Transcutaneous carbon dioxide application inhibits muscle atrophy after fracture in rats.

BACKGROUND: Muscle atrophy causes difficulty in resuming daily activities after a fracture. Because transcutaneous carbon dioxide (CO2) application has previously upregulated oxygen pressure in the local tissue, thereby demonstrating its potential in preventing muscle atrophy, here we investigated effects of CO2 application on muscle atrophy after femoral shaft fracture. METHODS: Thirty fracture model rats were produced and randomly divided into a no treatment (control group) and treatment (CO2 group) groups. After treatment, the soleus muscle was dissected at post-fracture days 0, 14, and 21. Evaluations were performed by measuring muscle weight and performing histological examination and gene expression analysis. RESULTS: Muscle weight was significantly higher in the CO2 group than in the control group. Histological analysis revealed that the muscle fiber cross-sectional area was reduced in both groups. Nevertheless, the extent of atrophy was lesser in the CO2 group. Muscle fibers in the control group tended to change into fast muscle fibers. Vascular staining revealed that more capillary vessels surrounded the muscle fibers in the CO2 group than in the control group. Messenger RNA (mRNA) analysis revealed that the CO2 group had a significantly enhanced expression of genes that were related to muscle synthesis. CONCLUSION: Transcutaneous CO2 application may be a novel therapeutic strategy for preventing skeletal muscle atrophy after fracture.

Effects of parathyroid hormone 1-34 on osteogenic and chondrogenic differentiation of human fracture haematoma-derived cells in vitro.

Parathyroid hormone (PTH) 1-34 has been shown to accelerate fracture healing. Previously, we reported that progenitor cells with osteogenic and chondrogenic potential exist in human fracture haematoma, suggesting that the fracture haematoma-derived progenitor cells (HCs) contribute to fracture healing. However, there has been no study investigating the effect of PTH on HCs. We investigated the effect of pulsatile and continuous PTH treatment on human fracture HCs in vitro. HCs were isolated from seven patients. The HCs were divided into four groups: growth medium; control [osteogenic medium (OM) without PTH]; PTH-C (OM with continuous PTH); and PTH-P (OM with pulsatile PTH) groups. Osteogenic differentiation potential and proliferation of HCs were compared among the four groups. For chondrogenesis, the HCs were divided into two groups: control [chondrogenic medium (CM) without PTH]; and PTH-C (CM with continuous PTH) groups, and chondrogenic differentiation potential was analysed. PTH treatment did not affect cell proliferation, regardless of the mode of administration. Osteogenic activity was also not significantly affected by continuous PTH treatment but significantly inhibited by pulsatile PTH treatment. Conversely, chondrogenic differentiation was significantly inhibited by continuous PTH treatment. Our results revealed that PTH treatment on HCs, either continuous or pulsatile, does not exhibit any positive effect, and indicates that exogenous PTH administration after fracture has no effect on HCs. PTH may not have a positive effect at the fracture site during the early stage of fracture healing in which haematoma formation occurs. Copyright © 2013 John Wiley & Sons, Ltd.

Topical cutaneous CO2 application by means of a novel hydrogel accelerates fracture repair in rats.

BACKGROUND: We previously demonstrated that topical cutaneous application of CO2, by means of a hydrogel in which the CO2 readily dissolves, increases blood flow and oxygen dissociation from hemoglobin in the soft tissues surrounding bone. In the present study, we utilized a rat fracture model to test the hypothesis that application of this treatment to fractured limbs would accelerate fracture repair. METHODS: A closed femoral shaft fracture was created in each rat. Topical cutaneous application of CO2 by means of a hydrogel was performed five times a week for up to four weeks in the CO2/hydrogel group (n = 60). Sham treatments were performed in the control group (n = 60). Radiographic, histological, immunohistochemical, laser Doppler perfusion imaging, real-time polymerase chain reaction, and biomechanical assessments were performed. RESULTS: Radiographic fracture union was evident at week 3 in twelve (86%) of fourteen animals in the CO2/hydrogel group compared with five (36%) of fourteen in the control group (p < 0.05; 95% CI [confidence interval] for the difference in union rate, 2.26% to 99.64%). Histological assessment revealed promotion of endochondral ossification in the CO2/hydrogel group. Immunohistochemical assessment at week 2 showed significantly greater capillary density in the CO2/hydrogel group (p < 0.05; 95% CI for the difference, 161 to 258 per mm(2)). Laser Doppler perfusion imaging demonstrated that the blood flow in the fractured limb was significantly greater at weeks 2 and 3 in the CO2/hydrogel group (p < 0.05; 95% CI for the difference, 8.4% to 22.4% and 6.7% to 19.0%, respectively). Gene expression of chondrogenic, osteogenic, and angiogenic markers was significantly greater in the CO2/hydrogel group at several time points. Ultimate stress, extrinsic stiffness, and failure energy (relative to the contralateral limb) were significantly greater in the CO2/hydrogel group at week 3 (p < 0.05; 95% CI for the difference, 24.8% to 67.5%, 4.0 % to 22.7%, and 9.6% to 58.8%, respectively). There were no significant differences between the groups with respect to any outcome measure at week 4. CONCLUSIONS: Topical cutaneous application of CO2 by means of a hydrogel accelerated fracture repair in association with the promotion of angiogenesis, blood flow, and endochondral ossification. .

Cubic Rashba spin-orbit interaction of a two-dimensional hole gas in a strained-Ge/SiGe quantum well.

The spin-orbit interaction (SOI) of a two-dimensional hole gas in the inversion symmetric semiconductor Ge is studied in a strained-Ge/SiGe quantum well structure. We observe weak antilocalization (WAL) in the magnetoconductivity measurement, revealing that the WAL feature can be fully described by the k-cubic Rashba SOI theory. Furthermore, we demonstrate electric field control of the Rashba SOI. Our findings reveal that the heavy hole (HH) in strained Ge is a purely cubic Rashba system, which is consistent with the spin angular momentum m(j) = ± 3/2 nature of the HH wave function.

Effect of low-intensity pulsed ultrasound on bone morphogenetic protein 7-induced osteogenic differentiation of human nonunion tissue-derived cells in vitro.

OBJECTIVES: Low-intensity pulsed ultrasound (US) has been shown to have positive effects on the healing of nonunions, and bone morphogenetic protein 7 (BMP-7) is known to be a strong stimulator of osteogenic differentiation. Recently, we showed that nonunion tissue contains multilineage mesenchymal progenitor cells, suggesting that nonunion tissue-derived cells may play an important role during the healing process of nonunions. In this study, we investigated whether low-intensity pulsed US promoted BMP-7-induced osteogenic differentiation of nonunion tissue-derived cells in vitro. METHODS: Nonunion tissue-derived cells were isolated from 7 patients. The cells were divided into two groups: (1) BMP-7 alone, consisting of nonunion tissue-derived cells cultured in osteogenic medium containing BMP-7 without low-intensity pulsed US treatment; and (2) BMP-7 + low-intensity pulsed US, consisting of nonunion tissue-derived cells cultured in osteogenic medium containing BMP-7 with low-intensity pulsed US treatment. The osteogenic differentiation potential and proliferation of nonunion tissue-derived cells were compared between the two groups. RESULTS: The alkaline phosphatase activity, gene expression levels of alkaline phosphatase and runt-related transcription factor 2, and mineralization were higher in the BMP-7 + low-intensity pulsed US group than in the BMP-7-alone group. There was no significant difference in cell proliferation between the two groups. CONCLUSIONS: These findings show a significant effect of low-intensity pulsed US on the osteogenic differentiation of nonunion tissue-derived cells induced by BMP-7. This study may provide substantial evidence for the clinical combined application of BMP-7 and low-intensity pulsed US for nonunion treatment.

In vitro hypertrophy and calcification of human fracture haematoma-derived cells in chondrogenic differentiation.

PURPOSE: The haematoma at a fracture site plays an important role in fracture healing. Previously, we demonstrated that a fracture haematoma contains multilineage mesenchymal progenitor cells. We postulated that the haematoma provided a source of chondrogenic cells for endochondral ossification during fracture healing and preservation of the cells contributed to biological fracture healing. In this study, we investigated whether haematoma-derived cells (HCs) could differentiate into hypertrophic chondrocytes and finally induce calcification of the extracellular matrix in vitro. METHODS: Fracture haematomas were obtained from four patients. HCs were cultured for five weeks under conditions that induce chondrogenic differentiation, followed by two weeks of hypertrophic induction using a pellet culture system. The pellets were analysed histologically and immunohistochemically. The gene expression levels of chondrogenic, hypertrophic, osteogenic, and angiogenic markers were measured by real-time PCR. RESULTS: The histological and immunohistochemical analyses revealed that HCs differentiated into chondrocytes and hypertrophic chondrocytes, followed by calcification of the extracellular matrix. This sequential differentiation was also reflected in the gene expression profiles. After chondrogenic induction, expression of osteogenic and angiogenic markers was not significantly upregulated. However, the expression of these markers was significantly upregulated following hypertrophic induction. These in vitro observations mimicked the process of endochondral ossification during fracture healing. CONCLUSIONS: Our results suggest that the fracture haematoma may offer a source of cells with chondrogenic potential that play key roles in endochondral ossification during fracture healing. These findings support the opinion that the haematoma should be preserved for biological fracture healing.

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.

Incidence of venous thromboembolism in fractures around and below the knee with physical prophylaxis.

Fractures occurring at the distal part of the lower extremities are recognized to have a relatively lower risk of venous thromboembolism (VTE); however, few detailed reports exist on the subject. The purpose of this study was to investigate the incidence of VTE in fractures around and below the knee. Overall, 109 consecutive patients with fractures around and below the knee who were surgically treated at the authors' hospital were analyzed retrospectively. Physical prophylaxis was performed in all patients. Until April 2009, VTE screening was performed by contrast-enhanced computed tomography or ultrasonography when the D-dimer value did not decline predictably, exceeded 20 µg/mL 5 days after trauma and surgery, or increased above 20 µg/mL after a period of decline. After April 2009, ultrasonography was routinely performed pre- and postoperatively irrespective of the D-dimer value. The patients were divided into 2 groups based on the absence or presence of accompanying injuries, including head, chest, abdominal, or spinal injury and other fractures of the pelvis and lower extremities. Overall, VTE and pulmonary thromboembolism were detected in 28 (25.7%) patients and 5 (4.6%) patients, respectively. All cases were asymptomatic. The VTE incidence rates were 8.6% (former screening) and 40% (newer screening) in patients with isolated fractures and 25% (former screening) and 41.7% (newer screening) in patients with accompanying injuries. The pulmonary thromboembolism incidence rates were 2.9% (former screening) and 0% (newer screening) in patients with isolated fractures and 3.2% (former screening) and 25.0% (newer screening) in patients with accompanying injuries. Surgeons should be vigilant for symptoms of VTE in patients with fractures occurring at the distal part of the lower extremities.

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.

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.

Incidence of venous thromboembolism in pelvic and acetabular fractures in the Japanese population.

BACKGROUND: There are no detailed reports of the incidence of venous thromboembolism (VTE) in pelvic and acetabular fractures in the Asian population. The purpose of this study was to investigate the incidence of VTE in pelvic and acetabular fractures in the Japanese population. METHODS: Forty-six Japanese patients with pelvic and acetabular fractures treated at our hospital from February 2004 to April 2011 were analyzed retrospectively. Until April 2009, VTE screening was performed by contrast-enhanced computed tomography (CT) or ultrasonography (US) when the D-dimer value did not decline predictably, still exceeded 20 µg/ml at 5 days after trauma and surgery, or increased >20 µg/ml after a period of decline. After April 2009, contrast-enhanced CT and US were performed routinely irrespective of the D-dimer value. Physical prophylaxis was performed in all patients. The effects of the presence of pelvic and acetabular fractures, fracture types, accompanying injuries, and screening strategies on the incidences of VTE and pulmonary thromboembolism (PTE) were investigated. RESULTS: Overall, 19 patients (41.3%) were diagnosed with VTE and PTE in ten (21.7%). All were asymptomatic. Compared with trauma patients without pelvic and acetabular fractures treated during the same period, significantly higher incidences of VTE and PTE were observed in patients with pelvic and acetabular fractures. No significant differences were observed in the incidences of VTE and PTE between pelvic and acetabular fractures or between patients with and without accompanying injuries. Compared with the previous screening strategy, the detection rates of VTE and PTE were higher for the newer screening strategy; however, the difference did not reach statistical significance. CONCLUSIONS: We should be vigilant for the high incidence of VTE, especially PTE, in patients with pelvic and acetabular fractures in the Japanese population.

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.

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.

Rashba spin-orbit coupling probed by the weak antilocalization analysis in InAlAs/InGaAs/InAlAs quantum wells as a function of quantum well asymmetry.

We have investigated the values of the Rashba spin-orbit coupling constant alpha in In(0.52)Al(0.48)As/In(0.53)Ga(0.47)As/In(0.52)Al(0.48)As quantum wells using the weak antilocalization (WAL) analysis as a function of the structural inversion asymmetry (SIA) of the quantum wells. We have found that the deduced alpha values have a strong correlation with the degree of SIA of the quantum wells as predicted theoretically. The good agreement between the theoretical and experimental values of alpha suggests that our WAL approach for deducing alpha values provides a useful tool in designing future spintronics devices that utilize the Rashba spin-orbit coupling.

Spin-filter device based on the Rashba effect using a nonmagnetic resonant tunneling diode.

We propose an electronic spin-filter device that uses a nonmagnetic triple barrier resonant tunneling diode (TB-RTD). This device combines the spin-split resonant tunneling levels induced by the Rashba spin-orbit interaction and the spin blockade phenomena between two regions separated by the middle barrier in the TB-RTD. Detailed calculations using the InAlAs/InGaAs material system reveal that a splitting of a peak should be observed in the I-V curve of this device as a result of the spin-filtering effect. The filtering efficiency exceeds 99.9% at the peak positions in the I-V curve.