Altered expression of microRNA during fracture healing in diabetic rats.

OBJECTIVES: Diabetes mellitus (DM) is known to impair fracture healing. Increasing evidence suggests that some microRNA (miRNA) is involved in the pathophysiology of diabetes and its complications. We hypothesized that the functions of miRNA and changes to their patterns of expression may be implicated in the pathogenesis of impaired fracture healing in DM. METHODS: Closed transverse fractures were created in the femurs of 116 rats, with half assigned to the DM group and half assigned to the control group. Rats with DM were induced by a single intraperitoneal injection of streptozotocin. At post-fracture days five, seven, 11, 14, 21, and 28, miRNA was extracted from the newly generated tissue at the fracture site. Microarray analysis was performed with miRNA samples from each group on post-fracture days five and 11. For further analysis, real-time polymerase chain reaction (PCR) analysis was performed at each timepoint. RESULTS: Microarray analysis showed that there were 14 miRNAs at day five and 17 miRNAs at day 11, with a greater than twofold change in the DM group compared with the control group. Among these types of miRNA, five were selected based on a comparative and extended literature review. Real-time PCR analysis revealed that five types of miRNA (miR-140-3p, miR-140-5p, miR-181a-1-3p, miR-210-3p, and miR-222-3p) were differentially expressed with changing patterns of expression during fracture healing in diabetic rats compared with controls. CONCLUSIONS: Our findings provide information to further understand the pathology of impaired fracture healing in a diabetic rat model. These results may allow the potential development of molecular therapy using miRNA for the treatment of impaired fracture healing in patients with DM.Cite this article: S. Takahara, S. Y. Lee, T. Iwakura, K. Oe, T. Fukui, E. Okumachi, T. Waki, M. Arakura, Y. Sakai, K. Nishida, R. Kuroda, T. Niikura. Altered expression of microRNA during fracture healing in diabetic rats. Bone Joint Res 2018;7:139-147. DOI: 10.1302/2046-3758.72.BJR-2017-0082.R1.

Efficient cell-seeding into scaffolds improves bone formation.

Bone marrow stromal cells (BMSCs)/beta-tricalcium phosphate (beta-TCP) composites have attracted a great deal of attention in bone tissue engineering. If more effective bone regeneration is to be achieved, efficient cell-seeding systems need to be clarified. In this study, we investigated the number of cells contained in composites, and the in vitro/vivo osteogenic differentiation capacity of composites using 4 conventional systems of seeding rat BMSCs into beta-TCP: soak, low-pressure, pipette, and syringe systems. The highest number of cells was contained in the composites from the syringe group. Moreover, after two-week osteogenic induction in vitro, the composites in the syringe group exhibited the highest osteogenic potential, which continued at 8 weeks after subcutaneous implantation in vivo. Our results indicated that efficient and appropriate cell-seeding could improve in vitro/vivo bone formation in composites and thus make a potential clinical contribution to successful bone tissue engineering.

Prevention of corticosteroid-induced osteonecrosis in rabbits by intra-bone marrow injection of autologous bone marrow cells.

OBJECTIVES: Femoral head osteonecrosis (ON) is a serious complication of steroid administration. We evaluated bone marrow transplantation (BMT) for preventing corticosteroid-induced ON. METHODS: Rabbits, injected with methylprednisolone (MPSL; 20 mg/kg), were divided into four groups: (i) MPSL alone; MPSL injection only, (ii) MPSL+needling; 2 days after MPSL injection, a hole (1.2 mm diameter) was drilled from the outer cortex 2.5 cm distal to the proximal end of the greater trochanter, (iii) MPSL+saline; 2 days after MPSL injection, 2 ml saline was injected directly into the bone marrow cavity, and (iv) MPSL+BMT; 2 days after MPSL injection, 1 x 10(7)/2 ml bone marrow cells (BMCs) were injected directly into the bone marrow cavity. Platelets, fibrinogen, prothrombin time and total cholesterol in peripheral blood were measured before and after treatment. Tissues were stained with haematoxylin and eosion and terminal deoxynucleotidyl-mediated deoxyuridine triphosphate nick-end labelling stain and immunostained for VEGF, while cell proliferation and viability of whole BMCs in the femur were analysed by cell cycle analysis and [(3)H]-thymidine uptake. RESULTS: The ON incidence in rabbits treated with MPSL alone, MPSL+needling and MPSL+saline was 72.7, 70.0 and 66.7%, respectively, while in the MPSL+BMT group, the incidence was 0%. Serological findings in the MPSL+BMT group were almost normalized. VEGF and TUNEL staining were reduced in the MPSL+BMT group compared with all other groups. There were significantly fewer BMCs in G1 phase from the MPSL+BMT group than the other groups, while uptake of [(3)H]-thymidine was significantly increased. CONCLUSION: Direct injection of autologous BMCs into femurs prevents corticosteroid-induced ON following treatment with high-dose, short-term steroids.

An in vitro study demonstrating that haematomas found at the site of human fractures contain progenitor cells with multilineage capacity.

We isolated multilineage mesenchymal progenitor cells from haematomas collected from fracture sites. After the haematoma was manually removed from the fracture site it was cut into strips and cultured. Homogenous fibroblastic adherent cells were obtained. Flow cytometry revealed that the adherent cells were consistently positive for mesenchymal stem-cell-related markers CD29, CD44, CD105 and CD166, and were negative for the haemopoietic markers CD14, CD34, CD45 and CD133 similar to bone-marrow-derived mesenchymal stem cells. In the presence of lineage-specific induction factors the adherent cells could differentiate in vitro into osteogenic, chondrogenic and adipogenic cells. Our results indicate that haematomas found at a fracture site contain multilineage mesenchymal progenitor cells and play an important role in bone healing. Our findings imply that to enhance healing the haematoma should not be removed from the fracture site during osteosynthesis.

Implication of spinal protein kinase C in the suppression of morphine-induced rewarding effect under a neuropathic pain-like state in mice.

We previously demonstrated that spinal protein kinase C (PKC) is involved in the development of a neuropathic pain-like state induced by sciatic nerve ligation, and the morphine-induced rewarding effect is attenuated by sciatic nerve ligation in rodents. Here we first investigated whether sciatic nerve injury could change the activity of a conventional PKC (cPKC) and an atypical PKC isoform PKCzeta in the mouse spinal cord. The second experiment was to investigate whether direct inhibition of spinal PKC by intrathecal (i.t.) administration of a specific PKC inhibitor, 2-[8-[(dimethylamino)methyl]-6,7,8,9-tetrahydropyrido[1,2-a]indol-3-yl]-3-(1-methyl-1H-indole-3-yl)maleimide (RO-32-0432), could affect the rewarding effect induced by morphine following sciatic nerve ligation in mice. We found here that the activities of both cPKC and PKCzeta in the spinal cord were clearly increased following sciatic nerve ligation. Furthermore, i.t. administration of RO-32-0432 reversed a long-lasting pain-like syndrome as indicated by thermal hyperalgesia following sciatic nerve ligation in mice. These data provide direct evidence that activated cPKC and PKCzeta in the spinal cord may contribute to the development and maintenance of neuropathic pain. In the present study, we confirmed that the morphine-induced place preference was significantly suppressed by sciatic nerve ligation. It should be mentioned that i.t. pretreatment with RO-32-0432 significantly reversed the attenuation of morphine-induced rewarding effect following sciatic nerve ligation. These results suggest that activation of PKCs, including cPKC and PKCzeta, within the spinal cord is directly responsible for the attenuation of the morphine-induced rewarding effect under a neuropathic pain-like state following sciatic nerve ligation in mice.

Increased QT dispersion does not reflect the increased regional variation of cardiac sympathetic nervous activity in hypertrophic cardiomyopathy.

BACKGROUND: QT dispersion (QTD) reflects regional variation of ventricular repolarization. However, the relationship between QTD and the regional variation of cardiac sympathetic nerve activity in hypertrophic cardiomyopathy (HCM) is not yet elucidated. METHODS: Cardiac sympathetic nerve activity was evaluated in 25 patients with HCM by iodine 123 metaiodobenzylguanidine (MIBG) myocardial scintigraphy. With planar MIBG imaging, heart and mediastinum ratios (H/M) at early (20 minutes) and delayed (3 hours) acquisition and the washout rate (WR) were calculated. Polar maps of left ventricular myocardium were divided into 20 segments. The SD of early uptake (EU-SD), delayed uptake (DU-SD), and WR (WR-SD) in 20 segments as indices of regional variation were calculated. QT intervals were corrected by use of the Bazett formula. RESULTS: Maximum QTc correlated positively with H/M early, WR, and left ventricular wall thickness (LVWT). Minimum QTc correlated positively with WR and LVWT. Corrected QTD (QTDc) correlated negatively with EU-SD, DU-SD, and WR-SD and positively with the interventricular septal thickness/posterior wall thickness ratio. Stepwise regression analysis revealed that the most powerful determinants for maximum QTc, minimum QTc, and QTDc were WR, LVWT, and EU-SD, respectively. CONCLUSIONS: QTD correlated negatively rather than positively with the regional variability index of cardiac sympathetic nerve activity. These results suggest that increased QTD in patients with HCM may not reflect increased heterogeneity of cardiac sympathetic nerve activity.

Significance of adrenomedullin under cardiopulmonary bypass in children during surgery for congenital heart disease.

To elucidate the effect of adrenomedullin (AM) on fluid homeostasis under cardiopulmonary bypass (CPB), we investigated the serial changes in plasma AM and other parameters related to fluid homeostasis in 13 children (average age, 28.2 months) with congenital heart disease during cardiac surgery under CPB. Arterial blood and urine samples were collected just after initiation of anesthesia, just before commencement of CPB, 10 min before the end of CPB, 60 min after CPB, and 24 h after operation. Plasma AM levels increased significantly 10 min before the end of CPB and decreased 24 h after operation. Urine volume increased transiently during CPB, which paralleled changes in AM. Simple regression analysis showed that plasma AM level correlated significantly with urinary vasopressin, urine volume, urinary sodium excretion, and plasma osmolarity. Stepwise regression analysis indicated that urine volume was the most significant determinant of plasma AM levels. Percent rise in AM during CPB relative to control period correlated with that of plasma brain natriuretic peptide (r = 0.57, P < 0.01). Our results suggest that AM plays an important role in fluid homeostasis under CPB in cooperation with other hormones involved in fluid homeostasis.

Systolic dysfunction and blood pressure responses to supine exercise in patients with hypertrophic cardiomyopathy.

Left ventricular function and blood pressure responses were evaluated in 56 patients with non-obstructive hypertrophic cardiomyopathy (HCM) and 12 control subjects by using a radionuclide ventricular function monitor during supine ergometer exercise. Patients with HCM were divided into 2 groups: (i) group A had no decrease in ejection fraction (EF) during exercise; and (ii) group B had a decrease in EF during exercise. During exercise, the change in end-diastolic volume did not differ between the 3 groups. In contrast, the change in end-systolic volume differed between the 3 groups (p<0.0001). The change in systolic blood pressure (SBP) also differed significantly between the 3 groups. The change in SBP in group B was smaller than that in the control group and group A, and changes in the EF and changes in the SBP between rest and peak exercise showed a significant correlation (p<0.005). These results suggest that exercise-induced systolic dysfunction in patients with non-obstructive HCM may contribute to abnormal blood pressure response in those patients.

Slow spin relaxation of Rb atoms confined in glass cells filled with dense 4He gas at 1.85 K

At temperatures below 2.1 K, long-lived gaseous Rb atoms in glass cells have been generated with a simple method: irradiating the cells, containing 4He gas and Rb metal, with a cw laser. The obtained atomic Rb density ( approximately 10(8) cm(-3)) decreases with a 1/e time constant of about 10 s at 1.85 K. We have performed optical pumping of the Rb atoms and measured the longitudinal electronic spin relaxation time at 1.85 K as well. For processes (such as Rb-He collisions) which do not remove the atomic Rb from the vapor, this relaxation time is found to be about 60+/-15 s.

Successful reoperation for diffuse supravalvular aortic stenosis.

We reoperated for diffuse supravalvular aortic stenosis using a modified technique of patch plasty described by Brom. A 36-year-old woman admitted to our hospital with a peak systolic pressure gradient of 92 mmHg across the ascending aorta had previously undergone Doty's operation at another hospital. Aortography showed an ascending aorta diffusely stenotic from the sinotubular junction to the aortic arch. We transected the ascending aorta just above the stenotic portion and incised the proximal wall to the sinus of Valsalva. Three patches were sewn to each sinus to expand them and the ascending aorta. The pressure gradient decreased postoperatively to 11 mmHg, and we conclude that this technique sufficiently relieves diffuse supravalvular aortic stenosis.

Mitral valve replacement in a patient with idiopathic thrombocytopenic purpura.

Reports of a cardiac operation in a patient with idiopathic thrombocytopenic purpura are scarce. Here we present a case of successful mitral valve replacement in a patient with idiopathic thrombocytopenic purpura. Preoperative treatment with high-dosage gamma-globulin successfully increased the platelet count from 50,000/microliter to 80,000/microliter. Twenty units of platelet-rich plasma were administered during and after the operation. No other blood products were used. The postoperative convalescence was uneventful. Perioperative management for patients with idiopathic thrombocytopenic purpura undergoing open-heart surgery is discussed.

[Four cases of the anomalous origin of the left coronary artery from the pulmonary artery].

We report perioperative management of 4 patients with anomalous origin of the left coronary artery from the pulmonary artery. This report involves with 3 infant cases and an adult. Two infants underwent coronary reimplantation procedure and Takeuchi's method was performed on the other infant. In all infant cases, mitral valve plasty was performed to correct mitral regurgitation secondary to papillary muscle dysfunction. The adult patient underwent CABG with ligation of LCA. General anesthesia was performed with high doses of fentanyl in all cases. We employed a relatively high PaCO2 and low FIO2 in order to maintain a high pulmonary vascular resistance. It aims to decrease the incidence of left to right shunt. We used epinephrine to wean one infant and the adult from cardiopulmonary bypass. Perioperative course was uneventful with the use of catecholamines and high doses of vasodilators for left ventricular dysfunction and coronary perfusion under mechanical ventilation.

Modulation of norepinephrine release by ATP-dependent K(+)-channel activators and inhibitors in guinea-pig and human isolated right atrium.

OBJECTIVE: The aim of this study was to show, whether ATP sensitive K+ channels (KATP channels), are involved in the modulation of norepinephrine (NE) release from the sympathetic nerves innervating the guinea-pig and human right atrium. METHODS: The resting and stimulation-evoked release of [3H]norepinephrine ([3H]NE) was measured from the isolated guinea-pig and human right atrium and the effect of activators and inhibitors of ATP sensitive K+ channels was studied. RESULTS: Cromakalim (30-300 microM), a KATP channel-agonist decreased concentration-dependently the stimulation-evoked release of NE from the guinea-pig atrium, an effect, antagonized by glibenclamide, a KATP channel-antagonist (30 microM). Diazoxide (30-300 microM), another activator of the KATP channels reduced the resting release of NE, and also attenuated the evoked release at a single concentration (100 microM), and this latter action was also counteracted by glibenclamide (30 microM). Pinacidil, increased dose-dependently the resting and stimulation-evoked release of NE in a glibenclamide-sensitive manner and reversed the inhibitory effect of cromakalim (100 microM), suggesting that it acts as an antagonist. Glibenclamide (30-300 microM), by itself enhanced the stimulation-evoked release of [3H]NE, and also increased the resting release of NE. On the other hand, 5-hydroxydecanoate, an ischemia-selective inhibitor of cardiac KATP channels did not change NE release. Adenosine, (30-300 microM), an A1-receptor agonist, clonidine (3 microM), an alpha 2-adrenoceptor agonist and oxotremorine, a muscarinic receptor agonist (30 microM) all reduced the evoked release of [3H]NE, but these effects were not modified by glibenclamide (300 microM), indicating that neuronal adenosine (A1), adrenergic (alpha 2) and muscarinic (M3) receptors do not act on KATP channels. In the human right atrium, cromakalim, and diazoxide did not affect significantly the release of [3H]NE. However, glibenclamide (30-300 microM) and pinacidil (30-300 microM) enhanced dose-dependently the evoked-release of NE, and pinacidil also augmented the resting release. CONCLUSIONS: Our results indicate that sympathetic nerve endings of the human and guinea-pig atrium are endowed with ATP-sensitive K+ channels. These channels responded to agonists and antagonists under the experimental conditions applied and they could modulate the release of NE thereby affecting the autonomic control of cardiac function under various physiological and pathophysiological conditions.