Aging and Autophagy: Roles in Musculoskeletal System Injury.

Aging is a multifactorial process that ultimately leads to a decline in physiological function and a consequent reduction in the health span, and quality of life in elderly population. In musculoskeletal diseases, aging is often associated with a gradual loss of skeletal muscle mass and strength, resulting in reduced functional capacity and an increased risk of chronic metabolic diseases, leading to impaired function and increased mortality. Autophagy is a highly conserved physiological process by which cells, under the regulation of autophagy-related genes, degrade their own organelles and large molecules by lysosomal degradation. This process is unique to eukaryotic cells and is a strict regulator of homeostasis, the maintenance of energy and substance balance. Autophagy plays an important role in a wide range of physiological and pathological processes such as cell homeostasis, aging, immunity, tumorigenesis and neurodegenerative diseases. On the one hand, under mild stress conditions, autophagy mediates the restoration of homeostasis and proliferation, reduction of the rate of aging and delay of the aging process. On the other hand, under more intense stress conditions, an inadequate suppression of autophagy can lead to cellular aging. Conversely, autophagy activity decreases during aging. Due to the interrelationship between aging and autophagy, limited literature exists on this topic. Therefore, the objective of this review is to summarize the current concepts on aging and autophagy in the musculoskeletal system. The aim is to better understand the mechanisms of age-related changes in bone, joint and muscle, as well as the interaction relationship between autophagy and aging. Its goal is to provide a comprehensive perspective for the improvement of diseases of the musculoskeletal system.

Three-Dimensional Tendon Scaffold Loaded with TGF-ß1 Gene Silencing Plasmid Prevents Tendon Adhesion and Promotes Tendon Repair.

Tendon adhesion formation is associated with the aberrant expression of many genes, and interfering with the expression of these genes can prevent adhesion and promote tendon repair. Recent studies have found that silencing the transforming growth factor ß-1 (TGF-ß1) gene can reduce the occurrence of tendon adhesions. The development of tissue engineering and three-dimensional (3D) printing technology have provided new solutions for tendon repair. In this study, TGF-ß1 gene silencing microRNA (miRNA) based RNAi plasmid was loaded on a 3D tendon scaffold using 3D printing technology. In vitro experiments confirmed the sustained release of plasmid and the good biocompatibility of the printed tendon scaffold. Subsequently, the TGF-ß1 gene silencing plasmid loaded tendon scaffold was implanted in a chicken tendon defect model to evaluate the effect of the scaffold in vivo. The results from biomechanical tests and histological examinations showed that the scaffold not only promoted tendon regeneration but also prevented tendon adhesion, which was conducive to the recovery of biofunction. Evaluation of protein expression showed that the loaded plasmids prevented tendon adhesion and promoted tendon functional repair via silencing of the TGF-ß1 gene.

Exosomes From M2 Macrophage Promote Peritendinous Fibrosis Posterior Tendon Injury via the MiR-15b-5p/FGF-1/7/9 Pathway by Delivery of circRNA-Ep400.

Achilles tendon rupture prognosis is usually unsatisfactory. After the tendon is injured, it may not function properly because of the fibrotic healing response, which restrains tendon motion. Inflammatory monocytes and tissue-resident macrophages are indispensable regulators in tissue repair, fibrosis, and regeneration. Exosomes from macrophages are crucial factors in tissue microenvironment regulation following tissue injury. This study therefore aimed to clarify the roles of macrophage exosomes in tendon injury (TI) repair. The results show that macrophages play a role after TI. M1 macrophages were increased relative to peritendinous fibrosis after TI. High-throughput sequencing showed abnormal expression of circular RNAs (circRNAs) between exosomes from M2 and M0 macrophages. Among the abnormal expressions of circRNA, circRNA-Ep400 was significantly increased in M2 macrophage exosomes. The results also show that M2 macrophage-derived circRNA-Ep400-containing exosomes are important for promoting peritendinous fibrosis after TI. Bioinformatics and dual-luciferase reporting experiments confirmed that miR-15b-5p and fibroblast growth factor (FGF)-1/7/9 were downstream targets of circRNA-Ep400. High circRNA-Ep400-containing exosome treatment inhibited miR-15b-5p, but promoted FGF1/7/9 expression in both fibroblasts and tenocytes. Furthermore, high circRNA-Ep400-containing exosome treatment promoted fibrosis, proliferation, and migration in both fibroblasts and tenocytes. Taken together, the results show that M2 macrophage-derived circRNA-Ep400-containing exosomes promote peritendinous fibrosis after TI via the miR-15b-5p/FGF-1/7/9 pathway, which suggests novel therapeutics for tendon injury treatment.

Hydroxyurea promotes TET1 expression and induces apoptosis in osteosarcoma cells.

Ten-eleven translocation (TET) proteins are abnormally expressed in various cancers. Osteosarcoma cells were treated with hydroxyurea to investigate the expression pattern of TET proteins in these cells. The expression of TET1 was increased in U2OS cells after treatment with hydroxyurea. In addition, hydroxyurea increased cell apoptosis and altered the cell cycle. TET proteins catalyze the oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC); therefore, 5mC and 5hmC levels were evaluated. Increased 5hmC levels were observed after the hydroxyurea treatment. Experiments examining cell apoptosis and the cell cycle after knockdown and overexpression of TET1 were conducted to further investigate whether TET1 expression affected cell growth. The overexpression of TET1 increased cell apoptosis and inhibited cell growth. Taken together, TET1 expression regulated proliferation and apoptosis in U2OS cells, changes that were associated with 5hmC levels.

Engineered tendon-fibrocartilage-bone composite and bone marrow-derived mesenchymal stem cell sheet augmentation promotes rotator cuff healing in a non-weight-bearing canine model.

Reducing rotator cuff failure after repair remains a challenge due to suboptimal tendon-to-bone healing. In this study we report a novel biomaterial with engineered tendon-fibrocartilage-bone composite (TFBC) and bone marrow-derived mesenchymal stem cell sheet (BMSCS); this construct was tested for augmentation of rotator cuff repair using a canine non-weight-bearing (NWB) model. A total of 42 mixed-breed dogs were randomly allocated to 3 groups (n = 14 each). Unilateral infraspinatus tendon underwent suture repair only (control); augmentation with engineered TFBC alone (TFBC), or augmentation with engineered TFBC and BMSCS (TFBC + BMSCS). Histomorphometric analysis and biomechanical testing were performed at 6 weeks after surgery. The TFBC + BMSCS augmented repairs demonstrated superior histological scores, greater new fibrocartilage formation and collagen fiber organization at the tendon-bone interface compared with the controls. The ultimate failure load and ultimate stress were 286.80 ± 45.02 N and 4.50 ± 1.11 MPa for TFBC + BMSCS group, 163.20 ± 61.21 N and 2.60 ± 0.97 MPa for control group (TFBC + BMSCS vs control, P = 1.12E-04 and 0.003, respectively), 206.10 ± 60.99 N and 3.20 ± 1.31 MPa for TFBC group (TFBC + BMSCS vs TFBC, P = 0.009 and 0.045, respectively). In conclusion, application of an engineered TFBC and BMSCS can enhance rotator cuff healing in terms of anatomic structure, collagen organization and biomechanical strength in a canine NWB model. Combined TFBC and BMSCS augmentation is a promising strategy for rotator cuff tears and has a high potential impact on clinical practice.

Polypyrrole-coated poly(l-lactic acid-co-ε-caprolactone)/silk fibroin nanofibrous nerve guidance conduit induced nerve regeneration in rat.

According to our previous study, polypyrrole (Ppy) possessed good conductivity and non-cytotoxicity. In this study, the surface of electrospun poly(l-lactic acid-co-ε-caprolactone)/silk fibroin (PLCL/SF) was coated with Ppy to fabricate Ppy-coated nerve guidance conduit (NGC). Firstly, the presence of Ppy on the prepared NGC was characterized and confirmed by scanning electron microscopy (SEM), X-ray photoelectron spectroscopic spectra (XPS) and Fourier transforms infrared spectroscopy (FTIR). Subsequently, Ppy-coated NGC was used to repair a 10 mm sciatic nerve gap in vivo. 4 and 12 weeks after implantation, the regenerated nerve tissues on defect sites were removed and sectioned for further evaluation. Histological analysis with hematoxylin-eosin (HE), toluidine blue (TB), and luxol fast blue (LFB) staining indicated that the coated Ppy could promote SCs proliferation in early post-surgery, and enhance myelin formation in later post-surgery. In consideration of immunofluorescence and morphology observation with SEM and TEM, it showed that the nerve regeneration of Ppy-coated NGC group was close to autograft group, which was better than PLCL/SF NGC. In addition, walking track analysis indicated that Ppy-coated NGC group showed a similar performance compared with the autograft group, and significantly better than PLCL/SF NGC group. These promising results showed the potential of Ppy-coated NGC in peripheral nerve regeneration.

New transgenic NIS reporter rats for longitudinal tracking of fibrogenesis by high-resolution imaging.

Fibrogenesis is the underlying mechanism of wound healing and repair. Animal models that enable longitudinal monitoring of fibrogenesis are needed to improve traditional tissue analysis post-mortem. Here, we generated transgenic reporter rats expressing the sodium iodide symporter (NIS) driven by the rat collagen type-1 alpha-1 (Col1α1) promoter and demonstrated that fibrogenesis can be visualized over time using SPECT or PET imaging following activation of NIS expression by rotator cuff (RC) injury. Radiotracer uptake was first detected in and around the injury site day 3 following surgery, increasing through day 7-14, and declining by day 21, revealing for the first time, the kinetics of Col1α1 promoter activity in situ. Differences in the intensity and duration of NIS expression/collagen promoter activation between individual RC injured Col1α1-hNIS rats were evident. Dexamethasone treatment delayed time to peak NIS signals, showing that modulation of fibrogenesis by a steroid can be imaged with exquisite sensitivity and resolution in living animals. NIS reporter rats would facilitate studies in physiological wound repair and pathological processes such as fibrosis and the development of anti-fibrotic drugs.

Ascorbic acid inhibits senescence in mesenchymal stem cells through ROS and AKT/mTOR signaling.

Mesenchymal stem cell (MSC) aging seriously affects its function in stem cell transplantation for treatment. Extensive studies have focused on how to inhibit senescence in MSCs. However, the mechanism of senescence in MSC was not clear. In this study, we used D-galactose to induce MSC aging. Then we found that the number of aging cells was increased compared with untreated MSCs. We discovered that ascorbic acid could inhibit the production of reactive oxygen species (ROS) and activation of AKT/mTOR signaling in MSCs caused by D-galactose. Especially, when treated together with a ROS scavenger or AKT inhibitor, the senescent cells were obviously decreased in D-galactose-induced MSCs. Taken together, we identify that ascorbic acid owns the potential to inhibit the senescence of MSCs through ROS and Akt/mTOR signaling. Together, our data supports that ascorbic acid can be used to prevent MSCs from senescence, which can enhance the efficiency of stem cell transplantation in the clinic.

Development of Nanofiber Sponges-Containing Nerve Guidance Conduit for Peripheral Nerve Regeneration in Vivo.

In the study of hollow nerve guidance conduit (NGC), the dispersion of regenerated axons always confused researchers. To address this problem, filler-containing NGC was prepared, which showed better effect in the application of nerve tissue engineering. In this study, nanofiber sponges with abundant macropores, high porosity, and superior compressive strength were fabricated by electrospinning and freeze-drying. Poly(l-lactic acid-co-ε-caprolactone)/silk fibroin (PLCL/SF) nanofiber sponges were used as filler to prepare three-dimensional nanofiber sponges-containing (NS-containing) NGC. In order to study the effect of fillers for nerve regeneration, hollow NGC was set as control. In vitro cell viability studies indicated that the NS-containing NGC could enhance the proliferation of Schwann cells (SCs) due to the macroporous structure. The results of hematoxylin-eosin (HE) and immunofluorescence staining confirmed that SCs infiltrated into the nanofiber sponges. Subsequently, the NS-containing NGC was implanted in a rat sciatic nerve defect model to evaluate the effect in vivo. NS-containing NGC group performed better in nerve function recovery than hollow NGC group. In consideration of the walking track and triceps weight analysis, NS-containing NGC was close to the autograft group. In addition, histological and morphological analyses with HE and toluidine blue (TB) staining, and transmission electron microscope (TEM) were conducted. Better nerve regeneration was observed on NS-containing NGC group both quantitatively and qualitatively. Furthermore, the results of three indexes' immuno-histochemistry and two indexes' immunofluorescence all indicated good nerve regeneration of NS-containing NGC as well, compared with hollow NGC. The results demonstrated NS-containing NGC had great potential in the application of peripheral nerve repair.

Identifying key genes in rheumatoid arthritis by weighted gene co-expression network analysis.

PURPOSE: This study aimed to identify rheumatoid arthritis (RA) related genes based on microarray data using the WGCNA (weighted gene co-expression network analysis) method. METHODS: Two gene expression profile datasets GSE55235 (10 RA samples and 10 healthy controls) and GSE77298 (16 RA samples and seven healthy controls) were downloaded from Gene Expression Omnibus database. Characteristic genes were identified using metaDE package. WGCNA was used to find disease-related networks based on gene expression correlation coefficients, and module significance was defined as the average gene significance of all genes used to assess the correlation between the module and RA status. Genes in the disease-related gene co-expression network were subject to functional annotation and pathway enrichment analysis using Database for Annotation Visualization and Integrated Discovery. Characteristic genes were also mapped to the Connectivity Map to screen small molecules. RESULTS: A total of 599 characteristic genes were identified. For each dataset, characteristic genes in the green, red and turquoise modules were most closely associated with RA, with gene numbers of 54, 43 and 79, respectively. These genes were enriched in totally enriched in 17 Gene Ontology terms, mainly related to immune response (CD97, FYB, CXCL1, IKBKE, CCR1, etc.), inflammatory response (CD97, CXCL1, C3AR1, CCR1, LYZ, etc.) and homeostasis (C3AR1, CCR1, PLN, CCL19, PPT1, etc.). Two small-molecule drugs sanguinarine and papaverine were predicted to have a therapeutic effect against RA. CONCLUSION: Genes related to immune response, inflammatory response and homeostasis presumably have critical roles in RA pathogenesis. Sanguinarine and papaverine have a potential therapeutic effect against RA.

[OBSERVATION OF EFFECTIVENESS OF HIP RESURFACING ARTHROPLASTY IN TREATMENT OF OSTEONECROSIS OF FEMORAL HEAD IN YOUNG AND MIDDLE-AGED PATIENTS].

OBJECTIVE: To evaluate the clinical and radiographic outcomes of hip resurfacing arthroplasty (HRA) for treating osteonecrosis of the femoral head (ONFH) in young and middle-aged patients. METHODS: Between January 2008 and April 2009, 34 patients with ONFH underwent HRA. There were 19 males and 15 females with an average age of 54 years (range, 33-59 years). Of 34 cases, 16 left hips and 18 right hips were involved, including 9 cases of alcohol-induced ONFH, 8 cases of steroid-induced ONFH, 7 cases of traumatic ONFH, and 10 cases of unexplained ONFH. According to modified Ficat classification system, 26 hips were rated as stage III, and 8 hips as stage IV. The Harris hip score (HHS) and modified University of California, Los Angeles (UCLA) activity score were used to evaluate the clinical results. Migration of prosthesis was assessed on the anteroposterior radiographs. The abduction angle was measured on the acetabular side. On the femoral side, varus-valgus shift was determined by measurement of stem-shaft angle. The axial collapse of femoral component was assessed with the component-lateral cortex ratio. RESULTS: Healing of incision by first intention was achieved in all patients without complications of infection and thrombosis of deep vein of lower extremities. Thirty-two patients were followed up 78 months on average (range, 70-84 months). No implant loosening, infection, femoral neck fracture, dislocation, and inflammatory pseudotumor were observed. At last follow-up, the HHS score was significantly increased to 95.22 +/- 1.47 from preoperative 50.10 +/- 2.27 (t=1.510, P=0.008). Modified UCLA activity score was significantly increased to 7.70 +/- 1.13 from preoperative 3.90 +/- 0.90 (t=0.830, P=0.003). The abduction angle, stem-shaft angle, and compotent-lateral cortex ratio showed no significant difference between at 3 days after operation and last follow-up (P>0.05). CONCLUSION: If the indication of operation is mastered strictly, HRA may be effective in treatment for ONFH at Ficat stage III or IV in young and middle-aged patients.

[Comparison of the effect of total hip arthroplasty through mini invasive direct anterior approach during learning curve period and posterolateral approach for the treatment of femoral head necrosis].

OBJECTIVE: To compare clinical results of treating femoral head necrosis staged Ficat III or IV with total hip arthroplasty (THA) between mini invasive direct anterior approach (DAA) and posterolateral approach. METHODS: From January 2008 to December 2009, 48 patients with femoral head necrosis staged Ficat III or IV treated with THA were compared and analyzed. There were 21 patients in mini invasive direct anterior approach group including 11 males and 10 females with an average age of (65.2±4.3) years old;while there were 27 patients in posterolateral approach group including 16 males and 11 females with an average age of (63.6±4.0) years old. Operative time, blood loss during operation, bed rest time and complications of two groups were observed and compared. Acetabular abduction and stem shaft angle were measured 1 month after operation and compared between two groups. Postoperative Harris Hip scoring and VAS scoring were applied for evaluating hip function and pain at 1, 6 months and 5 years after operation respectively. RESULTS: All patients were followed up for 48 to 73 months with an average of 60.4 months. Operative time, blood loss in DAA group was (78.30±5.08) min, (351.30±21.46) ml, respectively, in posterolateral approach group was (75.61±10.60) min, (362.20±26.15) ml, and no significant differences between two groups. Bed rest time in DAA group was (2.05±1.10) days, better than that of in posterolateral approach which was (3.30±1.35) days. No significant differences were found between two groups in acetabular abduction and stem shaft angle at 1 month after operation. There was no significant differences between two groups in HHS and VAS score at 1, 6 months and 5 years after operation. There was 1 case with injury of ascending branch of the lateral circumflex femoral artery, 1 case with great trochanter fracture and 1 case with superficial infection in DAA group, 1 case with dislocation in posterolateral group. No prosthesis loosening occurred in two groups. CONCLUSIONS: Both DAA and posterolateral approach are effective in treating femoral head necrosis staged Ficat III or IV, and could obtain excellent outcomes. However, DAA seemed to has disadvantage in learing curve compared posteriolateral approach in complex cases.

Are CXCL13/CXCR5/FAK critical regulators of MSCs migration and differentiation?

Osteonecrosis of the femoral head is a common and challenging disease worldwide. The traditional treatments, such as core decompression procedure and joint replacement, are not satisfactory due to the limited outcome, repetitive surgery and cost. In recent years, autologous mesenchymal stem cells (MSCs) implantation into the femoral head has emerged as a promising method. The homing and differentiation of MSCs is determined by chemokines and their receptors, specific signals present in the micro-environment of the damaged tissue. CXCL13/CXCR5, highly expressed in the osteoblast and MSCs, are tissue specific and selectively migrate MSCs, thereafter triggering phosphorylation of focal adhesionkinase through mitogen-activated protein kinase pathway. Considering these characteristics, we hypothesize that CXCL13/CXCR5/FAK are critical signals in the trafficking and differentiation of MSCs.

A new glutarimide derivative from marine sponge-derived Streptomyces anulatus S71.

Four glutarimide-derived compounds including a new 3-[2-[2-hydroxy-3-methylphenyl-5-(hydroxymethyl)]-2-oxoethyl] glutarimide (1) and three known 3-[2-(2-hyroxy-3,5- dimethylphenyl)-2-oxoethyl] glutarimide (2, actiphenol), 3-hydroxy-3-[2-(2-hydroxy-3,5-dimethylphenyl)-2-oxoethyl] glutarimide (3) and 3-[2-[2-hydroxy-3-(hydroxymethyl)-5-methylphenyl]-2-oxoethyl] glutarimide (4), along with a known indole alkaloid 3-(hydroxyacetyl) indole (5), were isolated from ethyl acetate extract of the fermentation broth of the marine sponge-derived Streptomyces anulatus S71. Their structures were deduced by extensive studies of NMR and mass spectra.

[Failure of internal fixation on displaced femoral neck fractures in adults under fifty-five years old].

OBJECTIVE: To investigate the failure of internal fixation on displaced femoral neck fractures in adults under fifty-five years old retrospectively inorder to pay more attention to the treatment of these fractures. METHODS: From Junary 2007 to June 2010,18 failed cases of internal fixation on displaced femoral neck fractures in adults under fifty-five years old were treated,there were 13 males and 5 females with an average age of (48.0 +/- 6.0) years old ranging from 27 to 55. Among them, 17 patients were treated with cannulated screws and 1 patient was treated with intramedullary nail; 16 patients were diagnosed as osteonecrosis and 2 patients as osteonecrosis associated with nonunion. RESULTS: The average time from internal fixation to failure was 23 months (ranged, 8 to 32 months). The quality of fracture reduction in Garden index was poor. The Harris Hip Score was (56.0 +/- 12.5) (ranged,33 to 80). Eight cases of osteonecrosis and 2 cases of nonunion combinated osteonecrosis were received total hip arthroplasty. Hip resurfacing arthroplasty were performed for other 5 osteonecrosis. Because of no evident clinical symptoms,the other 3 cases received conservative treatment. The patients with total hip arthroplasty and hip resurfacing arthroplasty were followed-up for 34 months ranging from 12 to 53 months. After operation,the Harris score was (94.0 +/- 3.0) ranged 89 to 96. CONCLUSION: Osteonecrosis is a common complication after internal fixation on displaced femoral neck fracture in adults under fifty-five years old. More attention should be paid to the treatment of displaced femoral neck fracture in those patients.

A comparative study of two methods for treating type III tibial eminence avulsion fracture in adults.

PURPOSE: Suture fixation is mostly used in arthroscopic treatment of tibial eminence avulsion fractures. However, no clinical studies of metal cable fixation have been reported. We hypothesised that cable fixation can provide equal stability and clinical outcome compared with Ethibond sutures. METHODS: Between 2007 and 2008, we treated 42 patients of adult type III tibial eminence avulsion fractures. Twenty-three patients were male, and 19 were female. All patients were confirmed by radiographs, MRI, and arthroscopy during surgery. Ligament injury and meniscus tears were excluded from this study. Twenty-two patients were treated with No. 2 Ethibond suture fixation (group I), and 20 were treated with cable fixation under arthroscopy (group II). Follow-up assessments included imaging evaluation, Lysholm knee score, International Knee Documentation Committee (IKDC) classification, and the Lachman test. RESULTS: Bone union was found in radiographic evaluation in all patients within 3 months. At the last follow-up, there was neither extension nor flexion limitation in any patient. There were no significant differences in the Lysholm score between the two groups at follow-up. All 42 patients were classified by the IKDC as normal or nearly normal. Stability based on the Lachman test showed two patients of grade II laxity in group I. At the final follow-up, all 42 patients had returned to their pre-injury activities. CONCLUSIONS: Cable fixation to treat type III tibial eminence avulsion fracture can provide a clinical outcome equal to that of Ethibond sutures. LEVEL OF EVIDENCE: IV.