METTL3-mediated m6A modification increases Hspa1a stability to inhibit osteoblast aging.

Senile osteoporosis is mainly caused by osteoblasts attenuation, which results in reduced bone mass and disrupted bone remodeling. Numerous studies have focused on the regulatory role of m6A modification in osteoporosis; however, most of the studies have investigated the differentiation of bone marrow mesenchymal stem cells (BMSCs), while the direct regulatory mechanism of m6A on osteoblasts remains unknown. This study revealed that the progression of senile osteoporosis is closely related to the downregulation of m6A modification and methyltransferase-like 3 (METTL3). Overexpression of METTL3 inhibits osteoblast aging. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) revealed that METTL3 upregulates the stability of Hspa1a mRNA, thereby inhibiting osteoblast aging. Moreover, the results demonstrated that METTL3 enhances the stability of Hspa1a mRNA via m6A modification to regulate osteoblast aging. Notably, YTH N6-methyladenosine RNA binding protein 2 (YTHDF2) participates in stabilizing Hspa1a mRNA in the METTL3-mediated m6A modification process, rather than the well-known degradation function. Mechanistically, METTL3 increases the stability of Hspa1a mRNA in a YTHDF2-dependent manner to inhibit osteoblast aging. Our results confirmed the significant role of METTL3 in osteoblast aging and suggested that METTL3 could be a potential therapeutic target for senile osteoporosis.

METTL3-mediated m6A modification of SOX4 regulates osteoblast proliferation and differentiation via YTHDF3 recognition.

N6-methyladenosine (m6A), the most prevalent internal modification in mRNA, is related to the pathogenesis of osteoporosis (OP). Although methyltransferase Like-3 (METTL3), an m6A transferase, has been shown to mitigate OP progression, the mechanisms of METTL3-mediated m6A modification in osteoblast function remain unclear. Here, fluid shear stress (FSS) induced osteoblast proliferation and differentiation, resulting in elevated levels of METTL3 expression and m6A modification. Through Methylated RNA Immunoprecipitation Sequencing (MeRIP-seq) and Transcriptomic RNA Sequencing (RNA-seq), SRY (Sex Determining Region Y)-box 4 (SOX4) was screened as a target of METTL3, whose m6A-modified coding sequence (CDS) regions exhibited binding affinity towards METTL3. Further functional experiments demonstrated that knockdown of METTL3 and SOX4 hampered osteogenesis, and METTL3 knockdown compromised SOX4 mRNA stability. Via RNA immunoprecipitation (RIP) assays, we further confirmed the direct interaction between METTL3 and SOX4. YTH N6-Methyladenosine RNA Binding Protein 3 (YTHDF3) was identified as the m6A reader responsible for modulating SOX4 mRNA and protein levels by affecting its degradation. Furthermore, in vivo experiments demonstrated that bone loss in an ovariectomized (OVX) mouse model was reversed through the overexpression of SOX4 mediated by adeno-associated virus serotype 2 (AAV2). In conclusion, our research demonstrates that METTL3-mediated m6A modification of SOX4 plays a crucial role in regulating osteoblast proliferation and differentiation through its recognition by YTHDF3. Our research confirms METTL3-m6A-SOX4-YTHDF3 as an essential axis and potential mechanism in OP.

Biomechanical Comparison of Anatomic Versus Lower of Anteromedial and Anterolateral Tibial Tunnels in Posterior Cruciate Ligament Reconstruction.

OBJECTIVE: In order to reduce the "killer turn" effect, various tibial tunnels have been developed. However, few studies investigated the biomechanical effects of different tibial tunnels during PCL reconstruction. This study aims to compare the time-zero biomechanical properties of anteromedial, anterolateral, lower anteromedial, and lower anterolateral tibial tunnels in transtibial posterior cruciate ligament (PCL) reconstruction under load-to-failure loading. METHODS: Porcine tibias and bovine extensor tendons were used to simulate in vitro transtibial PCL reconstruction. Forty bovine extensor tendons and 40 porcine tibias were randomly divided into four experimental groups: anteromedial tunnel group (AM group, n = 10), anterolateral tunnel group (AL group, n = 10), lower anteromedial tunnel group (L-AM group, n = 10), and lower anterolateral tunnel group (L-AL group, n = 10). The biomechanical test was then carried out in each group using the load-to-failure test. The ultimate load (in newtons), yield load (in newtons), tensile stiffness (in newtons per millimeter), load-elongation curve, failure mode, and tibial tunnel length (in millimeter) were recorded for each specimen. One-way analysis of variance (ANOVA) was used to compare the mean differences among the four groups. RESULTS: The biomechanical outcomes showed that there were no differences in the mean tensile stiffness and failure mode among four groups. The ultimate load and yield load of the L-AM group were significantly higher than those of other three groups (P < 0.05). For the AM group, its ultimate load is significantly higher than that of the L-AL group (P < 0.05), and its yield load is higher than that of the AL group and L-AL group (P < 0.05). However, we found no significant differences in either ultimate load or yield load between AL group and L-AL group (P > 0.05). There was significant statistical difference in the length of tibial tunnel between anatomic groups (AM and AL) and lower groups (L-AM and L-AL) (P < 0.05). CONCLUSION: Compared with the anteromedial, anterolateral, and lower anterolateral tibial tunnel, the lower anteromedial tibial tunnel showed better time-zero biomechanical properties including ultimate load and yield load in transtibial PCL reconstruction.

A New Technique of Achilles Tendon Rupture Repaired by Double Transverse Mini-incision to Avoid Sural Nerve Injury: A Consecutive Retrospective Study.

OBJECTIVE: Percutaneous suture is a classic technique used in Achilles tendon repair. However, the complication rates surrounding the sural nerve remain relatively high. Modified percutaneous repair technology can effectively avoid these complications; however, the surgical procedure is complicated. Hence, the present study was conducted to describe a redesigned repair technique for the Achilles tendon able to avoid sural nerve injury and reduce the complexity of the procedure. METHODS: Data of patients with acute primary Achilles tendon rupture at our hospital from January 2019 to May 2020 were included. Subjects with expectations for surgical scarring underwent a minimally invasive-combined percutaneous puncture technique. The surgical time, requirement for conversion to other technologies, and length of postoperative hospitalization were investigated to assess efficacy. The American Orthopedic Foot & Ankle Society (AOFAS) score and the Arner-Lindholm scale (A-L scale) were used to assess postoperative clinical outcomes (> 24 months). During the 2-year follow-up, MRI was performed to observe the healing of the Achilles tendon. In addition, subjective satisfaction with surgical scar healing was recorded. RESULTS: Twenty consecutive subjects with an average follow-up of 28.3 ± 4.5 months (range, 24-41) met the inclusion criteria. None of the 20 enrolled patients required a converted surgical approach. The mean surgical time was 26.9 ± 6.47 min (range, 20-44). None of the patients experienced dysesthesia or anesthesia around the sural nerve. No signs of postoperative infections were observed. MRI data showed that the wounds of the Achilles tendon healed completely in all the subjects. The AOFAS score increased from 55.6 ± 11.07 (range, 28-71) preoperatively to 97.8 ± 3.34 (range, 87-100) at the last follow-up. The A-L scale showed that 90% of the subjects (n = 18) presented as excellent and 10% of the subjects (n = 2) presented as good, with an excellent/good rate of 100%. Moreover, subjects' satisfaction for surgical scars was 9.1 ± 0.78 (upper limit, 10). CONCLUSIONS: The results indicate that this technique can achieve good postoperative function, a small surgical incision, and high scar satisfaction. In addition, this technique should be widely used in suturing Achilles tendon ruptures.

[Comparative study on posterior cruciate ligament reconstruction with autologous hamstring tendon and LARS artificial ligament in the treatment of KD-â ¢-M knee dislocation].

OBJECTIVE: To observe the curative effect of one-stage reconstruction of anterior cruciate ligament(ACL), posterior cruciate ligament (PCL) and medial collateral ligament (MCL) in patients with KD-Ⅲ-M knee injury, and to compare the operation time, hospitalization cost and curative effect after arthroscopic reconstruction of PCL with LARS artificial ligament and autogenous hamstring tendon, ACL reconstruction with autogenous hamstring tendon and MCL repair combined with limited incision. METHODS: From March 2016 to January 2019, a total of 36 patients met the criteria of this study. Twenty patients in group A were treated with autogenous hamstring tendon reconstruction of ACL and PCL and repair of MCL, including 17 males and 3 females, with an average age of (34.7±9.2) years old. Sixteen patients in group B with LARS artificial ligament reconstruction of PCL, with an autogenous hamstring tendon reconstruction of PCL and MCL repair as before as group B, including 15 males and 1 female, with an average age of (36.8±8.6) years old. The operation time, hospitalization time and total hospitalization cost were compared between the two groups. The preoperative and postoperative functions of the two groups were evaluated by Hospital for Sepcial Surgery (HSS) score and Lysholm score respectively, and the curative effects were compared within and between groups. RESULTS: All the patients in the two groups were followed up for at least 1 year. There were no complications such as infection and poor wound healing in both groups. There was significant difference in operation time between (120.25±9.55) min in group A and (106.63±8.85) min in group B (P<0.01). The average hospitalization days in group A was (10.60±1.64) days, while that in group B was (10.38±1.59) days. There was no significant difference between the two groups (P>0.05). The HSS score of group A increased from preoperative 32.95±5.03 to postoperative 84.70±5.72 (P<0.01), and that of group B increased from preoperative 33.81±4.10 to postoperative 85.00±5.25 (P<0.01). The Lyshlom score in group A increased from preoperative 21.10±3.46 to postoperative 80.25±5.75 (P<0.01), and in group B increased from preoperative 21.56±3.01 to postoperative 80.00±4.30(P<0.01). There was no significant difference in preoperative and postoperative scores between the two groups(P>0.05). CONCLUSION: There was no significant difference in the average hospitalization days between the two groups, but the operation time in group A was longerthan that in group B, and the hospitalization cost in group B was higher than that in group A. There was no difference in HSS score and Lysholm score before and follow-up for a certain period of time after operation.

MicroRNA-185 inhibits the growth and proliferation of osteoblasts in fracture healing by targeting PTH gene through down-regulating Wnt/ß -catenin axis: In an animal experiment.

Fracture healing is a repair process of a mechanical discontinuity loss of force transmission, and pathological mobility of bone. Increasing evidence suggests that microRNA (miRNA) could regulate chondrocyte, osteoblast, and osteoclast differentiation and function, indicating miRNA as key regulators of bone formation, resorption, remodeling, and repair. Hence, during this study, we established a right femur fracture mouse model to explore the effect microRNA-185 (miR-185) has on osteoblasts in mice during fracture healing and its underlying mechanism. After successfully model establishment, osteoblasts were extracted and treated with a series of mimics or inhibitors of miR-185, or siRNA against PTH. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot analysis were performed to determine the levels of miR-185, PTH, ß-catenin and Wnt5b. Cell viability, cycle distribution and apoptosis were detected by means of MTT and flow cytometry assays. Dual luciferase reporter gene assay verified that PTH is a target gene of miR-185. Osteoblasts transfected with miR-185 mimics or siRNA against PTH presented with decreased levels of PTH, ß-catenin and Wnt5b which indicated that miR-185 blocks the Wnt/ß -catenin axis by inhibiting PTH. Moreover, miR-185 inhibitors promoted the osteoblast viability and reduced apoptosis with more cells arrested at the G1 stage. MiR-185 mimics were observed to have inhibitory effects on osteoblasts as opposed to those induced by miR-185 inhibitors. Above key results indicated that suppression of miR-185 targeting PTH could promote osteoblast growth and proliferation in mice during fracture healing through activating Wnt/ß -catenin axis.

MRI analysis of tibial PCL attachment in a large population of adult patients: reference data for anatomic PCL reconstruction.

BACKGROUND: Consistent reference data used for anatomic posterior cruciate ligament (PCL) reconstruction is not well defined. Quantitative guidelines defining the location of PCL attachment would aid in performing anatomic PCL reconstruction. The purpose was to characterize anatomic parameters of the PCL tibial attachment based on magnetic resonance imaging (MRI) in a large population of adult knees. METHODS: The PCL tibial attachment site was examined in 736 adult knees with an intact PCL using 3.0-T proton density-weighted sagittal MRI. The outcomes measured were the anterior-posterior diameter (APD) of the tibial plateau; angle between the tibial plateau and the posterior tibial 'shelf' (the slope where the PCL tibial attachment site was) (PTS); length of the PTS; proximal, central, and distal PCL attachment positions as well as the width of the PCL attachment site; and vertical dimension of the PCL attachment site inferior from the tibial plateau. RESULTS: The average APD of the tibia plateau was 33.6 ± 3.5 mm, yielding significant differences between males (35.5 ± 3.0 mm) and females (31.6 ± 2.7 mm), P <.05, and there was a significantly decreasing trend with increasing age in males (P <.05). Mean angle between the tibial plateau and the PTS was 122.4° ± 8.1°, and subgroup analysis showed that the young group had a differently smaller angle (120.9° ± 7.5°) than the middle-aged (123.7° ± 8.2°) and the old (123.4° ± 7.7°) in males population, while there were no significant differences between sexes (P >.05). The proximal, central positions and width of the PCL attachment site were 13.4 ± 3.0 mm, 17.8 ± 3.0 mm and 9.6 ± 2.4 mm along the PTS, with significant differences between males and females (P <.05), and accounted for 60.0 % ± 9.1 %, 80.0 % ± 4.6 % and 43.3 % ± 9.7 % of the PTS respectively, with no significant differences between sexes and among age groups (all P >.05). CONCLUSIONS: This study provides reference data of the tibial PCL attachment based on MRI in the sagittal orientation. In analysis of retrospective data from a large population of adult patients, the quantitative values can be used as references to define the inserted angle and depth of the drill guide, and the exact position and size of the tibial PCL tunnel for performing arthroscopic anatomic PCL reconstruction.