Twenty-Four–Hour Ex Vivo Perfusion with Acellular Solution Enables Successful Replantation of Porcine Forelimbs

Abstract

Background: A critical barrier to successful limb replantation and allotransplantation is the maximum allowable limb ischemia time of 4 to 6 hours. The current gold standard is to preserve amputated limbs on an ice slurry. Experimental machine perfusion has yielded promising results as an alternative. In particular, hypothermic acellular perfusion has enabled preservation of amputated limbs for up to 12 hours thus far. Methods: Amputated forelimbs of Yorkshire pigs were preserved on static cold storage at 4°C for 4 hours (static cold storage group) or perfused at 8°C for 24 hours (perfusion group) with oxygenated modified STEEN Solution perfusate before replantation. Animals were followed up for 7 days after replantation. Results: Eight animals underwent replantation (cold storage group, n = 4; perfusion group, n = 4). Seventy-five and 100 percent of animals in the static cold storage and perfusion groups survived for 7 days, respectively. Glycogen and adenosine triphosphate remained stable throughout perfusion. Heart and respiratory rate after replantation were increased in the static cold storage group. There was increased damage in muscle biopsy specimens obtained from animals in the static cold storage group after 7 days when compared with those from animals in the perfusion group. Conclusions: Hypothermic acellular ex vivo perfusion of limbs for up to 24 hours enables tissue preservation comparable to that obtained with conventional static cold storage for 4 hours and may reduce muscle damage and systemic reactions on limb replantation. Translation to human limbs may help improve limb replantation and allotransplantation outcomes.

Successful 18‐h acellular extracorporeal perfusion and replantation of porcine limbs ‐ Histology versus nerve stimulation

“…The items can be separately analysed to determine which categories are most predictive for true muscle damage. The second point to be addressed is that ischaemia‐reperfusion injury seems to present as a heterogenous phenomenon in skeletal muscle [5,14]. The microscopical evaluation of muscle cross‐sections showed diffusely spread areas of ischaemic damage, surrounded by well‐preserved areas.…”

“…For instance, Krezdorn et al . [14] found significantly lower levels of potassium and myoglobin following porcine limb replantation after 24‐h ECP compared to 4‐h SCS, despite of increased levels during ECP. The shorter anaesthesia time in the SCS group in this experiment might have possibly influenced the CK levels in porcine blood, especially since pigs tend to develop muscle spasms during anaesthesia.…”

“…The paraffin‐embedded cross‐sections were cut at 3‐µm thick and stained with standard haematoxylin‐eosin and Masson trichrome. All slides were evaluated with light microscopy for ischaemia‐reperfusion induced alterations by a blinded pathologist [14]. A simplified version of the ‘Histologic injury severity score' (HISS) as presented by Muller et al.…”

“…During ECP, the tissue is continuously provided with a preservation solution containing oxygen and nutrients whilst toxic metabolites are removed [9–11]. ECP has shown promising results for composite tissue preservation and effectively increased tissue preservation to 12 or even 24 h in large‐animal studies [12–14]. These studies mostly relied on muscle histology outcomes and are limited by the lack of functional assessment.…”

Tissue Viability of Free Flaps after Extracorporeal Perfusion Using a Modified Hydroxyethyl Starch Solution

“…Extracorporeal perfusion (EP) has the potential to substantially increase the time of survival of tissue disconnected from blood circulation [12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27]. This is especially true in the case of major amputates since the separated limb contains a lot of muscle tissue that is very sensitive to ischemia, limiting the time of survival to 4 h [6].…”