Protecting Autologous Free Flaps From Ischemia/Reperfusion Damage With Cold Storage.

Status of the art Reconstructive microsurgery, via microvascular anastomosis of 0.5 mm to 3 mm vessel diameter, needing an operating microscope, allows autologous transplantation of tissue blocks (flaps) from a donor site to a distant recipient site. The procedure involves temporary ischemia. The absence of perfusion and the post-anastomosis reperfusion causes ischemia/reperfusion (I/R) damage. There is clinical evidence supporting an increased percentage of flap complications associated with the longer duration of the ischemia time. At present, although there are several preservation solutions, for solid organ preservation the University of Wisconsin (UW) solution is still considered the gold standard. Cold storage is also used in autologous liver bench surgery, but not for microvascular reconstruction. Unfortunately, this application of ischemia preservation did not occur in microvascular reconstruction because flaps were considered less prone to damage, having a less functional role, pertaining simply to "filling" or "reshaping". However, this is not the whole truth, if we consider for example the fibula flap, containing an abundance of muscle, which is a vulnerable tissue, and above all the fibula free flap requires a great deal of bone healing, which is necessary for the following dental implants. Moreover, the quality preservation of the original tissues is of paramount clinical relevance if we think about the importance of a soft breast, of a pliable facial reconstruction and, above all, of a functional recovery in a replanted finger, where fibrotic degeneration may cause a functional disaster in terms of range of motion and sensation recovery. To our knowledge, in reconstructive surgery, except for reconstructive transplantation (Vascularized Composite Allotransplantations, VCA), the utilization of preservation solution is very limited.

However, in reconstructive microsurgery and hand/finger replantation, the warm ischemia time can be significantly longer due to flap preparation and difficult implanting within the recipient site, or due to intraoperative technical problems, and some flaps include tissues more vulnerable to warm ischemia time; consequently, it could be useful to use the same procedures employed in VCA to limit I/R injury. A comparative evaluation of the effects of cold preservation of auto-transplanted tissues versus the traditional treatment in which no preservation is performed is non-existing in the medical literature. This lack of scientific knowledge warrants the organization of studies investigating these unexplored aspects of microvascular reconstructive surgical science.

Aims The research hypothesis determining the objectives of this project is that cold static storage of autologous free flaps might offer benefits in terms of flap survival and better preservation of all tissues within the flaps, reducing the incidence of complications, flap shrinkage and the amount of fibrosis induced by I/R damage. Indeed, despite a flow-through and patent microvascular anastomoses, microcirculation and perfusion of the flap can be impaired due to endothelial damage, possibly induced by the I/R traumatic experience that can lead to irreversible fibrosis ending in partial necrosis of the flap and could also explain the significant complication rate (36.1% in head and neck surgery and 35% in breast surgery according to recent studies) in terms of partial flap loss or other early and late complications (Clavien-Dindo grade III), which are more frequent than total flap loss, reported around 2-3% of free tissue transfers in the breast and head and neck regions (2% in breast microsurgical reconstruction and 1-3% in head and neck reconstructions based on published reports). The University of Wisconsin (UW) solution is still considered the gold standard among the preservation solutions in solid organ transplantation as well as in VCA. The UW solution was developed by the surgeon Folkert Belzer and the basic scientist James Southard at the University of Wisconsin in the 1980s and was introduced for clinical use in the United States in 1987. UW solution is characterized by a buffer system (phosphate buffer), with high potassium and low sodium as electrolytes, which mimics the intracellular milieu; in addition, it contains raffinose and lactobionic acid, as osmotic membrane-impermeable agents, molecules that have a large molecular weight (594 and 358 Da) and counteract the transmembranous water shift to prevent cellular edema.

Hydroxyethyl starch was also added as an oncotic substance to prevent expansion of the extracellular space. However, hydroxyethyl starch confers the relatively high viscosity (5.70 cp) to UW solution, which is approximately four times higher than water. Overall the electrolyte composition and the membrane-impermeable macromolecules in UW solution maintain the ionic and osmotic balance during hypothermia. Allopurinol was added as an inhibitor of xanthine oxidase, which blocks the generation of reactive oxygen species (ROS) via the hypoxanthinexanthine- uric acid pathway. The antioxidant glutathione was added to neutralize ROS that are generated during reoxygenation at the time of reperfusion. UW solution also includes adenosine, which serves as a substrate for the resynthesis of ATP on reperfusion. UW solution also omitted Ca2+ because of the extremely low physiological intracellular Ca2+ concentration and the known negative effects of elevated intracellular Ca2+ concentration for cell survival. At present, in literature cold storage for autologous microvascular free tissue transfers has not been reported. Therefore, this project aims to reduce the risks of total and partial free flap failure or fibrotic degeneration and consequent early and late complications, using perfusion of the flaps and their cold storage in UW solution to reduce the I/R damage. Indeed, although the total ischemia time is significantly shorter in autologous free flaps compared to those reported in solid organ transplantation, the longer warm ischemia times sometimes reported in reconstructive surgery should be avoided or somehow neutralized. Moreover, in hand and finger replantation, where surgery is not elective but emergency-based, ischemia times may often be significantly longer.

Sudden circulation interruption alters cellular oxygen and nutrient supply, inducing mitochondrial damage and affecting the metabolic function of the tissues. Reperfusion suddenly restores oxygen and nutrient supply and, following the ischemic time, represents a challenge for the flap tissues that may become particularly sensitive to ROS production. ROS trigger free radical attack to cellular membrane polyunsaturated fatty acids (PUFA) and induce lipid peroxidation, with the formation of unstable fatty acid hydro(pero)xides, eicosanoids, and non-enzymatic (free radical-induced) isoprostanes capable of further propagating free radical reactions thereby extending the damage. I/R damage has a described role in hampering flap integration within the recipient site, its normal and smooth healing with the highst possible degree of similarity to the native preoperative donor tissue. I/R damage determines fibrotic reactions and consequent damage to the neurosensory function due to interference of the scar tissue with the nerve network. In case of mandibular reconstruction, I/R damage is supposed to worsen the quality of bone stock in the reconstructied neomandibula, thereby reducing the possibility of osteointegrated dental implants. Purpose of the study is to evaluate the effect of cold preservation on reconstructive flaps for the breast and head \& neck areas, in terms of multiple clinical and radiological endpoints, with a focus on intraoperative evaluation of perfusion, and postoperative neurosensory recovery and quality of the bone in fibula-based mandibular reconstruction, in view of dental implant osteo-integration.

Project The present study is a RCT to evaluate the efficacy of cold preservation of free flaps from I/R damage in post-oncological microsurgical reconstructions. The study design will conform to the CONSORT guidelines for RCTs. The project has been approved by the local IRB. An anonymous database (only patient initials) will be created to record all the included patients' data and it will not be available to the blinded outcomes assessors. The recorded data will be ischemia time (the time between vessel division and reperfusion at the end of microvascular anastomoses), weight of the flap and duration of surgery, patients' demographics, BMI, smoking and comorbidities. Two randomization lists (breast and head \& neck) will be created through a randomization list generator, available online (random.org), with a 5 block assignment to Group A (flap preserved in UW solution and cold storage during the ischemia time) and Group B (flap without perfusion and cold storage). The randomization will be kept blinded to the outcome assessors and the radiologists. Once harvested and detached from their donor sites, the flaps of Group A will be flushed with 200 ml UW solution at 4°C into the flap artery within 2 minutes after vessel division.

Following the intra-flap injection of the first 150 ml, the last 50 ml of solution will be used to externally irrigate the outer flap, which will be stored at 4°C during the bench surgery and the anastomoses. Before its reperfusion, the flap will be flushed again through its artery with 0.9% saline solution. The flaps of Group B will be stored at room temperature during the bench surgery and the anastomosis time.