Downregulation of miRNA-127-5p aggravates spinal cord injury through activating MAPK1.

OBJECTIVE: To uncover the role of microRNA-127-5p (miRNA-127-5p) in aggravating motor dysfunction following spinal cord injury (SCI) by regulating the mitogen-activated protein kinase 1 (MAPK1) level. MATERIALS AND METHODS: In vivo SCI model in mice was established by constructing spinal cord hitting injury. Mice were classified into sham group, SCI group, SCI+miRNA-127-5p mimics group, and SCI+miRNA-127-5p mimics+MAPK1 group, respectively. Grip strengths of mouse pair forepaws, right forepaw, and left forepaw at different time points were determined. Expression levels of miRNA-127-5p and MAPK1 in mice of each group at post-SCI were detected. Potential binding sites in promoter regions of miRNA-127-5p and MAPK1 were predicted by bioinformatics and further confirmed by Dual-Luciferase reporter gene assay and Western blot. RESULTS: Grip strengths of SCI mice were much lower than those in sham group at different time points after SCI procedures. MiRNA-127-5p was markedly downregulated on the postoperative 3rd day in SCI group, and its level time-dependently decreased since after. In vivo overexpression of miRNA-127-5p in SCI mice improved their grip strengths from the postoperative 7th day. MAPK1 was the direct target of miRNA-127-5p. Transfection of miRNA-127-5p mimics downregulated protein level of MAPK1 in 293T cells. Overexpression of MAPK1 abolished the protective effect of miRNA-127-5p on motor function recovery following SCI. CONCLUSIONS: Downregulation of miRNA-127-5p aggravates SCI-induced motor dysfunction through negatively regulating MAPK1 level.

LncRNA XIXT promotes osteogenic differentiation of bone mesenchymal stem cells and alleviates osteoporosis progression by targeting miRNA-30a-5p.

OBJECTIVE: To uncover the role of XIXT in influencing the osteogenesis of hBMSCs by adsorbing microRNA-30a-5p (miRNA-30a-5p) to upregulate RUNX2. PATIENTS AND METHODS: The serum samples were collected from osteoporosis patients and normal people. hBMSCs were isolated from femoral head tissues. The serum levels of XIXT and miRNA-30a-5p were determined by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). The expression levels and activities of the osteogenic differentiation-related genes in hBMSCs after transfection of sh-XIXT, sh-RUNX2, miRNA-30a-5p mimic, and inhibitor were detected by qRT-PCR, Western blot, ALP activity assay, and alizarin red staining. The Dual-Luciferase Reporter Gene Assay was performed to confirm the binding of XIXT to miRNA-30a-5p, as well as the binding of miRNA-30a-5p to RUNX2. RESULTS: LncRNA XIXT was significantly downregulated, and miRNA-30a-5p was upregulated in the serum of osteoporosis patients. The osteogenic differentiation-related genes (ALP, RUNX2) and XIXT were markedly upregulated in a time-dependent manner, while miRNA-30a-5p level gradually decreased in hBMSCs with the prolongation of osteogenesis. The knockdown of XIXT inhibited the osteogenic differentiation of hBMSCs. In hBMSCs, XIXT regulated RUNX2 expression by targeting miRNA-30a-5p. The knockdown of miRNA-30a-5p partially reversed the inhibitory effect of XIXT on the osteogenesis of hBMSCs. However, the downregulated RUNX2 reversed the promotive effect of miRNA-30a-5p on the osteogenesis of hBMSCs. CONCLUSIONS: LncRNA XIXT upregulated RUNX2 by absorbing miRNA-30a-5p, and thus induced hBMSCs osteogenesis to alleviate osteoporosis.

[Knee stability after anterior cruciate ligament reconstruction with weight-bearing magnetic resonance imaging].

Objective: To investigate the application value of the weight-bearing magnetic resonance imaging (MRI) in evaluating the stability of knee joint after anterior cruciate ligament reconstruction. Methods: From July 2011 to August 2013, a total of 25 patients with anterior cruciate ligament rupture and reconstruction surgery in the Second Affiliated Hospital of Soochow University were enrolled in this study, including 18 males and 7 females, with an average age of (32±5) years. All the patients underwent the weight-bearing MRI, knee joint passive relaxation test (Kneelax 3), and Lysholm score before the surgery and 3 and 6 months after the surgery. The three examinations before and after the operation were analyzed by repeated measures of general linear model, and paired t test was used to compare the results before and after the operation. The correlation between the three preoperative examinations was statistically analyzed. Results: The relaxation data measured by Kneelax 3 after the surgery was significantly lower than that before the operation [(1.1±0.9) mm vs (6.1±1.3) mm, t=16.9, P<0.01]. The post-operative lateral tibial plateau anterior shift score was less than the pre-operative score [(3.0±0.7) mm vs (4.8±1.2) mm, t=6.2, P<0.01]. The post-operative Lysholm score was significantly higher than that before the operation (89±6 vs 64±14, t=-8.3, P<0.05). There was a negative correlation between the anterior displacement of the lateral platform and Lysholm score (r=-0.902, P<0.01). There was no correlation between anterior displacement and passive relaxation of the lateral platform in preoperative weight-bearing MRI. Conclusions: Anterior cruciate ligament reconstruction can improve the knee stability. The stability of knee joint can be evaluated by weight-bearing MRI. The anterior tibial displacement measured by the weight-bearing MRI is correlated with the clinical score.

ß4GalT1 promotes inflammation in human osteoarthritic fibroblast-like synoviocytes by enhancing autocrine TNF-α activity.

OBJECTIVE: Synovial inflammation plays an important role in the pathogenesis of osteoarthritis (OA), and ß4GalT1 has been reported to be involved in the inflammatory process. The aim of our study was to investigate the role of ß4GalT1 in the progression of inflammation and analyze the association between ß4GalT1 and tumor necrosis factor (TNF)-α in human OA fibroblast-like synoviocytes (FLS). PATIENTS AND METHODS: Primary cultured FLS isolated from OA synovial tissues were cultured, and the levels of ß4GalT1, TNF-α, MMP-3, p/t-ERK, p/t-JNK, and p/t-P38 were analyzed by Western blotting. An enzyme-linked immunosorbent assay (ELISA) was performed to measure the secretion of TNF-α, interleukin (IL)-1ß, and IL-6 in OA-FLS. Immunofluorescence staining was used to examine the co-localization of ß4GalT1 and TNF-α or THY1. RT-PCR was used to detect the transfection efficiency of ß4GalT1. RESULTS: The expression of ß4GalT1 was increased in OA-FLS. ß4GalT1 promoted cell invasion, MMP-3 production, and the secretion of TNF-α, IL-1ß, and IL-6. si-TNF-α attenuated the ß4GalT1-enhanced cell invasion and inflammatory factor secretion in OA-FLS. Furthermore, ß4GalT1 increased autocrine TNF-α signaling in OA-FLS. ß4GalT1 knockdown successfully decreased autocrine TNF-α activity, while ß4GalT1 overexpression increased autocrine TNF-α activity in OA-FLS. Moreover, ß4GalT1 enhanced the ERK, JNK, and P38 MAPK signaling pathways through the induction of autocrine TNF-α signaling in OA-FLS. CONCLUSIONS: ß4GalT1 may promote the inflammatory progression of OA-FLS by enhancing autocrine TNF-α signaling.

[The 467th case: proteinuria, periungual fibromas, and facial steatadenomas].

A 22-year-old manpresented as a refractory nephrotic syndrome with edema and proteinuria for more than one year. Physical examination revealed facial steatadenomas and periungual fibromas. Images were characterized by hamartomatous lesions in multiple organs, including the central nervous system, heart, lungs, liver, and kidneys. Gene tests verified TSC2 mutation and confirmed the diagnosis of tuberous sclerosis complex. The APOL1 mutation was positive in this patient, which indicated the possibility of steroid-resistant focal segmental glomerulonephritis. Thus, he was treated with sirolimus. Renal angiomyolipoma was shrunk, but proteinuria was not relieved (24h unine protein>10 g) and eventually led into renal insufficiency. Nondialytic therapy was initiated consequently. Losartan 50 mg/d was used to control proteinuria under the close watch of serum creatinine. A recent phone call on October 2018 failed to reachthe patient. Therefore, the follow-up information was not updated.

MiR-299-5p targets cell cycle to promote cell proliferation and progression of osteosarcoma.

OBJECTIVE: Osteosarcoma is a common primary bone tumor with high mortality. MicroRNA (miRNA, miR) is a small RNA with 20-25 nucleotides, which could regulate diverse biological processes by targeting 3'-UTR of gene to degrade it. MiR-299-5p has been reported to participate in the progression of many diseases, but the role in osteosarcoma is still uncertain. The aim of this work was to investigate the expression of miR-299-5p in osteosarcoma and its clinical significance. MATERIALS AND METHODS: The datasets of osteosarcoma miRNA was searched in Gene Expression Omnibus (GEO) datasets, including GSE65071, GSE39040, and GSE39055. Osteosarcoma U2 and MG-63 cells were cultured in our study. Cell proliferation level after transfection was detected by using Cell Counting Kit-8 (CCK8) and colon formation assay. Cell cycles were explored using flow cytometer and cell protein expression levels after that the transfection was detected by Western blotting. RESULTS: We found that ROC curve analysis showed that miR-299-5p was a sensitivity diagnostic criteria and GSEA indicated that miRNA-299-5p may regulate cell cycle. Gain of function assay showed that miR-299-5p promotes cell cycle transition and proliferation. Reversely, the opposite results were observed with loss of function assay. At last, Western blotting assay showed that miR-299-5p may promote cell cycle transition by regulating CDK family.

[0.5 Gy X-ray radiation promotes osteoblast differentiation by Wnt/ß-Catenin signaling].

Objective: To investigate the important roles of Wnt signaling in the processes of 0.5Gy X-ray promoting osteoblast differentiation, and make clear the molecular mechanisms involved. Methods: Flow cytometry was employed to detect the apoptosis after osteoblast exposure to 0.5 Gy X-ray radiation.The protein level of osteoblast differentiation markers, such as collagen Iα (Col1α), alkaline phosphatase (ALP), osteocalcin (OCN), were detected by Western-blot and ALP activity staining was performed. Real-time PCR and Western-blot were utilized to evaluate the variations of key factors in Wnt signaling pathways, while specific inhibitor of Wnt/ß-Catenin, XAV939 was used to block the Wnt signaling. Results: Low-dose (0.5 Gy) X-ray induced significant decline in MC3T3-E1 osteoblast apoptosis at three days after radiation.The dynamic variations in the expression of osteoblast differentiation markers, including Col1α, ALP, OCN, were observed after 0.5 Gy X-ray irradiation by Western blot analysis.The protein levels of Col1α have a reduction temporarily at 4 days of radiation (34.5%±5.8%, t=9.912, P<0.001), then a significant increase is detected at 10 day after radiation (162.5%±6.5%, t=2.673, P<0.05). OCN levels dropped by 83% (t=3.968, P<0.01) at 4 day after 0.5 Gy X-ray radiation, and raised at 10 day (39.5%±4.1%, t=3.219, P<0.05) and 14 day (79.4%±7.5%, t=6.708, P<0.001), respectively. ALP levels increased at 7 day (79.7%±22.3%, t=6.257, P<0.001) and 10 day(128.3%±6.1%, t=4.340, P<0.01)after radiation. At the same time, 0.5 Gy X-ray radiation can activate Wnt/GSK-3/ß-Catenin signaling.The mRNA levels of Wnt3a、LPR5 and TCF-4 increased by 1.7 fold (t=6.573, P<0.001), 1.1 fold (t=5.323, P<0.05) and 1.4 fold (t=3.054, P<0.05) at 7 day after radiation.In addition, p-GSK-3ß level reduced by 42.1% (t=4.460, P<0.01), and active ß-Catenin increased by 1.9 fold (t=3.528, P<0.05). However, the specific inhibitor of Wnt/ß-Catenin, XAV939 completely abrogated Wnt/ß-Catenin signaling and the increase in ALP expression and activity induced by 0.5 Gy X-ray radiation. Conclusion: These results demonstrated that low dose X-ray radiation promoted osteoblast survival at early differentiation, and promoted differentiation at middle and late stage, in which Wnt signaling participated the regulation processes.

[Surgery of ipsilateral Hawkins â ¢ talus neck and ankle joint fractures via internal and lateral approaches with Herbert screws].

Objective: To explore the manual operation skills of operative treatment of ipsilateral Hawkins Ⅲ talus neck and ankle joint fractures via internal and lateral approaches with Herbert screws, and to study the clinical results. Method: From Jan 2009 to Dec 2014, the clinical data of 13 patients with ipsilateral Hawkins Ⅲ talus neck and ankle joint fractres via internal and lateral approaches with Herbert screws were retrospectively analyzed in our department.There were 10 males and 3 female, ranging in age from 20 to 60 years with an average age of 31.5 years.The fractures occurred on the right side in 9 patients and on the left side in 4 patients.Three cases had the complication of medial malleolar fracture.Ten cases had the complication of medial and lateral malleolar fracture. Totally 11 cases were made calcaneal skeletal traction, and all the were made CT with three-dimensional image reconstruction.Two cases were treated with emergency operation.Eleven cases were treated with selective operation.The operation time was 5 hours-10 days after injury. The functional results were evaluated by American Orthopaedic Foot and Ankle Society (AOFAS). Result: The average duration of follow-up was 22.6 months (range, 14-65 months). There was skin necrosis in one cases, no incision infection, malunion and nonunion of the fractures and loss of reduction. At final follow-up, AOFAS ankle score was 75.2 (range, 42 to 93), higher than preoperative 39.2 (range, 23 to 60), the difference was statistically significant (P=0.023). The result was excellent in 4 cases, good in 5 cases, fair in 3 cases and 1 cases in poor, and the overall excellent or good rate was 69.2%. Avascular necrosis occurred in 3 cases (23.1%, 3/13). Traumatic arthritis was found in 5 cases (38.5%, 5/13), involved tibial astragaloid joint in 2 cases, involved subtalar joint in 1 case, involved tibial astragaloid joint and subtalar joint in 2 cases. Conclusion: The effect of surgical treatment for ipsilateral Hawkins Ⅲ talus neck and ankle joint fractures via internal and lateral approaches with Herbert screws is satisfactory.Correct operative approach and pay more attention to protect blood circulation of intraoperative, anatomical precision and strong reduction and fixation are the key to achieve and gain better long-term results for the surgical treatment of ipsilateral Hawkins Ⅲ talus neck and ankle joint fractures.

[Effects of 0.5 Gy X-ray radiation on the profile of gene expression in MC3T3-E1 osteoblasts].

Objective: To investigate the molecular mechanism of low-dose X-ray irradiation on osteoblasts by detecting the gene expression profiles of osteoblasts radiated with 0.5 Gy X-ray. Methods: Microarray analyses to value the changes of gene expression of MC3T3-E1 osteoblasts after 0.5 Gy X-ray irradiation were conducted.The end points included modulation of key markers, and pathway and gene ontology through transcriptomic profiling and bioinformatics analysis. Several major genes during osteoblasts differentiation were selected for Real-time PCR analysis. Results: 1 412 differentially expressed transcripts were identified between the radiated group and control group. Among the identified genes, 559 transcripts were up-regulated and 853 transcripts were down-regulated after irradiation.Real-time PCR analysis indicated that the mRNA expressions of Bglap-rs1, Col1a2, Tgf-ß1, Lrp5, Dvl1, Map4k5 were apparently higher than that of the controls (P<0.01), whereas the Id2 and Dkk1 expression decreased under the same condition(P<0.01). Western-blot analysis indicated TGF-ß1 and Lrp-5 protein expression increased and Dkk1 expression decreased(P<0.05). Pathway analysis and gene ontology analysis revealed that focal adhesion, extracellular signaling, cytoskeletal protein binding, Wnt signal pathway, IκB /NF-κB signal pathway and growth factor receptors activities. Conclusions: Low-dose X-ray radiation promoted osteoblasts differentiation in which focal adhesion, extracellular signaling, cytoskeletal protein binding, Wnt signal pathway, IκB /NF-κB signal pathway and growth factor activities might be involved.

Accuracy of low-field MRI on meniscal tears.

This study aimed to verify the accuracy of low-field-intensity magnetic resonance imaging (MRI) in diagnosing meniscus tears. A total of 171 patients were examined through low-field-intensity MRI to detect meniscus injuries. These patients were then diagnosed through arthroscopy. Examination results were recorded and compared. The accuracy of the diagnosis for internal and external meniscus tears through low-field-intensity MRI was 95.91% and 95.91%, respectively, the sensitivities were 95.60% and 96.47%, respectively, and the specificities were 96.25% and 95.35%, respectively. Low-field-intensity MRI is an accurate and cost-effective method for diagnosing meniscus tears.

Arthroscopic reconstruction of the anterior cruciate ligament with the LARS artificial ligament: thirty-six to fifty-two months follow-up study.

OBJECTIVES: This study aims to analyze the operating methods and fundamental clinical effects of the reconstruction of the anterior cruciate ligament (ACL) with the Ligament Advancement Reinforcement System (LARS) artificial ligaments using an arthroscopy. PATIENTS AND METHODS: Fifty-three patients with ACL rupture who were treated using LARS artificial ligaments were enrolled in this retrospective study. The mean age at the time of reconstruction was 31.2 y (range, 22-51y). Average time from injury to surgery was 18 d (range, 5-51 d). Average follow-up period was 45 mo (range 36-52 months). Follow-up examinations included the Lysholm Knee Score and the International Knee Documentation Committee (IKDC) score. RESULTS: The average Lysholm Knee Score was 53.1±5.8 preoperatively (range, 47-76) versus 93.2±3.4 three years after operation (range, 80-100). Fifty-one of 53 patents (96.2%) showed good or excellent results at final assessment. The final IKDC score 3 years after operation were normal in 28 patients (52.8%), nearly normal in 23 patients (43.4%), and abnormal in 2 patients (3.8%). No postoperative complications, such as infection, ligament rupture, or ligament cinch occurred. CONCLUSIONS: The results suggest that LARS artificial ligament appears to be an effective graft for ACL reconstruction leading to good knee function and stability. Long-term follow-up should be performed to confirm the durable stability of the knee and the tolerance of the knee to the LARS artificial ligament.

Mesenchymal stem cell-based repair of articular cartilage with polyglycolic acid-hydroxyapatite biphasic scaffold.

This study investigates the capacity of a composite scaffold composed of polyglycolic acid-hydroxyapatite (PGA-HA) and autologous mesenchymal stem cells (MSCs) to promote repair of osteochondral defects. MSCs from culture-expanded rabbits were seeded onto a PGA and HA scaffold. After a 72-hour co-culture period, the cell-adhered PGA and HA were joined together, forming an MSCs-PGA-HA composite. Full-thickness cartilage defects in the intercondylar fossa of the femur were then implanted with the MSC-PGA-HA composite, the PGA-HA scaffold only, or they were left empty (n=20). Animals were sacrificed 16 or 32 weeks after surgery and the gross appearance of the defects was evaluated. The specimens were examined histologically for morphologic features, and stained immunohistochemically for type 2 collagen. Specimens of the MSCs-PGA-HA composite implantation group demonstrated hyaline cartilage and a complete subchondral bone formation. At 16 weeks post-implantation, significant integration of the newly formed tissue with surrounding normal cartilage and subchondral bone was observed when compared to the two control groups. At 32 weeks, no sign of progressive degeneration of the newly formed tissue was found. A significant difference in histological grading score was found compared with the control groups. The novel MSCs-seeded, PGA-HA biphasic graft facilitated both articular cartilage and subchondral bone regeneration in an animal model and might serve as a new approach for clinical applications.

[Prediction of segmental collapse of femoral head after femoral neck fracture by scintimetry].

A follow-up study regarding the occurrence of segmental collapse of femoral head has been done in 53 patients of united femoral neck fracture including a periodic examination by 99mTc-MDP scintimetry. The results showed that the sequential variations in the nuclide uptake ratio of the femoral heads could reflect the histological repairing process and metabolic level in the femoral head. A sustainedly high nuclide uptake in 12 months after operation often denotes that there are defects in the repairing process of the ischemic femoral head, and that the segmental collapse of the femoral head will ensue.

[Early diagnosis by scintigraphy of segmental collapse of the femur head following femur neck fracture]. / Diagnostic précoce par scintigraphie des collapsus segmentaires de la tête fémorale après fracture du col fémoral.

In order to obtain an early prediction for segmental collapse of the femoral head after femoral neck fracture, we have studied in 53 cases of the femoral neck fracture using 99mTC-MDP scintimetry. According to the radionuclide uptake ratio of the femoral heads, we can estimate the gravity of the avascular necrosis of the femoral head after fracture and recognize the repair process in the necrotic head. Fifty-three cases of fresh fracture were examined by sequential scintigraphy before operation and during follow up examinations after operation. The radionuclide uptake were all increased in 3 to 4 months after operation as comparing with that done before operation. The uptake ratios in 37 cases decreased gradually and approached 1 in 12 months after operation. All of them have an excellent result during follow up examination 36 months after surgery. The uptake ratios in 19 cases were also increased after operation, but still maintained at a high level in 6 to 12 months. They all showed radiographical signs of segmental collapse 18 to 24 months after operation. These results showed that uptake ratio of the radionuclide bone imaging is able to predict the occurrence of segmental collapse of the femoral head after femoral neck fracture. The time of the diagnosis by scintigraphy for segmental collapse of the femoral head is earlier than that by radiography.

[Early diagnosis by scintigraphy of femur head necrosis after femur neck fracture]. / Diagnostic précoce par scintigraphie des nécroses de la tête fémorale après fracture du col.

Necrosis of the head of the femur with segmentary collapse after transcervical fractures occurs in 30 to 50% of the cases. Diagnosis of avascular necrosis should be made early before the head collapses. In a clinical and experimental study, we attempted to perform early screening for this complication. We studied 41 fractures of the neck of the femur with technitium scintigraphy with intravenous injections and compared uptake in pathological and normal heads. In the first group of 20 cases of fresh fractures, scintigraphy was performed before operation then repeated every three months to the 36th months later. High uptake we seen in 12 of the 20 cases then returned to normal 12 to 18 months later. Consolidation occurred in all 12 without necrosis. In 8 cases uptake was low initially then rose greatly and remained higher than normal. Necrosis occurred in 6 of these 8 cases. In a second group of 21 patients with a history of necrosis of the head of the femur, uptake was high. Pathologic examination of the surgical specimen after resection for prosthesis showed cell growth around the zone of necrosis. In conclusion, this technique, when possible, would appear to be highly useful to screen for necrosis early.

Hook-pin and compression screw in the treatment of femoral neck fractures. Clinical trial and biomechanical study.

One hundred and two patients with femoral neck fracture underwent combined hook-pin and compression screw fixation. Eighty-five patients were followed up for over 6 months. The fracture was united in 80 patients. The advantages of this method included less trauma, simple technique and firm internal fixation and early postoperative partial weight-bearing. Biomechanical study showed that this method is superior to Smith-Petersen nailing and two hook-pins methods. The sliding and continuous compression of this method promotes impaction and healing of the fracture.