MicroRNA‐1 (miR‐1) inhibits chordoma cell migration and invasion by targeting slug

Recent studies have revealed that expression of miRNA‐1 (miR‐1) is frequently down‐regulated in several cancer types including chordoma. Identifying and validating novel targets of miR‐1 is useful for understanding the roles of miR‐1 in chordoma. We aimed to further investigate the functions of miR‐1 in chordoma. Specifically, we assessed whether restoration of miR‐1 affects cell migration and invasion in chordoma, and focused on the miR‐1 potential target Slug gene. Migratory and invasive activities were assessed by wound healing and Matrigel invasion assays, respectively. Cell proliferation was determined by MTT assay. Slug expression was evaluated by Western blot, immunofluorescence, and immunohistochemistry. Restoration of miR‐1 expression suppressed the migratory and invasive activities of chordoma cells. Transfection of miR‐1 inhibited cell proliferation both time‐ and dose‐dependently in chordoma. MiR‐1 transfected cells showed inhibited Slug expression. Slug was over‐expressed in chordoma cell lines and advanced chordoma tissues. In conclusion, we have shown that miR‐1 directly targets the Slug gene in chordoma. Restoration of miR‐1 suppressed not only proliferation, but also migratory and invasive activities, and reduced the Slug expression in chordoma cells. These results collectively indicate that miR‐1/Slug pathway is a potential therapeutic target because of its crucial roles in chordoma cell growth and migration. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1075–1082, 2014.     

Evaluation of Hyphecan (1‐4,2‐acetamide‐deoxy‐B‐D‐glucan polymer) on wound healing in a rodent model

Objective: To evaluate the effect of an artificial skin Hyphecan (1‐4,2‐acetamide‐deoxy‐B ‐D ‐glucan polymer) on wound healing in a rodent model. Materials and Method: The prospective study was conducted at a basic science laboratory at a tertiary teaching hospital. Two 4 cm × 4 cm full‐thickness wounds were created on the dorsal surface of 10 Spraque–Dawley rats and covered with Hyphecan and Kaltostat, respectively. Wounds were examined and measured on days 4, 10, 21 and 28, and would continue after day 28 until healed up completely. Punch biopsies (3 mm) were taken on days 4, 10 and 28 for histological examination of the response of healing and repair. Results: Despite the fact that the wound healing rate was similar for both groups on days 4, 10, 21 and 28, the average healing time for the Hyphecan group (29.1 ± 1.7 days) was significantly shorter statistically (P = 0.03) than the Kaltostat group (30.7 ± 2.8 days). Conversely, the marked healing response elicited by Hyphecan on day 4 persisted on days 10 and 28 in contrast to Kaltostat, which had only a mild degree of healing response on days 10 and 28. The study suggests that wounds treated by Hyphecan heal faster than Kaltostat. Conclusion: The findings provide basic scientific evidence supporting the clinical use of Hyphecan in different wounds and might also reduce the cost of wound management as Hyphecan is cheaper than Kaltostat and requires a shorter treatment time.      

SDF‐1/CXCR4 Axis in Tie2‐Lineage Cells Including Endothelial Progenitor Cells Contributes to Bone Fracture Healing

CXC chemokine receptor 4 (CXCR4) is a specific receptor for stromal‐derived‐factor 1 (SDF‐1). SDF‐1/CXCR4 interaction is reported to play an important role in vascular development. On the other hand, the therapeutic potential of endothelial progenitor cells (EPCs) in fracture healing has been demonstrated with mechanistic insight of vasculogenesis/angiogenesis and osteogenesis enhancement at sites of fracture. The purpose of this study was to investigate the influence of the SDF‐1/CXCR4 pathway in Tie2‐lineage cells (including EPCs) in bone formation. We created CXCR4 gene conditional knockout mice using the Cre/loxP system and set two groups of mice: Tie2‐Cre^ER CXCR4 knockout mice (CXCR4^?/?) and wild‐type mice (WT). We report here that in vitro, EPCs derived from of CXCR4^?/? mouse bone marrow demonstrated severe reduction of migration activity and EPC colony‐forming activity when compared with those derived from WT mouse bone marrow. In vivo, radiological and morphological examinations showed fracture healing delayed in the CXCR4^?/? group and the relative callus area at weeks 2 and 3 was significantly smaller in CXCR4^?/? group mice. Quantitative analysis of capillary density at perifracture sites also showed a significant decrease in the CXCR4^?/? group. Especially, CXCR4^?/?group mice demonstrated significant early reduction of blood flow recovery at fracture sites compared with the WT group in laser Doppler perfusion imaging analysis. Real‐time RT‐PCR analysis showed that the gene expressions of angiogenic markers (CD31, VE‐cadherin, vascular endothelial growth factor [VEGF]) and osteogenic markers (osteocalcin, collagen 1A1, bone morphogenetic protein 2 [BMP2]) were lower in the CXCR4^?/? group. In the gain‐of‐function study, the fracture in the SDF‐1 intraperitoneally injected WT group healed significantly faster with enough callus formation compared with the SDF‐1 injected CXCR4^?/? group. We demonstrated that an EPC SDF‐1/CXCR4 axis plays an important role in bone fracture healing using Tie2‐Cre^ER CXCR4 conditional knockout mice. © 2014 American Society for Bone and Mineral Research.     

Distribution of TRAP‐positive cells and expression of HIF‐1α, VEGF,and FGF‐2 in the reparative reaction in patients with osteonecrosis of the femoral head

Osteogenesis and angiogenesis are closely associated with the reparative process in bone. In osteonecrosis of the femoral head (ONFH), although the progression of bone resorption by osteoclasts is considered to be followed by femoral head collapse, the reparative reaction remains unknown. In order to investigate the reparative reaction in patients with ONFH, the distribution of TRAP‐ positive cells and expression of HIF‐1α, VEGF, and FGF‐2 were observed in 51 hips in 42 patients. TRAP‐positive cells were detected around the teres insertion and retinaculum in the early radiologic stage, and increased around the new trabecular bone throughout the reparative interface zone in the late collapsed stage. HIF‐1α expression was detected at the fibrosis area and the transitional area, which included the proximal area of the reparative interface zone adjacent to the necrotic zone. VEGF was expressed at the edematous area of the reparative interface zone, while FGF‐2 was detected widely in the reparative interface zone and the normal zone. In the late radiologic stages, HIF‐1α, VEGF, and FGF‐2 were not detected in the necrotic zone, and they acted in angiogenesis in the reparative interface zone, while TRAP‐positive cells increased around the new bone formation in response to remodeling after the collapse. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27: 694–700, 2009     

Positive effect of IGF‐1 injection on gastrocnemius of rat during distraction osteogenesis

Distraction osteogenesis (DO) is used to form new bone between bone segments to lengthen the callus. Skeletal muscles frequently fail to adapt to distraction, which causes complications. Insulin‐like growth factor‐1 (IGF‐1) has been implicated as a central regulator of muscle repair. We hypothesized that IGF‐1 injection could reduce muscle complications in DO. A total of 102 Sprague‐Dawley rats received DO or did not were randomly assigned into saline, IGF‐1 and normal groups. On the day before the distraction, the rats in the IGF‐1 group were injected with IGF‐1. The gastrocnemius muscles of the rats were harvested at the 0, 1st, 4th, 7th, and 10th days of distraction. The weight of the muscles, cross‐sectional area (CSA) of the muscle fibers, collagen volume fraction (CVF), maximum limit load (MLL), maximum contraction forces, and gene expression of Akt, MyoD, myogenin, myostatin, and collagen I were analyzed. The results indicated that IGF‐1 injection had increased the weights, CSA of the muscle fibers, MLL and force generation of the gastrocnemius. Also, Akt, MyoD, and myogenin were upregulated, and myostatin was downregulated in the IGF‐1 group. Injection of IGF‐1 could attenuate the gastrocnemius atrophy, prevent fibrosis, increase MLL, and regulate the related mRNA. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:1424–1432, 2015.     

Immunotherapy synergizes with debridement and antibiotic therapy in a murine 1‐stage exchange model of MRSA implant‐associated osteomyelitis

Methicillin‐resistant Staphylococcus aureus (MRSA) reinfection following revision surgery remains a major orthopaedic problem. Toward the development of immunotherapy with anti‐glucosaminidase monoclonal antibodies (anti‐Gmd), we aimed to: (i) develop a murine 1‐stage exchange model of bioluminescent MRSA (USA300LAC::lux) contaminated femoral implants; and (ii) utilize this model to demonstrate the synergistic effects of combination vancomycin and anti‐Gmd therapy on reinfection and bone healing. Following an infection surgery, the original plate and two screws were removed on day 7, and exchanged with sterile implants. Mice were randomized to five groups: (i) no infection control; (ii) infected placebo; (iii) anti‐Gmd; (iv) vancomycin; and (v) combination therapy. Bioluminescent imaging (BLI) was performed on days 0, 1, 3, 5, 7, 8, 10, 12, and 14. Mice were euthanized on day 14 (day 7 post‐revision), and efficacy was assessed via colony forming units (CFU) on explanted hardware, micro‐CT, and histology. As monotherapies, anti‐Gmd inhibited Staphylococcus abscess communities, and vancomycin reduced CFU on the implants. However, only combination therapy prevented increased BLI post‐revision surgery, with a significant 6.5‐fold reduction on day 10 (p < 0.05 vs. placebo), and achieved sterile implant levels by day 12. Synergistic effects were also apparent from reduced osteolysis and increased new bone formation around the screws only observed following combination therapy. Taken together, we find that: (i) this murine femoral plate 1‐stage revision model can efficiently evaluate therapies to prevent reinfection; and (ii) immunotherapy plays a distinct role from antibiotics to reduce reinfection following revision surgery, such that synergy to achieve osseointegration is possible. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1590–1598, 2018.