Bradykinin protects nucleus pulposus cells from tert-butyl hydroperoxide-induced damage and delays intervertebral disc degeneration.

Intervertebral disc degeneration (IVDD) is a leading cause of degenerative spinal disorders, involving complex biological processes. This study investigates the role of the kallikrein-kinin system (KKS) in IVDD, focusing on the protective effects of bradykinin (BK) on nucleus pulposus cells (NPCs) under oxidative stress. Clinical specimens were collected, and experiments were conducted using human and rat primary NPCs to elucidate BK's impact on tert-butyl hydroperoxide (TBHP)-induced oxidative stress and damage. The results demonstrate that BK significantly inhibits TBHP-induced NPC apoptosis and restores mitochondrial function. Further analysis reveals that this protective effect is mediated through the BK receptor 2 (B2R) and its downstream PI3K/AKT pathway. Additionally, BK/PLGA sustained-release microspheres were developed and validated in a rat model, highlighting their potential therapeutic efficacy for IVDD. Overall, this study sheds light on the crucial role of the KKS in IVDD pathogenesis and suggests targeting the B2R as a promising therapeutic strategy to delay IVDD progression and promote disc regeneration.

ERK5 negatively regulates Kruppel-like factor 4 and promotes osteogenic lineage cell proliferation in response to MEK5 overexpression or fluid shear stress.

Aim of the study: Fluid shear stress (FSS) plays a critical role in osteoblast proliferation via extracellular signal-regulated kinase 5 (ERK5). Kruppel-like factor 4 (KLF4) knockout robustly enhances bone formation due to increased osteoblast differentiation and mineralization. However, the effect of KLF4 on osteoblast proliferation is unresolved. Therefore, the aim of our study was to investigate the effect of KLF4 on osteogenic lineage cell proliferation and the relationship between KLF4 and ERK5. Materials and methods: MC3T3-E1 cells were treated with FSS and/or KLF4 siRNA, cell viability was accessed by Edu labeling and CCK-8 assay, and proliferative gene expression were assessed by PCR array. Bone marrow stromal cells (BMSCs) were infected with adenovirus expressing KLF4 and/or constitutively active MEK5, cell viability was evaluated using crystal violet staining, colony formation assay, and cell WST1 assay. The levels of KLF4 and ERK5 phosphorylation were identified through qRT-PCR and western blot, respectively. Results: KLF4 expression was significantly down-regulated by FSS exposure, however, this was reversed by ERK5 siRNA. KLF4 overexpression inhibited colony formation efficiency and cell viability in BMSCs. Adenoviruses expressing constitutively active MEK5 increased ERK5 phosphorylation, which inhibited KLF4 expression, and promoted BMSC proliferation. FSS-induced osteoblast proliferation also involved elevation of Cyclin B2 and Cdc14b as well as repressed expression of P27. Conclusions: KLF4 negatively regulates osteogenic lineage cell proliferation, and ERK5 negatively regulates KLF4 expression and promotes osteogenic lineage cell proliferation.

Fluid Shear Stress Suppresses Osteoclast Differentiation in RAW264.7 Cells through Extracellular Signal-Regulated Kinase 5 (ERK5) Signaling Pathway.

BACKGROUND Although extracellular signal-regulated kinase 5 (ERK5) is known to be critical for osteoclast differentiation, there are few studies on how fluid shear stress (FSS) regulates osteoclast differentiation through the ERK5 signaling pathway. We examined the expression of nuclear factor of activated T cells c1 (NFATc1) in RAW264.7 cells and its downstream factors, including cathepsin K (CTSK), tartrate-resistant acid phosphatase (TRAP), matrix metalloproteinases-9 (MMP-9) and their relationship with ERK5. MATERIAL AND METHODS RAW264.7 cells were treated with RANKL, XMD8-92 (ERK5 inhibitor), and then loaded onto 12 dyn/cm² FSS for 4 days. Endpoints measured were osteoclast differentiation, bone resorption, and TRAP activity. Cell viability was detected by using the Cell Counting Kit-8 (CCK-8) assay. Western blot was used to analyze protein expression of phosphorylated-ERK5 (p-ERK5), NFATc1, CTSK, TRAP, and MMP-9. RESULTS FSS inhibited osteoclast differentiation and expression of NFATc1, CTSK, TRAP, and MMP-9; cell viability was not affected. ERK5 expression increased by FSS but not by RANKL, and it was blocked by XMD8-92. Furthermore, FSS suppressed osteoclast differentiation in RAW264.7 cells through ERK5 pathway. CONCLUSIONS Our findings demonstrated that FSS inhibited osteoclast differentiation in RAW264.7 cells via the ERK5 pathway through reduced NFATc1 expression and its downstream factors MMP-9, CTSK, and TRAP.

Proximal, Distal, and Combined Fixation Within the Tibial Tunnel in Transtibial Posterior Cruciate Ligament Reconstruction: A Time-Zero Biomechanical Study In Vitro.

PURPOSE: To compare the time-zero biomechanical properties of 3 graft fixation techniques (proximal, distal, and combined fixation) within the tibial tunnel in transtibial posterior cruciate ligament (PCL) reconstruction. METHODS: Porcine tibias and bovine extensor tendons were used to simulate a transtibial PCL reconstruction in vitro. Load-to-failure testing was carried out in 3 groups: distal fixation alone (group I, n = 10), proximal fixation alone (group II, n = 10), and combined fixation (group III, n = 10). The load-elongation curve, tensile stiffness (in newtons per millimeter), ultimate load (in newtons), yield load (in newtons), energy absorbed to failure (in joules), and failure mode were recorded. RESULTS: All graft-tibia complexes failed because the grafts slipped past the interference screws. The tensile stiffness, yield load, and energy absorption in group I were significantly lower than those in group II and group III (tensile stiffness, 19.25 ± 9.68 N/mm in group I vs 34.92 ± 16.48 N/mm in group II [P = .016] and 32.31 ± 13.79 N/mm in group III [P = .041]; yield load, 265.36 ± 144.52 N in group I vs 398.23 ± 57.04 N in group II [P = .006] and 424.94 ± 74.00 N in group III [P = .001]; and energy absorption, 5.16 ± 2.35 J in group I vs 19.95 ± 3.48 J in group II [P < .001] and 21.09 ± 4.29 J in group III [P < .001]). No statistically significant differences in biomechanical properties were found between group II and group III (P > .05). CONCLUSIONS: Compared with distal fixation in transtibial PCL reconstruction, proximal fixation and combined fixation showed superior time-zero biomechanical properties. CLINICAL RELEVANCE: Proximal fixation and combined fixation produced superior biomechanical properties to distal fixation in transtibial PCL reconstruction.

[Effect of knocking down Piezo1 mechanically sensitive protein on migration of MC3T3-E1 osteoblast cells].

Objective: To discuss the effect of Piezo1 mechanically sensitive protein in migration process of mouse MC3T3-E1 osteoblast cells. Methods: The 5th-10th generation mouse MC3T3-E1 osteoblasts were divided into Piezo1-small interfering RNA (siRNA) transfection group (group A), negative control group (group B), and blank control group (group C). Piezo1-siRNA or negative control siRNA was transfected into mouse MC3T3-E1 osteoblasts by siRNA transfection reagent, respectively; group C was only added with siRNA transfection reagent; and the cell morphology was observed under inverted phase contrast microscope and fluorescence microscope, and the transfection efficiency was calculated. The expression of Piezo1 protein was detected by immunofluorescence staining and Western blot. Transwell cell migration assay and cell scratch assay were used to detect the migration of MC3T3-E1 osteoblasts after Piezo1-siRNA transfection. Results: After 48 hours of transfection, group A showed a slight increase in cell volume and mutant growth, but cell colonies decreased, suspension cells increased and cell fragments increased when compared with untransfected cells. Under fluorescence microscope, green fluorescence was observed in MC3T3-E1 osteoblasts of group B, and the transfection efficiency was 68.56%±4.12%. Immunofluorescence staining and Western blot results showed that the expression level of Piezo1 protein in group A was significantly lower than that in groups B and C ( P<0.05); there was no significant difference between group B and group C ( P>0.05). Transwell cell migration assay and cell scratch assay showed that the number of cells per hole and the scratch healing rate of cells cultured for 1-4 days in group A were significantly lower than those in groups B and C ( P<0.05); there was no significant difference between group B and group C ( P>0.05). Conclusion: Piezo1 knocked down by siRNA can inhibit the migration ability of MC3T3-E1 osteoblast cells.

Evaluation of the permissible maximum angle of the tibial tunnel in transtibial anatomic posterior cruciate ligament reconstruction by computed tomography.

INTRODUCTION: Excessive angle of the tibial tunnel may cause breakage of the posterior cortex in transtibial anatomic posterior cruciate ligament (PCL) reconstruction. However, a few studies have determined the permissible maximum angle of the tibial tunnel. The purpose of this study was to determine the permissible maximum angle of the tibial tunnel relative to the tibial plateau in transtibial anatomic PCL reconstruction and characterize the anatomic parameters of the tibial PCL attachment position. MATERIALS AND METHODS: Computed tomography (CT) scans of a consecutive series of 408 adult knees with normal PCL attachment were measured. The parameters measured were the permissible maximum angle (PMA) of the 10 mm-diameter tibial tunnel relative to the tibial plateau, the distance from the anterior orifice of the tibial tunnel to the tibial tuberosity (OTD), the anterior-posterior diameter (APD) of the tibial plateau, the distance from the center of PCL attachment site to the posterior edge of the tibial plateau (PPED), and the angle between the tibial plateau and the posterior tibial slope where the PCL insertion site was (PSA). Subgroup analysis was performed to determine the correlations between parameters, and sex, age, and height. The measurement reliability was evaluated by intraclass correlation coefficients (ICCs). RESULTS: The average value of PMA was 48.2 ± 5.4°, and it was not affected by sex, age, and height (P > 0.05). The values of OTD, APD, PPED, PSA, and height were significantly higher in males than females (OTD, P < 0.01; APD, P < 0.01; PPED, P < 0.01; PSA, P = 0.019; height, P < 0.01). With regard to age, we stratified the cases into three groups: the young (18-30 years old), the middle-aged (31-45 years old), and the elderly (46-60 years old). The mean value of OTD, APD, and height were significantly lower in the elderly than that in the middle-aged (P < 0.01, P < 0.01, P < 0.01, respectively). With regard to height, we stratified the cases into three groups: ~ 1.65 m (1), 1.66 ~ 1.75 m (2), and 1.76 m ~ (3). The mean value of OTD, APD, and PPED significantly increased with height, P < 0.05. The mean value of PSA was significant higher in II group than that in I group (P = 0.034). CONCLUSIONS: There should be a limit to the angle of the tibial tunnel in transtibial anatomic PCL reconstruction to prevent the fracture of posterior tunnel wall. The permissible maximum angle (PMA) of the 10 mm-diameter tibial tunnel relative to the tibial plateau was 48.2°. Besides, the determination of the value of OTD, APD, PPED, and PSA could provide a clinical reference to insertion site, depth, and angle of the tibial drill guide in PCL reconstruction.

Evaluation of the theoretical optimal angle of the tibial tunnel in transtibial anatomic posterior cruciate ligament reconstruction by computed tomography.

BACKGROUND: "Killer turn" effect is a critical explanation for the recurrent posterior laxity following transtibial posterior cruciate ligament (PCL) reconstruction, which affected by the angle of the tibial tunnel. Meanwhile, excessive tunnel angle would have an adverse impact on the healing of tendon to bone. The purpose was to evaluate the theoretical optimal angle of the tibial tunnel in transtibial anatomic PCL reconstruction. METHODS: The measurements were performed on CT sagittal plane, including the thickness of cancellous bone (L1), the theoretical optimal angle of the tibial tunnel (TOA, which was measured between tibial plateau and the extension cord connecting the center of PCL insertion site with a point 5 mm superior from marrow cavity vertex), L2 - the distance from anterior tunnel aperture to anterior end of tibial plateau, L3 - the distance from anterior tunnel aperture to tibial tuberosity (lowest edge of patellar ligament attachment). RESULTS: The value of TOA and L3 were 35.4 ± 7.9 ° and 26.8 ± 11.4 mm, respectively. L1 and L2 were higher in males than females (L1, P = 0.002; L2, P = 0.046). Regarding age, L1, TOA, L2 and L3 were higher in the 46-60 years group than 31-45 years group (P = 0.02, P = 0.001, P = 0.038, P = 0.032, respectively). With regard to height, L1 was lower in group I - < 1.66 m than group II - 1.66 to 1.75 m and group III - > 1.75 m (I v II, P = 0.015, I v III, P = 0.026). L2 was also lower in group I than group II and group III (I v II, P = 0.026, I v III, P = 0.006). TOA and L3 showed no significant differences among sex and height groups (P > 0.05). CONCLUSIONS: TOA (35.4 ° ± 7.9 °) and L3 (26.8 ± 11.4 mm) could be used as a reference for ideal tibial tunnel placement in transtibial anatomic PCL reconstruction, so as to prevent recurrent PCL laxity and ensure good graft healing. However, further clinical validation is needed.