Comparative study of bone substitute materials on transplanted tissue in a rat model of vascularized composite allograft

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Although bone substitutes are widely used in craniofacial surgery, their immunological effects in the setting of VCA remain inadequately investigated. In this study, fifteen Sprague–Dawley rats underwent reciprocal abdominal wall flap allotransplantation, with flaps exchanged between paired animals. Reconstruction was performed under five conditions: control, titanium mesh, polycaprolactone (PCL), poly-L-lactic acid with unsintered hydroxyapatite (PLLA-uHA), and silicone. All recipients received cyclosporine-based immunosuppressive therapy. Graft survival, collagen matrix deposition, and immunohistochemical expression of interleukin-1β, tumor necrosis factor-α, and interferon-γ were evaluated over eight weeks. All silicone grafts failed within 3 weeks as a result of hematoma and vascular compromise. In surviving groups, collagen deposition and pro-inflammatory cytokine expression were significantly greater in the control and titanium groups than in the PCL and PLLA-uHA groups (p < 0.01). Titanium supported stable matrix deposition without persistent cytokine elevation, whereas absorbable substitutes elicited chronic inflammation and impaired tissue integration. These findings suggest that titanium provides the most reliable skeletal support in craniofacial VCA, ensuring favorable collagen deposition with minimal immune activation. Vascularized composite allotransplantation Bone substitute materials Immune response Foreign body reaction Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Vascularized composite allotransplantation (VCA) provides a unique reconstructive solution for patients with extensive facial [ 1 ] , upper extremity, or other complex tissue loss [ 2 – 3 ] , enabling simultaneous restoration of function and appearance. Despite substantial technical advances [ 4 ] , its long-term success remains limited by complex immunologic factors. Unlike solid organ transplantation, VCA involves multiple immunogenic components, such as skin, muscle, bone, vessels, and nerves, with each entity eliciting distinct immune responses [ 5 ] . The skin is particularly immunogenic and is often the earliest site of rejection. In spite of systemic immunosuppression, chronic inflammation and fibrosis continue to compromise graft stability [ 6 ] . Although systemic immunosuppressants such as tacrolimus (FK-506) suppress acute rejection, localized immune activation caused by tissue injury or implanted materials may persist [ 7 ] . The local microenvironment, especially the interface between the graft tissue and biomaterials, plays a crucial role in modulating inflammatory signaling and influencing graft remodeling [ 8 ] . Structural implants further complicate this immune environment. Titanium and polymer-based substitutes such as poly L-lactic acid (PLLA) and polycaprolactone (PCL) are widely used for skeletal reconstruction [ 9 – 10 ] , yet their immunologic compatibility in allotransplantation remains unclear. Materials that elicit macrophage activation and cytokine release may exacerbate inflammation and impede bone healing [ 10 ] , whereas those that maintain a stable interface may support remodeling and graft survival under immunosuppression. This study investigated the biological and immune responses elicited by titanium- and polymer-based substitutes in a rat model of VCA [ 11 ] . Through histologic and cytokine analyses, we aimed to elucidate how different implant materials influence acute rejection and tissue integration, thereby providing insights into the selection of bone substitutes that minimize immune-mediated graft failure during reconstructive transplantation [ 12 – 14 ] . Methods Animal Model and Study Design Fifteen male Sprague–Dawley rats aged nine weeks and weighing between 280–330 g were used in this study. The animals were maintained at a constant room temperature of approximately 28.5°C and were provided with sterilized feed. All the animals were housed and managed at the Laboratory Animal Facility of the Asan Institute for Life Sciences, Asan Medical Center. The study was conducted with the approval of the Institutional Animal Care and Use Committee of the Clinical Research Center, Asan Medical Center. All experimental animals were cared for in accordance with institutional guidelines Bone Substitute Materials and Experimental Groups Four types of bone-substitute materials were used for comparison: titanium mesh (Titanium), polycaprolactone mesh (PCL), poly L-lactic acid–unsintered hydroxyapatite composite mesh (PLLA-uHA), and silicone sheets (Silicone). Five experimental groups were established to evaluate material-specific differences under identical surgical conditions. The overall transplantation and fixation procedures were performed using the same protocol in all groups, differing only in the type of bone substitute material implanted at the graft–recipient interface: Group I (Control): No bone substitute inserted. Group II (Titanium Group): Titanium mesh. Group III (PCL Group): Polycaprolactone mesh. Group IV (PLLA-uHA Group): Poly L-lactic acid–unsintered hydroxyapatite mesh. Group V (Silicone Group): Silicone sheet. Each group included three rats (n = 3), and all animals underwent surgery at nine weeks of age under identical anesthetic and perioperative conditions. Surgical Procedure Anesthesia was induced with 4% isoflurane and maintained with 2–3% inhalation throughout the procedure. In both donor and recipient rats, an abdominal flap measuring 3 × 3 cm was designed in the lower abdomen. Following a skin incision and soft-tissue dissection, the flap was elevated from the abdominal wall, preserving only the femoral artery and vein as the vascular pedicles. Once the preparation of both the donor and recipient was completed, the vascular pedicles were transected and the flaps were exchanged between the two animals (Fig. 1 ). The transplanted flap was anastomosed to the recipient’s femoral artery and vein under microscopic guidance. After achieving hemostasis, the designated bone substitute material was fixed to the flap bed according to the experimental protocol. Finally, the transplanted flap was sutured using 4 − 0 Vicryl. Postoperative Management Postoperatively, all animals received ketorolac and ampicillin for 5 days for analgesia and infection prophylaxis. Analgesics were administered before recovery from anesthesia to minimize postoperative discomfort, in full accordance with institutional animal care guidelines. Cyclosporine was administered subcutaneously at 16 mg/kg daily for the first 15 days, followed by 8 mg/kg daily until euthanasia at 8 weeks. Graft perfusion, wound healing, and skin color were evaluated daily (Fig. 2 ). The animals were sacrificed at 4 and 8 weeks postoperatively for sample collection Histological Evaluation At 8 weeks postoperatively, the animals were euthanized, and the vascularized composite allografts were harvested for histological analysis. Immediately after collection, the tissue samples were fixed overnight in 4% paraformaldehyde (Sigma, USA) at 4°C to ensure adequate preservation, followed by decalcification. The fixed specimens were then embedded in paraffin, and 5 µm sections were prepared. Sections were deparaffinized in xylene, rehydrated through graded ethanol, and stained with hematoxylin and eosin (H&E) for general morphology and with Masson’s trichrome (MT) staining to evaluate collagen matrix deposition. Collagen formation was quantified using ImageJ software (version 1.53e, National Institutes of Health, Bethesda, MD, USA) by calculating the percentage of blue-stained area relative to the total tissue area. Immunohistochemical staining was performed on formalin-fixed, paraffin-embedded sections to identify interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ). After deparaffinization and rehydration, antigen retrieval was achieved by heating the slides in citrate buffer (pH 6.0). Endogenous peroxidase activity was quenched with 3% hydrogen peroxide, and nonspecific binding was blocked with normal serum. The sections were then incubated overnight at 4°C with primary antibodies against IL-1β, TNF-α, and IFN-γ. After washing, the slides were treated with biotinylated secondary antibodies and subsequently processed using the streptavidin–biotin complex (SABC) method. Visualization was performed using a diaminobenzidine (DAB) chromogen, followed by hematoxylin counterstaining, dehydration, and mounting. Positively stained cells were counted in three randomly selected high-power fields per section to quantify cytokine expression. Statistical Analysis Statistical analysis was performed using the Kruskal–Wallis test followed by the Bonferroni-corrected Mann–Whitney U tests, with p < 0.05 being considered statistically significant. Results Composite tissue allotransplantation was successfully performed in all five experimental groups (n = 3 per group). Each graft measured 3 cm × 3 cm and comprised full-thickness skin, subcutaneous tissue, fascia, and underlying muscle, a dimension exceeding the critical threshold for spontaneous survival without vascular anastomosis. Microsurgical anastomosis between the donor and recipient femoral vessels was achieved in all rats. One rat in the PCL group developed graft necrosis within the first postoperative week owing to thrombus formation at the anastomotic site, which was considered a surgical complication unrelated to the implanted material. In the silicone group, all grafts exhibited progressive necrosis within three weeks, accompanied by extensive hematoma and seroma formation beneath the implant. Pedicle occlusion was confirmed at euthanasia, and these animals were excluded from subsequent analyses. At the 8-week follow-up, viable grafts were observed in the control (n = 3), titanium (n = 3), PCL (n = 2), and PLLA-uHA (n = 3) groups (Table. 1). No.1 No.2 No.3 Control POD#56 POD#56 POD#56 Tttanium POD#56 POD#56 POD#56 PCL POD#6 POD#56 POD#56 PLLA-uHA POD#56 POD#56 POD#56 Silicone POD#11 POD#14 POD#21 Histological evaluation using hematoxylin and eosin (H&E) staining revealed progressive granulation-tissue formation, fibroblast proliferation, and collagen deposition across all surviving grafts. Despite mild variability, overall tissue architecture was preserved, and no remarkable morphological differences were observed among the control, titanium, PCL, and PLLA-uHA groups at low magnification. In contrast, the silicone group showed extensive necrosis and dense inflammatory infiltration, consistent with gross findings. Masson’s trichrome (MT) staining demonstrated distinct differences in collagen fiber density and organization among groups (Fig. 3 ). Quantitative analysis confirmed significant intergroup variation (p = 0.000), with mean collagen-area ratios of 60.93 ± 1.91 (control), 62.28 ± 2.75 (titanium), 52.61 ± 0.98 (PCL), 49.77 ± 1.70 (PLLA-uHA), and 63.48 ± 1.14 (silicone). Collagen deposition was significantly greater in the control and titanium groups than in the PCL and PLLA-uHA groups (p = 0.002–0.004), suggesting that titanium supports a more active extracellular-matrix formation and efficient tissue remodeling. Polymer-based groups displayed less organized collagen networks, indicating delayed granulation-tissue maturation. Immunohistochemical analysis corroborated these findings (Fig. 4 ). IL-1β staining, which reflects early inflammatory cytokine activity, was markedly elevated in all groups during the initial postoperative phase. However, by 8 weeks, expression stabilized in the control and titanium groups, whereas IL-1β levels remained persistently high in the PCL and PLLA-uHA groups (p = 0.000). The mean IL-1β values were 6.84 ± 1.52, 4.03 ± 2.76, 15.94 ± 2.54, 14.43 ± 2.71, and 3.03 ± 0.15 for the control, titanium, PCL, PLLA-uHA, and silicone groups, respectively. A comparable pattern was observed for TNF-α, a key mediator of inflammatory cell recruitment and tissue injury. Expression was transient and relatively low in the control and titanium groups but remained elevated in the PCL and PLLA-uHA groups (p = 0.000). The mean TNF-α values were 4.78 ± 0.68 (control), 5.07 ± 0.59 (titanium), 8.29 ± 1.11 (PCL), 9.14 ± 2.27 (PLLA-uHA), and 4.93 ± 0.49 (silicone). These findings indicate that polymer-based materials induced prolonged inflammatory signaling, whereas titanium maintains stable cytokine expression with minimal chronic inflammation. In contrast, interferon-γ (IFN-γ), a marker of adaptive immune activation and acute rejection—showed no substantial increase in any group (p = 0.017). The mean IFN-γ values were 4.36 ± 0.63 (control), 4.51 ± 0.39 (titanium), 4.36 ± 0.27 (PCL), 3.91 ± 0.22 (PLLA-uHA), and 4.70 ± 0.18 (silicone). Although minor differences were noted between the titanium and PLLA-uHA groups (p = 0.002) and between the PCL and PLLA-uHA groups (p = 0.004), no evidence of acute immune rejection was observed. Collectively, these results indicate that titanium promoted superior graft stability and tissue integration with reduced inflammatory cytokine activity. In contrast, polymer-based materials such as PCL and PLLA-uHA elicited persistent local inflammation, disrupted collagen organization, and delayed wound maturation. Silicone implants caused early graft failure, possibly as a result of mechanical obstruction and hematoma formation rather than immune-mediated mechanisms. These findings underscore that the choice of bone substitute material critically influences local inflammatory responses and matrix remodeling in vascularized composite allotransplantation. Discussion This study investigated the relationship between bone substitute materials and acute rejection in a rat model of vascularized composite allotransplantation (VCA). The initial hypothesis, which states that direct contact between certain implanted materials and the graft bed might increase the incidence of acute rejection, was not clearly supported. However, grafts containing absorbable materials showed increased and persistent inflammatory responses, suggesting that material degradation may affect local immune activation. These findings likely reflect early inflammatory phases rather than established rejection, considering the limited two-month observation period and the small number of experimental animals per group. Absorbable implants, commonly composed of biodegradable polymers or ceramics, are widely used in orthopedics, dentistry, and reconstructive surgery. Although these materials offer the advantage of gradual resorption without the need for removal, the degradation process can induce local inflammatory and immune reactions. Such responses involve macrophage recruitment and the release of inflammatory cytokines, including TNF-α and IL-1β. The intensity and duration of inflammation depend on material properties such as size, morphology, chemical composition, and degradation rate [ 11 ] . Rapid degradation may produce excessive by-products that exceed the body’s clearance capacity and intensify inflammation. In this investigation, polymer-based substitutes demonstrated sustained cytokine expression and less organized collagen deposition, consistent with prolonged local inflammatory reactions [ 12 – 13 ] . Previous studies have reported inconsistent results regarding the biocompatibility of biodegradable materials [ 16 ] . Some studies reported minimal immune reactions, whereas others observed significant inflammatory or foreign-body responses depending on material composition and microstructure. Most notably, hydroxyapatite-containing calcium phosphate ceramics, have been reported to induce inflammation under specific conditions related to particle composition and morphology, sometimes leading to implant loosening and impaired integration [ 16 ] . Non-resorbable materials may cause inflammatory responses when they fragment into small particles that are further degraded by cellular and physical processes. These mechanisms may explain the increased inflammation observed in grafts containing HA-based bone substitutes in this study [ 17 ] . If local inflammation around bone substitutes persists, it can negatively influence graft survival in the VCA. Chronic inflammatory activity may contribute to repeated episodes of acute rejection, which are known to progress to chronic rejection despite temporary clinical improvement after immunosuppressive therapy [ 18 – 19 ] . Therefore, the prevention of acute rejection by reducing local inflammation remains essential. Although stronger systemic immunosuppression could theoretically reduce immune reactions, such regimens carry well-recognized risks including infection, malignancy, and metabolic complications [ 20 ] . Among the materials tested in this study, titanium exhibited inflammatory responses comparable to those of the control group, indicating lower local immune activity and better tissue stability than absorbable materials. Several factors should be considered when interpreting these results. The sample size was small, and the follow-up period was limited to eight weeks, which may not fully reflect long-term immune or remodeling processes. Moreover, the study compared immune responses within graft tissues but did not assess systemic immune activity in recipient organs. The inclusion of both local and systemic evaluations could help distinguish graft-specific reactions from generalized immune responses. In addition, the uniform use of immunosuppressive therapy may have influenced bone healing, as previous studies have shown that agents such as tacrolimus or cyclosporine can impair peri-implant bone integration and reduce osseointegration. Despite these limitations, this study provides foundational evidence that the choice of bone substitute material influences inflammatory response patterns in vascularized composite allotransplantation. Titanium and absorbable substitutes demonstrated distinctly different local tissue reactions, emphasizing the need for further investigations of material-dependent immune interactions during reconstructive transplantation. In a rat model of abdominal wall flap allotransplantation, titanium facilitated favorable collagen matrix deposition and stable cytokine profiles, while absorbable substitutes (PCL and PLLA-uHA) induced persistent inflammatory activity and reduced matrix remodeling. The use of silicone was associated with early graft loss. These findings suggest that titanium appears to be the most reliable bone substitute material for craniofacial VCA requiring skeletal support. Declarations Competing Interests Statement The authors declare no competing interests. Author contributions Jeong conceived and designed the study, performed the surgical experiments, collected and analyzed the data, and drafted the initial manuscript. Jang contributed to manuscript revision and figure preparation, assisted with English editing, and coordinated the submission process. Lee contributed to manuscript editing and revision, reviewed the figures and data presentation, and provided feedback throughout the submission process. Choi supervised the study, provided overall guidance, and approved the final version of the manuscript for submission. Acknowledgments This study was supported by a grant (2012 − 461, 2013 − 461, 2015–4610) from the Asan Institute for Life Sciences, Asan Medical Center, Seoul, Korea. Data Availability Statement The datasets generated and analyzed during the current study are not publicly available due to institutional policy but are available from the corresponding author on reasonable request. References Vyas K et al (2022) Facial transplantation. Facial Plast Surg Clin North Am 30:255–269. https://doi.org/10.1016/j.fsc.2022.01.011 Shpigel M et al (2022) Special considerations in face transplantation: a systematic review. J Craniofac Surg 33:1352–1357. https://doi.org/10.1097/scs.0000000000008484 Nassimizadeh M, Nassimizadeh AK, Power D (2014) Hand transplant surgery. Ann R Coll Surg Engl 96:571–574. https://doi.org/10.1308/003588414X13946184902767 Anggelia MR et al (2022) Cell therapy in vascularized composite allotransplantation. Biomed J 45:454–464. https://doi.org/10.1016/j.bj.2022.01.005 Moris D, Cendales LC (2021) Sensitization and desensitization in vascularized composite allotransplantation. Front Immunol 12:682180. https://doi.org/10.3389/fimmu.2021.682180 Fischer S et al (2014) Acute rejection in vascularized composite allotransplantation. Curr Opin Organ Transpl 19:531–544. https://doi.org/10.1097/mot.0000000000000140 Kollar B et al (2019) The significance of vascular alterations in acute and chronic rejection for vascularized composite allotransplantation. J Vasc Res 56:163–180. https://doi.org/10.1159/000500958 Etra JW, Raimondi G, Brandacher G (2018) Mechanisms of rejection in vascular composite allotransplantation. Curr Opin Organ Transpl 23:28–33. https://doi.org/10.1097/mot.0000000000000490 Siemionow M et al (2016) The reversed paradigm of chimerism induction: donor conditioning with recipient-derived bone marrow cells as a novel approach for tolerance induction in vascularized composite allotransplantation. Microsurgery 36:676–683. https://doi.org/10.1002/micr.30041 Gibon E et al (2017) The biological response to orthopedic implants for joint replacement. II: polyethylene, ceramics, PMMA, and the foreign body reaction. J Biomed Mater Res B Appl Biomater 105:1685–1691. https://doi.org/10.1002/jbm.b.33676 LeGeros RZ (2008) Calcium phosphate-based osteoinductive materials. Chem Rev 108:4742–4753. https://doi.org/10.1021/cr800427g Huang Y, Wu C, Zhang X, Chang J, Dai K (2018) Regulation of immune response by bioactive ions released from silicate bioceramics for bone regeneration. Acta Biomater 66:81–92. https://doi.org/10.1016/j.actbio.2017.08.044 Hady TF et al (2021) Uniform 40-µm-pore diameter precision templated scaffolds promote a pro-healing host response by extracellular vesicle immune communication. J Tissue Eng Regen Med 15:24–36. https://doi.org/10.1002/term.3160 Morra M (2007) Biomolecular modification of implant surfaces. Expert Rev Med Devices 4:361–372. https://doi.org/10.1586/17434440.4.3.361 Hollinger JO, Battistone GC (1986) Biodegradable bone repair materials. Synthetic polymers and ceramics. Clin Orthop Relat Res 290–305. https://www.ncbi.nlm.nih.gov/pubmed/3522015 Costantino MD et al (2020) Inflammatory response to magnesium-based biodegradable implant materials. Acta Biomater 101:598–608. https://doi.org/10.1016/j.actbio.2019.10.014 Lange T et al (2011) Size dependent induction of proinflammatory cytokines and cytotoxicity of particulate beta-tricalciumphosphate in vitro. Biomaterials 32:4067–4075. https://doi.org/10.1016/j.biomaterials.2011.02.039 Zheng X et al (2017) Effect of FK-506 (tacrolimus) therapy on bone healing of titanium implants: a histometric and biomechanical study in mice. Eur J Oral Sci 125:28–33. https://doi.org/10.1111/eos.12320 Duarte PM et al (2001) The effect of an immunosuppressive therapy and its withdrawal on bone healing around titanium implants. A histometric study in rabbits. J Periodontol 72:1391–1397. https://doi.org/10.1902/jop.2001.72.10.1391 Kim NK, Nam W, Kim HJ (2015) Comparison of miniplates and biodegradable plates in reconstruction of the mandible with a fibular free flap. Br J Oral Maxillofac Surg 53:223–229. https://doi.org/10.1016/j.bjoms.2014.11.010 Tables Table 1 is available in the Supplementary Files section. Additional Declarations The authors declare no competing interests. Supplementary Files table1.png Table 1. Experimental design showing the five study groups. Abdominal wall flap allotransplantation was performed in all animals (n = 15). Each group consisted of three rats, and bone substitute materials were placed beneath the graft bed in the experimental groups. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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15:33:22","extension":"png","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":239026,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7989788/v1/8dca11e5742a740c57b3be10.png"},{"id":94986773,"identity":"f397c181-daa4-44c6-ac8a-0d953d2202f9","added_by":"auto","created_at":"2025-11-03 07:00:46","extension":"png","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":354810,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7989788/v1/cf58da029b6d3f6aab4c592f.png"},{"id":94874991,"identity":"a4c7a1cc-28be-4400-b521-db3ffae9179b","added_by":"auto","created_at":"2025-10-31 15:33:22","extension":"png","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":411841,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7989788/v1/713d323a6baaf7cd497c4912.png"},{"id":94874988,"identity":"3fa88ff7-d58c-49a2-a49c-482259e5805e","added_by":"auto","created_at":"2025-10-31 15:33:21","extension":"xml","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":59953,"visible":true,"origin":"","legend":"","description":"","filename":"rs79897880structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7989788/v1/7a626f924ddebf37814de296.xml"},{"id":94874986,"identity":"5d407822-8423-4e62-b255-0048ab4509ae","added_by":"auto","created_at":"2025-10-31 15:33:21","extension":"html","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":65925,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7989788/v1/ea766f8c4b1c397db7badc42.html"},{"id":94986660,"identity":"89a34749-178b-4f98-a64b-cf6fb0ceb530","added_by":"auto","created_at":"2025-11-03 07:00:32","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1722936,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eSurgical design of the abdominal wall flap and schematic of reciprocal transplantation\u003c/em\u003e. Schematic illustration of the 3 × 3 cm abdominal wall flap harvested from paired rats, exchanged, and transplanted into the same anatomical location of each counterpart. Vascular continuity was restored by microsurgical anastomosis of the femoral vessels.\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-7989788/v1/3c9c05a39449c7be08db9768.png"},{"id":94874977,"identity":"d43cca00-45e4-4e66-a61b-7d7bcf1ef115","added_by":"auto","created_at":"2025-10-31 15:33:21","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":531364,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eGross outcomes of graft survival across groups\u003c/em\u003e. Representative clinical photographs of grafts from each group at immediate postoperative(A), after 7 days (B), 2 weeks (C), 3 weeks (D), 4 weeks (E) and 8 weeks (F). Silicone group grafts failed early due to hematoma and vascular compromise.\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7989788/v1/cd3b0ac97320380167111f85.jpg"},{"id":94874979,"identity":"1e76565f-62e8-411a-90a1-3a7c13e84256","added_by":"auto","created_at":"2025-10-31 15:33:21","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":941803,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eCollagen matrix deposition assessed by Masson’s trichrome staining.\u003c/em\u003e Representative trichrome-stained sections of grafts from each group at postoperative week 8. Collagen fibers are stained blue. Control and titanium groups showed abundant collagen matrix deposition with organized extracellular matrix formation. PCL and PLLA-uHA groups exhibited reduced collagen deposition with less organized matrix architecture. Silicone group specimens could not be evaluated due to early graft failure. Scale bars = 100 μm.\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7989788/v1/92dcf546f7fb30576f18cf2a.jpg"},{"id":94874990,"identity":"07a6f190-5e86-4fe2-b32a-2e6d04db8f8a","added_by":"auto","created_at":"2025-10-31 15:33:21","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":819293,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eImmunohistochemical analysis of inflammatory cytokine expression.\u003c/em\u003e Representative sections and quantitative analyses of IL-1β, TNF-α, and IFN-γ expression in graft tissues at postoperative week 8. Control and titanium groups showed relatively low expression of IL-1β and TNF-α, whereas PCL and PLLA-uHA groups demonstrated significantly higher expression, indicating persistent local inflammation. IFN-γ expression did not differ significantly among groups. Quantitative results are presented as mean ± SD from high-power fields. Scale bars = 100 μm.\u003c/p\u003e","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7989788/v1/990653b306677fc885e492d6.jpg"},{"id":95000833,"identity":"08c13a72-1a4f-4827-a622-6b934ae6b6b2","added_by":"auto","created_at":"2025-11-03 09:00:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4204507,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7989788/v1/20511773-beed-4450-a990-2ab49fde60d8.pdf"},{"id":94874976,"identity":"448a6349-8ddc-4e0e-8554-80ae1c8f0f38","added_by":"auto","created_at":"2025-10-31 15:33:21","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":7556,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable 1.\u003c/strong\u003e Experimental design showing the five study groups. Abdominal wall flap allotransplantation was performed in all animals (n = 15). Each group consisted of three rats, and bone substitute materials were placed beneath the graft bed in the experimental groups.\u003c/p\u003e","description":"","filename":"table1.png","url":"https://assets-eu.researchsquare.com/files/rs-7989788/v1/cea841baf0dfd007cdab9f01.png"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eComparative study of bone substitute materials on transplanted tissue in a rat model of vascularized composite allograft\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eVascularized composite allotransplantation (VCA) provides a unique reconstructive solution for patients with extensive facial\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e, upper extremity, or other complex tissue loss\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e, enabling simultaneous restoration of function and appearance. Despite substantial technical advances \u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e, its long-term success remains limited by complex immunologic factors. Unlike solid organ transplantation, VCA involves multiple immunogenic components, such as skin, muscle, bone, vessels, and nerves, with each entity eliciting distinct immune responses\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. The skin is particularly immunogenic and is often the earliest site of rejection. In spite of systemic immunosuppression, chronic inflammation and fibrosis continue to compromise graft stability\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAlthough systemic immunosuppressants such as tacrolimus (FK-506) suppress acute rejection, localized immune activation caused by tissue injury or implanted materials may persist\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. The local microenvironment, especially the interface between the graft tissue and biomaterials, plays a crucial role in modulating inflammatory signaling and influencing graft remodeling\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eStructural implants further complicate this immune environment. Titanium and polymer-based substitutes such as poly L-lactic acid (PLLA) and polycaprolactone (PCL) are widely used for skeletal reconstruction\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e, yet their immunologic compatibility in allotransplantation remains unclear. Materials that elicit macrophage activation and cytokine release may exacerbate inflammation and impede bone healing\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e, whereas those that maintain a stable interface may support remodeling and graft survival under immunosuppression. This study investigated the biological and immune responses elicited by titanium- and polymer-based substitutes in a rat model of VCA\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. Through histologic and cytokine analyses, we aimed to elucidate how different implant materials influence acute rejection and tissue integration, thereby providing insights into the selection of bone substitutes that minimize immune-mediated graft failure during reconstructive transplantation\u003csup\u003e[\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eAnimal Model and Study Design\u003c/h2\u003e\u003cp\u003eFifteen male Sprague\u0026ndash;Dawley rats aged nine weeks and weighing between 280\u0026ndash;330 g were used in this study. The animals were maintained at a constant room temperature of approximately 28.5\u0026deg;C and were provided with sterilized feed. All the animals were housed and managed at the Laboratory Animal Facility of the Asan Institute for Life Sciences, Asan Medical Center.\u003c/p\u003e\u003cp\u003eThe study was conducted with the approval of the Institutional Animal Care and Use Committee of the Clinical Research Center, Asan Medical Center. All experimental animals were cared for in accordance with institutional guidelines\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eBone Substitute Materials and Experimental Groups\u003c/h3\u003e\n\u003cp\u003eFour types of bone-substitute materials were used for comparison: titanium mesh (Titanium), polycaprolactone mesh (PCL), poly L-lactic acid\u0026ndash;unsintered hydroxyapatite composite mesh (PLLA-uHA), and silicone sheets (Silicone).\u003c/p\u003e\u003cp\u003eFive experimental groups were established to evaluate material-specific differences under identical surgical conditions. The overall transplantation and fixation procedures were performed using the same protocol in all groups, differing only in the type of bone substitute material implanted at the graft\u0026ndash;recipient interface:\u003c/p\u003e\u003cp\u003eGroup I (Control): No bone substitute inserted.\u003c/p\u003e\u003cp\u003eGroup II (Titanium Group): Titanium mesh.\u003c/p\u003e\u003cp\u003eGroup III (PCL Group): Polycaprolactone mesh.\u003c/p\u003e\u003cp\u003eGroup IV (PLLA-uHA Group): Poly L-lactic acid\u0026ndash;unsintered hydroxyapatite mesh.\u003c/p\u003e\u003cp\u003eGroup V (Silicone Group): Silicone sheet.\u003c/p\u003e\u003cp\u003eEach group included three rats (n\u0026thinsp;=\u0026thinsp;3), and all animals underwent surgery at nine weeks of age under identical anesthetic and perioperative conditions.\u003c/p\u003e\n\u003ch3\u003eSurgical Procedure\u003c/h3\u003e\n\u003cp\u003eAnesthesia was induced with 4% isoflurane and maintained with 2\u0026ndash;3% inhalation throughout the procedure. In both donor and recipient rats, an abdominal flap measuring 3 \u0026times; 3 cm was designed in the lower abdomen. Following a skin incision and soft-tissue dissection, the flap was elevated from the abdominal wall, preserving only the femoral artery and vein as the vascular pedicles. Once the preparation of both the donor and recipient was completed, the vascular pedicles were transected and the flaps were exchanged between the two animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The transplanted flap was anastomosed to the recipient\u0026rsquo;s femoral artery and vein under microscopic guidance. After achieving hemostasis, the designated bone substitute material was fixed to the flap bed according to the experimental protocol. Finally, the transplanted flap was sutured using 4\u0026thinsp;\u0026minus;\u0026thinsp;0 Vicryl.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003ePostoperative Management\u003c/h3\u003e\n\u003cp\u003ePostoperatively, all animals received ketorolac and ampicillin for 5 days for analgesia and infection prophylaxis. Analgesics were administered before recovery from anesthesia to minimize postoperative discomfort, in full accordance with institutional animal care guidelines. Cyclosporine was administered subcutaneously at 16 mg/kg daily for the first 15 days, followed by 8 mg/kg daily until euthanasia at 8 weeks. Graft perfusion, wound healing, and skin color were evaluated daily (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The animals were sacrificed at 4 and 8 weeks postoperatively for sample collection\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eHistological Evaluation\u003c/h3\u003e\n\u003cp\u003eAt 8 weeks postoperatively, the animals were euthanized, and the vascularized composite allografts were harvested for histological analysis. Immediately after collection, the tissue samples were fixed overnight in 4% paraformaldehyde (Sigma, USA) at 4\u0026deg;C to ensure adequate preservation, followed by decalcification. The fixed specimens were then embedded in paraffin, and 5 \u0026micro;m sections were prepared. Sections were deparaffinized in xylene, rehydrated through graded ethanol, and stained with hematoxylin and eosin (H\u0026amp;E) for general morphology and with Masson\u0026rsquo;s trichrome (MT) staining to evaluate collagen matrix deposition. Collagen formation was quantified using ImageJ software (version 1.53e, National Institutes of Health, Bethesda, MD, USA) by calculating the percentage of blue-stained area relative to the total tissue area.\u003c/p\u003e\u003cp\u003eImmunohistochemical staining was performed on formalin-fixed, paraffin-embedded sections to identify interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ). After deparaffinization and rehydration, antigen retrieval was achieved by heating the slides in citrate buffer (pH 6.0). Endogenous peroxidase activity was quenched with 3% hydrogen peroxide, and nonspecific binding was blocked with normal serum. The sections were then incubated overnight at 4\u0026deg;C with primary antibodies against IL-1β, TNF-α, and IFN-γ. After washing, the slides were treated with biotinylated secondary antibodies and subsequently processed using the streptavidin\u0026ndash;biotin complex (SABC) method. Visualization was performed using a diaminobenzidine (DAB) chromogen, followed by hematoxylin counterstaining, dehydration, and mounting. Positively stained cells were counted in three randomly selected high-power fields per section to quantify cytokine expression.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eStatistical analysis was performed using the Kruskal\u0026ndash;Wallis test followed by the Bonferroni-corrected Mann\u0026ndash;Whitney U tests, with p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 being considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eComposite tissue allotransplantation was successfully performed in all five experimental groups (n\u0026thinsp;=\u0026thinsp;3 per group). Each graft measured 3 cm \u0026times; 3 cm and comprised full-thickness skin, subcutaneous tissue, fascia, and underlying muscle, a dimension exceeding the critical threshold for spontaneous survival without vascular anastomosis. Microsurgical anastomosis between the donor and recipient femoral vessels was achieved in all rats. One rat in the PCL group developed graft necrosis within the first postoperative week owing to thrombus formation at the anastomotic site, which was considered a surgical complication unrelated to the implanted material. In the silicone group, all grafts exhibited progressive necrosis within three weeks, accompanied by extensive hematoma and seroma formation beneath the implant. Pedicle occlusion was confirmed at euthanasia, and these animals were excluded from subsequent analyses. At the 8-week follow-up, viable grafts were observed in the control (n\u0026thinsp;=\u0026thinsp;3), titanium (n\u0026thinsp;=\u0026thinsp;3), PCL (n\u0026thinsp;=\u0026thinsp;2), and PLLA-uHA (n\u0026thinsp;=\u0026thinsp;3) groups (Table. 1).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNo.1\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNo.2\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNo.3\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePOD#56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePOD#56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePOD#56\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTttanium\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePOD#56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePOD#56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePOD#56\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePCL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePOD#6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePOD#56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePOD#56\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePLLA-uHA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePOD#56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePOD#56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePOD#56\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSilicone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePOD#11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePOD#14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePOD#21\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eHistological evaluation using hematoxylin and eosin (H\u0026amp;E) staining revealed progressive granulation-tissue formation, fibroblast proliferation, and collagen deposition across all surviving grafts. Despite mild variability, overall tissue architecture was preserved, and no remarkable morphological differences were observed among the control, titanium, PCL, and PLLA-uHA groups at low magnification. In contrast, the silicone group showed extensive necrosis and dense inflammatory infiltration, consistent with gross findings.\u003c/p\u003e\u003cp\u003eMasson\u0026rsquo;s trichrome (MT) staining demonstrated distinct differences in collagen fiber density and organization among groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Quantitative analysis confirmed significant intergroup variation (p\u0026thinsp;=\u0026thinsp;0.000), with mean collagen-area ratios of 60.93\u0026thinsp;\u0026plusmn;\u0026thinsp;1.91 (control), 62.28\u0026thinsp;\u0026plusmn;\u0026thinsp;2.75 (titanium), 52.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.98 (PCL), 49.77\u0026thinsp;\u0026plusmn;\u0026thinsp;1.70 (PLLA-uHA), and 63.48\u0026thinsp;\u0026plusmn;\u0026thinsp;1.14 (silicone). Collagen deposition was significantly greater in the control and titanium groups than in the PCL and PLLA-uHA groups (p\u0026thinsp;=\u0026thinsp;0.002\u0026ndash;0.004), suggesting that titanium supports a more active extracellular-matrix formation and efficient tissue remodeling. Polymer-based groups displayed less organized collagen networks, indicating delayed granulation-tissue maturation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eImmunohistochemical analysis corroborated these findings (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). IL-1β staining, which reflects early inflammatory cytokine activity, was markedly elevated in all groups during the initial postoperative phase. However, by 8 weeks, expression stabilized in the control and titanium groups, whereas IL-1β levels remained persistently high in the PCL and PLLA-uHA groups (p\u0026thinsp;=\u0026thinsp;0.000). The mean IL-1β values were 6.84\u0026thinsp;\u0026plusmn;\u0026thinsp;1.52, 4.03\u0026thinsp;\u0026plusmn;\u0026thinsp;2.76, 15.94\u0026thinsp;\u0026plusmn;\u0026thinsp;2.54, 14.43\u0026thinsp;\u0026plusmn;\u0026thinsp;2.71, and 3.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15 for the control, titanium, PCL, PLLA-uHA, and silicone groups, respectively.\u003c/p\u003e\u003cp\u003eA comparable pattern was observed for TNF-α, a key mediator of inflammatory cell recruitment and tissue injury. Expression was transient and relatively low in the control and titanium groups but remained elevated in the PCL and PLLA-uHA groups (p\u0026thinsp;=\u0026thinsp;0.000). The mean TNF-α values were 4.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68 (control), 5.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.59 (titanium), 8.29\u0026thinsp;\u0026plusmn;\u0026thinsp;1.11 (PCL), 9.14\u0026thinsp;\u0026plusmn;\u0026thinsp;2.27 (PLLA-uHA), and 4.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49 (silicone). These findings indicate that polymer-based materials induced prolonged inflammatory signaling, whereas titanium maintains stable cytokine expression with minimal chronic inflammation.\u003c/p\u003e\u003cp\u003eIn contrast, interferon-γ (IFN-γ), a marker of adaptive immune activation and acute rejection\u0026mdash;showed no substantial increase in any group (p\u0026thinsp;=\u0026thinsp;0.017). The mean IFN-γ values were 4.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63 (control), 4.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39 (titanium), 4.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27 (PCL), 3.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22 (PLLA-uHA), and 4.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18 (silicone). Although minor differences were noted between the titanium and PLLA-uHA groups (p\u0026thinsp;=\u0026thinsp;0.002) and between the PCL and PLLA-uHA groups (p\u0026thinsp;=\u0026thinsp;0.004), no evidence of acute immune rejection was observed.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eCollectively, these results indicate that titanium promoted superior graft stability and tissue integration with reduced inflammatory cytokine activity. In contrast, polymer-based materials such as PCL and PLLA-uHA elicited persistent local inflammation, disrupted collagen organization, and delayed wound maturation. Silicone implants caused early graft failure, possibly as a result of mechanical obstruction and hematoma formation rather than immune-mediated mechanisms. These findings underscore that the choice of bone substitute material critically influences local inflammatory responses and matrix remodeling in vascularized composite allotransplantation.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study investigated the relationship between bone substitute materials and acute rejection in a rat model of vascularized composite allotransplantation (VCA). The initial hypothesis, which states that direct contact between certain implanted materials and the graft bed might increase the incidence of acute rejection, was not clearly supported. However, grafts containing absorbable materials showed increased and persistent inflammatory responses, suggesting that material degradation may affect local immune activation. These findings likely reflect early inflammatory phases rather than established rejection, considering the limited two-month observation period and the small number of experimental animals per group.\u003c/p\u003e\u003cp\u003eAbsorbable implants, commonly composed of biodegradable polymers or ceramics, are widely used in orthopedics, dentistry, and reconstructive surgery. Although these materials offer the advantage of gradual resorption without the need for removal, the degradation process can induce local inflammatory and immune reactions. Such responses involve macrophage recruitment and the release of inflammatory cytokines, including TNF-α and IL-1β. The intensity and duration of inflammation depend on material properties such as size, morphology, chemical composition, and degradation rate\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. Rapid degradation may produce excessive by-products that exceed the body\u0026rsquo;s clearance capacity and intensify inflammation. In this investigation, polymer-based substitutes demonstrated sustained cytokine expression and less organized collagen deposition, consistent with prolonged local inflammatory reactions\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003ePrevious studies have reported inconsistent results regarding the biocompatibility of biodegradable materials\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. Some studies reported minimal immune reactions, whereas others observed significant inflammatory or foreign-body responses depending on material composition and microstructure. Most notably, hydroxyapatite-containing calcium phosphate ceramics, have been reported to induce inflammation under specific conditions related to particle composition and morphology, sometimes leading to implant loosening and impaired integration\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. Non-resorbable materials may cause inflammatory responses when they fragment into small particles that are further degraded by cellular and physical processes. These mechanisms may explain the increased inflammation observed in grafts containing HA-based bone substitutes in this study\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIf local inflammation around bone substitutes persists, it can negatively influence graft survival in the VCA. Chronic inflammatory activity may contribute to repeated episodes of acute rejection, which are known to progress to chronic rejection despite temporary clinical improvement after immunosuppressive therapy\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Therefore, the prevention of acute rejection by reducing local inflammation remains essential. Although stronger systemic immunosuppression could theoretically reduce immune reactions, such regimens carry well-recognized risks including infection, malignancy, and metabolic complications\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. Among the materials tested in this study, titanium exhibited inflammatory responses comparable to those of the control group, indicating lower local immune activity and better tissue stability than absorbable materials.\u003c/p\u003e\u003cp\u003eSeveral factors should be considered when interpreting these results. The sample size was small, and the follow-up period was limited to eight weeks, which may not fully reflect long-term immune or remodeling processes. Moreover, the study compared immune responses within graft tissues but did not assess systemic immune activity in recipient organs. The inclusion of both local and systemic evaluations could help distinguish graft-specific reactions from generalized immune responses. In addition, the uniform use of immunosuppressive therapy may have influenced bone healing, as previous studies have shown that agents such as tacrolimus or cyclosporine can impair peri-implant bone integration and reduce osseointegration. Despite these limitations, this study provides foundational evidence that the choice of bone substitute material influences inflammatory response patterns in vascularized composite allotransplantation. Titanium and absorbable substitutes demonstrated distinctly different local tissue reactions, emphasizing the need for further investigations of material-dependent immune interactions during reconstructive transplantation. In a rat model of abdominal wall flap allotransplantation, titanium facilitated favorable collagen matrix deposition and stable cytokine profiles, while absorbable substitutes (PCL and PLLA-uHA) induced persistent inflammatory activity and reduced matrix remodeling. The use of silicone was associated with early graft loss. These findings suggest that titanium appears to be the most reliable bone substitute material for craniofacial VCA requiring skeletal support.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting Interests Statement\u003c/h2\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003ch2\u003eAuthor contributions\u003c/h2\u003e\n\u003cp\u003eJeong conceived and designed the study, performed the surgical experiments, collected and analyzed the data, and drafted the initial manuscript.\u003c/p\u003e\n\u003cp\u003eJang contributed to manuscript revision and figure preparation, assisted with English editing, and coordinated the submission process.\u003c/p\u003e\n\u003cp\u003eLee contributed to manuscript editing and revision, reviewed the figures and data presentation, and provided feedback throughout the submission process.\u003c/p\u003e\n\u003cp\u003eChoi supervised the study, provided overall guidance, and approved the final version of the manuscript for submission.\u003c/p\u003e\n\u003ch2\u003eAcknowledgments\u003c/h2\u003e\n\u003cp\u003eThis study was supported by a grant (2012\u0026thinsp;\u0026minus;\u0026thinsp;461, 2013\u0026thinsp;\u0026minus;\u0026thinsp;461, 2015\u0026ndash;4610) from the Asan Institute for Life Sciences, Asan Medical Center, Seoul, Korea.\u003c/p\u003e\n\u003ch2\u003eData Availability Statement\u003c/h2\u003e\n\u003cp\u003eThe datasets generated and analyzed during the current study are not publicly available due to institutional policy but are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eVyas K et al (2022) Facial transplantation. Facial Plast Surg Clin North Am 30:255\u0026ndash;269. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.fsc.2022.01.011\u003c/span\u003e\u003cspan address=\"10.1016/j.fsc.2022.01.011\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShpigel M et al (2022) Special considerations in face transplantation: a systematic review. J Craniofac Surg 33:1352\u0026ndash;1357. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1097/scs.0000000000008484\u003c/span\u003e\u003cspan address=\"10.1097/scs.0000000000008484\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNassimizadeh M, Nassimizadeh AK, Power D (2014) Hand transplant surgery. Ann R Coll Surg Engl 96:571\u0026ndash;574. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1308/003588414X13946184902767\u003c/span\u003e\u003cspan address=\"10.1308/003588414X13946184902767\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAnggelia MR et al (2022) Cell therapy in vascularized composite allotransplantation. Biomed J 45:454\u0026ndash;464. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.bj.2022.01.005\u003c/span\u003e\u003cspan address=\"10.1016/j.bj.2022.01.005\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMoris D, Cendales LC (2021) Sensitization and desensitization in vascularized composite allotransplantation. Front Immunol 12:682180. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fimmu.2021.682180\u003c/span\u003e\u003cspan address=\"10.3389/fimmu.2021.682180\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFischer S et al (2014) Acute rejection in vascularized composite allotransplantation. Curr Opin Organ Transpl 19:531\u0026ndash;544. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1097/mot.0000000000000140\u003c/span\u003e\u003cspan address=\"10.1097/mot.0000000000000140\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKollar B et al (2019) The significance of vascular alterations in acute and chronic rejection for vascularized composite allotransplantation. J Vasc Res 56:163\u0026ndash;180. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1159/000500958\u003c/span\u003e\u003cspan address=\"10.1159/000500958\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEtra JW, Raimondi G, Brandacher G (2018) Mechanisms of rejection in vascular composite allotransplantation. Curr Opin Organ Transpl 23:28\u0026ndash;33. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1097/mot.0000000000000490\u003c/span\u003e\u003cspan address=\"10.1097/mot.0000000000000490\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSiemionow M et al (2016) The reversed paradigm of chimerism induction: donor conditioning with recipient-derived bone marrow cells as a novel approach for tolerance induction in vascularized composite allotransplantation. Microsurgery 36:676\u0026ndash;683. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/micr.30041\u003c/span\u003e\u003cspan address=\"10.1002/micr.30041\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGibon E et al (2017) The biological response to orthopedic implants for joint replacement. II: polyethylene, ceramics, PMMA, and the foreign body reaction. J Biomed Mater Res B Appl Biomater 105:1685\u0026ndash;1691. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/jbm.b.33676\u003c/span\u003e\u003cspan address=\"10.1002/jbm.b.33676\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLeGeros RZ (2008) Calcium phosphate-based osteoinductive materials. Chem Rev 108:4742\u0026ndash;4753. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/cr800427g\u003c/span\u003e\u003cspan address=\"10.1021/cr800427g\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHuang Y, Wu C, Zhang X, Chang J, Dai K (2018) Regulation of immune response by bioactive ions released from silicate bioceramics for bone regeneration. Acta Biomater 66:81\u0026ndash;92. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.actbio.2017.08.044\u003c/span\u003e\u003cspan address=\"10.1016/j.actbio.2017.08.044\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHady TF et al (2021) Uniform 40-\u0026micro;m-pore diameter precision templated scaffolds promote a pro-healing host response by extracellular vesicle immune communication. J Tissue Eng Regen Med 15:24\u0026ndash;36. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/term.3160\u003c/span\u003e\u003cspan address=\"10.1002/term.3160\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMorra M (2007) Biomolecular modification of implant surfaces. Expert Rev Med Devices 4:361\u0026ndash;372. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1586/17434440.4.3.361\u003c/span\u003e\u003cspan address=\"10.1586/17434440.4.3.361\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHollinger JO, Battistone GC (1986) Biodegradable bone repair materials. Synthetic polymers and ceramics. Clin Orthop Relat Res 290\u0026ndash;305. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/pubmed/3522015\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/pubmed/3522015\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCostantino MD et al (2020) Inflammatory response to magnesium-based biodegradable implant materials. Acta Biomater 101:598\u0026ndash;608. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.actbio.2019.10.014\u003c/span\u003e\u003cspan address=\"10.1016/j.actbio.2019.10.014\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLange T et al (2011) Size dependent induction of proinflammatory cytokines and cytotoxicity of particulate beta-tricalciumphosphate in vitro. Biomaterials 32:4067\u0026ndash;4075. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.biomaterials.2011.02.039\u003c/span\u003e\u003cspan address=\"10.1016/j.biomaterials.2011.02.039\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZheng X et al (2017) Effect of FK-506 (tacrolimus) therapy on bone healing of titanium implants: a histometric and biomechanical study in mice. Eur J Oral Sci 125:28\u0026ndash;33. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/eos.12320\u003c/span\u003e\u003cspan address=\"10.1111/eos.12320\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDuarte PM et al (2001) The effect of an immunosuppressive therapy and its withdrawal on bone healing around titanium implants. A histometric study in rabbits. J Periodontol 72:1391\u0026ndash;1397. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1902/jop.2001.72.10.1391\u003c/span\u003e\u003cspan address=\"10.1902/jop.2001.72.10.1391\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKim NK, Nam W, Kim HJ (2015) Comparison of miniplates and biodegradable plates in reconstruction of the mandible with a fibular free flap. Br J Oral Maxillofac Surg 53:223\u0026ndash;229. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.bjoms.2014.11.010\u003c/span\u003e\u003cspan address=\"10.1016/j.bjoms.2014.11.010\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Asan Medical Center","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Vascularized composite allotransplantation, Bone substitute materials, Immune response, Foreign body reaction","lastPublishedDoi":"10.21203/rs.3.rs-7989788/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7989788/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eVascularized composite allotransplantation (VCA) enables the reconstruction of extensive craniofacial and extremity defects but carries a higher risk of rejection than solid organ transplantation, primarily due to the immunogenicity of the skin and composite tissues. Although bone substitutes are widely used in craniofacial surgery, their immunological effects in the setting of VCA remain inadequately investigated.\u003c/p\u003e\u003cp\u003eIn this study, fifteen Sprague\u0026ndash;Dawley rats underwent reciprocal abdominal wall flap allotransplantation, with flaps exchanged between paired animals. Reconstruction was performed under five conditions: control, titanium mesh, polycaprolactone (PCL), poly-L-lactic acid with unsintered hydroxyapatite (PLLA-uHA), and silicone. All recipients received cyclosporine-based immunosuppressive therapy. Graft survival, collagen matrix deposition, and immunohistochemical expression of interleukin-1β, tumor necrosis factor-α, and interferon-γ were evaluated over eight weeks.\u003c/p\u003e\u003cp\u003eAll silicone grafts failed within 3 weeks as a result of hematoma and vascular compromise. In surviving groups, collagen deposition and pro-inflammatory cytokine expression were significantly greater in the control and titanium groups than in the PCL and PLLA-uHA groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Titanium supported stable matrix deposition without persistent cytokine elevation, whereas absorbable substitutes elicited chronic inflammation and impaired tissue integration.\u003c/p\u003e\u003cp\u003eThese findings suggest that titanium provides the most reliable skeletal support in craniofacial VCA, ensuring favorable collagen deposition with minimal immune activation.\u003c/p\u003e","manuscriptTitle":"Comparative study of bone substitute materials on transplanted tissue in a rat model of vascularized composite allograft","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-31 15:33:16","doi":"10.21203/rs.3.rs-7989788/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f34eb07a-5978-49c1-b10a-de228c8735c8","owner":[],"postedDate":"October 31st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-31T15:33:17+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-31 15:33:16","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7989788","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7989788","identity":"rs-7989788","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}