Ryosuke Ikeguchi

Co-infusion of donor bone marrow with host mesenchymal stem cells treats GVHD and promotes vascularized skin allograft survival in rats.

We investigated the effect of autologous mesenchymal stem cells (MSC) on multiple unmodified donor bone marrow (BM) infusions and vascularized skin graft outcome. BM-derived rat MSC were examined for phenotype and function. MSC/MSC-conditioned-medium suppressed IFN-gamma production by T cells and modified DC function. Infusions of MSC with one-time BM improved vascularized skin graft survival, while with one-two-times BM reversed graft versus host disease (GVHD). Mixed chimerism was enhanced in recipients given two-four-times BM with MSC infusions. Interestingly, four-times BM infusions with MSC delayed GVHD onset, reduced host tissue damage and enhanced vascularized skin allograft survival compared to four-times BM alone. These data demonstrate that, the co-infusion of MSC with unmodified BM limit the toxicity of allogeneic BM transplantation, enhance mixed chimerism and improve vascularized skin graft survival. These findings provide insights for the development of autologous MSC-based BM transplantation and prevention of graft rejection or treatment of autoimmunity.

Long-Term Survival of Limb Allografts Induced by Pharmacologically Conditioned, Donor Alloantigen-Pulsed Dendritic Cells Without Maintenance Immunosuppression

Background. We showed recently that limb allograft survival could be enhanced by administration of alloantigen (Ag)-pulsed immature dendritic cells (DC) after transplantation. Since indefinite graft survival was not achieved, we have further modified the DC by pharmacologic (rapamycin; Rapa) conditioning and ascertained their influence on graft survival, without continued immunosuppressive therapy. Methods. We compared the ability of donor Ag-pulsed, Rapa-conditioned rat myeloid DC (Rapa DC) and control DC (CTR DC) to inhibit alloreactive T-cell responses after limb transplantation in antilymphocyte serum (ALS)-treated recipients given a short postoperative course of cyclosporine (CsA). Results. Both DC populations expressed similar levels of major histocompatibility complex (MHC) II, CD40 and CD54, but Rapa DC expressed lower CD86. After toll-like receptor activation, both populations produced minimal interleukin (IL)-12p70, but Rapa DC secreted lower levels of IL-6 and IL-10. The capacity of DCs to stimulate T-cell proliferation in mixed leukocyte reactions was very low. Pulsing of the DC with donor Ag did not alter their phenotype or function. Interestingly, posttransplant administration of donor Ag-pulsed Rapa DC to rats given perioperative ALS and 21 days CsA significantly delayed graft rejection and promoted long-term (>125 days) graft survival. AlloAg-pulsed Rapa DC induced T-cell hyporesponsiveness and promoted the generation of IL-10-secreting CD4+ T cells upon ex vivo challenge. Conclusions. Infusion of donor Ag-pulsed, Rapa-conditioned DC after composite tissue transplantation can prevent rejection of the grafts, including skin, across a full MHC mismatch and in the absence of continued immunosuppressive therapy.

Treatment of posttraumatic painful neuromas at the digit tip using neurovascular island flaps.

PURPOSE We report the treatment of 9 patients with 9 painful neuromas at the tips of the digits using reverse pedicled island flaps containing subcutaneous nerves that were connected to the digital nerve stumps after removal of the neuromas. METHODS There were 9 patients (7 men and 2 women) with painful cutaneous neuromas at the tips of the digits. The ages of the patients at time of surgery were 21 to 66 years (mean, 46 y). All neuromas were formed in the palmar digital nerves in the hand. Preoperative and postoperative status of the patients with neuromas were assessed and graded using a system modified from previously described grading systems for patients with neuromas. Recovery of sensation in the transplanted skin islands was assessed using the Semmes-Weinstein monofilament test. RESULTS In the preoperative assessment of patients using the grading system for neuromas, 3 patients were assessed as grade 4 (severe), 5 patients as grade 3 (moderate), and the remaining patient as grade 2 (mild). After surgery, Tinel sign disappeared completely in 6 of 9 patients. According to the grading system for neuromas, 6 patients were assessed as grade 1 (normal), and the other 3 patients were assessed as grade 2 (mild) postoperatively. Using the Semmes-Weinstein monofilament test for assessing recovery of sensation of the skin islands, 3 patients recognized the 2.83 monofilament (normal), 2 patients recognized the 3.22 monofilament (loss of tactile sense), and the remaining 4 patients recognized the 3.66 monofilament applied (loss of tactile sense). CONCLUSIONS This skin island approach provided us with good pain control and recovery of hand function after painful neuromas. TYPE OF STUDY/LEVEL OF EVIDENCE Therapeutic IV.

Nerve regeneration promoted in a tube with vascularity containing bone marrow-derived cells.

Bone marrow-derived cells (BMCs) are multipotent cells that have the potential to differentiate into bone, cartilage, fat, muscle, or neuronal lineages such as neurons and glial cells. A silicone tube model containing reverse-pedicled sural vessels was created in the sciatic nerves of Lewis rats. About 1 × 107 BMCs, removed from the bone marrow of synergetic rat femurs and cultured in vitro, were transplanted into the 15-mm-long chambers of the silicone tubes. Nerve regeneration in vessel-containing tubes that had received BMCs was significantly greater at 12 and 24 weeks after surgery than in tubes that did not receive cells. Transplantation of fibroblasts instead of BMCs into the vessel-containing tube resulted in reduced axonal regeneration, which was inferior to regeneration in the vessel-containing tube that did not receive cells. Polymerase chain reaction (PCR) studies revealed that in vessel-containing tubes containing transplanted BMCs, about 29% of cells in the regenerated nerve originated from BMCs. Cells identified by in situ hybridization and PKH26 prelabeling as being of BMC origin stained positively for S100 and GFAP. Transplanted BMCs differentiated into cells with phenotypes similar to those of Schwann cells under the influence of neurochemical factors and survived by obtaining nutrients from vessels that had been preinserted into the tube. They thus functioned similarly to Schwann cells, promoting nerve regeneration.

Regeneration of osteonecrosis of canine scapho-lunate using bone marrow stromal cells: Possible therapeutic approach for Kienböck disease

We evaluated the ability of canine bone marrow stromal cells (cBMSCs) to regenerate bone in a cavity of the scapholunate created by curretage and freeze–thawing with liquid nitrogen (LN). Autologous BMSCs were harvested from the iliac crest and expanded in vitro. Their potential to differentiate into osteo-, chondro-, and adipogenic lineages was confirmed using a standard differentiation induction assay. LN-treated scapholunates showed no regeneration of bone tissue when the cavity was left alone, demonstrating severe collapse and deformity as observed in human Kienböck disease. A combination of β-tri-calcium phosphate and a vascularized bone graft with autologous fibroblasts failed to regenerate bone in the LN-treated cavity. When the same procedure was performed using BMSCs, however, LN-treated scapholunates showed no collapse and deformity, and the cavity was completely filled with normal cancerous bone within 4 weeks. These results suggested the potential of using BMSCs to treat Kienböck disease.

The efficacy of a scaffold-free Bio 3D conduit developed from human fibroblasts on peripheral nerve regeneration in a rat sciatic nerve model

Background Although autologous nerve grafting is the gold standard treatment of peripheral nerve injuries, several alternative methods have been developed, including nerve conduits that use supportive cells. However, the seeding efficacy and viability of supportive cells injected in nerve grafts remain unclear. Here, we focused on a novel completely biological, tissue-engineered, scaffold-free conduit. Methods We developed six scaffold-free conduits from human normal dermal fibroblasts using a Bio 3D Printer. Twelve adult male rats with immune deficiency underwent mid-thigh-level transection of the right sciatic nerve. The resulting 5-mm nerve gap was bridged using 8-mm Bio 3D conduits (Bio 3D group, n = 6) and silicone tube (silicone group, n = 6). Several assessments were conducted to examine nerve regeneration eight weeks post-surgery. Results Kinematic analysis revealed that the toe angle to the metatarsal bone at the final segment of the swing phase was significantly higher in the Bio 3D group than the silicone group (-35.78 ± 10.68 versus -62.48 ± 6.15, respectively; p < 0.01). Electrophysiological studies revealed significantly higher compound muscle action potential in the Bio 3D group than the silicone group (53.60 ± 26.36% versus 2.93 ± 1.84%; p < 0.01). Histological and morphological studies revealed neural cell expression in all regions of the regenerated nerves and the presence of many well-myelinated axons in the Bio 3D group. The wet muscle weight of the tibialis anterior muscle was significantly higher in the Bio 3D group than the silicone group (0.544 ± 0.063 versus 0.396 ± 0.031, respectively; p < 0.01). Conclusions We confirmed that scaffold-free Bio 3D conduits composed entirely of fibroblast cells promote nerve regeneration in a rat sciatic nerve model.

Comparison between partial ulnar and intercostal nerve transfers for reconstructing elbow flexion in patients with upper brachial plexus injuries

Background There have been several reports that partial ulnar transfer (PUNT) is preferable for reconstructing elbow flexion in patients with upper brachial plexus injuries (BPIs) compared with intercostal nerve transfer (ICNT). The purpose of this study was to compare the recovery of elbow flexion between patients subjected to PUNT and patients subjected to ICNT. Methods Sixteen patients (13 men and three women) with BPIs for whom PUNT (eight patients) or ICNT (eight patients) had been performed to restore elbow flexion function were studied. The time required in obtaining M1, M3 (Medical Research Council scale grades recovery) for elbow flexion and a full range of elbow joint movement against gravity with the wrist and fingers extended maximally and the outcomes of a manual muscle test (MMT) for elbow flexion were examined in both groups. Results There were no significant differences between the PUNT and ICNT groups in terms of the age of patients at the time of surgery or the interval between injury and surgery. There were significantly more injured nerve roots in the ICNT group (mean 3.6) than in the PUNT group (mean 2.1) (P = 0.0006). The times required to obtain grades M1 and M3 in elbow flexion were significantly shorter in the PUNT group than in the ICNT group (P = 0.04 for M1 and P = 0.002 for M3). However, there was no significant difference between the two groups in the time required to obtain full flexion of the elbow joint with maximally extended fingers and wrist or in the final MMT scores for elbow flexion. Conclusions PUNT is technically easy, not associated with significant complications, and provides rapid recovery of the elbow flexion. However, separation of elbow flexion from finger and wrist motions needed more time in the PUNT group than in the ICNT group. Although the final mean MMT score for elbow flexion in the PUNT group was greater than in the ICNT group, no statistically significant difference was found between the two groups.

Reconstruction of a phalangeal bone using a vascularised metacarpal bone graft nourished by a dorsal metacarpal artery.

SUMMARY We report on a patient with an infected nonunion of the left little-finger phalanges following a gunshot injury. The defect was treated by transplanting a partial fifth metacarpus, vascularised by the fourth dorsal metacarpal vessels. Bone union was obtained 6 months after surgery and no signs of infection were found at the site of the nonunion. Although the range of the interphalangeal joints of the finger was limited, the patient was satisfied because the preserved little finger had a metacarpophalangeal (MP) joint with unrestricted motion.

Basic fibroblast growth factor promotes nerve regeneration in a C--ion-implanted silicon chamber

We reported previously that a silicone tube whose inner surface has been implanted with negatively charged carbon ions (C-) enables a nerve to regenerate across a 15-mm inter-stump gap. In this study, we investigated whether a C- -ion-implanted tube pretreated with basic fibroblast growth factor promotes peripheral nerve regeneration. The C- -ion-implanted tube significantly accelerated nerve regeneration, and this effect was enhanced by basic fibroblast growth factor.

Successful storage of peripheral nerve before transplantation using green tea polyphenol : An experimental study in rats

Green tea polyphenol is known to act as a buffer, reducing biological responses to oxidative stress. Several effects of polyphenol have been reported, such as protection of tissue from ischemia, antineoplasmic and anti-inflammatory effects, and suppression of arteriosclerosis. In this study, we investigated whether peripheral nerve segments could be kept viable in a polyphenol solution for 1 month. Sciatic nerve segments, 20 mm long, were harvested from Lewis rats and treated in three different ways before transplanting to recipient Lewis rats to bridge sciatic nerve gaps created by removal of 15-mm-long nerve segments. Group F: nerve segments were transplanted immediately after harvesting. Group P: nerve segments were transplanted after they had been stored in Dulbeccos Modified Eagles Medium (DMEM) containing polyphenol for 7 days at 4 degrees C and then in DMEM for 21 days at 4 degrees C. Group M: nerve segments were stored in DMEM solution alone for 28 days at 4 degrees C. Viability of the nerve segments was assessed by vital staining (calcein-AM/ethidium homodimer), by electron microscopy and by genomic studies before transplantation. Nerve regeneration was evaluated using electrophysiological and morphological studies 12 and 24 weeks after transplantation. Neural cell viability of the preserved nerve segments was confirmed in group P, in which the nerve regeneration was similar to that in group F and superior to that in group M. Peripheral nerve segments can be successfully preserved for 1 month using green tea polyphenol.