro by staphylococcus epidermidis RP-62. One week later, the 18 surviving animals underwent reoperation for graft removal and were randomized into three groups for in situ replacement: group I (control, n = 6) received a new gelatin-sealed graft; group II (n = 6) received a non–antibiotic-treated cryopreserved allograft; and group III (n = 6) received an antibiotic-treated cryopreserved allograft. Control grafts and allografts were removed 4 weeks after the initial intervention for quantitative bacteriologic analysis and histologic analysis. Bacteriologic results were expressed as colony-forming units per square centimeter of graft material. Qualitative bacteriologic analysis was also obtained from perigraft fluid and tissue. Results: All of the initially implanted grafts and all of the control grafts (group I) were infected at the time of removal. In group II, three out of six allografts were not totally incorporated, whereas in group III incorporation was always complete, with a significantly decreased inflammatory reaction. All of the antibiotic-treated allografts were sterile, whereas three untreated allografts grew bacteria. Conclusions: In this model, cryopreserved aortic allografts were more resistant to reinfection than synthetic grafts after in situ replacement of an infected prosthetic graft. However, the antibiotic loading of the cryopreserved aortic allograft appears to be essential to obtain optimal therapeutic effects. (J Vasc Surg 1998;27:689-98.)     

Prognosis after aortic root replacement with cryopreserved allografts in adults

BACKGROUND: Aortic root replacement with cryopreserved allografts is associated with excellent hemodynamics, little endocarditis, low thromboembolic event rates, and no need for anticoagulation. There is, however, concern regarding the long-term durability of this valve substitute, especially in younger patients. Meta-analysis and microsimulation were used to calculate age-specific long-term prognosis after allograft aortic root replacement based on current evidence. METHODS: Our center's experience with cryopreserved allograft aortic root replacement in 165 adult patients was combined in a meta-analysis with reported and individual results from four other hospitals. Using this information, the microsimulation model predicted age- and gender-specific total and reoperation-free and event-free life expectancy. RESULTS: The pooled results comprised 629 patients with a total follow-up of 1860 patient-years (range 0 to 12.8 years). Annual risks were 0.6% for thromboembolism, 0.05% for bleeding, 0.5% for endocarditis, and 0.5% for nonstructural valve failure. Structural allograft failure requiring reoperation occurred in 15 patients, and a patient age-specific Weibull function was constructed accordingly. Calculated total life expectancy varied from 27 years in a 25-year-old to 12 years in a 65-year-old male; corresponding actual lifetime risk of reoperation was 89% and 35%, respectively. CONCLUSIONS: Cryopreserved aortic allografts have an age-related limited durability. This results in a considerable lifetime risk of reoperation, especially in young patients. The combination of meta-analysis and microsimulation provides an appropriate tool for estimating individualized long-term outcome after aortic valve replacement and can be useful both for patient counseling and prognostic research purposes.     

Early results of cryopreserved pulmonary allografts as aortic valve substitute.

Excellent clinical results with pulmonary autografts and experimental evidence that the pulmonary valve can withstand the higher stress in the systemic circulation led us to use the cryopreserved pulmonary allograft for aortic valve replacement. From September 1988 to March 1991, 45 consecutive patients (59.9 +/- 12.0 years, 25 men and 20 women) received a cryopreserved pulmonary allograft in the aortic position from our hospital based valve bank. All allografts were inserted freehand in the subcoronary position. There were 3 in-hospital deaths (7%) and 1 patient had severe valvular incompetence immediately postoperatively requiring reoperation after 4 weeks. Forty-one patients were followed at 3-6 month interval for 14.7 +/- 7.8 months (3-28 months) and valve performance was assessed routinely by means of color flow Doppler echocardiography: 34 patients (83%) had no or trivial aortic valve regurgitation. Valvular incompetence class II was present in 2 patients (5%) whereas 3 (7%) demonstrated class II-III. Severe aortic regurgitation (class III-IV) could be detected in 2 patients (5%). Both had to undergo reoperation 4 months and 15 months, respectively, postoperatively. Macroscopic and histological evaluation of the explanted valves demonstrated absence of significant degeneration. We assume that a mismatch in size between allograft and aortic annulus could have lead to dilatation of the allograft valve ring and consequently to valvular incompetence. Pulmonary cryopreserved allografts achieve acceptable short-term results which can be improved if initial technical problems can be avoided.     

Mechanical testing of cryopreserved aortic allografts. Comparison with xenografts and fresh tissue

Reports indicate that cryopreserved aortic valve allografts have a better long-term survivability than other bioprostheses, such as the porcine xenograft. Unlike xenografts, allograft valves do not require treatment with glutaraldehyde and may therefore retain much of their original mechanical function. The effects of cryopreservation on the mechanical integrity of collagen fibers and mucopolysaccharides, however, are still largely unknown. We therefore compared the mechanical behavior of cryopreserved allograft leaflet material to that of fresh tissue and xenografts by measuring their bending stiffness (nine strips of tissue) and their uniaxial tensile stress/strain and stress/relaxation behavior (six strips of each tissue type). The bending tests showed no significant difference between the pliability of cryopreserved allografts and fresh pig aortic valve tissue, but the xenograft material was significantly stiffer than both (p less than 0.001). The mean circumferential tensile elastic moduli of the allografts, fresh tissue, and xenografts at a stress of 300 kPa were 9.1 +/- 5.4 MPa, 13.0 +/- 1.7 MPa, and 12.5 +/- 3.0 MPa, respectively, and were not significantly different from each other. We also found that the transition from a low to a high modulus on the stress/strain curves, a measure of extensibility, occurs at 23%, 22%, and 12% strain for the three materials. There was no significant difference between the allograft and the fresh tissues, but the xenograft material was less extensible than the other two (p less than 0.001). The xenograft tissue also had significantly lower rates of stress relaxation than the other two materials (p less than 0.005). Thus no detectable differences were found between the mechanical behavior of the cryopreserved allograft aortic leaflets and fresh tissue, whereas the xenograft material was less extensible and less capable of relaxing than both the allograft and fresh tissue. The ability of allografts valves to respond to tensile and flexural stresses in a manner similar to that of the natural aortic valve may therefore contribute to their good in vivo survivability.     

Technical details with the use of cryopreserved arterial allografts for aortic infection: influence on early and midterm mortality.

PURPOSE: In situ repair with cryopreserved vascular allografts improves the results in the surgical treatment of aortic infection. This study evaluated the technical pitfalls with the use of allografts that influence early and midterm mortality. METHODS: Between 1990 and 1999, 49 patients, 21 (43%) with a mycotic aneurysm and 28 (57%) with a prosthetic graft infection of the thoracic and abdominal aorta including pelvic and groin vessels, underwent in situ repair with cryopreserved arterial allografts. Seventeen patients (35%) had aortobronchial, aortoesophageal, or aortoenteric fistulas. RESULTS: Allograft-related technical problems occurred in eight patients (16%) in this series, and they included: intraoperative rupture caused by allograft friability; allograftenteric fistula from ligated allograft side branches rupturing 8, 18, and 48 months after implantation; anastomotic failure caused by inappropriate mechanical stress; anastomotic stricture after partial replacement of infected prosthetic grafts; allograft failure caused by inappropriate wound drainage; and recurrence of infection after inappropriate duration of antifungal treatment. Seven of the eight technical problems (87%) occurred in the first 10 patients (80%) in this series. There was one technical failure in the remaining 39 patients (2.6%; P =.0002) because of various technical adaptations, such as critical selection of allografts, use of allograft strips supporting large anastomoses, sealing with antibiotic-impregnated fibrin glue, and change in technique of allograft side-branch ligature. The 30-day mortality rate was 6% for the whole series; however, it was 2.6% for last 39 patients, with no recurrence of infection or allograft-related late death. CONCLUSIONS: In situ repair with cryopreserved arterial allografts achieves excellent early and late results in the treatment of aortic infection. However, distinct allograft-related technical problems had to be overcome to improve the outcome of patients with major vascular infections.     

Immunogenicity of decellularized cryopreserved allografts in pediatric cardiac surgery: comparison with standard cryopreserved allografts

BACKGROUND: Recognition of the immunogenicity of standard cryopreserved allografts has led to the development of new decellularized allografts (CryoValve SG; CryoLife, Inc, Kennesaw, Ga). This preliminary study examined the HLA antibody response to these decellularized allografts and compared it with the response to standard allograft material. METHODS: We prospectively measured the frequency of panel-reactive HLA class I (HLA-A, HLA-B, and HLA-C) and class II (HLA-DR/DQ) alloantibodies in 14 children (age 8.5 +/- 7.9 years) receiving decellularized, cryopreserved allografts, including 6 undergoing allograft patch insertion and 8 with a valved pulmonary allograft. We compared them with 20 historical control subjects (age 1.7 +/- 2.4 years) undergoing implantation of standard cryopreserved allografts, 8 with valves and 12 with allograft patch. All patients had panel-reactive antibody levels measured before and at 1, 3, and 12 months after the operation. HLA class I and class II panel-reactive antibody levels were determined with a sensitive flow cytometry technique. RESULTS: We found panel-reactive antibody levels in decellularized allografts to be elevated slightly from preoperative levels for both class I and class II antibodies at 1, 3, and 12 months (P >.05). The panel-reactive antibody level for both class I and class II antibodies were significantly lower for decellularized allografts as compared to standard allografts. Functionally, the allografts were similar with decellularized valved grafts showing a peak echo-determined systolic gradient of 13 +/- 15 mm Hg at 8 +/- 2.6 months postoperatively as compared to a gradient of 24 +/- 18 mm Hg measured 12 +/- 6 months postoperatively in standard allografts (P =.11). CONCLUSIONS: Decellularized grafts elicited significantly lower levels of class I and class II HLA antibody formation at 1, 3, and 12 months after implantation than did standard cryopreserved allografts. Early hemodynamic function of decellularized grafts was similar to that of standard cryopreserved allograft valves. Further experience is necessary to determine whether the reduced immunogenicity of decellularized allografts will truly allow tissue ingrowth and improved long-term durability in patients.     

Successful tracheal transplantation using cryopreserved allografts in a rat model.

OBJECTIVES: The purpose of this study was to determine the appropriate cryopreservation period of tracheal allografts based on morphological and immunological findings and to test the possibility of tracheal transplantation in rats using cryopreserved allografts without immunosuppression. METHODS: Morphological and immunological studies were performed to compare the differences between non-cryopreserved grafts and cryopreserved grafts. Orthotopic tracheal transplantation using cryopreserved allografts, non-cryopreserved allografts, and non-cryopreserved autografts was performed and the rejection score of each group was evaluated. RESULTS: Epithelial cells were lost when the grafts were cryopreserved for more than 20 days. Immunohistochemical staining of the trachea revealed that the MHC classII antigen was expressed on normal epithelium. These findings suggest that cryopreservation for more than 20 days decreased the antigeneicity of allografts because of epithelial desquamation. All rats that received allografts cryopreserved for more than 20 days survived until the scheduled sacrifice day. Microscopically, cryopreserved allografts that had been preserved for more than 20 days had a significantly lower rejection score than that of non-cryopreserved allografts (P < 0.05). CONCLUSIONS: We conclude that the appropriate period for cryopreservation of allografts would be 20 days or more, because cryopreservation for more than 20 days depleted epithelium, which possessed the MHC classII antigen. Therefore, a longer period of cryopreservation decreases the antigeneicity of allografts. Rat tracheal transplantations using cryopreserved allografts is possible without immunosuppression when the grafts have been cryopreserved for more than 20 days.     

Transplantation of cryopreserved osteochondral Dowel allografts for repair of focal articular defects in an ovine model.

The purpose of this study was to test whether successful cryopreservation of osteochondral tissue is possible and whether, with the appropriate surgical procedure, it can be used for the successful repair of focal articular defects within joints. Fresh (nonfrozen) and snap-frozen (plunged in liquid nitrogen and thawed in a water bath at 37 degrees C, repeated three times) autografts were used as positive and negative controls, respectively. Snap-frozen, frozen (fresh tissue placed in a freezer at -80 degrees C), and cryopreserved (immersed in 10% dimethyl sulfoxide for 30 minutes and then frozen at 1 degrees C/min to -80 degrees C) allografts were transplanted into the knees of adult sheep. Outcomes were evaluated 3, 6, and 12 months after transplantation. The morphological, histological, biochemical, and biomechanical behaviors and characteristics of the graft cartilage, the host cartilage adjacent to the grafts, and the opposing tibial cartilage were assessed. Freezing protocols that yielded poor chondrocyte recovery after thawing (frozen and snap-frozen) resulted in poor overall graft outcome. The cryopreservation protocol, however, resulted in intermediate recovery (50%) of chondrocytes and in intermediate overall graft outcome compared with fresh autografts. The membrane integrity of the allograft chondrocytes immediately following cryopreservation was identified as the most reliable predictor of long-term outcome of the graft. Further improvements in cryopreservation technique may lead to an effective method of banking osteochondral tissue for successful transplantation for the repair of focal defects and larger joint reconstructions.     

Studies in thoracic aortic graft infections: the development of a porcine model and a comparison of collagen-impregnated dacron grafts and cryopreserved allografts.

OBJECTIVE: A porcine model of thoracic aortic graft infection was created, and various anatomic sites and the timing of inoculation of the graft to induce infection were investigated. Ultimately, the ability of cryopreserved allograft to resist infection was compared with that of collagen-impregnated Dacron graft. METHODS: Yorkshire pigs (n = 16) underwent placement of an expanded polytetrafluoroethylene patch graft in the ascending aorta and the left atrial appendage (phase I). Eight animals were immediately given a 50-mL bolus (1 x 10(8) cfu/mL) of Staphylococcus aureus whereas the other 8 received the infusion 24 hours later. Animals were put to death 8 weeks later and the grafts were sterilely explanted and analyzed via microbiologic culture and standard histologic procedures for evidence of infection. The results displayed that the aortic graft and a delay of induced bacteremia of 24 hours were more reliable methods of producing infection. During phase II, 13 pigs were randomized to receive either a collagen-impregnated Dacron graft (n = 6) or a cryopreserved allograft (n = 7) in the ascending aortic position only and infusion of S aureus 24 hours after the operation. The experiment then proceeded to completion. RESULTS: Phase I results displayed that use of an aortic graft and induced bacteremia 24 hours after the operation was a more reliable and reproducible method of producing infection. In phase II, graft infection was present in 38.5% (5/13) of animals, with only 16.7% (1/6) in the collagen-impregnated Dacron graft group and 57.2% (4/7) in the cryopreserved allograft group becoming infected. There was no significant difference between the collagen-impregnated Dacron graft and cryopreserved allograft groups in the incidences of thoracic aortic graft infections (P =.27, Fisher exact test). CONCLUSIONS: This novel porcine model of thoracic aortic graft infection is a reproducible method for the investigation of thoracic aortic graft infections. The phase I study investigated the timing of the induced bacteremia and the most susceptible position of a graft. Phase II demonstrated that collagen-impregnated Dacron grafts are equivalent, if not superior, to cryopreserved allografts in resisting central vascular graft infections in the ascending aorta.     

Results of endoluminal grafting in an experimental aortic aneurysm model

We studied the impact of an endoluminally placed stented aortic graft on the geometry of a surgically created abdominal aortic dilation (AAD) in nonatherosclerotic mongrel dogs. Patulous iliac vein patch infrarenal aortoplasty produced a fusiform AAD, doubling the aorta diameter. Lumbar and mesenteric aortic tributaries were preserved and no mural thrombus formed. AADs created in 23 dogs were endoluminally excluded through transfemoral placement of a thin-wall Dacron graft 4 ± 2 months later. Balloon-expandable stents were used to anchor each end of the graft to the aorta. The graft was crimped radially in its body and longitudinally at its ends to provide longitudinal and radial expandability in these respective zones. Serial color duplex, angiography, and direct caliper measurements were made. Before graft placement, a 19% ± 11% diameter growth was observed. At graft placement, flow arrest immediately occurred in the space between the graft and the AAD intima in all cases. Although microscopic recanalization of the thrombus in this space was seen at sacrifice 6 and 12 months later, no macroscopic duplex flow was imaged. A 10% ± 11% reduction in AAD diameter was measured at 6 months (p < 0.001), with no further reduction at 12 months. Graft dimensions remained stable. No anastomotic leaks developed. AAD growth stopped during the first year after effective endoluminal exclusion in normotensive dogs despite patent side branches (<1.5 mm internal diameter) and no mural thrombus at the time of graft placement. Whether microscopic recanalization of the thrombus that forms outside the graft has an impact after 1 year remains to be seen. (J Vasc Surg 1996;23:819-31.)     

Antigenicity of fresh and cryopreserved rat valve allografts

Aortic valve allografts have demonstrated excellent clinical performance, but the importance of antigenic differences between donor and recipient is largely unknown. To determine the antigenicity of aortic valve grafts, rat aortic valves with a short portion of thoracic aorta were transplanted into the abdominal aorta of recipient rats. Valves were used immediately after harvest (fresh) or following cryopreservation. Three weeks after this procedure, the recipient rats received a skin graft from a rat of a strain syngeneic to that of the aortic valve donor. Additional groups of rats were subjected to sham operation (sham) followed three weeks later by skin grafting. Recipient rats were of the Lewis strain. Donor rats were of the Lewis, F344 (weakly allogeneic, RT1-compatible, non-RT1-incompatible), LBN F1 (moderately allogeneic, one-haplotype-identical and one-haplotype-incompatible at both the RT1 and non-RT1 loci), or BN (strongly allogeneic, RT1 and non-RT1-incompatible) strain. Time to skin graft rejection was measured. Among rats receiving the F344 grafts, the time to skin graft rejection (mean +/- SD) was sham: 9.1 +/- 1.0 days, fresh: 7.1 +/- 1.2 days, cryopreserved: 6.9 +/- 0.7 days. Among rats receiving the LBN F1 grafts, the corresponding times were sham: 7.8 +/- 0.8 days, fresh: 5.6 +/- 0.5 days, cryopreserved: 5.4 +/- 0.5 days. Among rats receiving the BN grafts, the corresponding times were sham: 7.1 +/- 0.3, fresh: 4.5 +/- 1.0 days, and cryopreserved: 4.3 +/- 0.7 days. Significant differences (P less than 0.05) existed between sham and fresh and between sham and cryopreserved, but not between fresh and cryopreserved. Significant differences (P less than 0.05) also existed between each histocompatibility grouping. It is concluded that aortic valve allografts in rats are antigenic and produce recipient sensitization. Cryopreservation does not diminish this sensitization. The degree of antigenicity is related to the degree of histoincompatibility between donor and recipient. Both RT1 and non-RT1 antigens appear to play a role in this process.     

Bacterial colonization of bone allografts related to increased interval between death and procurement: an experimental study in rats

Whereas organs from donors must be removed almost immediately after death to maximize organ viability in the recepient, there is a slightly longer window for tissue allograft recovery. To determine the maximum safe interval after death within which bone allografts may be harvested for clinical use, an experimental model was devised using adult Sprague-Dawley (SD) rats and duplicating cadaveric storage techniques. Allografts were procured at increasing time intervals after death. The grafts were then transplanted to 80 living SD rats, and the animals killed at 7 weeks to evaluate any increase in the risk of infection and bacterial colonization. None of the allografts procured within 48 h after death were colonized with bacteria, while 12% of grafts procured at 96 h and 50% of allografts procured at 1 week were colonized. The results suggest that it may be possible to extend the safe period within which cadaveric tissue may be procured for transplantation to up to 96 h following death, provided scrupulous measures to prevent and detect microorganism contamination are followed.     

Peripheral nerve allografts: Survey of present state in an experimental model of the rat

Rejection and regeneration processes in peripheral nerve allografts are analyzed in this review of a series of experiments with special reference to the possible clinical application of peripheral nerve allografting in clinical reconstructive surgery. A long segment of the sciatic nerve (2.5 cm) was grafted between congenic rat strains across a maximal genetic barrier; immunohistologically, donor- and recipient-derived structures can be differentiated. If allografting was performed without immunosuppression, a rejection response with consecutive regeneration of minor quality was observed. Under immunosuppression with cyclosporin A no rejection response was observed and regeneration quality was comparable to control autografts. The persistence of donorderived Schwann cells in the immunosuppressed allografts can be demonstrated immunohistologically. After discontinuation of immunosuppression a rejection response is exerted. We conclude that Schwann cells are eliminated from peripheral nerves during rejection. Consecutive regeneration of minor quality is possible, which implies the ingrowth of recipient-derived Schwann cells into the rejected allograft. Under immunosuppression, allogenic Schwann cells survive and actively promote regeneration. They are still immunologically competent and can exert rejection when immunosuppression is discontinued. A certain degree of replacement of donorderived Schwann cells seems possible. © 1994 Wiley-Liss, Inc.     

Calcification tendency of various biological aortic valves in an experimental animal model

The present study aimed at investigating the influence of fixation methods on the ultrastructure and calcium content of five different aortic biovalves. Biovalves subcutaneously implanted in Wistar rats for 12 weeks demonstrated significant differences in their calcium contents. Using Scheffe tests, two different groups of biovalves could be distinguished: (i) valves with high calcium content, such as Toronto-SPV (165 +/- 42 mg/g dry tissue) and Intact (193 +/- 36 mg/g dry tissue), and (ii) valves with low calcium content, such as Mosaic (2.7 +/- 1.8 mg/g dry tissue), Freestyle (2.3 +/- 1.1 mg/g dry tissue) and Hancock-II (3.6 +/- 1.2 mg/g dry tissue) (P < 0.05). All biovalves with an ultrastructurally preserved endothelium exhibited a low calcification tendency. The data suggest that if the endothelium is lost as a result of the fixation procedure, then calcification can only be prevented by appropriate anticalcification methods.     

Different healing rates of bone autografts, syngeneic grafts, and allografts in an experimental rat model

Matching of donors and recipients for tissue antigens is vitally important for successful transplantation of essentially all organs and tissues, the major exception being bone. The importance of tissue-typing for the healing of bone allografts remains, however, a controversial issue as development of both humoral and cell-mediated immunity against the grafted bone has been observed in some experimental systems. In the present study, we compared the healing patterns of frozen antigen-mismatched allografts, frozen antigen-matched allografts (syngeneic grafts), and fresh cortical bone autografts in an experimental rat model. Histomorphometry of the graft-host interface revealed that new bone formation started significantly earlier in autografts than in allografts or syngeneic grafts. By 2 weeks, the level of new bone formation in the syngeneic grafts had reached that in autografts. Antigen-mismatched allografts, however, continued to exhibit a retarded formation of new bone throughout the union process. These histomorphometric observations were confirmed by molecular biologic analyses for the mRNA levles of type I collagen, which increased earlier and reached a higher level in autografts than in allografts. Use of syngeneic grafts resulted in a longer persistence of type I collagen mRNA expression in the healing tissue than in antigen-mismatched allografts. No apparent differences were seen between allografts and autografts in the expression of type III collagen. No cartilage-specific type JI collagen mRNA was observed, indicating that antigen-mismatching or preservation by freezing did not alter the basic mechanism of the interface healing process, although it did slow down the beginning of the process. The experiments suggest that a major antigen mismatch between donor and recipient affects the temporal gene expression of extracellular bone matrix and delays new bone formation at the graft-host interface of cortical bone allografts.