Cobalt-Doped Mesoporous Silica Coated Magnetic Nanoparticles Promoting Accelerated Bone Healing in Distraction Osteogenesis.

Introduction: Large bone abnormalities are commonly treated using distraction osteogenesis (DO), but it is not suitable for a long-term application; therefore, there is an urgent need for adjuvant therapy that can accelerate bone repair. Methods: We have synthesized mesoporous silica-coated magnetic nanoparticles doped with cobalt ions (Co-MMSNs) and assessed their capacity to quicken bone regrowth in a mouse model of DO. Furthermore, local injection of the Co-MMSNs significantly accelerated bone healing in DO, as demonstrated by X-ray imaging, micro-CT, mechanical tests, histological evaluation, and immunochemical analysis. Results: In vitro, the Co-MMSNs exhibited good biocompatibility and induced angiogenic gene expression and osteogenic development in bone mesenchymal stem cells. And the Co-MMSNs can promote bone regeneration in a rat DO model. Discussion: This study demonstrated the significant potential of Co-MMSNs to shorten the DO treatment duration and effectively reduce the incidence of complications.

Tumor microenvironment-responsive nanohybrid for hypoxia amelioration with photodynamic and near-infrared II photothermal combination therapy.

Phototherapy, particularly photothermal therapy (PTT) and photodynamic therapy (PDT), has been widely investigated for tumor treatment. However, the limited tissue penetration depth of light in the near-infrared I (NIR-I) region and the hypoxic tumor microenvironment (TME) severely constrain their clinical applications. To address these challenges, in the present study, we developed a chlorin e6 (Ce6) and MnO2-coloaded, hyaluronic acid (HA)-coated single-walled carbon nanohorns (SWNHs) nanohybrid (HA-Ce6-MnO2@SWNHs) for PDT and PTT combination therapy of tumor. HA-Ce6-MnO2@SWNHs responded to the mild acidic TME to ameliorate tumor hypoxia, thus enhancing tumor PDT. Moreover, HA-Ce6-MnO2@SWNHs had a high photothermal conversion efficiency at 1064 nm (55.48%), which enabled deep tissue penetration (3.05 cm) and allowed for highly efficient tumor PTT in near-infrared II (NIR-II) window. PDT and PTT combination therapy with HA-Ce6-MnO2@SWNHs achieved a good therapeutic efficacy on 4T1 tumor-bearing mice, eradicating the primary tumors and suppressing cancer recurrence. Our study provides a promising strategy for developing a hypoxia relief and deep tissue penetration phototherapy platform by using SWNHs for highly effective tumor PDT and NIR-II PTT combination therapy. STATEMENT OF SIGNIFICANCE: The hypoxic tumor microenvironment (TME) and the limited penetration of the NIR-I light in biological tissues compromise the efficacy of photothermal therapy (PTT) and photodynamic therapy (PDT) on tumors. Here, we developed a chlorin e6 (Ce6) and MnO2-coloaded, hyaluronic acid (HA)-coated single-walled carbon nanohorns (SWNHs) nanohybrid (HA-Ce6-MnO2@SWNHs) for PDT and PTT combination therapy of tumors. The nanohybrid could efficiently accumulate in tumors through CD44-mediated active targeting. The sequential MnO2-enhanced PDT and efficient NIR-II PTT had a remarkable therapeutic effect by eliminating the primary tumor and simultaneously inhibiting tumor recurrence.

Accelerated Bone Regeneration by Astragaloside IV through Stimulating the Coupling of Osteogenesis and Angiogenesis.

Both osteoblasts and preosteoclasts contribute to the coupling of osteogenesis and angiogenesis, regulating bone regeneration. Astragaloside IV (AS-IV), a glycoside of cycloartane-type triterpene derived from the Chinese herb Astragalus membranaceus, exhibits various biological activities, including stimulating angiogenesis and attenuating ischemic-hypoxic injury. However, the effects and underlying mechanisms of AS-IV in osteogenesis, osteoclastogenesis, and bone regeneration remain poorly understood. In the present study, we found that AS-IV treatment inhibited osteoclastogenesis, preserved preosteoclasts, and enhanced platelet-derived growth factor-BB (PDGF-BB)-induced angiogenesis. Additionally, AS-IV promoted cell viability, osteogenic differentiation, and angiogenic gene expression in bone marrow mesenchymal stem cells (BMSCs). The activation of AKT/GSK-3ß/ß-catenin signaling was found to contribute to the effects of AS-IV on osteoclastogenesis and osteogenesis. Furthermore, AS-IV accelerated bone regeneration during distraction osteogenesis (DO), as evidenced from the improved radiological and histological manifestations and biomechanical parameters, accompanied by enhanced angiogenesis within the distraction zone. In summary, AS-IV accelerates bone regeneration during DO, by enhancing osteogenesis and preosteoclast-induced angiogenesis simultaneously, partially through AKT/GSK-3ß/ß-catenin signaling. These findings reveal that AS-IV may serve as a potential bioactive molecule for promoting the coupling of osteogenesis and angiogenesis, and imply that AKT/GSK-3ß/ß-catenin signaling may be a promising therapeutic target for patients during DO treatment.

Jingshu Keli for treating cervical spondylotic radiculopathy: The first multicenter, randomized, controlled clinical trial.

BACKGROUND: Jingshu Keli (or Jingshu granules), a traditional Chinese medicine, are widely used for treating cervical spondylotic radiculopathy in China; however, no randomized, double-blind, controlled study has verified their effectiveness. PURPOSE: To evaluate the efficacy and safety of Jingshu Keli for the treatment of cervical spondylotic radiculopathy in a randomized controlled trial. DESIGN: From August 2015 to July 2017, a multicenter, randomized, double-blind, placebo-controlled trial was conducted at 13 large- and medium-sized hospitals in China. PATIENT SAMPLE: A total of 360 and 120 patients were initially enrolled in the Jingshu and control groups, respectively; 386 patients completed the study, with 299 in the Jingshu group and 87 in the control group. OUTCOME MEASURES: The main index for evaluating the curative effect was the pain score on a visual analogue scale (VAS; 0-100 points). METHODS: All patients were administered a bag of Jingshu Keli or placebo 3 times a day for 4 weeks, and were interviewed at the second and fourth weeks. The decrease in pain scores and rate of change in pain scores after treatment were calculated, related laboratory indices were reviewed, and adverse reactions were recorded. RESULTS: In the Per Protocol Set (PPS) analysis, the baseline pain VAS scores in the control and Jingshu groups were 49.31 â€‹± â€‹6.97 and 50.06 â€‹± â€‹7.33, respectively, with no significant difference between the groups (P â€‹> â€‹0.05). While there were no differences at 2 weeks between groups, at four weeks the pain VAS scores in the control and Jingshu groups decreased by 12.86 â€‹± â€‹13.45 and 22.72 â€‹± â€‹15.08, respectively relative to the values at baseline, with significant group differences (P â€‹< â€‹0.0001). While there were similar significant differences between the groups (P â€‹< â€‹0.0001) in the Full Analysis Set (FAS) analyses neither group achieved the minimal clinically important difference at any time point. CONCLUSIONS: Jingshu Keli are effective for the treatment of cervical spondylotic radiculopathy. TRANSLATIONAL POTENTIAL STATEMENT: This is the first prospective, multicenter, randomized, double-blind, placebo-controlled clinical trial that confirmed the clinical efficacy and safety of Jingshu Keli for treating cervical spondylotic radiculopathy, which can provide evidence for clinical treatment.

Postconditioning With Red-Blue Light Therapy Improves Survival of Random Skin Flaps in a Rat Model.

BACKGROUND: Random skin flap ischemic necrosis is a serious challenge in reconstructive surgery. Photobiomodulation is a noninvasive effective technique to improve microcirculation and neovascularization. Photobiomodulation with red or blue light has been separately proven to partially prevent skin flap necrosis, but the synergistic effect of red and blue light not been elucidated. Our experiment evaluated the impact of postconditioning with red-blue light therapy on the viability of random flaps. METHODS: Thirty Sprague-Dawley male rats (male, 12 weeks) with a cranially based random pattern skin flap (3 × 8 cm) were divided into 3 groups: control group, red light group, and red-blue light group. On postoperative day 7, flap survival was observed and recorded using transparent graph paper, flaps were obtained and stained with hematoxylin and eosin, and microvessel density was measured. Micro-computed tomography was used to measure vascular volume and vascular length. On days 0, 3, and 7 after surgery, blood flow was measured by laser Doppler. To investigate the underlying mechanisms, the amount of nitric oxide (NO) metabolites in the flap tissue was assessed on days 3, 5, and 7 after surgery. RESULTS: The mean percentage of skin flap survival was 59 ± 10% for the control group, 69 ± 7% for the red light group, and 79 ± 9% for the red-blue light group (P < 0.01). The microvessel density was 12.3 ± 1.2/mm2 for the control group, 31.3 ± 1.3/mm2 for the red light group, and 36.5 ± 1.4/mm2 for the red-blue light group (P < 0.01). Both vascular volume and total length in the red-blue light group showed significantly increased compared with the red light and control group (P < 0.01). Blood flow in the red-blue light treated flap showed significantly increased at postsurgery days 3 and 7 compared with the red light and control group (P < 0.01). The level of the NO metabolites was significantly increased in flap tissues belonging to the red-blue light group compared with the other 2 groups (P < 0.01). CONCLUSIONS: This study showed that postconditioning with red-blue light therapy can enhance the survival of random skin flap by improving angiogenesis and NO releasing.

Docosahexaenoic Acid Improves Diabetic Wound Healing in a Rat Model by Restoring Impaired Plasticity of Macrophage Progenitor Cells.

BACKGROUND: Chronic inflammation associated with delayed diabetic wound healing is induced by disturbed polarization of macrophages derived mainly from predisposed progenitor cells in bone marrow. Docosahexaenoic acid plays a critical role in regulating the function of macrophage progenitor cells. The authors evaluated whether docosahexaenoic acid accelerates diabetic wound healing in rats. METHODS: Streptozotocin-induced diabetic rats divided into control and docosahexaenoic acid-treated groups (n = 10) were subjected to paired dorsal skin wounds. Docosahexaenoic acid (100 mg/kg per day) was orally supplemented 2 weeks before wounding until termination. The wound healing process was recorded 0, 7, and 14 days after wounding. At day 7, blood perfusion was measured by laser Doppler perfusion imaging; angiogenesis was compared using immunofluorescent CD31 and α-smooth muscle actin staining; macrophage polarization was detected using immunofluorescence for CD68, CD206, and inducible nitric oxide synthase. Hematoxylin and eosin staining was used to examine wound healing at day 14. Activation status of macrophages derived from bone marrow cells in normal, diabetic, and docosahexaenoic acid-treated diabetic rats was determined in vitro using Western blotting and enzyme-linked immunosorbent assay. RESULTS: Docosahexaenoic acid significantly accelerated wound healing 7 and 14 days (p < 0.01) after wounding. Increased vessel densities (1.96-fold; p < 0.001) and blood perfusion (2.56-fold; p < 0.001) were observed in docosahexaenoic acid-treated wounds. Immunofluorescence revealed more CD206 and fewer inducible nitric oxide synthase-positive macrophages (p < 0.001) in treated wounds. Furthermore, macrophages derived from diabetic rats expressed higher levels of inducible nitric oxide synthase and tumor necrosis factor-α and lower arginase-1 and interleukin-10 (p < 0.05). CONCLUSION: Docosahexaenoic acid accelerates diabetic wound healing at least in part by restoring impaired plasticity of macrophage progenitor cells.

Is EDTA Irrigation Effective in Reducing Bacterial Infection in a Rat Model of Contaminated Intra-articular Knee Implants?

BACKGROUND: To mitigate the possibility of infection after arthroplasty, intraoperative irrigation is essential to remove contaminating bacteria. Previous studies have demonstrated that irrigation with an EDTA solution before wound closure is superior to irrigation with normal saline in removing contaminating bacteria in a rat model of open fractures. However, the effectiveness of an EDTA solution in a model with a contaminated intra-articular implant remains unclear. QUESTIONS/PURPOSES: (1) Does irrigation with an EDTA solution decrease the proportion of culture-positive joints compared with normal saline, benzalkonium chloride, and povidone iodine? (2) Is an EDTA solution toxic to cells resident in joints including chondrocytes, osteoblasts, and synovial fibroblasts? (3) Does irrigation with an EDTA solution have adverse effects including arthrofibrosis and hypocalcemia? METHODS: We first established a model of contaminated intra-articular implants. Female Sprague-Dawley rats (n = 30 for each treatment group) underwent knee arthrotomy and implantation of a femoral intramedullary wire with 1 mm of intra-articular communication. To simulate bacterial contamination, the inserted wire was inoculated with either Staphylococcus aureus or Escherichia coli. After 1 hour, the wound and implant were irrigated with normal saline, benzalkonium chloride, povidone iodine, or an EDTA solution (1 mM). The animals were euthanized 1 week later, and the distal femur, knee capsule, and implanted wire were harvested for bacterial culture using standard techniques. In this study, we used a well-established animal model of an intra-articular implant and inoculated the implant to simulate the clinical setting of intraoperative contamination. The proportion of culture-positive joints in normal saline, benzalkonium chloride, povidone-iodine, and EDTA groups were compared. The viable cell numbers (chondrocytes, osteoblasts, and synovial fibroblasts) were counted and compared after treatment with either solution. Measurement of blood calcium level and histological examination of the joint were performed to rule out hypocalcemia and arthrofibrosis after EDTA irrigation. RESULTS: With S. aureus inoculation, EDTA irrigation resulted in fewer culture-positive joints than normal saline (37% [11 of 30] versus 70% [21 of 30]; p = 0.019), benzalkonium chloride (83% [25 of 30]; p < 0.001), and povidone iodine (83% [25 of 30]; p < 0.001) irrigation. Likewise, infection rates for implant inoculation with E. coli were also lower in the EDTA irrigation group (13% [four of 30]) than in the normal saline (60% [18 of 30]; p < 0.001), benzalkonium chloride (77% [23 of 30]; p < 0.001), and povidone iodine (80% [24 of 30]; p < 0.001) groups. Between normal saline control and EDTA, there were no differences in cell viability in chondrocytes (normal saline: 98% ± 18%; EDTA: 105% ± 18%; p = 0.127), osteoblasts (normal saline: 102 ± 19%, EDTA: 103 ± 14%; p = 0.835), and synovial fibroblasts (normal saline: 101% ± 21%, EDTA: 110% ± 13%; p = 0.073). EDTA irrigation did not result in hypocalcemia (before irrigation: 2.21 ± 0.32 mmol/L, after irrigation: 2.23 ± 0.34 mmol/L; p = 0.822); and we observed no arthrofibrosis in 30 histologic samples. CONCLUSIONS: In a rat model of a bacteria-contaminated intra-articular implants, intraoperative irrigation with 1 mmol/L of an EDTA solution was superior to normal saline, 0.03% benzalkonium chloride, and 0.3% povidone iodine in preventing surgical-site infection and caused no adverse effects including death of resident cells, arthrofibrosis, and hypocalcemia. Future studies should seek to replicate our findings in other animal models, perhaps such as dog and goat. CLINICAL RELEVANCE: If other animal models substantiate the efficacy and safety of the EDTA solution, clinical trials would be warranted to determine whether the use of an EDTA irrigation solution might reduce the risk of periprosthetic joint infections in patients compared with traditional irrigation solutions.

Catalpol promotes the osteogenic differentiation of bone marrow mesenchymal stem cells via the Wnt/ß-catenin pathway.

BACKGROUND: Rehmanniae Radix is a traditional herbal medicine in East Asia that has been widely used to treat patients with osteoporosis. However, the effect of catalpol, the primary active principle component of Rehmanniae Radix, on the function of bone marrow mesenchymal stem cells (BMSCs) and the underlying molecular mechanisms associated with its activity remain poorly understood. METHODS: The effect of catalpol on the proliferation of BMSCs was evaluated using a Cell Counting Kit-8 assay. Alkaline phosphatase (ALP) staining, ALP activity and Alizarin Red staining were performed to elucidate the effect of catalpol on the osteogenesis of BMSCs. qRT-PCR, Western blotting and immunofluorescence were performed to evaluate the expression of osteo-specific markers and the Wnt/ß-catenin signalling-related genes and proteins. Moreover, a rat critical-sized calvarial defect model and a rat ovariectomy model were used to assess the effect of catalpol on bone regeneration in vivo. RESULTS: Catalpol significantly enhanced osteoblast-specific gene expression, alkaline phosphatase activity and calcium deposition in BMSCs in vitro. This phenomenon was accompanied by an upregulation of Wnt/ß-catenin signalling. In addition, the enhanced osteogenesis due to catalpol treatment was partially reversed by a Wnt/ß-catenin antagonist. Furthermore, catalpol increased the bone healing capacity of BMSCs in a rat critical-sized calvarial defect model and attenuated bone loss in a rat ovariectomy model. CONCLUSIONS: These data suggest that catalpol enhances the osteogenic differentiation of BMSCs, partly via activation of the Wnt/ß-catenin pathway. Catalpol may provide a new strategy for bone tissue engineering and can be a potential agent for the treatment of postmenopausal osteoporosis.

Dual Chemodrug-Loaded Single-Walled Carbon Nanohorns for Multimodal Imaging-Guided Chemo-Photothermal Therapy of Tumors and Lung Metastases.

Tumor combination therapy using nano formulations with multimodal synergistic therapeutic effects shows great potential for complete ablation of tumors. However, targeting tumor metastases with nano structures is a major obstacle for therapy. Therefore, developing a combination therapy system able to target both primary tumors and their metastases at distant sites with synergistic therapy is desirable for the complete eradication of tumors. To this end, a dual chemodrug-loaded theranostic system based on single walled carbon nanohorns (SWNHs) is developed for targeting both primary breast tumors and their lung metastases. Methods: SWNHs were first modified simultaneously with poly (maleic anhydride-alt-1-octadecene) (C18PMH) and methoxypolyethyleneglycol-b-poly-D, L-lactide (mPEG-PLA) via hydrophobic-hydrophobic interactions and π-π stacking. Then cisplatin and doxorubicin (DOX) (2.9:1 molar ratio) were sequentially loaded onto the modified nanohorns in a noninterfering way. After careful examinations of the release profiles of the loaded drugs and the photothermal performance of the dual chemodrug-loaded SWNHs, termed SWNHs/C18PMH/mPEG-PLA-DOX-Pt, the dual drug chemotherapeutic and chemo-photothermal synergetic therapeutic effects on tumor cells were evaluated. Subsequently, the in vivo behavior and tumor accumulation of the drug-loaded SWNHs were studied by photoacoustic imaging (PAI). For chemo-photothermal therapy of tumors, 4T1 tumor bearing mice were intravenously injected with SWNHs/C18PMH/mPEG-PLA-DOX-Pt at a dose of 10 mg/kg b.w. (in SWNHs) and tumors were illuminated by an 808 nm laser (1W/cm2 for 5 min) 24 h post-injection. Results: DOX and cisplatin were loaded onto the modified SWNHs with high efficiency (44 wt% and 66 wt%, respectively) and released in a pH-sensitive, tandem and sustainable manner. The SWNHs/C18PMH/mPEG-PLA-DOX-Pt had a hydrodynamic diameter of 182 ± 3.2 nm, were highly stable in physiological environment, and had both dual drug chemotherapeutic (CI = 0.439) and chemo-photothermal synergistic antitumor effects (CI = 0.396) in vitro. Moreover, the dual drug-loaded SWNHs had a long blood half-life (10.9 h) and could address both the primary breast tumors and their lung metastases after intravenous administration. Consequently, chemo-photothermal combination therapy ablated the primary tumors and simultaneously eradicated the metastatic lung nodules. Conclusion: Our study demonstrates that SWNHs/C18PMH/mPEG-PLA-DOX-Pt is highly potent for chemo-photothermal combination therapy of primary tumors and cocktail chemotherapy of their metastases at a distant site.

Effect of SDF-1/Cxcr4 Signaling Antagonist AMD3100 on Bone Mineralization in Distraction Osteogenesis.

Distraction osteogenesis (DO) is a widely applied technique in orthopedics surgery, which involves rapid stem cell migration, homing, and differentiation. Interactions between the chemokine receptor Cxcr4 and its ligand, stromal derived factor-1 (SDF-1), regulate hematopoietic stem cell trafficking to the ischemic area and induce their subsequent differentiation. Here, we examined SDF-1 expression and further investigated the role of SDF-1/Cxcr4 signaling antagonist AMD3100 during bone regeneration in rat DO model. The results showed that expression levels of SDF-1 and osteogenic genes were higher in DO zones than in the fracture zones, and SDF-1 expression level was the highest at the termination of the distraction phase. Radiological, mechanical, and histological analyses demonstrated that the local administration of AMD3100 (400 µM) to DO rats significantly inhibited new bone formation. In the rat bone marrow mesenchymal stem cells culture, comparing to the group treated with osteogenic induction medium, AMD3100 supplement led to a considerable decrease in the expression of alkaline phosphatase and early osteogenic marker genes. However, the amount of calcium deposits in rat MSCs did not differ between the groups. Therefore, our study demonstrated that the DO process induced higher expression of SDF-1, which collated to rapid induction of callus formation. Local application of SDF-1/Cxcr4 signaling antagonist AMD3100 significantly inhibited bone mineralization and osteogenesis in DO, which may represent a potential therapeutic approach to the enhancement of bone consolidation in patients undergoing DO.

Staphylococcal enterotoxin C2 expedites bone consolidation in distraction osteogenesis.

Distraction osteogenesis (DO) technique could be used to manage large-size bone defect successfully, but DO process usually requires long duration of bone consolidation. Innovative approaches for augmenting bone consolidation are of great need. Staphylococcal enterotoxin C2 (SEC2) has been found to suppress osteoclastogenesis of mesenchymal stem cells in vitro. In this study, we investigated the effect of SEC2 on proliferation and osteogenic differentiation of rat bone marrow derived mesenchymal stem cells (rBMSCs). Further, we locally administrated SEC2 (10 ng/ml) or PBS into the distraction gap in Sprague-Dawley male rat DO model every 3 days till termination at 3 and 6 weeks. The regenerates were subjected to X-rays, micro-computed tomography, mechanical testing, histology, and immunohischemistry examinations to assess new bone quality. SEC2 had no effect on cell viability. The calcium deposition was remarkably increased and osteogenic marker genes were significantly up-regulated in rBMSCs treated with SEC2. In rat DO model, SEC2 group had higher bone volume/total tissue volume in the regenerates. At 6 weeks, mechanical properties were significantly higher in SEC2-treated tibiae comparing to the control group. Histological analysis confirmed that the new bone had improved quality in SEC2 treated group, where the osteocalcin and osterix expression in the regenerates was up-regulated, indicating faster bone formation. The current study demonstrated that SEC2 local injection promotes osteogenesis and enhanced bone consolidation in DO. The findings support application of SEC2 as a potential novel strategy to expedite bone consolidation in patients undergoing DO treatment. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1215-1225, 2017.