Pathological Characteristics of Muscle Rejection and Dysfunction in a Swine Vascularized Composite Allotransplantation Model and a Scoring Proposal: A Pilot Study

Muscle plays a pivotal role in the functional reconstruction of deformities associated with vascularized composite allotransplantation (VCA). Although the 2007 Banff working classification has clarified on skin allograft pathology, the existing literature lacks comprehensive studies on muscle rejection in VCA.1,2 The skin is recognized as the body’s most immunogenic tissue, however, relying solely on skin rejection may not entirely capture the overall degree of VCA graft rejection, particularly with the advent of local immunosuppression. Rejection in muscular tissue will ultimately lead to a nonreversible loss of function. The Büttemeyer grading system, initially devised for cardiomyocyte rejection over 2 decades ago, is still considered the standard to evaluate muscle rejection.3 However, its applicability to VCA is questionable, as it does not incorporate VCA-specific factors such as apraxia and dysfunction. Therefore, we consider a tailored classification system of muscle rejection in VCA as a necessity. Nevertheless, the limited availability of VCA muscle samples in published literature to delineate the pathological features, coupled with the challenges in acquiring these samples at various stages of muscle rejection in VCA, presents significant hurdles to VCA muscle rejection classification. Our study used a heterotopic porcine hindlimb VCA model, meticulously described in previous literature.4,5 The flap contains skin (monitor island), underlying thigh muscles, knee joint, distal femur, and proximal tibia. The skin island measures around 12 × 8 cm. We analyzed the muscle from 19 individual pigs, encompassing 17 recipients and 2 donors. Recipient groups included no treatment, oral systemic tacrolimus for 14 days, single dose or repeated dose of a local tacrolimus-encapsulated hydrogel and in situ forming implant loaded with rapamycin. The details of the immunosuppressant have been detailed in Table 1 and our previous publication.5 The clinical endpoint was determined by extensive necrosis of the graft’s skin (macroscopic grade III-IV rejection) or 90 d after operation. Muscle samples in the VCA graft have been retrieved for histological analysis at the endpoint. Because of various treatments and survival durations in our study, the muscle samples displayed diverse pathological characteristics. By comparing histological morphology and established muscle damage classification systems, we divided the features of muscle rejection and dysfunction in VCA and proposed a histological scoring system (Table 1; Figure S1A, SDC,https://links.lww.com/TP/D154). The histologic features were scored in a semiquantitative manner based on the expertise of pathologists involved in the study. TABLE 1. - Histopathologic evaluation and scoring of muscle rejection and dysfunction in VCA Pig No. ID Treatment Survival time (POD) Skin Muscle Macro Skin Grad Banff SkinGrad SLI Leukocyte infiltration Muscular histopathology Vasc. dam Fatty infil Total Score PMN Ly/PC M Eo Var.size MyoD Fibrosis Int.edema 1 6483 graft No treatment 7 III II 2 3 1 1 0 2 3 0 3 3 0 18 2 6484 graft No treatment 7 III III 4 3 1 3 0 2 2 0 2 0 0 17 3 6596 graft No treatment 9 III-IV IV 4 3 1 2 2 NA 3 0 3 3 0 21 4 6597 graft No treatment 8 III IV 4 3 1 1 2 NA 3 0 3 3 0 20 5 6918 graft Oral systemic tacrolimus for 14 d 48 III III 4 3 2 2 2 0 3 0 3 2 3 24 6 6916 graft Oral systemic tacrolimus for 14 d 55 III IV 4 2 2 2 2 1 2 0 1 0 3 19 7 6574 graft Oral systemic tacrolimus for 14 d 27 III I 3 0 2 2 1 2 0 1 0 0 2 13 8 304 graft Oral systemic tacrolimus for 14 d 56 III-IV IV 4 3 1 2 0 0 3 0 1 3 3 20 9 6933 graft TGMS-TAC*S 51 III IV 4 1 3 2 1 2 3 1 2 0 3 22 10 6913 graft TGMS-TAC*S 39 III-IV II 3 0 2 2 0 0 1 3 0 0 3 14 11 7023 graft TGMS-TAC*S 34 III II 4 0 1 2 0 1 2 0 2 0 3 15 12 8803 graft TGMS-TAC*R 88 III IV 4 3 2 1 0 NA 3 0 0 2 3 19 13 8764 graft TGMS-TAC*R 91 II I 1 0 1 0 0 NA NA 0 0 0 3 5 14 8804 graft TGMS-TAC*R 89 II II 2 0 1 1 1 3 3 0 0 0 3 14 15 8790 graft TGMS-TAC*R 90 II I 1 0 1 1 1 1 2 0 0 0 3 10 16 315 graft Rapa-ISFI 65 III-IV IV 4 2 3 2 0 0 3 0 3 3 3 23 17 337 graft Rapa-ISFI 36 III-IV III 4 0 3 2 1 0 2 0 3 0 2 17 18 6471 donor Healthy donor 0 Null Null 0 0 0 0 0 0 0 0 0 0 0 0 19 299 donor Healthy donor 0 Null Null 0 0 0 0 0 0 0 0 0 0 0 0 Cuffs were defined as circumferential inflammatory cells immediately surrounding a blood vessel.2 Absent, 0; mild, 1; moderate, 2; severe, 3.Banff skin Grad, Banff 2007 skin grading1; Eo, eosinophils; Fatty infil, fatty infiltration between myofibers; Int. Edema, interstitial edema; Ly/PC, lymphocytes and plasma cells; M, macrophages; Macro Skin Grad, macroscopic skin grading2; MyoD, myofiber damage; PMN, polymorphic nuclear cells/neutrophils; POD, postoperative days; Rapa-ISFI: in situ forming implant loaded with rapamycin on POD15 (intragraft injection of 1.25 mL/kg in situ forming implants loaded with 5 mg of rapamycin per 0.31 mL); SLI, severity of leukocyte infiltration (0: no cuffs; 1: <5 cuffs, ≤2 cells thick in any direction; 2: <5 cuffs, >2 cells thick in any direction; 3: 5–15 cuffs, any thickness; 4: >15 cuffs, any thickness or diffuse leukocyte infiltration [average over at least 3 × 20 fields]); TAC, tacrolimus; TGMS, triglycerol monostearate; TGMS*R, local repeated dose of TGMS hydrogel loaded with TAC on POD0, 30, 60; TGMS-TAC*S, local single dose of TGMS hydrogel loaded with TAC on POD0 (intragraft injection of 140 mg TGMS-TAC [7 mg/mL] per kg of graft weight); Vasc. Dam, vascular damage; Var. Size, variation in fiber size; VCA, vascularized composite allotransplantation. A prominent feature observed was the infiltration of leukocytes, particularly lymphocytes, from perivascular spaces into the interstitial space and myofibers, representing a hallmark of rejection. This infiltration appeared diffuse or patchy and was frequently accompanied by interstitial edema, myofibrillar damage and in severe cases extensive necrosis. Additionally, the accumulation of leukocytes around vessels contributed to vascular damage, including endotheliitis and vascular thrombosis, resulting in hemorrhage. These pathological findings collectively suggest an acute process of rejection. Moreover, in certain samples, the presence of mononuclear infiltrates, fibrosis, myotube formation, and fatty infiltration were found, suggesting chronic rejection and dysfunctional changes. The representative histopathologic images of each characteristic used in our classification, along with those from each pig, are provided as a supplemental material (Figure S1A and S1B, SDC,https://links.lww.com/TP/D154) in the BORIS Portal (Bern Open Repository and Information System, https://doi.org/10.48620/413). Additionally, the original raw digital scanned histological images of these muscle samples are available in the repository. Because the inherent challenges associated with establishing a large-animal VCA model and the difficulty of performing muscle biopsies in a large-animal VCA graft, the limited sample size is a significant limitation of our study. Nevertheless, the 17 grafts in our study represent a decent number for large-animal VCA studies and may allow a proposal of scoring system of muscle rejection even if would not yet allow widespread application. Our focus was primarily on delineating the characteristics of muscular rejection and providing a corresponding scoring proposal to serve as a reference for the pathological classification of muscle rejection and dysfunction, thus paving the way for future research to refine and expand upon our proposed classification system. Enhanced comprehension of muscle rejection in VCA, coupled with a comparison of skeletal and cardiac muscle rejection, will enrich both knowledge and clinical practice. We hope that further studies and collaborative efforts expand on these data and help to establish definitive grading criteria for muscle rejection in VCA. ACKNOWLEDGMENTs The authors thank all the participants involved in this project for their support.