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Background: Although decellularized nerve grafts are often used as a bridging material in nerve defect repair, the lack of perfusion support in this procedure limits the regeneration capacity. To address this, we applied vascularized biogenic conduits, which are fibrous membranes prefabricated around the silicone rod that contain rich vascularity and macrophages, to nerve defect repair procedures using decellularized nerve grafts. The purpose of this study is to investigate the capacity of combining a vascularized biogenic conduit and a decellularized nerve graft for peripheral nerve regeneration using a 10-mm nerve defect model in rats., Materials and Methods: Sixteen adult male rats (F344 rats, 10-12 weeks, 200-250 g) were used in this study. For the prefabrication of vascularized biogenic conduits, a silicone rod was transplanted next to the sciatic nerve. After 8 weeks, this silicone rod was enveloped in connective tissue, called a vascularized biogenic conduit. The first rat was used to investigate the histological characteristics of vascularized biogenic conduits through immunofluorescence studies. The remaining 15 rats were divided into three groups to evaluate the efficacy of the combination of a decellularized nerve graft and a vascularized biogenic conduit: a decellularized nerve graft (DNG) group, a decellularized nerve graft with a vascularized biogenic conduit (DNG-w-VBC) group, and an autologous nerve graft (ANG) group. Eight weeks after nerve graft surgery, the assessment results of both functional recovery (electrophysical studies and target muscle atrophy) and morphological recovery (total number, diameter, and myelin thickness of the regenerated axons) of the regenerated nerves were examined., Results: Immunofluorescence studies revealed that the VBC contains extracellular matrix, vascular tissue, and macrophages. The results of the DNG-w-VBC group were superior to the DNG group in electrophysiological studies (CMAP; 6.29 ± 0.80% vs. 4.02 ± 3.35%, MNCV; 50.6 ± 8.4% vs. 25.7 ± 15.6%, p < .05, respectively), regenerated axon number (11,348 ± 812 vs. 7697 ± 2197, p < .05), and mean axon diameter (2.72 ± 0.33 μm vs. 1.64 ± 0.12 μm, p < .05)., Conclusions: Our study confirms that vascularized biogenic conduits supply vascularity and macrophages to nerve defect sites. Combining vascularized biogenic conduits with decellularized nerve grafts to treat nerve defects offers superior functional and morphological recovery of regenerated axons., (© 2021 Wiley Periodicals LLC.)

Introduction: A Bio 3D printed nerve conduit was reported to promote nerve regeneration in a 5 mm nerve gap model. The purpose of this study was to fabricate Bio 3D nerve conduits suitable for a 10 mm nerve gap and to evaluate their capacity for nerve regeneration in a rat sciatic nerve defect model., Materials and Methods: Eighteen F344 rats with immune deficiency (9-10 weeks old; weight, 200-250 g) were divided into three groups: a Bio 3D nerve conduit group (Bio 3D, n = 6), a nerve graft group (NG, n = 6), and a silicon tube group (ST, n = 6). A 12-mm Bio 3D nerve conduit or silicon tube was transplanted into the 10-mm defect of the right sciatic nerve. In the nerve graft group, reverse autografting was performed with an excised 10-mm nerve segment. Assessments were performed at 8 weeks after the surgery., Results: In the region distal to the suture site, the number of myelinated axons in the Bio 3D group were significantly larger compared with the silicon group (2,548 vs. 950, p < .05). The myelinated axon diameter (MAD) and the myelin thickness (MT) of the regenerated axons in the Bio 3D group were significantly larger compared with those of the ST group (MAD: 3.09 vs. 2.36 μm; p < .01; MT: 0.59 vs. 0.40 μm, p < .01)., Conclusions: This study indicates that a Bio 3D nerve conduit can enhance peripheral nerve regeneration even in a 10 mm nerve defect model., (© 2019 Wiley Periodicals, Inc.)

Background: The reduction of systemic immunosuppressive agents is essential for the expansion of vascularized composite allotransplantation (VCA) in a clinical setting. The purpose of this study is to compare human-induced pluripotent stem cell-derived mesenchymal stem cells (iMSCs) with four other types of mesenchymal stem cells (human bone marrow-derived MSCs [BMMSCs], human adipose-derived MSCs [ADMSCs], rat BMMSCs, and rat ADMSCs) in vitro, and to investigate the in vivo immunomodulatory effect of iMSCs in a rat VCA model., Materials and Methods: One Brown Norway (BN) rat, 2 Lewis (LEW) rats, and 1 Wistar rat were used in the mixed lymphocyte reaction (MLR), and 9 BN rats and 3 LEW rats (for donors), and 24 LEW rats (for recipients) were used in the VCA model. The abovementioned five types of MSCs were imaged to examine their morphology and were also tested for suppressor function using a MLR. The 24 recipient LEW rats were divided randomly into four groups, and subjected to orthotopic hind limb transplantation. The three control groups were the Iso group, in which transplantation was performed on from three to six LEW rats without immunosuppressive treatment (n = 6); the FK group, in which transplantation was performed from BN rats to LEW rats and recipient rats were treated with tacrolimus alone (FK 506, 0.2 mg/kg, days 0-6 postoperatively, intraperitoneally) (n = 6); and the UT group, in which transplantation was performed from BN rats to LEW rats without any immunosuppressive treatment (n = 6). The experimental group was the iMSC group, in which transplantation was performed from BN rats to LEW rats and recipient rats were treated with tacrolimus (FK 506, 0.2 mg/kg, days 0-6 postoperatively, intraperitoneally) and injected with iMSCs (2 × 10 6 cells, day 7, intravenously) (n = 6). Hind limb survival was assessed by daily inspection of gross appearance until 50 days postoperatively. Histology of the skin and muscle biopsy were investigated on day 14 postoperatively. A time series of the plasma cytokine level (before transplantation, and at 10, 14, and 17 days after transplantation) was also analyzed., Results: The size of adherent and trypsinized iMSCs was 67.5 ± 8.7 and 9.5 ± 1.1 μm, respectively, which was the smallest among the five types of MSCs (p < .01). The absorbance in MLR was significantly smaller with rat ADMSCs (p = .0001), human iMSCs (p = .0006), rat BMMSCs (p = .0014), human ADMSCs (p = .0039), and human BMMSCs (p = .1191) compared to without MSCs. In vivo, iMSC treatment prolonged hind limb survival up to 12.7 days in macroscopic appearance, which is significantly longer than that of the FK group (p < .01). Histology of the skin and muscle biopsy revealed that mononuclear cell infiltration was significantly reduced by iMSC injection (p < .01). iMSC treatment also affected proinflammatory cytokines (interferon-gamma (IFNγ) and tumor necrosis factor α (TNFα)) and the anti-inflammatory cytokine (interleukin-10 (IL-10)) of the recipient plasma. The IFNγ levels at Δ14 and the TNFα levels at Δ14 and Δ17 of the iMSC group were significantly lower than those of the FK group (p = .0226, .0004, and .004, respectively). The IL-10 levels at Δ10 and Δ14 of the iMSC group were significantly higher than those of the FK group (p = .0013 and .0374, respectively)., Conclusions: iMSCs induce T cell hyporesponsiveness to prolong hind limb survival in a rat VCA model. This immunomodulatory property against acute rejection could provide one of the promising strategies capable of enabling the toxicities of immunosuppressants to be avoided in clinical settings., (© 2019 Wiley Periodicals, Inc.)

Background: Skin is considered to be the most antigenic component of all vascularized composite allotransplantation tissues. However, no studies have used methods other than histological assessment to analyze the relative antigenicity of various components. In this study, we analyzed gene expression to investigate the relative antigenicity of each component in the transplanted limb., Methods: Seven Brown Norway rats and 31 Lewis rats were assigned to two groups: an allograft group and a syngeneic (control) group. Brown Norway rats were used as the allogeneic donors, and Lewis rats were used as the syngeneic donors and recipients. About 13 recipients in the allograft group and 12 recipients in the control group were analyzed. Histological assessment was performed in 5 of the recipients in each group, and microRNA expression was analyzed in the remaining recipients, except for 1 recipient in the syngeneic group., Results: In the allograft group, the relative microRNA-146a expression was significantly higher in skin (2.34 ± 0.44) than in muscle (1.25 ± 0.22; p = .034) and bone (1; p = .0081). In the allograft group, microRNA-155 expression was significantly higher in skin (1.91 ± 0.18) than in bone (1; p = .010). Histological assessment showed that some skin tissue in the allograft group showed evidence of severe acute rejection., Conclusions: The microRNA-146a and microRNA-155 seemed to reflect the relative antigenicity during acute rejection of transplanted limbs. Skin seemed to be more antigenic than muscle and bone in both the histological assessment and gene expression analysis., (© 2018 Wiley Periodicals, Inc.)

Background: Limb transplantation is considered to be a treatment option for amputees. Visual skin inspection and histological assessment are used to assess rejection, but these techniques are largely subjective. Using a rat model, we examined the potential of several microRNAs (miRNAs) to be used as objective and minimally invasive biomarkers of acute rejection of transplanted limbs., Methods: Three Brown Norway rats and 15 Lewis rats were assigned to 2 groups. In the allograft group, 3 Brown Norway rats were used as the donors and 6 Lewis rats as the recipients. In the syngeneic (control) group, 9 Lewis rats were used as the donors and recipients. The hind limbs of the donors were orthotopically transplanted to the recipients. Plasma samples were obtained from all recipients before surgery and on posttransplantation days 3, 7, 10, and 14. All recipients were euthanized on day 14, and skin tissues were harvested for histological assessment., Results: On posttransplantation days 10 and 14, the plasma expression of miRNA-146a, miRNA-155, and miRNA-182 was significantly upregulated in the allograft group compared with the syngeneic control group. Of these 3 miRNAs, miRNA-182 had the highest sensitivity and specificity; however the cutoff point for miRNA-182 was close to the baseline and miRNA-155 was considered to be most suitable for identifying rejection. Most skin samples in the allograft group were classified as exhibiting grade III rejection on day 14., Conclusions: Some miRNAs are upregulated during acute rejection of transplanted limbs. These miRNAs are potential biomarkers for the objective, early, minimally invasive diagnosis of rejection., (© 2017 Wiley Periodicals, Inc.)

Background: Recent studies have indicated that bone marrow-derived stromal cells (BMSCs) have immunomodulatory properties that suppress the T cell responses that cause graft rejection. The purpose of this study is to evaluate the effect of recipient BMSCs intravenous infusion for immunomodulation in a rat vascularized composite allotransplantation model., Methods: A total of nine Wistar (WIS) rats and thirty Lewis (LEW) rats were used. BMSCs were harvested from three LEW rats. Twenty-four LEW rats were used as recipients and divided randomly into four groups: BMSC group, FK group, UT group, and Iso group. In the BMSC group, orthotopic rat hind limb transplantation was performed between WIS donor and LEW recipient rats. Recipient rats were injected intravenously with 2 × 10 6 recipient BMSCs on day 6, and with 0.2 mg/kg/day tacrolimus administered over 7 days (n = 6). In the FK group, recipient rats were treated with tacrolimus alone (n = 6). Rats in the UT group received no immunosuppressive treatment (n = 6). In the Iso group, transplantation was performed from three LEW donor rats to six LEW recipient rats without any immunosuppressive treatment (n = 6). Graft survival was assessed by daily inspection and histology. The immunological reactions of recipients were also evaluated., Results: The graft survival of recipient rats in the BMSC group (24.5 days) was significantly prolonged in comparison with that of the FK group (18 days) (P < .01). Cytokine expression analysis of the skin of grafted limbs showed that BMSCs treatment significantly decreased IFN-γ mRNA expression of the BMSC group (0.138 ± 0.045) in comparison with that of the FK group (1.049 ± 0.167) (P = .0001). Recipient rats in the BMSC group had significantly reduced serum IFN-γ cytokine levels (1.571 ± 0.779 pg/ml) in comparison with that of the FK group (7.059 ± 1.522 pg/ml) (P = .001). In in vitro study, BMSCs induce T cell hyporesponsiveness in a mixed lymphocyte reaction., Conclusion: BMSCs induce T cell hyporesponsiveness and prolong graft survival in the rat vascularized composite allotransplantation model. BMSCs exhibit immunomodulatory properties against acute rejection that can be realized without the need for significant recipient immunosuppression., (© 2016 Wiley Periodicals, Inc.)

Previously, we showed that undifferentiated bone marrow stromal cell (uBMSC) implantation and vessel insertion into a nerve conduit facilitated peripheral nerve regeneration in a rodent model. In this study, we investigated the efficacy of the uBMSC-laden vessel-containing conduit in repair of segmental nerve defects, using a canine model. Eight beagle dogs were used in this study. Thirty-millimeter ulnar nerve defects were repaired with the conduits (right forelimbs, n = 8) or autografts (left forelimbs, n = 7). In the conduit group, the ulnar artery was inserted into the l-lactide/ε-caprolactone tube, which was filled with autologous uBMSCs obtained from the ilium. In the autograft group, the reversed nerve segments were sutured in situ. At 8 weeks, one dog with only nerve repair with the conduit was sacrificed and the regenerated nerve in the conduit underwent immunohistochemistry for investigation of the differentiation capability of the implanted uBMSCs. In the remaining seven dogs, the repaired nerves underwent electrophysiological examination at 12 and 24 weeks and morphometric measurements at 24 weeks. The wet weight of hypothenar muscles was measured at 24 weeks. At 8 weeks, almost 35% of the implanted uBMSCs expressed glial markers. At 12 weeks, amplitude (0.4 ± 0.4mV) and conduction velocity (18.9 ± 14.3m/s) were significantly lower in the conduit group than in the autograft group (3.2 ± 2.5 mV, 34.9 ± 12.1 m/s, P < 0.05). Although the nerve regeneration in the conduit group was inferior when compared with the autograft group at 24 weeks, there were no significant differences between both groups, regarding amplitude (10.9 ± 7.3 vs. 25.3 ± 20.1 mV; P = 0.11), conduction velocity (23.5 ± 8.7 vs 31.6 ± 20.0m/s; P = 0.35), myelinated axon number (7032 ± 4188 vs 7165 ± 1814; P = 0.94), diameter (1.73 ± 0.31 vs 2.09 ± 0.39μm; P = 0.09), or muscle weight (1.02 ± 0.40 vs 1.19 ± 0.26g; P = 0.36). In conclusion, this study showed that vessel-containing tubes with uBMSC implantation may be an option for treatment of peripheral nerve injuries. However, further investigations are needed. © 2015 Wiley Periodicals, Inc. Microsurgery 36:316-324, 2016., (© 2015 Wiley Periodicals, Inc.)