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Cryopreservation of composite tissue transplants.

ABSTRACT: Composite tissue allotransplantation holds great promise for upper extremity reconstruction but is limited by donor part availability. Cryopreservation may increase the availability of donor parts and even reduce antigenicity. The purpose of the study was to evaluate the viability of cryopreserved composite tissues and to demonstrate the feasibility of microvascular isotransplantation of cryopreserved composite flaps. Twenty epigastric flaps were harvested from Lewis rats. Ten flaps were analyzed fresh. Ten flaps were perfused with dimethyl sulfoxide (DMSO)/trehelose cryoprotectant agent (CPA), frozen by controlled cooling to -140 degrees C, and stored for 2 weeks. Flaps were evaluated by factor VIII endothelial staining and MTT tetrazolium salt assay. For the in vivo phase, 30 flaps were harvested. Ten were transplanted fresh to isogenetic recipient animals, ten were perfused with CPA and transplanted, and ten were cryopreserved for 2 weeks, thawed, and transplanted. All cryopreserved samples displayed intact vascular endothelia on factor VIII staining. On MTT analysis, the epithelial viability index for the cryopreserved samples was not significantly different from fresh controls (p = 0.12). All freshly transplanted flaps (10/10) were viable at 60 days. Nine of ten flaps in the perfused/transplanted group were viable at 60 days. Survival of cryopreserved/transplanted flaps ranged from 5 to 60 days. The skin and vascular endothelial components of composite tissue flaps appear to retain their viability after cryopreservation. The in vivo studies demonstrate that the long-term survival of cryopreserved composite tissue transplants is feasible and support an indirect injury, rather than direct injury from freezing or cryoprotectant agents, as the mechanism of flap loss.

SUBMITTER: Rinker B

PROVIDER: S-EPMC2528968 | biostudies-other | 2008 Mar

REPOSITORIES: biostudies-other

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Project description:BackgroundVascularized composite tissue allotransplantation (VCA) to replace limbs or faces damaged beyond repair is now possible. The resulting clear benefit to quality of life is a compelling reason to attempt this complex procedure. Unfortunately, the high doses of immunosuppressive drugs required to protect this type of allograft result in significant morbidity and mortality giving rise to ethical concerns about performing this surgery in patients with non-life-threatening conditions. Here we tested whether we could suppress anti-graft immune activity by using a safe β2 -adrenergic receptor (AR) agonist, terbutaline, to mimic the natural immune suppression generated by nervous system-induced signalling through AR.MethodsA heterotopic hind limb transplantation model was used with C57BL/6 (H-2b) as recipients and BALB/c (H-2d) mice as donors. To test the modulation of the immune response, graft survival was investigated after daily intraperitoneal injection of β2 -AR agonist with and without tacrolimus. Analyses of immune compositions and quantification of pro-inflammatory cytokines were performed to gauge functional immunomodulation. The contributions to allograft survival of β2 -AR signalling in donor and recipient tissue were investigated with β2 -AR-/- strains.ResultsTreatment with the β2 -AR agonist delayed VCA rejection, even with a subtherapeutic dose of tacrolimus. β2 -AR agonist decreased T-cell infiltration into the transplanted grafts and decreased memory T-cell populations in recipient's circulation. In addition, decreased levels of inflammatory cytokines (IFN-γ, IL-6, TNF-α, CXCL-1/10 and CCL3/4/5/7) were detected following β2 -AR agonist treatment, and there was a decreased expression of ICAM-1 and vascular cell adhesion molecule-1 in donor stromal cells.Conclusionsβ2 -AR agonist can be used safely to mimic the natural suppression of immune responses, which occurs during adrenergic stress-signalling and thereby can be used in combination regimens to reduce the dose needed of toxic immunosuppressive drugs such as tacrolimus. This strategy can be further evaluated for feasibility in the clinic.

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Cryopreservation of composite tissue transplants.

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