Low-energy shock waves promote the cisplatin chemosensitivity of human osteosarcoma MNNG/HOS cells via the P2X7/Akt/mTOR pathway.

Background: The current dilemma of osteosarcoma treatment is the resistance of chemotherapeutic drugs after long-term usage, which also introduces life-threatening side effects. Methods and results: To minimize chemoresistance in osteosarcoma patients, the authors applied shock waves (SWs) to human osteosarcoma MNNG/HOS cells, then evaluated the cell viability and extracellular ATP levels, and further investigated the effect of SWs on cisplatin (DDP) cytotoxicity in MNNG/HOS cells. The authors' results showed that 400 SW pulses at 0.21 mJ/mm2 exhibited little influence on the MNNG/HOS cell viability. In addition, this SW condition significantly promoted the extracellular ATP release in MNNG/HOS cells. Importantly, low-energy SWs obviously increased Akt and mammalian target of rapamycin (mTOR) phosphorylation and activation in MNNG/HOS cells, which could be partially reversed in the presence of P2X7 siRNA. The authors also found that low-energy SWs strongly increased the DDP sensitivity of MNNG/HOS cells in the absence of P2X7. Conclusions: For the first time, the authors found that SW therapy reduced the DDP resistance of MNNG/HOS osteosarcoma cells when the ATP receptor P2X7 was downregulated. SW therapy may provide a novel treatment strategy for chemoresistant human osteosarcoma.

Noninvasive Imaging OX40+ Activated T Cells Provides Early Warning of Rheumatoid Arthritis.

PURPOSE: The goal of this study was to develop an imaging probe-IRDye-680RD-OX40 mAb-that can be used for noninvasive imaging and optical imaging of rheumatoid arthritis (RA). OX40/OX40 ligand (OX40L) interactions have been shown to exert potent costimulatory effects on T cell activation. Detectable change in T cell activation profiles was observed in early RA. METHODS: OX40 expression pattern was analyzed by flow cytometry. N-hydroxysuccinimide (NHS) esters are used to label proteins selectively on free amino groups of OX40 monoclonal antibody (mAb). Characterization of IRDye-680RD-OX40 mAb was measured and a fluorescence spectrum gathered. Cell binding assay was also performed between activated and naïve murine T cells. Longitudinal near-infrared fluorescence (NIRF) imaging of the probe was performed on day 8, day 9, day 10, and day 11 of adjuvant-induced arthritis (AIA) mouse model. Paw thickness and body weight were compared between the OX40 mAb and IgG injection groups. RESULTS: NIRF imaging with IRDye-680RD-OX40 mAb revealed strong OX40-positive responses with high specificity. Flow analysis showed that OX40 was specifically expressed on the surface of T cells in RP and spleen of AIA model. The AIA group was significantly differentiated from the control group at all time points with imaging monitoring. The region of interest (ROI) was in line with ex vivo imaging and biodistribution study. This study highlights the potential utility of the OX40 NIRF imaging as a new strategy for RA prediction and T cell monitoring. CONCLUSION: The results provide evidence that IRDye-680RD-OX40 mAb detects organized T cells activation in early RA. The optical probe was capable of detection of RA pathogenesis. It identified transcriptional responses to RA that mediate its immune functions. Thus, it may be an ideal probe for RA imaging.

Treatment of pediatric tibial shaft nonunion using ipsilateral free non-vascularized fibular graft.

BACKGROUND: Although tibial shaft fractures are the third most common long bone fractures in children after the forearm and femur, nonunion of these fractures are rare in the pediatric population. CASE REPORT: Despite seldom seen, tibial nonunion is very complex and it is also a devastating complication of tibial fracture especially when infected. Numerous methods have been employed to treat pediatric tibial nonunion, but there is no consensus. Here, we present a case of a child with right tibial shaft fracture nonunion. We treated this patient with ipsilateral free non-vascularized fibular graft. RESULTS: Both the nonunion site and fibular donor site united well with good function in the injured extremity and no adverse events. CONCLUSION: We recommend the use of ipsilateral free non-vascularized fibular graft for the treatment of pediatric tibial shaft nonunion.

Functional outcomes and adverse effects following three interventions for displaced midshaft clavicular fractures: A Bayesian network meta-analysis of randomized controlled trials.

INTRODUCTION: The efficacy of the most commonly used interventions for clavicle fractures remains controversial. These interventions are: open reduction and plate fixation (ORPF), non-surgical intervention (NSI), and use of an intramedullary nail (IMN). In adult patients with clavicle fractures, choosing which intervention might be best is challenging. MATERIALS AND METHODS: PubMed, Journals@Ovid Full Text, Cochrane Central Register of Controlled Trials (CENTRAL), Ovid MEDLINE, and Embase were performed to search English-language studies from the inception to February 2020. Randomized controlled trials (RCTs) comparing any of these three interventions were included. Patient and baseline characteristics, nonunion, major complications, Constant-Murley score (CMS), and Disabilities of the Arm, Shoulder and Hand score (DASH) were extracted. Then, we evaluated the functional outcomes and adverse effects after use of these three interventions for the management of displaced midshaft clavicle fractures in a Bayesian network meta-analysis. RESULTS: A Bayesian random-effects model was conducted, and nonunion and major complications were evaluated with: risk ratio (RR) and 95% confidential interval (CI); while CMS and DASH were evaluated with mean differences (MD) and the corresponding 95% confidential interval CI. The rank probability of each endpoint was assessed on the basis of the surface area under the cumulative ranking curve (SUCRA). DISCUSSION: ORPF is most likely to be successful in achieving objective functional outcomes as captured by the CMS, and IMN demonstrates significant efficacy for subjective functional outcomes, as captured by DASH scores. Compared with the other interventions examined, IMN was associated with decreased risk for adverse effects. LEVELS OF EVIDENCE: I; meta-analysis.

Comparison of plate fixation vs. intramedullary fixation for the management of mid-shaft clavicle fractures: A systematic review and meta-analysis of randomised controlled trials.

A number of meta-analyses have compared clinical outcomes following plate vs. intramedullary fixation for midshaft clavicle fractures (MSCF), but with conflicting results. There is a requirement for updated level-1 evidence to guide clinicians managing MSCF. The aim of the present systematic review and meta-analysis was to compare clinical outcomes following plate vs. intramedullary fixation of MSCF. The PubMed, Scopus, BioMed Central, Cochrane Central Register of Controlled Trials and Google Scholar databases were searched for records added until 1st July 2019. A total of 10 randomised controlled trials (RCTs) were included. Shoulder function was assessed using the Constant-Murley Shoulder Outcome questionnaire and the Disabilities of the Arm, Shoulder and Hand questionnaire (DASH). There was no statistically significant difference in Constant-Murley scores between plate and intramedullary fixation [Mean difference (MD)=0.75; 95% CI: -2.49 to 3.99; P=0.65; I2=85%]. Similarly, there was no statistically significant difference in DASH scores between the two groups (MD=1.55; 95% CI: -1.12 to 4.23; P=0.26; I2=89%). There was no statistically significant difference in complications requiring non-routine surgery between plate and intramedullary fixation [risk ratio (RR)=1.80, 95%CI: 0.80-4.05, P=0.15; I2=0%]. There was an increased risk of complications that did not require non-routine surgery with plate fixation as compared to intramedullary fixation (RR=2.38, 95%CI: 1.22-4.62, P=0.01; I2=70%). Plate fixation was also associated with an increased risk of infection and complications of cosmetic dissatisfaction. The present results indicated no difference in long-term functional outcomes between plate and intramedullary fixation of MSCF. Plate fixation was associated with an increased risk of complications not requiring non-routine surgery. Further high-quality RCTs shall strengthen the evidence on this subject.

Nasal delivery of a CRMP2-derived CBD3 adenovirus improves cognitive function and pathology in APP/PS1 transgenic mice.

Calcium dysregulation is a key pathological event in Alzheimer's disease (AD). In studying approaches to mitigate this calcium overload, we identified the collapsin response mediator protein 2 (CRMP2), an axonal guidance protein that participates in synapse dynamics by interacting with and regulating activity of N-methyl-D-aspartate receptors (NMDARs). We further identified a 15 amino acid peptide from CRMP2 (designated CBD3, for calcium-binding domain 3), that reduced NMDAR-mediated Ca2+ influx in cultured neurons and post-synaptic NMDAR-mediated currents in cortical slices. Whether targeting CRMP2 could be therapeutically beneficial in AD is unknown. Here, using CBD3, we tested the utility of this approach. Employing the APP/PS1 mouse model of AD which demonstrates robust pathophysiology including Aß1-42 deposition, altered tau levels, and diminished cognitive functions, we asked if overexpression of CBD3 could rescue these events. CBD3 was engineered into an adeno-associated vector and nasally delivered into APP/PS1 mice and then biochemical (immunohistochemistry, immunoblotting), cellular (TUNEL apoptosis assays), and behavioral (Morris water maze test) assessments were performed. APP/PS1 mice administered adeno-associated virus (AAV, serotype 2) harboring CBD3 demonstrated: (i) reduced levels of Aß1-42 and phosphorylated-tau (a marker of AD progression), (ii) reduced apoptosis in the hippocampus, and (iii) reduced cognitive decline compared with APP/PS1 mice or APP/PS1 administered a control virus. These results provide an instructive example of utilizing a peptide-based approach to unravel protein-protein interactions that are necessary for AD pathology and demonstrate the therapeutic potential of CRMP2 as a novel protein player in AD.

Cyclic adenosine phosphate improves functional recovery after spinal cord injury via activating unfolded protein response.

The current study aimed to evaluate the role and underlying mechanism of cyclic adenosine phosphate (cAMP) on the functional recovery of spinal cord injury (SCI). Basso, Beattie and Bresnahan (BBB) scoring and inclined plane test indicated that cAMP treatment improved the functional recovery of SCI rats. Real time PCR and western blot analysis showed the mRNA and protein levels of IRE1, PERK, and ATF6 were increased in the SCI rats than those of sham control. However, higher levels of IRE1, PERK, and ATF6 were indicated after cAMP treatment. Meanwhile, more apoptotic cells were observed in the SCI rats, as evidenced by TUNEL staining and increased expression of GRP78, CHOP, and caspase12. In contrast, the expression of GRP78, CHOP, and caspase12 was decreased in SCI rats after cAMP treatment. In summary, we showed novel data that cAMP reduced cell apoptosis and functional recover after SCI mainly via activating UPR.

The term "floating" used in traumatic orthopedics.

BACKGROUND: The term "floating" is used in orthopedic literature to describe certain patterns of skeletal injuries that share one common character which is disruption and discontinuity of bones above and below a joint. The first time used in orthopedic literature being in late 1970 to describe a type of elbow injury. Later the word was used increasingly and applied to a variety of injuries affecting the knee, shoulder, hip, forearm, hand, and ankle. Currently, there are about 12 different skeletal injuries described as floating. OBJECTIVES: The aim of this article was to define the term "floating" used in traumatic orthopedics and to discuss its history, mechanism of injury in each region, treatment and outcomes based on the currently available literature. As there were many separate articles describing different sites of floating injuries, this review aimed to summarize all floating injuries into 1 article.

IGF-1R inhibitor PQ401 inhibits osteosarcoma cell proliferation, migration and colony formation.

IGF-1R is expressed abnormally in osteosarcoma (OS) and could participate in its progression. In this study, we aimed to explore the effect of the IGF-1R inhibitor PQ401 as a treatment for OS. The relative expression of IGF-1R in OS patient tumors and the U2OS cell line were determined by qRT-PCR and by accessing information in a public database. Inhibition of cell proliferation by PQ401 was determined by MTT assay. Cell migration under low concentration treatment of PQ401 was carried out by transwell and wound healing assays. PQ401 induction of OS cell apoptosis was investigated by flow cytometry. Tumorigenesis under PQ401 treatment was evaluated by a colony formation assay. Finally, downstream blockage of the IGF-1R pathway was verified by western blotting. Our results show that the expression of IGF-1R was remarkably higher in OS cells, particularly in U2OS, than in other cancer-type cell lines. The inhibition of the IGF-1R pathway by PQ401 exhibited significant anticancer activity in the U2OS cell line in not only proliferation but also migration and colony formation. In addition, PQ401 is a strong inducer of OS cell apoptosis. Furthermore, western blotting was used to demonstrate that the IGF-1R related downstream pathway, including total ERK1/2, was significantly inhibited by PQ401. Thus, IGF-1R inhibition may represent a novel treatment for OS.

Anti-Ma2-associated limbic encephalitis with coexisting chronic inflammatory demyelinating polyneuropathy in a patient with non-Hodgkin lymphoma: A case report.

RATIONALE: We report the rare case of a 74-year-old man with anti-Ma2-associated paraneoplastic neurologic syndrome (PNS), and review and analyze the clinical manifestations, diagnosis, and treatment of the disease. PATIENT CONCERNS: The patient presented with a 5-month history of muscle weakness, progressive body aches, and weakness and numbness in both lower extremities. Before his hospitalization, he had experienced cognitive function decline; ptosis, inward gaze, and vertical gaze palsy in the right eye; and occasional visual hallucinations. Brain and spinal cord magnetic resonance imaging (MRI) yielded normal results. Anti-Ma2 antibodies were detected in both serum and cerebrospinal fluid. A 4-hour electroencephalogram showed irregular sharp slow waves and δ waves in the temporal region. Electromyography showed peripheral nerve demyelination. Positron-emission tomography/computed tomography (PET-CT) examination revealed hypermetabolism in the lymph nodes of the whole body. Biopsy of the lymph nodes showed non-Hodgkin lymphoma. DIAGNOSIS: A clinical diagnosis of lymphoma and PNS was made. INTERVENTIONS: The patient was treated with intravenous dexamethasone (15 mg/day) for 3 days. LESSONS: We have presented a rare case of a PNS involving both the central and peripheral nervous systems. The clinical features of this case indicated anti-Ma2-associated encephalitis and chronic inflammatory demyelinating polyneuropathy. PET-CT played a critical role in enabling early diagnosis and prompt treatment in this case.

Gene markers of fracture healing in early stage and the regulatory mechanism during the process using microarray analysis.

BACKGROUND: The aim of this study was to explore crucial markers and uncover the regulatory mechanisms of fracture healing in the early stage. METHODS: Gene expression profile of GSE45156 was downloaded, in which 3 fractured samples and 3 unfractured samples were used in our present study. Based on the threshold value, differentially expressed genes (DEGs) were selected between two kinds of samples using limma package in R. Enrichment analysis of these DEGs was performed by DAVID software. Furthermore, protein-protein interaction (PPI) network was established integrating information in STRING database, and visualized by Cytoscape software. RESULTS: We identified a set of 960 DEGs including 509 up-regulated and 451 downregulated genes. Biological processes involving RNA splicing and cell cycle were significantly enriched for the up-regulated genes such as Snrpd2, Eftud2, Plk1 and Bub1b, whereas skeletal system development and bone development processes were predominant for down-regulated genes like Ubc. In the constructed PPI network, all the five genes were the predominant nodes, of which Snrpd2 was linked to Eftud2, while Bub1b was to interact with Plk1. CONCLUSION: Five candidate genes crucial for indicating the process of fracture in early stage were identified. Eftud2, Snrpd2, Bub1b and Plk1 might function through the involvement of cell-cycle-related BP, while Ubc might influence the protein degradation during bone development. However, more experimental validations are needed to confirm these results.

Shock wave-induced ATP release from osteosarcoma U2OS cells promotes cellular uptake and cytotoxicity of methotrexate.

BACKGROUND: Osteosarcoma is the most prevalent primary malignant bone tumor, but treatment is difficult and prognosis remains poor. Recently, large-dose chemotherapy has been shown to improve outcome but this approach can cause many side effects. Minimizing the dose of chemotherapeutic drugs and optimizing their curative effects is a current goal in the management of osteosarcoma patients. METHODS: In our study, trypan blue dye exclusion assay was performed to investigate the optimal conditions for the sensitization of osteosarcoma U2OS cells. Cellular uptake of the fluorophores Lucifer Yellow CH dilithium salt and Calcein was measured by qualitative and quantitative methods. Human MTX ELISA Kit and MTT assay were used to assess the outcome for osteosarcoma U2OS cells in the present of shock wave and methotrexate. To explore the mechanism, P2X7 receptor in U2OS cells was detected by immunofluorescence and the extracellular ATP levels was detected by ATP assay kit. All data were analyzed using SPSS17.0 statistical software. Comparisons were made with t test between two groups. RESULTS: Treatment of human osteosarcoma U2OS cells with up to 450 shock wave pulses at 7 kV or up to 200 shock wave pulses at 14 kV had little effect on cell viability. However, this shock wave treatment significantly promoted the uptake of Calcein and Lucifer Yellow CH by osteosarcoma U2OS cells. Importantly, shock wave treatment also significantly enhanced the uptake of the chemotherapy drug methotrexate and increased the rate of methotrexate-induced apoptosis. We found that shock wave treatment increased the extracellular concentration of ATP and that KN62, an inhibitor of P2X7 receptor reduced the capacity methotrexate-induced apoptosis. CONCLUSIONS: Our results suggest that shock wave treatment promotes methotrexate-induced apoptosis by altering cell membrane permeability in a P2X7 receptor-dependent manner. Shock wave treatment may thus represent a possible adjuvant therapy for osteosarcoma.

Incidence and risk factors for venous thromboembolism following surgical treatment of fractures below the hip: a meta-analysis.

Venous thromboembolism (VTE) is a common complication after surgical treatment of fractures, which is associated with significant morbidity and mortality. Identifying the risk factors for VTE is important for preventive strategies to reduce the incidence of VTE. Therefore, we conducted a meta-analysis to evaluate the incidence of VTE and the risk factors influencing the development of VTE in patients who underwent surgery for fractures below the hip. PubMed, Embase, Web of Science, SinoMed (Chinese BioMedical Literature Service System, China) and CNKI (National Knowledge Infrastructure, China) databases were systematically searched to identify cohort or case-control studies that investigated the incidence and risk factors for VTE following surgical treatment of fractures below the hip. VTE risk ratios (RRs) were pooled by use of a fixed-effect model or a random-effect model, depending on the heterogeneity among the included studies. Heterogeneity between the studies was assessed by I2 statistics. Twenty-three studies with a total of 191 294 patients who met the inclusion criteria were included in this meta-analysis. Our results demonstrated that age (≥60 years) (RR = 1·85, 95% confidence interval (CI): 1·34, 2·55; P = 0·000), previous VTE(RR = 5·25, 95% CI: 2·77, 9·96; P = 0·000), heart failure (RR = 1·74, 95% CI: 1·34, 2·27; P = 0·000), current smoking status (RR = 1·23, 95% CI: 1·07, 1·41; P = 0·004), hypertension (RR = 1·62, 95% CI: 1·27, 2·06; P = 0·000), hyperlipidaemia (RR = 2·16, 95% CI: 1·79, 2·62; P = 0·000), diabetes mellitus (RR = 1·46, 95% CI: 1·27, 1·68; P = 0·000), obesity (RR = 1·58, 95% CI: 1·35,·1·85; P = 0·000), multiple fractures (RR = 2·14, 95% CI: 1·00, 4·60; P = 0·050), varicose veins (RR = 3·07, 95% CI: 1·12, 8·47; P = 0·030), prolonged operation time (weighted mean differences (WMD) = 1·22, 95% CI: 0·63, 1·81; P = 0·000) and prolonged bed rest time (WMD = 3·12, 95% CI: 2·96, 3·29; P = 0·000) were associated with an increased risk of developing VTE. The other variables, including age (<60 years), previous smoking, immobility, pregnancy, cancer, open fractures and combination with trauma were not identified as significant risk factors for VTE. Almost all the risk factors mentioned above are in line with the known risk factors for VTE following surgery for fractures below the hip. Thus, surgeons should pay close attention to patients with these medical conditions in order to reduce the incidence of VTE following surgical treatment of fractures below the hip.

Posterior dislocation of the hip with bilateral femoral fractures: An unusual combination.

Posterior hip dislocation with concomitant femoral fracture is very rare. Here, we report a rare case of a 43-year-old man who was injured in a car accident. The patient sustained right posterior hip dislocation with concomitant right acetabular transverse and posterior wall fracture, ipsilateral femoral shaft fracture, and contralateral proximal femoral fracture (AO type 31-A3). Closed reduction of the hip was attempted, but failed. The acetabular fracture and posterior hip dislocation were reduced and acetabular fracture was fixed using plates through the Kocher-Langenbeck approach. The ipsilateral femoral fracture was treated with closed reduction and intramedullary nailing. The contralateral femoral fracture was treated with closed reduction and Gamma 3 nailing. Postoperative X-rays revealed reduction of the fractures. The patient achieved bone union and recovered function of the hip 4 months after surgery.

A novel technique to prevent guide wire related complications while inserting the 4.0 mm cannulated screws.

BACKGROUND: Cannulated screws (4.0 mm) provide inter-fragmentary compression and stability to fractures. A guide wire is used to define the screw trajectory and hold the fracture fragment while the screw is being inserted. The cannulated shaft typically accommodates a 1.25 mm guide pin. Since the guide pin is very slender and undergoes elastic deformation during insertion, there is a high probability of pin breakage. METHODS: The authors have devised a new way to place the 4.0 mm cannulated screws in a manner that prevents the intraoperative complication of guide wire breakage. For this technique, predrilling was achieved using a 2.0 mm K-wire which was subsequently replaced with a 1.25 mm guide pin under the protection of sleeve. 4.0 mm cannulated screws were then inserted into a defined trajectory over the guide pin. RESULTS: Using the technique, over 20 patients were managed in our department over a period of two years without any complications. CONCLUSION: We have observed that patients treated with this method experience short operation time, combined with good clinical outcome and we recommend its use in cases where cannulated screw use is warranted.

Donor Age and Cell Passage Affect Osteogenic Ability of Rat Bone Marrow Mesenchymal Stem Cells.

Tissue engineering allows the restoration of pathologically damaged tissues such as cartilage and bone using bio-scaffolds containing functionally active cells. Bone-marrow-derived mesenchymal stem cells (BMSCs) are a promising source of cells for tissue engineering due to their multilineage differentiation potential. However, proliferative and osteogenic abilities of BMSCs, and quantity of stem cells decreases in the bone marrow in aged population. We cultured BMSCs isolated from rats of various ages and evaluated their morphology, activity, and differentiation potential. Cultured BMSCs formed monolayer of fibroblast-like cells and maintained their characteristic morphology for 7-10 generations. Flow cytometry showed that aging of the cultured cell population correlated with the decrease in the expression of mesenchymal and hematopoietic surface markers, such as CD44, CD45, CD90, and CD29. We detected strong correlation between the age of BMSC donor and ALP activity in BMSC culture induced with low doses of dexamethasone and vitamin C. Cells from 2- and 6-week-old donor SD rats exhibited markedly increased ALP activity that coincided with increased bone content and strong positive staining of mineralized nodules. In contrast, BMSCs isolated from 10-month-old donors showed the lowest ALP activity, and decreased bone content and mineralized nodules formation. Our results demonstrate that the increase in donor age negatively affects proliferation and differentiation capacity of BMSCs in culture.

Shockwaves induce osteogenic differentiation of human mesenchymal stem cells through ATP release and activation of P2X7 receptors.

Shockwave treatment promotes bone healing of nonunion fractures. In this study, we investigated whether this effect could be due to adenosine 5'-triphosphate (ATP) release-induced differentiation of human mesenchymal stem cells (hMSCs) into osteoprogenitor cells. Cultured bone marrow-derived hMSCs were subjected to shockwave treatment and ATP release was assessed. Osteogenic differentiation and mineralization of hMSCs were evaluated by examining alkaline phosphatase activity, osteocalcin production, and calcium nodule formation. Expression of P2X7 receptors and c-fos and c-jun mRNA was determined with real-time reverse transcription polymerase chain reaction and Western blotting. P2X7-siRNA, apyrase, P2 receptor antagonists, and p38 MAPK inhibitors were used to evaluate the roles of ATP release, P2X7 receptors, and p38 MAPK signaling in shockwave-induced osteogenic hMSCs differentiation. Shockwave treatment released significant amounts (≈ 7 µM) of ATP from hMSCs. Shockwaves and exogenous ATP induced c-fos and c-jun mRNA transcription, p38 MAPK activation, and hMSC differentiation. Removal of ATP with apyrase, targeting of P2X7 receptors with P2X7-siRNA or selective antagonists, or blockade of p38 MAPK with SB203580 prevented osteogenic differentiation of hMSCs. Our findings indicate that shockwaves release cellular ATP that activates P2X7 receptors and downstream signaling events that caused osteogenic differentiation of hMSCs. We conclude that shockwave therapy promotes bone healing through P2X7 receptor signaling, which contributes to hMSC differentiation.