PKC signal amplification suppresses non-small cell lung cancer growth by promoting p21 expression and phosphorylation.

Protein kinase C (PKC) activation was previously associated with oncogenic features. However, small molecule inhibitors targeting PKC have so far proved ineffective in a number of clinical trials for cancer treatment. Recent progresses have revealed that most PKC mutations detected in diverse cancers actually lead to loss-of-function, thus suggesting the tumor-suppressive roles of PKC proteins. Unfortunately, the development of chemicals to enhance PKC activity is lagging behind relative to its small molecular inhibitors. Here, we report that a bisindolylmaleimide derivative (3,4-bis(1-(prop-2-ynyl)-1H-indol-3-yl)-1 H-pyrrole-2,5-dione, BD-15) significantly inhibited cell growth in non-small cell lung cancer (NSCLC). Mechanistically, BD-15 treatment resulted in markedly enhanced phosphorylation of PKC substrates and led to cell cycle arrest in G2/M. Further, BD-15 treatment upregulated p21 protein levels and enhanced p21 phosphorylation. BD-15 also promoted caspase3 cleavage and triggered cellular apoptosis. In xenograft mouse models, BD-15 exerted anti-tumor effects to suppress in vivo tumor formation. Collectively, our findings revealed the tumor-suppressive roles of BD-15 through enhancing PKC signaling and thus leading to upregulation of p21 expression and phosphorylation.

High-purity weight-bearing magnesium screw: Translational application in the healing of femoral neck fracture.

Magnesium (Mg)-based metals can be used as next-generation fracture internal fixation devices due to their specific properties. We used vascularized bone grafting fixed by degradable pure Mg screws and obtained satisfactory results in the treatment of osteonecrosis of the femoral head. However, the mechanical properties of these screws make them weaker than those made of traditional metals. In particular, one of the main challenges of using screws made of Mg-based metals is their application in fixation at important weight-bearing sites in the human body. Femoral neck fracture is a common clinical injury. In this injury, the large bearing stress at the junction requires a fixation device with extremely high mechanical strength. Surgery and appropriate internal fixation can accelerate the healing of femoral neck fractures. Traditional internal fixation devices have some disadvantages after surgery, including stress shielding effects and the need for secondary surgery to remove screws. On the basis of previous work, we developed high-strength pure Mg screws for femoral neck fractures. In this study, we describe the first use of high-purity Mg to prepare large-size weight-bearing screws for the fixation of femoral neck fractures in goats. We then performed a 48 weeks follow-up study using in vivo transformation experiments. The results show that these biodegradable high-purity Mg weight-bearing screws had sufficient mechanical strength and a degradation rate compatible with bone repair. Furthermore, good bone formation was achieved during the degradation process and reconstruction of the bone tissue and blood supply of the femoral head and femoral neck. This study provides a basis for future research on the clinical transformation of biodegradable high-purity Mg weight-bearing screws.

Exposure to tobacco smoke increases bone loss in spontaneously hypertensive rats.

PURPOSE: To define if exposure to tobacco smoke (TS) could induce reduction of bone mass and impairment of bone architecture, features observed in osteoporosis in normotensive rats and the influence of TS exposure on the osteoporotic features exhibited in the spontaneously hypertensive (SH) rats. METHODS: Normotensive Wistar Kyoto (WKY) and SH rats were exposed to filtered air or TS for 8 weeks, then their proximal femurs were extracted for micro-computed tomography (micro-CT) assessment, histological and immune-histological examinations to quantify the adverse influence of TS exposure on the bone mass and density, as well as bone architecture. RESULTS: We found that TS exposure not only induced significant decreases in bone mineral density (BMD), bone volume (BV), cortical and trabecular thickness (Ct.Th and Tb.Th), trabecular surface area (Tb.Ar), expression of hypoxia-inducible factor-1α (HIF-1α) in the trabecular marrow, delayed ossification of cartilage, as well as statistical increases in trabecular separation (Tb.SP) and the number of trabecular marrow adipocytes in both WKY and SH rats, but also exacerbated multiple features of osteoporosis exhibited in SH rats, including decreased BMD, Ct.Th, Tb.Ar, HIF-1α expression, delayed cartilage ossification, and increased Tb.SP. CONCLUSIONS: Our results show that TS exposure can reduce bone mass and impair bone architecture and exacerbate multiple features of osteoporosis exhibited in SH rats.

Combined Treatment with an Anticoagulant and a Vasodilator Prevents Steroid-Associated Osteonecrosis of Rabbit Femoral Heads by Improving Hypercoagulability.

Steroid-associated osteonecrosis of the femoral head remains a challenging problem in orthopedics worldwide. One pathomechanism is ischemia of the femoral head, as a result of thrombus formation and vasoconstriction. The present study investigates the effects of combination prevention with enoxaparin and EGb 761 on steroid-associated ONFH in rabbits. Rabbits were randomly divided into 5 groups (control group, model group, enoxaparin group, ginkgo group, and combination group). With the exception of the control group, the groups of rabbits were modeled with lipopolysaccharide and methylprednisolone acetate. Starting with modeling, the enoxaparin group and ginkgo group were injected with 1 µg/kg/day enoxaparin subcutaneously and orally given 40 mg/kg/day EGb 761 for 4 weeks, respectively; the combination group received both treatments. After modeling for 6 weeks, the hematology data indicated prolonged PT and APTT in the three prevention groups. The micro-CT examination revealed higher bone density and better structure; histomorphometry revealed significant pathological changes. Immunohistochemistry revealed higher expression of BMP-2 and VEGF, thus revealing better osteogenesis and angiogenesis activities. Among the three prevention groups, the combination group had the most efficient results. In conclusion, the combined prevention with an anticoagulant and a vasodilator has the potential to decrease the incidence of steroid-associated ONFH in rabbits.

Ion channel functional protein kinase TRPM7 regulates Mg ions to promote the osteoinduction of human osteoblast via PI3K pathway: In vitro simulation of the bone-repairing effect of Mg-based alloy implant.

Mg-based alloys, as the potential orthopaedic implant, can self-degrade to avoid second operation for its remove, and enable to promote bone repair; however, the underlying molecular mechanisms remain unclear. In the present study, we examined the effect of Mg ions on osteogenesis, chemotaxis and anti-alkaline stress in hFOB1.19 human osteoblast cells to simulate bone-repairing effect of a biodegradable Mg-based alloy implant in vitro, and explored the regulatory role of the transient receptor potential melastatin 7 (TRPM7)/phosphoinositide 3-kinase (PI3K) signalling pathway in the process of Mg ion-induced bone repair by knockdown of TRPM7 and antagonizing PI3K activity. Results indicate that Mg ions up-regulated the expression of Runx2 and alkaline phosphatase (ALP) through TRPM7/PI3K signalling pathway, which could significantly enhance the osteogenic activity of human osteoblasts. Furthermore, the expression levels of MMP2, MMP9 and vascular endothelial growth factor (VEGF) were increased by TRPM7/PI3K signalling pathway, which recruits osteoblasts from low- to high-Mg ion environments by inducing cell migration. Although an alkaline environment has antibacterial effects, alkaline stress can cause cytotoxicity and induce cell death. Finally, we found that Mg ions could activate PI3K phosphorylation to promote cell growth and survival, protecting cells against the alkaline-stress-induced cytotoxicity caused by the degradation of Mg-based alloy implants. Our study not only revealed the molecular mechanism of Mg in promoting bone repair but also explained the protective effects of Mg ions on osteoblasts in an alkaline environment, which provides a theoretical basis and new directions for the application of Mg-based alloy implant material in orthopaedics fixations and osteosarcoma treatment. STATEMENTS OF SIGNIFICANCE: As a potential biomaterial for orthopaedic implant, biodegradable magnesium has several advantages including self-degradation and bone repair promotion; however, the underlying mechanisms and effective concentration by which molecular regulates the bone repair remain unclear. The present study revealed that Mg ion and its effective concentration for activating PI3K phosphorylation via TRPM7, which causes three processes affecting bone repair, namely, osteoblast recruitment, osteogenesis and resistance to alkaline stress in human osteoblast. Therefore, our results have provided insight into the underlying molecular biological basis, and guidance for manipulating degradation rate, such as surface modification, of orthopaedic Mg-based implants.

Current status on clinical applications of magnesium-based orthopaedic implants: A review from clinical translational perspective.

As a new generation of medical metallic material, magnesium (Mg) and its alloys with or without surface coating have attracted a great deal of attention due to its biodegradability and potential for avoiding a removal operation after the implant has fulfilled its function for surgical fixation of injured musculoskeletal tissues. Although a few clinical cases on Mg-based orthopaedic implants were reported more than a century ago, it was not until recently that clinical trials using these implants with improved physicochemical properties were carried out in Germany, China and Korea for bone fracture fixation. The promising results so far suggest a bright future for biodegradable Mg-based orthopaedic implants and would warrant large scale phase II/III studies. Given the increasing interest on this emerging biomaterials and intense effort to improve its properties for various clinical applications, this review covers the evolution, current strategies, and future perspectives in the development of Mg-based orthopaedic implants. We also highlight a few clinical cases performed in China that may be unfamiliar to the general orthopaedic community.

Vascularized bone grafting fixed by biodegradable magnesium screw for treating osteonecrosis of the femoral head.

Hip-preserving surgery with vascularized bone graft implantation has been widely practiced in treating osteonecrosis of the femoral head (ONFH). However, the current approach presents a drawback, in which the implanted bone graft without screw fixation may slip or exhibit a certain degree of displacement postoperatively. This study was designed to investigate the application potential of biodegradable magnesium (Mg) screws for the fixation of vascularized bone graft in ONFH patients. Forty-eight patients were randomly divided into two groups: the Mg screw group (vascularized bone grafting fixed by Mg screws) and the control group (vascularized bone grafting without fixation). During 12 month follow-up period after surgery, treatment outcomes in patients were assessed by multiple imaging techniques including x-ray and computed tomography (CT) scanning as well as functional recovery Harris hip score (HHS). The temporal changes in serum levels of Mg, Ca, and P as well as in vivo degradation rate of Mg screws were determined. The absence of potential adverse effects induced by degradation products from Mg screws on surrounding bone tissue was validated via CT imaging analysis. HHS was significantly improved in the Mg screw group when compared to the control group. X-ray imaging analysis showed that the screw shape did not show significant alteration due to the diameter of Mg screws measured with approximate 25% reduction within 12 months post-surgically. The postoperative serum levels of Ca, Mg, and P, which are relevant for liver and kidney function, were all within normal physiological range in all patients of both groups. The use of biodegradable Mg screws may provide a promising bone graft-screw fixation route in treating ONFH and present considerable potential for orthopedic applications.

Combined vascularized iliac and greater trochanter graftings for reconstruction of the osteonecrosis femoral head with collapse: reports of three cases with 20 years follow-up.

In this report, we present the long-term results of using combined vascularized iliac and greater trochanter graftings for reconstruction of the osteonecrosis of the femoral head (ONFH) with collapse in three patients. Necrosis over two-thirds of the femoral head and collapse were observed in these patients, with Harris hip scores (HHS) of 46, 38, and 49 points, respectively. When the patients underwent the femoral head reconstruction procedures, the ages of the patients ranged from 20 to 28 years old. The patients were followed-up for 20-24 years. X-ray examinations showed no progress of necrosis or deformity in the femoral head of patients after surgery, with the exception of bone absorption in one patient with persistence of mild pain. The HHS in the three patients were 84, 65, and 86 points at the end of follow-up, respectively. These results show that the vascularized iliac and greater trochanter graftings may be a valuable option for reconstruction of the ONFH with collapse in younger patients.

[Nifedipine attenuates vascular inflammation via inhibin NF-κB activity].

OBJECTIVE: To explore the effects and related mechanism of nifedipine on vascular inflammation induced by cuff placement. METHODS: Adult male C57BL/6J mice (10 to 12 weeks of age) were assigned to control (no cuff placement without nifedipine), cuff placement (cuff placement without nifedipine) and treatment (cuff placement with nifedipine 1 or 5 mg×kg(-1)×d(-1)) groups. Activity of NF-κB in injured artery was measured 5 days after operation. MCP-1 expression and nuclear translocation of NF-κB were examined in injured artery 7 days after operation. RESULTS: DNA-binding activity of NF-κB was significantly increased in the injured artery 5 days after cuff placement which could be downregulated by nifedipine 5 mg×kg(-1)×d(-1). MCP-1 mRNA expression in the injured arteries was increased 7 days after cuff placement and which could be significantly attenuated by nifedipine 5 mg×kg(-1)×d(-1). Cuff placement decreased the cytoplasmic level of p50, IκBα, IκBß, and increased the nuclear level of p50. Nifedipine 5 mg×kg(-1)×d(-1) significantly attenuated these changes. CONCLUSION: Our results suggest that high dose nifedipine could suppresses expression of MCP-1 induced by injured arteries via the inhibin NF-κB DNA binding activity, thereby attenuating vascular inflammation.

[Experimental study on lyophilization of platelets].

The aim of this study was to search a procedure of platelet lyophilization and find a way of long-term storage of human platelets at normal temperature with smaller size and lighter weight, to be convenient to transport at long distance thus to meet the demands in accidents and war time. Human platelets were pretreated by freezing, the first and the second desiccation, and were added with reversible activation-inhibitors of platelets, DMSO and trehalose, then were rehydrated. At the same time, the recovery rate of platelets, platelet maximal aggregation induced by thrombin, coagulation of platelets, CD62p expression and PAC-1 expression were assayed. The results indicated that the recovery rate of the platelets was 56.29%. The platelet maximal aggregation induced by thrombin had no significant difference between lyophilized platelets and the fresh platelet-rich plasma (FPRP), but the aggregation of platelets induced by ADP or propyl gallate was decreased by 49.34% and 26.25%. Coagulation of the lyophilized platelets was not significantly different from FPRP. CD62p expression of the lyophilized platelets (42.36%) was higher than that in FPRP while PAC-1 expression was 2.12%. CD62p re-expression rate induced by thrombin was 50.88% and PAC-1 re-expression was 54.55%. It is concluded that the ability of recovered lyophilized platelets added with reversible activation-inhibitors, DMSO and trehalose to aggregate and coagulate has showed no significant difference as compared with FPRP. The reversible activation-inhibitors can decrease CD62p expression of lyophilized platelets, and may enhance their survival ability and prolongate survival time. Therefore the efficiency of lyophilizing platelets can be improved based on this freeze-drying procedure.

[Aggregation after rehydration of lyophilized platelets].

This study was aimed to investigate the aggregation of rehydrated-lyophilized platelets. The aggregation rate of fresh and rehydrated-lyophilized platelets were measured by using thrombin, ristocetin, ADP and collagen as inductors and APACT2 aggregameter; the effects of intra- and extra-cellular trehalose on maximum aggregation rate of rehydrated-lyophilized platelets were detected by using ADP as an inductor. The results showed that the aggregation rate of fresh platelets was all about 100%, while aggregation rate of rehydrated lyophilized platelets was (70.17 +/- 7.36)%, (15.3 +/- 2.81)%, (68.67 +/- 6.86)%, (64.67 +/- 11.6)% respectively, when the concentration of thrombin, ristocetin, ADP and collagen was 1 U/ml, 1.6 mg/ml, 20 micromol/L and 2 microg/ml. The maximum aggregation rates of rehydrated-lyophilized platelets in intra- and extra-cellular trehalose, extracellular trehalose and blank control groups were (66.0 +/- 4.69)%, (25.3 +/- 2.42)% and (11.5 +/- 1.87)% (P < 0.01), meanwhile there was significant difference of rehydrated-lyophilized platelet aggregation rate between intra- and extra-cellular trehalose and extracellular trehalose groups (P < 0.01). It is concluded that the concentrations of thrombin (1 U/ml), ristocetin (1.6 mg/ml), ADP (20 micromol/L) and collagen (2 microg/ml) are optimal for platelets aggregation tests, the internal and extracellular trehalose significantly enhance the aggregation of rehydrated-lyophilized platelets.

[Process of human platelets loaded with rehalose before lyophilization].

The aim of this research was to study the technology and methods of loading lyoprotectant-trehalose into cytoplasm of human platelets before lyophilization, to optimize experimental conditions of loading trehalose, to investigate the changes of platelets response to agonists and activation after incubation of platelets for 4 hours at 37 degrees C in the presence of lyoprotectant-trehalose, to protract the figures of loading efficiency and intracellular trehalose concentration versus incubation time, temperature and external trehalose concentration, to optimize loading parameters. The response of platelets to different agonists--thrombin, ADP, collagen and ristocetin were measured respectively by APACT2 aggregometer before and after loading trehalose into platelets; the expressions of CD62p and PAC-1 on platelet membranes in the presence and absence of reversible platelets activation inhibitors were measured by flow cytometry respectively before and after loading trehalose into cytoplasm of platelets. The results showed that the loading efficiency was linear to incubation time (2 hours later) and incubation temperature (rang from 30 degrees C to 40 degrees C), respectively. The loading efficiency almost reached 60% when the platelets were incubated at 37 degrees C for 4 hours. The intracellular trehalose concentration was higher with the increase of the extracellular trehalose concentration (< 50 mmol/L). Compared to untreated groups, the values of MPV and aggregation to different agonists in treated groups showed no significant difference, respectively (P > 0.01). After incubation of platelets for 4 hours, the expression of CD62p increased to some extent, however, the expression of CD62p decreased again when the reversible platelets activation inhibitor PGE-1 and adenosine were added to the incubation buffer. It is concluded that 37 degrees C, 4 hours and the extracellular trehalose concentration < 50 mmol/L are the optimal conditions for loading with trehalose. The processing of loading with trehalose before platelet lyophilization has no significant effects on response of platelets to agonists and activation.

[Optimization on trehalose loading technique as protective conditioning for lyophilization of human platelets].

This study was aimed to further optimize trehalose loading technique including loading temperature, loading time, loading solution and loading concentration of trehalose, based on the established parameters. Loading efficiency in plasma was compared with that in buffer at 37 degrees C; the curves of intracellular trehalose concentration versus loading time at 37 degrees C and 16 degrees C were measured; curves of mean platelet volume (MPV) versus loading time and loading concentration were investigated and compared. According to results obtained, the loaing time, loading temperature, loading solution and trehalose concentration were ascertained for high loading efficiency of trehalose into human platelet. The results showed that the loading efficiency in plasma was markedly higher than that in buffer at 37 degrees C, the loading efficiency in plasma at 37 degrees C was significantly higher than that at 16 degrees C and reached 19.51% after loading for 4 hours, but 6.16% at 16 degrees C. MPV at 16 degrees C was increased by 43.2% than that at 37 degrees C, but had no distinct changes with loading time and loading concentration. In loading at 37 degrees C, MPV increased with loading time and loading concentration positively. Loading time and loading concentration displayed synergetic effect on MPV. MPV increased with loading time and concentration while trehalose loading concentration was above 50 mmol/L. It is concluded that the optimization parameters of trehalose loading technique are 37 degrees C (temperature), 4 hours (leading time), plasma (loading solution), 50 mmol/L (feasible trehalose concentration). The trehalose concentration can be adjusted to meet the requirement of lyophilization.