Akihiro Koh

The effect of transcutaneous application of carbon dioxide (CO2) on skeletal muscle

In Europe, carbon dioxide therapy has been used for cardiac disease and skin problems for a long time. However there have been few reports investigating the effects of carbon dioxide therapy on skeletal muscle. Peroxisome proliferators-activated receptor (PPAR)-gamma coactivator-1 (PGC-1α) is up-regulated as a result of exercise and mediates known responses to exercise, such as mitochondrial biogenesis and muscle fiber-type switching, and neovascularization via up-regulation of vascular endothelial growth factor (VEGF). It is also known that silent mating type information regulation 2 homologs 1 (SIRT1) enhances PGC-1α-mediated muscle fiber-type switching. Previously, we demonstrated transcutaneous application of CO(2) increased blood flow and a partial increase of O(2) pressure in the local tissue known as the Bohr effect. In this study, we transcutaneously applied CO(2) to the lower limbs of rats, and investigated the effect on the fast muscle, tibialis anterior (TA) muscle. The transcutaneous CO(2) application caused: (1) the gene expression of PGC-1α, silent mating type information regulation 2 homologs 1 (SIRT1) and VEGF, and increased the number of mitochondria, as proven by real-time PCR and immunohistochemistry, (2) muscle fiber switching in the TA muscle, as proven by isolation of myosin heavy chain and ATPase staining. Our results suggest the transcutaneous application of CO(2) may have therapeutic potential for muscular strength recovery resulting from disuse atrophy in post-operative patients and the elderly population.

Ankle Arthrodesis Using Antegrade Intramedullary Nail for Salvage of Nonreconstructable Tibial Pilon Fractures

The treatment of nonreconstructable tibial pilon fractures for which the optimal timing for reduction and fixation has been missed is challenging. Ankle arthrodesis may be a treatment option in such cases. We treated 2 patients with nonreconstructable tibial pilon fractures using ankle arthrodesis with an antegrade intramedullary nail. Our method included exposing of the ankle joint through a lateral approach; excising the distal fibula, comminuted fragments, and remaining articular cartilage; inserting an intramedullary nail in an antegrade fashion from the proximal tibia into the talus; insertion of 2 interlocking screws in the talus and the proximal tibia; and autologous bone grafting using the excised distal fibula. At latest follow-up at 2 and 1 year respectively, fusion was complete, and both patients were pain free and could walk without support. When ankle arthrodesis is performed for a tibial pilon fracture, an intramedullary nail is thought to be superior to a plate, which is bulky and may impede soft tissue healing. Moreover, insertion of an intramedullary nail in an antegrade fashion can preserve the subtalar joint, and is therefore preferred over placement in a retrograde fashion. Ankle arthrodesis using this technique can be a useful salvage procedure for a nonreconstructable tibial pilon fracture.

A Novel System for Transcutaneous Application of Carbon Dioxide Causing an “Artificial Bohr Effect” in the Human Body

Background Carbon dioxide (CO2) therapy refers to the transcutaneous administration of CO2 for therapeutic purposes. This effect has been explained by an increase in the pressure of O2 in tissues known as the Bohr effect. However, there have been no reports investigating the oxygen dissociation of haemoglobin (Hb) during transcutaneous application of CO2 in vivo. In this study, we investigate whether the Bohr effect is caused by transcutaneous application of CO2 in human living body. Methods We used a novel system for transcutaneous application of CO2 using pure CO2 gas, hydrogel, and a plastic adaptor. The validity of the CO2 hydrogel was confirmed in vitro using a measuring device for transcutaneous CO2 absorption using rat skin. Next, we measured the pH change in the human triceps surae muscle during transcutaneous application of CO2 using phosphorus-31 magnetic resonance spectroscopy (31P-MRS) in vivo. In addition, oxy- and deoxy-Hb concentrations were measured with near-infrared spectroscopy in the human arm with occulted blood flow to investigate O2 dissociation from Hb caused by transcutaneous application of CO2. Results The rat skin experiment showed that CO2 hydrogel enhanced CO2 gas permeation through the rat skin. The intracellular pH of the triceps surae muscle decreased significantly 10 min. after transcutaneous application of CO2. The NIRS data show the oxy-Hb concentration decreased significantly 4 min. after CO2 application, and deoxy-Hb concentration increased significantly 2 min. after CO2 application in the CO2-applied group compared to the control group. Oxy-Hb concentration significantly decreased while deoxy-Hb concentration significantly increased after transcutaneous CO2 application. Conclusions Our novel transcutaneous CO2 application facilitated an O2 dissociation from Hb in the human body, thus providing evidence of the Bohr effect in vivo.

Low-intensity pulsed ultrasound enhances BMP-7-induced osteogenic differentiation of human fracture hematoma-derived progenitor cells in vitro.

Objective To investigate the effect of the combined application of bone morphogenetic protein-7 (BMP-7) and low-intensity pulsed ultrasound (LIPUS) on human fracture hematoma-derived progenitor cells (HCs). Methods HCs were isolated from 6 patients. The cells were then divided into 4 groups and cultured: (1) control group, HCs cultured in growth medium without LIPUS; (2) LIPUS group, HCs cultured in growth medium with LIPUS; (3) BMP-7 group, HCs cultured in osteogenic medium containing BMP-7 without LIPUS; and (4) BMP-7 + LIPUS group, HCs cultured in osteogenic medium with LIPUS. Osteogenic differentiation potential and proliferation of HCs were compared among 4 groups. Results Alkaline phosphatase activity, the expression of osteogenic genes, and the mineralization of HCs in BMP-7 + LIPUS group were shown to be significantly increased compared with the other groups. However, LIPUS did not affect the proliferation of HCs in the presence or absence of BMP-7. Conclusions These findings demonstrated for the first time the significant effect of LIPUS on the osteogenic differentiation of HCs in the presence of BMP-7. This study may provide significant evidence for the clinical combined application of BMP-7 and LIPUS for the treatment of acute bone fractures.

Differential gene expression and immunolocalization of insulin‐like growth factors and insulin‐like growth factor binding proteins between experimental nonunions and standard healing fractures

Insulin‐like growth factors (IGF‐I/II) are important growth factors in bone, and their actions are regulated by six IGF binding proteins (IGFBPs). However, little is known about their exact functions in fracture healing. The aim of this study was to compare the gene expression and immunolocalization of IGFs and IGFBPs between standard healing fractures and nonunions using rat experimental models. Standard healing fractures and nonunions produced by periosteal cauterization at the fracture site were created in rat femurs. At postfracture days 3, 7, 10, 14, 21, and 28, total RNA was extracted from the callus of the healing fractures and the fibrous tissue of the nonunions, and gene expression were analyzed by real‐time PCR. Additionally, immunolocalization of these proteins was studied by immunohistochemistry at postfracture days 7, 14, and 21. In nonunions, the gene expression of IGF‐I/II and IGFBP‐6 was significantly higher, and that of IGFBP‐5 was significantly lower at several time points. The immunolocalization of IGF‐I/II and IGFBP‐5 was widely distributed in both models. In contrast, that of IGFBP‐6 was barely detected in the fracture callus. In conclusion, our results suggest that IGFs/IGFBPs may have important roles not only in fracture healing but also in nonunion formation.

Venous thromboembolism in Japanese patients with fractures of the pelvis and/or lower extremities using physical prophylaxis alone

Purpose. To investigate the rate of venous thromboembolism (VTE) in Japanese patients with fractures of the pelvis and/or lower extremities using physical prophylaxis alone. Methods. Records of 66 men and 60 women aged 15 to 95 (mean, 57) years with fractures of the pelvis and/or lower extremities were retrospectively reviewed. They were screened for VTE based on D-dimer values. Contrast-enhanced computed tomography and/or ultrasonography were performed when the D-dimer value did not decline predictably or exceeded 20 μg/ml even 5 days after injury or surgery. Physical prophylaxis for VTE in terms of graduated compression stockings and intermittent pneumatic compression were applied for all patients. Results. Of the 126 patients, 24 were detected to have VTE (10 of 29 with multiple fractures and 14 of 97 with single fractures). Six patients were detected to have asymptomatic pulmonary thromboembolism (PTE), whereas 20 patients were detected to have deep vein thrombosis (bilaterally in 7). The rates of VTE were high in patients with multiple fractures (35%), pelvic fractures (18%), and femoral shaft fractures (50%). The rate of PTE was high in patients with pelvic fractures (12%). Conclusion. The rate of VTE in the Japanese patients was similar to that in western populations. Our screening method was useful for preventing fatal PTEs. Surgeons should be vigilant for VTE during the first 2 weeks after injury, especially in patients with multiple and pelvic fractures.

Increase in carbon dioxide accelerates the performance of endurance exercise in rats

Endurance exercise generates CO2 via aerobic metabolism; however, its role remains unclear. Exogenous CO2 by transcutaneous delivery promotes muscle fibre-type switching to increase endurance power in skeletal muscles. Here we determined the performance of rats running in activity wheels with/without transcutaneous CO2 exposure to clarify its effect on endurance exercise and recovery from muscle fatigue. Rats were randomised to control, training and CO2 groups. Endurance exercise included activity-wheel running with/without transcutaneous CO2 delivery. Running performance was measured after exercise initiation. We also analysed changes in muscle weight and muscle fibres in the tibialis anterior muscle. Running performance improved over the treatment period in the CO2 group, with a concomitant switch in muscle fibres to slow-type. The mitochondrial DNA content and capillary density in the CO2 group increased. CO2 was beneficial for performance and muscle development during endurance exercise: it may enhance recovery from fatigue and support anabolic metabolism in skeletal muscles.

Technique to Prepare the Bed for Autologous Bone Grafting in Nonunion Surgery

This article describes a technique for preparing the bed for autologous bone grafting in nonunion surgery. The procedure is divided into 2 steps. First, both ends of the fracture fragments are chipped into small pieces using an osteotome and hammer without peeling off the periosteum, creating pathways into the bone marrow. Second, cancellous bone harvested from the iliac crest is grafted into the aperture created by the previous bone chipping treatment. The technique is easy to perform and is a promising approach for enhancing bone healing in nonunion and delayed union.