BACKGROUND:Existing studies have made significant progress in PIWI-interacting RNAs(piRNAs)against osteoporosis,but the specific targets and related mechanisms by which piRNAs exert their functions remain to be explored.OBJECTIVE:To investigate the effects and downstream mechanisms of piR-7472 on the differentiation of mouse osteoblasts(MC3T3-E1 cells).METHODS:(1)Twelve C57/BL6J mice were randomly divided into a sham-operated and an ovariectomized group,with six mice in each group.Changes in bone mass and the expression of piR-7472 were detected using Micro-CT and RT-qPCR,respectively,at 8 weeks after surgery.(2)MC3T3-E1 cells were divided into NC mimics group,piR-7472 mimics group,NC inhibitor group,and piR-7472 inhibitor group.The mRNA expression of piR-7472,osteopontin,type I collagen,Runt-related transcription factor 2,and potassium voltage-gated channel modifier subfamily F member 1 were detected by RT-qPCR after 7 days of osteogenic induction.The protein expression of osteopontin,Runt-related transcription factor 2,bone morphogenetic protein 2,and potassium voltage-gated channel modifier subfamily F member 1(KCNF1)was detected using western blot assay.The expression of alkaline phosphatase was detected by alkaline phosphatase staining after 14 days of osteogenic induction,and the number of mineralized nodules was detected by alizarin red staining after 21 days of induction.Whether piR-7472 could bind to KCNF1 was observed by the dual luciferase reporter gene assay.RESULTS AND CONCLUSION:(1)Bone mineral density,bone volume fraction,bone trabecular thickness,bone trabecular number were significantly decreased and bone trabecular separation was significantly increased in ovariectomized mice,and piR-7472 in bone tissue was significantly down-regulated in osteoporotic mice.(2)Compared with the NC group,the mRNA expression of osteopontin,type I collagen,and Runt-related transcription factor 2 were significantly increased,the protein expression of osteopontin,Runt-related transcription factor 2,and bone morphogenetic protein 2 were significantly elevated,and the levels of mineralized deposition and alkaline phosphatase were increased in the piR-7472 mimics group.Compared with the NC inhibitor group,the mRNA expression of osteopontin,type I collagen,and Runt-related transcription factor 2 was significantly downregulated,the protein expression of osteopontin,Runt-related transcription factor 2,and bone morphogenetic protein 2 were significantly decreased,and the levels of mineralized deposition and alkaline phosphatase were reduced in the piR-7472 inhibitor group.(3)piR-7472 was found to interact with the potassium voltage-gated channel modifier subfamily F member 1 as predicted by the miRanda database.The dual luciferase reporter gene assay revealed that piR-7472 mimics could bind to and promote the expression of KCNF1.To conclude,piR-7472 can promote osteogenic differentiation of osteogenic precursor cells MC3T3-E1,and its mechanism of action may be achieved by promoting the expression of KCNF1.

BACKGROUND:The use of autologous or allogeneic transplantation materials for tissue repair and reconstruction is limited and carries limitations in clinical applications.Transgenic stem cells and tissue engineering materials offer new therapeutic possibilities.OBJECTIVE:To investigate the in vitro biological characteristics of enhanced green fluorescent protein recombinant lentivirus transfected into rabbit bone marrow mesenchymal stem cells and the biommineralization characteristics of transgenic tissue engineering materials constructed using decalcified bone matrix.METHODS:Rabbit bone marrow mesenchymal stem cells were obtained through adherent culture and density gradient centrifugation.The fifth-generation bone marrow mesenchymal stem cells were transfected with enhanced green fluorescent protein recombinant lentivirus at the optimal multiplicity of infection(MOI=100).The differences in cell proliferation capacity,cell phenotype,cell cycle,alkaline phosphatase expression,osteogenic transcription factor(Runx2)expression,and osteocalcin gene expression after osteogenesis were observed between the transfected cells and non-transfected cells in vitro.The micromorphology and elemental energy spectrum analysis of the transgenic tissue engineering materials constructed from cells and decalcified bone matrix scaffold were also observed.RESULTS AND CONCLUSION:Bone marrow mesenchymal stem cells transfected with enhanced green fluorescent protein recombinant lentivirus showed that cell proliferation at 24 and 48 hours after transfection was slower than that of untransfected cells(P<0.05).After 72 hours,the cell phenotype remained unchanged,and the expressions of alkaline phosphatase,Runx2,and osteocalcin after osteogenic induction were not significantly different from those of untransfected bone marrow mesenchymal stem cells(P>0.05).The transgenic tissue engineering materials constructed in vitro using enhanced green fluorescent protein recombinant lentivirus transfected bone marrow mesenchymal stem cells and decalcified bone matrix showed good biocompatibility on the decalcified bone matrix scaffold.Based on fluorescence expression intensity,it was estimated that the target gene would exert its maximum biological function in about 2 weeks,and calcium phosphate deposits would continue to appear,demonstrating superior biomineralization characteristics.

Objective:To explore the expression of nectin-4 and vanin-1 in esophageal squamous cell carcinoma(ESCC)and its influence on the malignant biological behaviors of ESCC cells,as well as the underlying mechanisms.Methods:Transcriptome sequencing combined with GO and KEGG enrichment analysis was used to identify the downstream target gene(vanin-1)regulated by nectin-4.The mRNA expression of vanin-1 in ESCC tissues was studied using the Timer2.0 database,and the mRNA and protein expression of vanin-1 in normal esophageal epithelial HET-1 and ESCC cells was detected by qPCR and Western blot,identifying ESCC KYSE-410 and KYSE-510 cells with the most significant differential expression.The expression of vanin-1 in KYSE-410 and KYSE-510 cells was knocked down using siRNA.The effects of vanin-1 knockdown on cell proliferation,migration,and invasion were measured using CCK-8 assay,wound healing assay,and Transwell chamber assay.Furthermore,KEGG and GO enrichment analyses were conducted for vanin-1-related signaling pathways.Immunohistochemistry was performed to compare the expression of vanin-1 between ESCC tissues and adjacent non-tumor tissues.Results:Timer2.0 database analysis and qPCR results showed that vanin-1 was highly expressed in both ESCC tissues and cell lines(both P<0.01).WB assay also confirmed high expression of vanin-1 protein in ESCC cells(P<0.01).siRNA successfully knocked down vanin-1 expression in KYSE-410 and KYSE-510 cells.Knockdown of vanin-1 significantly inhibited the proliferation,migration,and invasion capabilities of KYSE-410 and KYSE-510 cells(P<0.05 or P<0.01 or P<0.001 or P<0.000 1).KEGG and GO enrichment analysis suggested that vanin-1 might function through pathways related to pantothenic acid and coenzyme A synthesis metabolism.Immunohistochemistry results indicated that vanin-1 was highly expressed in ESCC tissues(P<0.000 1).Conclusion:Vanin-1 is highly expressed in ESCC tissues and promotes the proliferation,migration,and invasion of KYSE-410 and KYSE-510 cells through the nectin-4/vanin-1 axis.Targeting vanin-1 might offer a new therapeutic strategy for ESCC.

BACKGROUND:Existing studies have made significant progress in PIWI-interacting RNAs(piRNAs)against osteoporosis,but the specific targets and related mechanisms by which piRNAs exert their functions remain to be explored.OBJECTIVE:To investigate the effects and downstream mechanisms of piR-7472 on the differentiation of mouse osteoblasts(MC3T3-E1 cells).METHODS:(1)Twelve C57/BL6J mice were randomly divided into a sham-operated and an ovariectomized group,with six mice in each group.Changes in bone mass and the expression of piR-7472 were detected using Micro-CT and RT-qPCR,respectively,at 8 weeks after surgery.(2)MC3T3-E1 cells were divided into NC mimics group,piR-7472 mimics group,NC inhibitor group,and piR-7472 inhibitor group.The mRNA expression of piR-7472,osteopontin,type I collagen,Runt-related transcription factor 2,and potassium voltage-gated channel modifier subfamily F member 1 were detected by RT-qPCR after 7 days of osteogenic induction.The protein expression of osteopontin,Runt-related transcription factor 2,bone morphogenetic protein 2,and potassium voltage-gated channel modifier subfamily F member 1(KCNF1)was detected using western blot assay.The expression of alkaline phosphatase was detected by alkaline phosphatase staining after 14 days of osteogenic induction,and the number of mineralized nodules was detected by alizarin red staining after 21 days of induction.Whether piR-7472 could bind to KCNF1 was observed by the dual luciferase reporter gene assay.RESULTS AND CONCLUSION:(1)Bone mineral density,bone volume fraction,bone trabecular thickness,bone trabecular number were significantly decreased and bone trabecular separation was significantly increased in ovariectomized mice,and piR-7472 in bone tissue was significantly down-regulated in osteoporotic mice.(2)Compared with the NC group,the mRNA expression of osteopontin,type I collagen,and Runt-related transcription factor 2 were significantly increased,the protein expression of osteopontin,Runt-related transcription factor 2,and bone morphogenetic protein 2 were significantly elevated,and the levels of mineralized deposition and alkaline phosphatase were increased in the piR-7472 mimics group.Compared with the NC inhibitor group,the mRNA expression of osteopontin,type I collagen,and Runt-related transcription factor 2 was significantly downregulated,the protein expression of osteopontin,Runt-related transcription factor 2,and bone morphogenetic protein 2 were significantly decreased,and the levels of mineralized deposition and alkaline phosphatase were reduced in the piR-7472 inhibitor group.(3)piR-7472 was found to interact with the potassium voltage-gated channel modifier subfamily F member 1 as predicted by the miRanda database.The dual luciferase reporter gene assay revealed that piR-7472 mimics could bind to and promote the expression of KCNF1.To conclude,piR-7472 can promote osteogenic differentiation of osteogenic precursor cells MC3T3-E1,and its mechanism of action may be achieved by promoting the expression of KCNF1.

BACKGROUND:The use of autologous or allogeneic transplantation materials for tissue repair and reconstruction is limited and carries limitations in clinical applications.Transgenic stem cells and tissue engineering materials offer new therapeutic possibilities.OBJECTIVE:To investigate the in vitro biological characteristics of enhanced green fluorescent protein recombinant lentivirus transfected into rabbit bone marrow mesenchymal stem cells and the biommineralization characteristics of transgenic tissue engineering materials constructed using decalcified bone matrix.METHODS:Rabbit bone marrow mesenchymal stem cells were obtained through adherent culture and density gradient centrifugation.The fifth-generation bone marrow mesenchymal stem cells were transfected with enhanced green fluorescent protein recombinant lentivirus at the optimal multiplicity of infection(MOI=100).The differences in cell proliferation capacity,cell phenotype,cell cycle,alkaline phosphatase expression,osteogenic transcription factor(Runx2)expression,and osteocalcin gene expression after osteogenesis were observed between the transfected cells and non-transfected cells in vitro.The micromorphology and elemental energy spectrum analysis of the transgenic tissue engineering materials constructed from cells and decalcified bone matrix scaffold were also observed.RESULTS AND CONCLUSION:Bone marrow mesenchymal stem cells transfected with enhanced green fluorescent protein recombinant lentivirus showed that cell proliferation at 24 and 48 hours after transfection was slower than that of untransfected cells(P<0.05).After 72 hours,the cell phenotype remained unchanged,and the expressions of alkaline phosphatase,Runx2,and osteocalcin after osteogenic induction were not significantly different from those of untransfected bone marrow mesenchymal stem cells(P>0.05).The transgenic tissue engineering materials constructed in vitro using enhanced green fluorescent protein recombinant lentivirus transfected bone marrow mesenchymal stem cells and decalcified bone matrix showed good biocompatibility on the decalcified bone matrix scaffold.Based on fluorescence expression intensity,it was estimated that the target gene would exert its maximum biological function in about 2 weeks,and calcium phosphate deposits would continue to appear,demonstrating superior biomineralization characteristics.

Thoracic ossification of the ligamentum flavum (TOLF) is a pathological heterotopic ossification disease in which the fibrous tissue of the ligamentum flavum of the thoracic spine converts into bony tissue, often leading to thoracic spinal stenosis and compression of the thoracic spinal cord nerve. When TOLF patients present with symptoms of spinal cord nerve compression, surgical treatment is usually required, and traditional open surgery is more invasive and carries a higher risk of spinal cord nerve injury. In recent years, domestic and foreign researchers have tried to apply spinal endoscopic techniques such as microendoscopy, percutaneous foraminoscopy, and unilateral biportal endoscopy for the treatment of TOLF, which can maximize the preservation of normal bone while achieving adequate decompression of the spinal cord nerve, with less damage to spinal stability, and have the advantages of less surgical trauma, less bleeding, and faster postoperative recovery. Due to the special anatomical structure of the thoracic vertebra, spinal endoscopic techniques should focus on safety and it is recommended that they are performed in experienced centers, and surgical indications should be strictly controlled.

Thoracic ossification of the ligamentum flavum (TOLF) is a pathological heterotopic ossification disease in which the fibrous tissue of the ligamentum flavum of the thoracic spine converts into bony tissue, often leading to thoracic spinal stenosis and compression of the thoracic spinal cord nerve. When TOLF patients present with symptoms of spinal cord nerve compression, surgical treatment is usually required, and traditional open surgery is more invasive and carries a higher risk of spinal cord nerve injury. In recent years, domestic and foreign researchers have tried to apply spinal endoscopic techniques such as microendoscopy, percutaneous foraminoscopy, and unilateral biportal endoscopy for the treatment of TOLF, which can maximize the preservation of normal bone while achieving adequate decompression of the spinal cord nerve, with less damage to spinal stability, and have the advantages of less surgical trauma, less bleeding, and faster postoperative recovery. Due to the special anatomical structure of the thoracic vertebra, spinal endoscopic techniques should focus on safety and it is recommended that they are performed in experienced centers, and surgical indications should be strictly controlled.

Objective:To introduce the surgical method of Ortho-Bridge system (OBS) in the treatment of distal femoral fractures in elderly patients and investigate its clinical effect.Methods:From January 2018 to July 2021, 24 elderly patients who suffered distal femoral fractures were treated with bilateral OBS. There were 8 males and 16 females aging from 62 to 87 years, with an average age of 72.6 years. It included 15 cases of simple distal femoral fractures. According to AO classification, there were one case of A1, two of A2, five of A3, two of C1, three of C2 and two of C3. Nine cases of femoral periprosthetic fractures after total knee arthroplasty (TKA) were classified as type II according to rorabeck's classification. After operation, all patients were guided to perform knee joint functional exercise and to measure the range of motion of the knee joint. Then imaging examinations were used to evaluate the fracture healing and measure the femoral-tibial and femoral angles. The American Hospital for Special Surgery (HSS) knee joint scoring system was used to evaluate the knee function.Results:All 24 patients successfully completed the operation. The operation time was 84-115 min, with an average of 96.6 min; the intraoperative blood loss was 150-335 ml, with an average of 240 ml. All patients were followed up for 8-17 months, with an average of 13.6 months. Except for 1 case of nonunion due to few primary bone grafts, which required secondary bone grafting, the other 23 cases achieved bone union. The healing time was 3.5-6 months, with an average of 4.6 months. At 1, 3, and 6 months after operation and at the last follow-up, the flexion angles of knee were 92.2°±10.2°, 98.6°±13.3°, 106.4°±13.7°, 115.7°±15.3°, and the extension angles were -4.7°±4.1°, -1.2°±4.2°, 0.7°±4.5°, 1.8°±4.6°, respectively; and all differences were statistically significant ( F=17.03 and 12.68, P<0.001). The knee flexion and extension angles at the last follow-up were greater than 1, 3, and 6 months after operation, and the differences were statistically significant ( P<0.001). The femoral-tibial angle was 171.2°±2.4° and 170.7°±3.2°, and the femoral angle was 80.3°±1.7° and 79.6°±2.1°, respectively, at the immediate postoperative and last follow-up, with no significant difference. The HSS scores at 1, 3, 6 months after operation and at the last follow-up were 71.5±7.5, 74.6± 9.3, 78.9±10.4 and 84.7±9.4 respectively, with significant difference ( F=9.17, P<0.001). At the last follow-up, the HSS score was higher than that at 1, 3, and 6 months after the operation, and the differences were statistically significant ( P<0.001), and the knee function was evaluated according to the HSS scoring system: excellent in 12 cases, good in 9, fair in 3, with an excellent and good rate of 88% (21/24). There was no OBS crack or fixation failure in all patients, and no prosthetic loosening and instability occurred in patients with periprosthetic femoral fractures after TKA. Statistical analysis of the data at the last follow-up between the distal femoral fracture group and the periprosthetic femoral fracture group after TKA showed that the knee flexion function and HSS score of the periprosthetic femoral fracture group after TKA (126.8°±3.7°, 92.2±4.1) were both better than the simple distal femur fracture group (108.9°±15.7°, 80.2±8.8). The difference was statistically significant ( t=4.22, 4.52, P<0.05). One patient had incision fat liquefaction and healed after debridement; bone nonunion occurred in 1 case, which healed after iliac bone grafting. Conclusion:Double OBS has a good clinical effect in the treatment of distal femoral fractures in the elderly, especially in patients with periprosthetic femoral fractures after TKA.

Objective:To compare the clinical efficacy between a bidirectional-traction reduction device and a traction table in the treatment of femoral neck fracture with femoral neck system (FNS).Methods:A retrospective study was conducted in the 46 patients with femoral neck fracture who had been treated at Department of Orthopedics, The First Central Hospital of Baoding from January 2020 to January 2021. There were 19 males and 27 females, aged from 30 to 64 years (average, 47.1 years). According to the Garden classification, 29 cases were type Ⅲ and 17 type Ⅳ. By the reduction method, the patients were assigned into an observation group ( n=24) in which the reduction was assisted by a bidirectional-traction reduction device and a control group ( n=22) in which the reduction was assisted by a traction table. FNS fixation was conducted in both groups. The 2 groups were compared in terms of operation time, reduction time, fluoroscopy frequency, intraoperative blood loss, femoral neck shortening at immediate postoperation and 12 months postoperation, Harris scores of the affected hip at 3, 6, and 12 months postoperation, and incidence of lower extremity venous thrombosis. Results:There were no significant differences in age, gender or fracture type between the 2 groups, showing they were comparable ( P>0.05). The observation group needed significantly less operation time [57.5 (54.0, 64.5) min], reduction time [(16.3±3.0) min] and fluoroscopy frequency [(20.5±4.6) times] than the control group did [85.0 (71.3, 92.0) min, (21.0±6.0) min and (29.7±4.7) times, respectively] (all P<0.05). There was no significant difference in intraoperative blood loss between 2 groups ( P>0.05). All patients were followed up for 12 to 22 months (average, 15.5 months). There was no significant difference in femoral neck shortening between the 2 groups at immediate postoperation or 12 months postoperation ( P>0.05). The Harris score of the affected hip in the observation group was significantly better than that in the control group at 3 months after surgery ( P<0.05), but such a significant difference was not observed at 6 or 12 months postoperation ( P>0.05). The incidence of thrombotic complications in the observation group (12.5%, 3/24) was significantly lower than that in the control group (40.9%, 9/22) ( P<0.05). Conclusions:In the FNS treatment of femoral neck fracture, compared with a traction table, reduction assisted by a bidirectional-traction reduction device is more advantageous because it is simpler and less time-consuming, incurs less fluoroscopy and leads to better early functional recovery of the affected hip and lower incidence of thrombotic complications.

Objective:To compare the biomechanical parameters of Ortho-Bridge system (OBS) and locking compression plate+locking attachment plate (LCP+LAP) in the fixation of femoral periprosthetic type B1 fracture.Methods:The same periprosthetic type B1 fracture of human femur were made, including simple fracture model and comminuted fracture model, 12 in each. And the simple fracture models were randomly divided into 6 pieces of OBS system fixation group and 6 pieces of LCP+LAP system fixation group, and the complex fracture models were also randomly divided into 6 pieces of OBS system fixation group and 6 pieces of LCP + LAP system fixation group. Then the four groups of models were tested by axial compression and torsion tests, and the stiffness of the models under axial compression and torsion angle of the models under torsion test were collected. The axial compression failure test was carried out to collect the vertical load of the ultimate failure test. The axial stiffness, torsion angle and axial failure load of OBS and LCP+LAP fixed simple and comminuted fractures were statistically analyzed by t test. Results:For the test of fixed simple fracture, there was no significant difference ( t=0.535, P=0.522) in the axial stiffness between the OBS group (868.87±157.14 N) and the LCP+LAP group (904.53±44.76 N), whereas the results of torsion test showed that the LCP+LAP group had a higher torsion angle 7.17°±0.52° than the OBS group 5.45°±0.44° ( t=5.616, P<0.001); When fixing comminuted fractures, the OBS group had a higher axial stiffness (145.33±10.34 N) than the LCP+LAP group (84.15±8.94 N) ( t=10.961, P<0.001), but the LCP+LAP group had a higher torsion angle 7.75°±1.17° than the OBS group 5.23°±0.31° ( t=4.652, P=0.001). Ultimate failure test data showed that the failure pressure of OBS fixed group (4 967.49±132.88 N) was higher than LCP+LAP group (3 967.41±145.16 N) ( t=12.447, P<0.001). In the LCP+LAP group, there was destruction of the contact cortex at the fracture site, while in the OBS group, there was destruction of the contact cortex at the fracture site as well as fractures around the proximal fixation screw. Conclusion:OBS group has similar axial compression resistance to LCP+LAP group, but better torsion resistance than LCP+LAP group when it is used to fix B1 simple fracture around femoral prosthesis. When comminuted fracture is fixed, the axial compression resistance and torsion resistance of OBS group are better than LCP+LAP group. The stress is dispersed during OBS fixation, which can better avoid the failure of internal fixation during early functional exercise.