Yukitoshi Kaizawa

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.

A MODIFIED TENSION BAND WIRING TECHNIQUE FOR TREATMENT OF THE BONY MALLET FINGER

PURPOSE To report the outcomes of mallet fractures treated with our modified tension band wiring technique. METHODS Eleven men and two women (mean age; 33 years) with mallet fractures in which happened more than five weeks before surgery, or with fracture fragments involving more than 2/3 or less than 1/3 of the distal phalanx articular surface or with previous surgical intervention, were subjected to this study. The fracture fragment was fixed with a modified tension band wiring technique using a stainless steel wire and an injection needle. RESULTS All patients achieved bone union in nine weeks in average. All patients had no pain except one with mild pain. No patient showed a gap or step-off greater than 1 mm. CONCLUSIONS Our tension band wiring technique can be used regardless of the size of the dorsal fracture fragment or the interval between injury and surgery.

A new cervical nerve root avulsion model using a posterior extra-vertebral approach in rats

Background The nerve root avulsion injury causes decrease of motor neurons in the spinal ventral horn. To investigate the motoneuron death after avulsion injury in rats, the intradural root avulsion procedure is usually used, although it is technically demanding and associated with a risk of unexpected spinal cord damage. We have developed a new cervical nerve root avulsion procedure in rats and investigated the validity of our procedure. Methods Our procedure is using a posterior approach and pulling the C6 nerve root outside the vertebral foramen without intradural procedures. The lateral third of the lateral mass is needed to be resected before pulling the nerve root. The accomplishment of our procedure is judged by confirmation of the bifurcated stump of the avulsed nerve root and the leakage of the spinal fluid from vertebral foramen. At first, four Sprague–Dawley (SD) rats were used for the examination of C6 motor neuron distribution in the normal spinal cord. Then, 40 SD rats were divided into following four groups and the survival rate of motor neuron was examined. (A) an intradural avulsion group, (B) an intradural rhizotomy group, (C) our extravertebral avulsion group, and (D) an extravertebral rupture group. Another 26 SD rats were used for the examination of histomorphorogic changes in the spinal cord after our extra-vertebral avulsion procedure. Results At 28 days after injury, the percentage of surviving motor neurons in groups A (39.0 ± 2.1%) and C (47.5 ± 7.1%) were significantly lower than those in groups B (77.1 ± 12.3%) and D (98.9 ± 9.9%). Compared with other groups, our procedure was easier and associated with less unexpected spinal cord damage. Although the length of the distal stump of the extravertebrally avulsed ventral rootlets was varied between 1.5 and 3.2 mm, this difference did not affect motoneuron death. The extravertebral avulsion injury showed intraspinal bleeding along the motoneuron axons, glial reaction and macrophage infiltration in the lesioned side of the ventral horn. Conclusions Our extravertebral avulsion procedure is simple and reproducible. It would become a useful tool for the study of cervical nerve root avulsion injury.

Plasma micrornas Are Potential Biomarkers of Acute Rejection After Hindlimb Transplantation in Rats

Background The development of effective immunosuppressive regimens has resulted in many cases of successful hand transplantation. Visual skin inspection and histological evaluation are used to assess the rejection of hand transplants, but these methods are largely subjective. In this study, we aimed to determine the potential of microRNAs (miRNAs) as biomarkers for acute rejection in vascularized composite allotransplants. Methods In allograft group, 7 male Brown-Norway rats (RT1n) were used as donors and 13 male Lewis rats (RT1l) were used as recipients. In control group, 8 Lewis rats were used as donors and recipients. The hindlimbs of donor rats were transplanted orthotopically to recipient rats. Skin changes were noted daily. Skin biopsies were obtained from 5 recipients and evaluated histologically. Plasma samples were obtained from the other 8 recipients before transplant and 7, 10, and 14 days posttransplant and used to measure miRNA expression. Results Skin changes occurred at a mean of 11.0 days posttransplant. Rejection in most skin biopsies taken 7 and 10 days posttransplant was histologically classified as grade 0, whereas that in most biopsies taken 14 days posttransplant was classified as grade 3. We found that expression of miRNA-146a and miRNA-155 was significantly upregulated at 10 and 14 days posttransplant compared with that at 7 days posttransplant. In control group, there were no significant changes in plasma miRNAs expressions. Conclusions The upregulation of plasma miRNA-146a and miRNA-155 was detected before the histological evaluation methods could diagnose complete rejection in the rat hindlimb transplantation model. Plasma miRNA-146a and miRNA-155 may be potential biomarkers of acute rejection after vascularized composite allotransplantation.

The neuroprotective effect of erythropoietin on spinal motor neurons after nerve root avulsion injury in rats.

PURPOSE The rapid death of many spinal motor neurons after nerve root avulsion injury results in limited functional recovery following replantation surgery of avulsed nerves into the spinal cord. Therefore, we investigated the neuroprotective effect of erythropoietin (EPO) on motor neurons after nerve root avulsion injury using a rat model. METHODS After C6 nerve root avulsion injury, EPO (2680 U/kg) was injected subcutaneously once a day for 3 consecutive days with various starting time points. At 28 and 56 days after injury, histological and immunohistological investigations were performed. RESULTS EPO-treated rats showed a significant increase in the number of surviving motor neurons at day 28 when the initial dose was started within 96 h after injury. In EPO-treated rats, superoxide formation in the motor neurons and proliferation of microglia were markedly suppressed in the acute phase. GAP-43-positive surviving motor neurons were significantly increased in EPO-treated rats at day 28. However, at 56 days after surgery, EPO-treated rats showed a much greater decrease of surviving motor neurons compared with those at day 28. CONCLUSION The neuroprotective effect of EPO is not long lasting, but may prolong the time before replantation surgery.

Motor and Sensory Cortical Changes after Contralateral Cervical Seventh Nerve Root (CC7) Transfer in Patients with Brachial Plexus Injuries

BACKGROUND Previous animal studies demonstrated that the sensory and motor functions in ipsilesional upper limbs that had been reconstructed by CC7 transfer eventually associated with the contralesional brain cortices that had originally mediated the functions of the ipsilesional upper limbs before brachial plexus injury (BPI). Our hypothesis was that the same findings would be seen in humans. METHODS Four patients with total BPI treated with CC7 transfer were included. Changes in the locations of the activated areas in the primary motor (M1) and somatosensory (S1) cortices corresponding to the motor outputs to and sensory inputs from the ipsilesional limbs were investigated using functional near-infrared spectroscopy (fNIRS) 2-3 years and 6-7 years after surgery. RESULTS One patient was excluded from the evaluation of motor function after CC7 transfer. The motor and sensory functions of the ipsilesional upper limb in all patients were still controlled by the ipsilesional brain hemisphere 2-3 years after CC7 transfer. The reconstructed motions of the ipsilesional upper limbs correlated with the contralesional M1 in one patient and the bilateral M1s in another patient (both of whom demonstrated good motor recovery in the ipsilesional upper limbs) and with the ipsilesional M1 in a third patient with poor motor recovery in the ipsilesional upper limb. Sensory stimulation of the ipsilesional hands 6-7 years after CC7 transfer activated the contralesional S1 in two patients who achieved good sensory recovery in the ipsilesional hands but activated the ipsilesional S1 in the other two patients with poor sensory recovery of the ipsilesional hands. CONCLUSIONS Transhemispheric transposition of the activated brain cortices associated with the recovery of motor and sensory functions of the ipsilesional upper limbs was seen in patients with CC7 transfer as has been reported for animal models of CC7 transfer.

Recipient bone marrow-derived stromal cells prolong graft survival in a rat hind limb allotransplantation model

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.

A nerve conduit containing a vascular bundle and implanted with bone marrow stromal cells and decellularized allogenic nerve matrix.

Cells, scaffolds, growth factors, and vascularity are essential for nerve regeneration. Previously, we reported that the insertion of a vascular bundle and the implantation of bone marrow-derived mesenchymal stem cells (BM-MSCs) into a nerve conduit promoted peripheral nerve regeneration. In this study, the efficacy of nerve conduits containing a vascular bundle, BM-MSCs, and thermally decellularized allogenic nerve matrix (DANM) was investigated using a rat sciatic nerve model with a 20-mm defect. Lewis rats were used as the sciatic nerve model and for the preparation of BM-MSCs, and Dark Agouti rats were used for the preparation of the DANM. The revascularization and the immunogenicity of the DANM were investigated histologically. The regeneration of nerves through nerve conduits containing vessels, BM-MSCs, and DANM (VBD group) was evaluated based on electrophysiological, morphometric, and reinnervated muscle weight measurements and compared with that of vessel-containing conduits that were implanted with BM-MSCs (VB group). The DANM that was implanted into vessel-containing tubes (VCTs) was revascularized by neovascular vessels that originated from the inserted vascular bundle 5–7 days after surgery. The number of CD8+ cells found in the DANM in the VCT was significantly smaller than that detected in the untreated allogenic nerve segment. The regenerated nerve in the VBD group was significantly superior to that in the VB group with regard to the amplitude of the compound muscle action potential detected in the pedal adductor muscle; the number, diameter, and myelin thickness of the myelinated axons; and the tibialis anterior muscle weight at 12 and 24 weeks. The additional implantation of the DANM into the BM-MSC-implanted VCT optimized the axonal regeneration through the conduit. Nerve conduits constructed with vascularity, cells, and scaffolds could be an effective strategy for the treatment of peripheral nerve injuries with significant segmental defects.

MicroRNAs are potential objective and early biomarkers for acute rejection of transplanted limbs in a rat model

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.

Percutaneous Fixation for Scaphoid Nonunion with Bone Grafting Through the Distal Insertion Hole of a Fully Threaded Headless Screw.

BACKGROUND Percutaneous scaphoid screw fixation is a popular treatment for acute scaphoid fractures with no or minimal displacement. For treating scaphoid nonunions, however, open reduction and internal fixation with bone grafting is still the most popular treatment. Percutaneous fixation with bone grafting through the screw insertion hole has received little attention, although it minimizes damage to the surrounding tissues. We report excellent results of six scaphoid nonunions treated by retrograde percutaneous fixation with curettage and bone grafting through the distal insertion hole of a fully threaded headless screw. METHODS Six scaphoid nonunions with substantial bone loss were treated, including one revision case. All nonunions were located at the middle third of the scaphoid. The mean patient age at operation was 26 years, and the mean interval between fracture and surgery was 7 months. In the revision case, the interval between the primary and revision surgery was 6 months. In one case, curettage alone was performed before retrograde insertion of the headless screw. In the other cases including the revision, curettage and bone grafting with a bone biopsy needle was required through a distal insertion hole. RESULTS The mean follow-up was 11.3 months. Radiologically, union was achieved at averaged 12 weeks postoperatively. At the final follow-up, there was significant improvement in the wrist extension range of movement (from 65.8° to 80.8°) and grip strength (from 69.5% to 93.0% of the unaffected side). Five patients were free of pain, and one experienced mild pain only during heavy manual labor. The mean VAS, DASH, and Cooney wrist scores were 0.3, 1 and 99, respectively. All patients returned to their work or athletic activities. CONCLUSIONS Retrograde percutaneous fixation with bone grafting through the distal insertion hole of a fully threaded headless screw is a promising option for surgical treatment of scaphoid nonunions.