Development of a rat forelimb vascularized composite allograft (VCA) perfusion protocol

Abstract

Vascularized composite allografts (VCAs) refer to en bloc heterogenous tissue that is transplanted to restore form and function after amputation or tissue loss. Rat limb VCA has emerged as a robust translational model to study the pathophysiology of these transplants. However, these models have predominately focused on hindlimb VCAs which does not translate anatomically to upper extremity transplantation, whereas the majority of clinical VCAs are upper extremity and hand transplants. This work details our optimization of rat forelimb VCA procurement and sub-normothermic machine perfusion (SNMP) protocols, with results in comparison to hindlimb perfusion with the same perfusion modality. Results indicate that compared to hindlimbs, rat forelimbs on machine perfusion mandate lower flow rates and higher acceptable maximum pressures. Additionally, low-flow forelimbs have less cellular damage than high-flow forelimbs based on oxygen uptake, edema, potassium levels, and histology through 2 hours of machine perfusion. These results are expected to inform future upper extremity VCA preservation studies.

Successful Extension of Vascularized Composite Allograft Perfusion Cold Storage to 24 h in a Rat Hindlimb Transplant Model

“…Furthermore, an optimal flow rate/pattern has not yet been defined, as it can vary depending on the perfusion solution, tissue, and characteristics of the perfusion system itself. 11 , 32 , 33 However, our HMP model shows that it is possible to achieve complete distribution of KPS-1 solution without significantly increasing edema in rat hindlimb VCAs. Therefore, there is potential to further improve this system and its storage outcomes through the addition of cell death inhibitors and other therapeutic agents.…”

“…Our study showed insignificant amounts of edema, apoptosis, and necrosis using KPS-1 even after prolonged storage at 24 h. Numerous other studies have shown much shorter storage times and greater amounts of apoptosis, necrosis, and edema using alternative perfusion solutions such as BSA- or DMEM-based solutions. 11,32,33 Although there is currently no universally accepted perfusion solution for VCA transplantation, our study indicates there is the potential for KPS-1 to fill this gap.…”

“…8 This treatment is currently limited due to the high susceptibility of soft tissue to ischemia reperfusion injury (IRI) and lifelong immunosuppression requirement for patients, making recipients prone to opportunistic infections, chronic kidney failure, and many other long-term complications. [9][10][11][12][13][14] To date, relatively few VCA transplants have been performed: approximately 150-200 VCA transplants worldwide. 15,16 Currently, the acceptable ex vivo storage time is limited to 6 h, but the detrimental effects of prolonged ischemic storage can be observed as early as 3 h. [17][18][19][20] As a result, some have advocated the transport of the donor and recipient to the same site to minimize cold ischemia time, which is often logistically challenging, if not impossible, with a multiorgan donor.…”

“…Studies investigating other perfusion solutions such as BSA- or DMEM-based perfusates have shown much shorter storage times and high rates of apoptosis, necrosis, and excessive edema; therefore, we chose an alternate perfusion solution that has already been established in the clinical setting for extending cold storage times in the setting of kidney transplantation. 11 This study evaluated the ability of heparinized UW KPS-1 solution to preserve donor rat hindlimbs subjected to 24-h ex vivo cold perfusion storage without significant edema, compared with heparinized normal saline (NS).…”

Towards Optimizing Sub-Normothermic Machine Perfusion in Fasciocutaneous Flaps: A Large Animal Study

“…Therefore, for clinical applications, the perfusate should be micro-filtrated and discarded, and should not be recirculated. We used biochemical metrics such as potassium, lactate, pH, and oxygen consumption by translation from other VCA models [31,32,43]. These metabolic outcomes have been shown to be relevant in solid organ preservation [35,44], but other parameters may be suitable for fasciocutaneous flaps.…”