Machine learning and weighted gene co-expression network analysis identify a three-gene signature to diagnose rheumatoid arthritis.

Background: Rheumatoid arthritis (RA) is a systemic immune-related disease characterized by synovial inflammation and destruction of joint cartilage. The pathogenesis of RA remains unclear, and diagnostic markers with high sensitivity and specificity are needed urgently. This study aims to identify potential biomarkers in the synovium for diagnosing RA and to investigate their association with immune infiltration. Methods: We downloaded four datasets containing 51 RA and 36 healthy synovium samples from the Gene Expression Omnibus database. Differentially expressed genes were identified using R. Then, various enrichment analyses were conducted. Subsequently, weighted gene co-expression network analysis (WGCNA), random forest (RF), support vector machine-recursive feature elimination (SVM-RFE), and least absolute shrinkage and selection operator (LASSO) were used to identify the hub genes for RA diagnosis. Receiver operating characteristic curves and nomogram models were used to validate the specificity and sensitivity of hub genes. Additionally, we analyzed the infiltration levels of 28 immune cells in the expression profile and their relationship with the hub genes using single-sample gene set enrichment analysis. Results: Three hub genes, namely, ribonucleotide reductase regulatory subunit M2 (RRM2), DLG-associated protein 5 (DLGAP5), and kinesin family member 11 (KIF11), were identified through WGCNA, LASSO, SVM-RFE, and RF algorithms. These hub genes correlated strongly with T cells, natural killer cells, and macrophage cells as indicated by immune cell infiltration analysis. Conclusion: RRM2, DLGAP5, and KIF11 could serve as potential diagnostic indicators and treatment targets for RA. The infiltration of immune cells offers additional insights into the underlying mechanisms involved in the progression of RA.

Intracellular Staphylococcus aureus infection in human osteoblasts: circRNA expression analysis.

Staphylococcus aureus (S. aureus) has the ability to invade human cortical bones and cause intracellular infections in osteoblasts, which may lead to a long-term infection that is difficult to eliminate. It is critical to identify the underlying mechanisms of the osteoblast response to the intracellular S. aureus. More recently, multiple circular RNA (circRNA) functions have been identified, including serving as protein scaffolds or miRNA sponges and being translated into polypeptides. The role that circRNAs play in intracellular S. aureus infection of osteoblasts has not, to our knowledge, been investigated. Here, we established an intracellular infection model of S. aureus in osteoblasts and compared the circRNA expression of osteoblasts between the infected and control groups using RNA sequencing technology, by which a significant difference was found. In total, 117 upregulated and 125 down-regulated differentially expressed circRNAs (DEcircRNAs) were identified, and reverse transcription-quantitative PCR was employed to validate the results of RNA sequencing. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses demonstrated that DEcircRNAs were enriched in processes associated with macromolecule modification, cellular component organization or biogenesis, and intracellular non-membrane-bound organelles. Finally, a potentially important network of circRNA-miRNA-mRNA based on the DEcircRNAs was constructed. Overall, this study revealed the circRNA expression profile of human osteoblasts infected by intracellular S. aureus for the first time, and identified the circRNAs that may contribute to the pathogenesis of infectious diseases caused by intracellular S. aureus infection in human osteoblasts.

Global Transcriptomic Study of Circular-RNA Expression Profile in Osteoclasts Infected by Intracellular Staphylococcus aureus.

Osteomyelitis is difficult to cure, and the rapidly rising morbidity is a thorny problem accompanied by a large number of joint replacement applications. Staphylococcus aureus is the main pathogen of osteomyelitis. Circular RNAs (circRNAs), as emerging noncoding RNAs, play important roles in multiple physiopathological processes which could provide novel insights into osteomyelitis. However, little is known about the roles of circRNAs in the pathogenesis of osteomyelitis. Osteoclasts, considered bone sentinels, are the resident macrophages in bone and may play the immune defense roles in osteomyelitis. It has been reported that S. aureus can survive in osteoclasts, but the function of osteoclast circRNAs in response to intracellular S. aureus infection remains unclear. In this study, we investigated the profile of circRNAs in osteoclasts infected by intracellular S. aureus through high-throughput RNA sequencing. In total, 24 upregulated and 62 downregulated differentially expressed circRNAs were identified and subsequently analyzed to demonstrate their potential functions. On this basis, three circRNAs (chr4:130718154-130728164+, chr8:77409548-77413627-, and chr1:190871592-190899571-) were confirmed as potential novel biomarkers for the diagnosis of osteomyelitis through the murine model of osteomyelitis. Most importantly, we verified that the circRNA chr4:130718154-130728164+ named circPum1 could regulate the host autophagy to affect the intracellular infection of S. aureus through miR-767. In addition, circPum1 could serve as a promising serum biomarker in osteomyelitis patients caused by S. aureus infection. Taken together, this study provided the first global transcriptomic profile analysis of circRNAs in osteoclasts infected by intracellular S. aureus and first proposed a novel perspective for the pathogenesis and immunotherapy of S. aureus-induced osteomyelitis from the term of circRNAs.

Mechanisms of promoting the differentiation and bone resorption function of osteoclasts by Staphylococcus aureus infection.

Bone infection is a common and serious complication in the field of orthopedics, which frequently leads to excessive bone destruction and fracture nonunion. Staphylococcus aureus (S. aureus) infection affects bone cell function which, in turn, causes bone destruction. Bone is mainly regulated by osteoblasts and osteoclasts. Osteoclasts are the only cell type with bone resorptive function. Their over-activation is closely associated with excessive bone loss. Understanding how S. aureus changes the functional state of osteoclasts is the key to effective treatment. By reviewing the literature, this paper summarizes several mechanisms of bone destruction caused by S. aureus influencing osteoclasts, thereby stimulating new ideas for the treatment of bone infection.

Profile analysis of circRNAs in human THP-1 derived macrophages infected with intracellular Staphylococcus aureus.

BACKGROUND: Intracellular Staphylococcus aureus (S. aureus) infection is generally persistent, recurrent and difficult to treat due to the poor availability of antibiotics within macrophages cells and the lack of ideal diagnostic markers. Circular RNAs (circRNAs), with covalently closed circular structures, exists in the serum stably and is not easily degraded by nucleases. Besides, circRNAs play a pivotal in the eukaryotic regulation of genes expression and served as biomarkers in variety disease including microbial infections. However, the function of host circRNAs in intracellular S. aureus infection remains largely unclear. METHODS: In this study, the circRNAs expression profile was investigated by RNA sequencing technology in both S. aureus-infected THP-1 derived macrophages and mock control cells. The differentially expressed circRNAs (DE circRNAs) with a fold-change >1.5 (p < 0.05) are analyzed using functional pathway clustering prediction. Then, RT-qPCR was performed to verify the top 2 up-regulated circRNAs in the THP-1 cell and human serum samples so as to evaluate the value of circRNAs for S. aureus diagnosis. RESULTS: An intracellular survival THP-1 derived macrophages model of S. aureus infection was established. A total of 5,299 circRNAs were identified in human THP-1 derived macrophages infected with intracellular S. aureus. There were 61 DE circRNAs with a fold-change >1.5 (p < 0.05) after S. aureus infection. Among them, 22 circRNAs were up-regulated while 39 circRNAs down-regulated. GO and KEGG pathway analysis demonstrated that DE circRNAs were enriched in the processes such as Neurotrophin, Pyruvate metabolism and Notch signaling pathway. Moreover, hsa_circ_0000311 and chr13:43500472-43544806-(novel) were verified to be significantly upregulated in THP-1 derived macrophages and human serum samples between two groups. Finally, the networks of circRNA-miRNA-mRNA based on these two circRNAs were constructed respectively. CONCLUSION: Our study provides the first profile analysis of host circRNAs involved in intracellular S. aureus infection, which may serve as biomarkers for S. aureus diagnosis and contribute to the understanding of S. aureus evasion mechanisms.

Identification of key serum biomarkers for the diagnosis and metastatic prediction of osteosarcoma by analysis of immune cell infiltration.

BACKGROUND: The role of circular RNAs (circRNAs) and microRNAs (miRNAs) in osteosarcoma (OS) development has not been fully elucidated. Further, the contribution of the immune response to OS progression is not well defined. However, it is known that circRNAs and miRNAs can serve as biomarkers for the diagnosis, prognosis, and therapy of many cancers. Thus, the aim of this study was to identify novel key serum biomarkers for the diagnosis and metastatic prediction of OS by analysis of immune cell infiltration and associated RNA molecules. METHODS: Human OS differentially expressed circRNAs (DEcircRNAs), differentially expressed miRNAs (DEmiRNAs), and differentially expressed mRNAs (DEmRNAs) were identified by analysis of microarray data downloaded from Gene Expression Omnibus (GEO) datasets. Further, characteristic patterns of OS-infiltrating immune cells were analyzed. On this basis, we identified statistically significant transcription factors. Moreover we performed pathway enrichment analysis, constructed protein-protein interaction networks, and devised competitive endogenous RNA (ceRNA) networks. Biological targets of the ceRNA networks were evaluated and potential OS biomarkers confirmed by RT-qPCR analysis of the patients' serum. RESULTS: Seven differentially expressed circRNAs, 166 differentially expressed miRNAs, and 175 differentially expressed mRNAs were identified. An evaluation of cellular OS infiltration identified the highest level of infiltration by M0 macrophages, M2 macrophages, and CD8+ T cells, with M0 macrophages and CD8+ T cells as the most prominent. Significant patterns of tumor-infiltrating immune cells were identified by principal component analysis. Moreover, 185 statistically significant transcription factors were associated with OS. Further, in association with immune cell infiltration, hsa-circ-0010220, hsa-miR-326, hsa-miR-338-3p, and FAM98A were identified as potential novel biomarkers for OS diagnosis. Of these, FAM98A had the most promise as a diagnostic marker for OS and OS metastasis. Most importantly, a novel diagnostic model consisting of these four biomarkers (hsa-circ-0010220, hsa-miR-326, hsa-miR-338-3p, and FAM98A) was established with a 0.928 AUC value. CONCLUSIONS: In summary, potential serum biomarkers for OS diagnosis and metastatic prediction were identified based on an analysis of immune cell infiltration. A novel diagnostic model consisting of these four promising serum biomarkers was established. Taken together, the results of this study provide a new perspective by which to understand immunotherapy of OS.

Capsular Healing in Interportal and Periportal Capsulotomy Methods of Hip Arthroscopy.

OBJECTIVE: To evaluate the midterm outcomes and the capsular healing in patients who had interportal capsulotomy versus periportal capsulotomy of hip arthroscopy. METHODS: Retrospectively reviewed 33 patients with labral tear received hip arthroscopy, with an average age of 41 (27-67) years, including 13 cases of Cam deformity and three cases of Pincer deformity. All patients had positive sign of flexion adduction internal rotation or flexion abduction external rotation. With MRI and radiographic (CT, X plain) imageological examination. MRI showed that all patients had labral tear. Radiographic finding (CT, X plain) showed the pathological changes of acetabular and femoral neck osteophyte. One group with 23 patients were treated with periportal capsulotomy. Another group with 10 patients were treated with interportal capsulotomy. All patients did not close the capsule. Clinical outcomes were measured with the Hip Outcome Score Activities of Daily Living (HOS-ADL) and the modified Harris Hip Score (mHHS), patient satisfaction measured with visual analogue scale (VAS). The healing of the capsule was evaluated by MRI. MRI showed continuous capsular indicated healing, discontinuous capsular indicated unhealing. Postoperatively 6 months, mHHS and HOS-ADL were obtained. Randomized controlled trials were used in this study for analysis. RESULTS: All patients were followed up with average time of 9.3 months(3-29 months). The postoperative symptoms were obviously relieved, the VAS decreased from (4.9 ± 0.6) to (1.2 ± 0.2) after 3 months postoperative. Follow up 6 months post-operation, patients in the interportal group, the mHHS and HOS-ADL scores improvement were respectively 69.4 ± 9.3 & 70 ± 8.8 pre-operation, and 92.5 ± 5.0 & 86.6 ± 5.4 post-operation (P < 0.05); Patients in the periportal group, the mHHS and HOS-ADL scores improvement were respectively 69.9 ± 15.8, 68.1 ± 15.0 pre-operation, and 90.1 ± 9.3 & 86.7 ± 7.9 post-operation (P < 0.05).The differences were statistically significant. Six months after operation, MRI showed that 23 patients with periportal capsulotomy, the capsule have healed, without other complications. Three of the ten patients with interportal capsulotomy were healed and seven were not. CONCLUSION: Interportal and periportal capsulotomy had good outcomes. The technique of periportal capsulotomy had little damage to the joint capsule. Although the capsule did not close, the capsule healed well in postoperative follow-up. The nonunion rate of the joint capsule was high in the interportal capsulotomy without close the capsule.

Construction of an autophagy interaction network based on competitive endogenous RNA reveals the key pathways and central genes of SARS-CoV-2 infection in vivo.

As of April 1, 2021, more than 2.8 million people have died of SARS-CoV-2 infection. In addition, the mutation of virus strains that have accompanied the pandemic has brought more severe challenges to pandemic control. Host microRNAs (miRNAs) are widely involved in a variety of biological processes of coronavirus infection, including autophagy in SARS-CoV-2 infection. However, the mechanisms underlying miRNAs involved in autophagy in SARS-CoV-2 infection have not been fully elucidated. In this study, the miRNA and messenger RNA (mRNA) expression profiles of patients with SARS-CoV-2 infection were investigated based on raw data from Gene Expression Omnibus (GEO) datasets, and potential novel biomarkers of autophagy were revealed by bioinformatics analyses. We identified 32 differentially expressed miRNAs and 332 differentially expressed mRNAs in patients with SARS-CoV-2 infection. Cytokine receptor related pathways were the most enriched pathways for differentially expressed miRNAs identified by pathway analysis. Most importantly, an autophagy interaction network, which was associated with the pathological processes of SARS-CoV-2 infection, especially with the cytokine storm, was constructed. In this network, hsa-miR-340-3p, hsa-miR-652-3p, hsa-miR-4772-5p, hsa-miR-192-5p, TP53INP2, and CCR2 may be biomarkers that predict changes in mild SARS-CoV-2 infection. Some molecules, including hsa-miR-1291 and CXCR4, were considered potential targets to predict the emergence of severe symptoms in SARS-CoV-2 infection. To our knowledge, this study provided the first profile analysis of an autophagy interaction network in SARS-CoV-2 infection and revealed several novel autophagy-related biomarkers for understanding the pathogenesis of SARS-CoV-2 infection in vivo.

Twelve New Genomic Loci Associated With Bone Mineral Density.

Aiming to identify more genomic loci associated with bone mineral density (BMD), we conducted a joint association analysis of 2 genome-wide association study (GWAS) by the integrative association method multi-trait analysis of GWAS (MTAG). The first one is the single GWAS of estimated heel BMD (eBMD) in the UK biobank (UKB) cohort (N = 426,824), and the second one is the GWAS meta-analysis of total body BMD (TB-BMD) in 66,628 participants from 30 studies. Approximate conditional association analysis was performed in the identified novel loci to identify secondary association signal. Statistical fine-mapping was conducted to prioritize plausible credible risk variants (CRVs). Candidate genes were prioritized based on the analyses of cis- expression quantitative trait locus (cis-eQTL) and cis-protein QTL (cis-pQTL) information as well as the functional category of the SNP. By integrating the information carried in over 490,000 participants, this largest joint analysis of BMD GWAS identified 12 novel genomic loci at the genome-wide significance level (GWS, p = 5.0 × 10-8), nine of which were for eBMD and four were for TB-BMD, explaining an additional 0.11 and 0.23% heritability for the two traits, respectively. These loci include 1p33 (lead SNP rs10493130, peBMD = 3.19 × 10-8), 5q13.2 (rs4703589, peBMD = 4.78 × 10-8), 5q31.3 (rs9324887, pTB-BMD = 1.36 × 10-9), 6p21.32 (rs6905837, peBMD = 3.32 × 10-8), 6q14.1 (rs10806234, peBMD = 2.63 × 10-8), 7q21.11 (rs10806234, pTB-BMD = 3.37 × 10-8), 8q24.12 (rs11995866, peBMD = 6.72 × 10-9), 12p13.31 (rs1639122, peBMD = 4.43 × 10-8), 12p12.1 (rs58489179, peBMD = 4.74 × 10-8), 12q24.23 (rs75499226, peBMD = 1.44 × 10-8), 19q13.31 (rs7255083, pTB-BMD = 2.18 × 10-8) and 22q11.23 (rs13056137, pTB-BMD = 2.54 × 10-8). All lead SNPs in these 12 loci are nominally significant in both original studies as well as consistent in effect direction between them, providing solid evidence of replication. Approximate conditional analysis identified one secondary signal in 5q13.2 (rs11738874, pconditional = 5.06 × 10-9). Statistical fine-mapping analysis prioritized 269 CRVs. A total of 65 candidate genes were prioritized, including those with known biological function to bone development (such as FGF1, COL11A2 and DEPTOR). Our findings provide novel insights into a better understanding of the genetic mechanism underlying bone development as well as candidate genes for future functional investigation.

Proximal femoral nail anti-rotation (PFNA) and hemi-arthroplasty in the treatment of elderly intertrochanteric fractures.

To determine reasonable treatment of intertrochanteric fractures with proximal femoral nail anti-rotation (PFNA) or hemi-arthroplasty (HA) in elderly patients. Between January 2009 and June 2013, a total of 367 patients were admitted to the Orthopedics Department of The Second Affiliated Hospital of Soochow University. Patient data were retrospectively analyzed and included 160 males and 207 females. The ages of the patients were between 60 and 97 years and the average age was 72 +/- 3.9 years. According to the Evans-Jensen classification scheme, the fracture types were type IA (n = 18), type IB (n =3 1), type II (n=154), and type III (n = 164). A comparison between the two surgical methods (PFNA and HA) included the duration of surgery, intra-operative blood loss, post-operative weight-bearing time, implant complications, and the Harris hip score. The data were analyzed after 14-50 months (average 24 months) of follow-up. The gender and age of the patients did not differ significantly between the two methods of treatment; however, the duration of surgery between the PFNA hemi-arthroplasty groups did differ (hemi-arthroplasty required less time), the intra-operative blood loss in the PFNA group was significantly less than the hemi-arthroplasty group, and the post-operative weight-bearing time was significantly shorter in the hemi-arthroplasty group than the PFNA group. A retrospective study was conducted in 367 patients during the 42-month study period (January 2009-June 2013) to observe the efficacy of PFNA and hemi-arthroplasty. Complete data were available for analysis. There are significant advantages and disadvantages with respect to the two surgical treatment modalities. For elderly patients with unstable fractures, severe osteoporosis, and pre-operative mobility, hemi-arthroplasty is preferred because hemi-arthroplasty has fewer disadvantages compared to PFNA, which is not suitable for full weight bearing and bone union. PFNA for the treatment of intertrochanteric fractures has been increasingly accepted and widely used; however the use of arthroplasty remains controversial (3). Conservative treatment for intertrochanteric fractures in elderly patients has become a main trend and often takes longer, gives rise to more complications, and has mortality rates higher than surgical treatment.

Human type H vessels are a sensitive biomarker of bone mass.

Vascularization is fundamental for bone formation and bone tissue homeostasis. However, in human subjects, a direct molecular relationship has not been identified between angiogenesis and agents that promote bone disease or factors related to age. Osteopenia is a condition in which bone mineral density is lower than normal, and it represents a sign of normal aging. Here we tested whether the type H vessel, which was recently identified as strongly positive for CD31 and Endomucin (CD31hiEmcnhi) in mice, is an important indicator of aging and osteopenia in human subjects. We found that age-dependent losses of type H vessels in human bone sections conform to the observations in aged mice. The abundance of human type H vessels and osteoprogenitors may be relevant to changes in the skeletal microarchitecture and advanced osteopenia. Furthermore, ovariectomized mice, a widely used model for postmenopausal osteoporosis, exhibited significantly reduced type H vessels accompanied by reduced osteoprogenitors, which is consistent with impaired bone microarchitecture and osteoporosis, suggesting that this feature is an indicator of bone mass independent of aging. More importantly, administration of desferrioxamine led to significantly increased bone mass via enhanced angiogenesis and increased type H vessels in ovariectomized mice. Altogether, these data represent a novel finding that type H vessels are regulated in aged and osteopenia subjects. The abundance of human type H vessels is an early marker of bone loss and represents a potential target for improving bone quality via the induction of type H vessels.

Association of Irisin and CRP Levels with the Radiographic Severity of Knee Osteoarthritis.

OBJECTIVE: Irisin, a recently identified myokine, is implicated in protecting mice from obesity. This study was designed to examine the relation of irisin levels in serum and synovial fluid (SF) with the radiographic severity of osteoarthritis (OA). METHODS: Our study included 215 patients with knee OA. Irisin levels in serum and SF were evaluated using an enzyme-linked immunosorbent assay. The progression of OA was assessed using Kellgren-Lawrence grading system. RESULTS: Knee OA patients had lower serum irisin concentrations and increased serum C-reactive protein (CRP) levels compared with healthy controls. There were markedly decreased irisin levels in both the serum and the SF, as well as increased serum CRP levels of knee OA patients with Kellgren and Lawrence (KL) grade 4 compared with patients classified as KL grade 2 and 3. Furthermore, patients with KL grade 3 showed markedly reduced serum and SF levels of irisin, as well as increased serum CRP levels compared with patients classified as KL grade 2. Irisin levels in serum and SF of knee OA patients were negatively correlated with disease severity evaluated by KL grading criteria. CONCLUSION: Irisin levels in the serum and SF of knee OA patients were negatively correlated with disease severity evaluated by the radiographic KL grading criteria.

Apigenin inhibits the proliferation and invasion of osteosarcoma cells by suppressing the Wnt/ß-catenin signaling pathway.

Osteosarcoma (OS) is the most common type of bone cancer. Even with early diagnosis and aggressive treatment, the prognosis for OS is poor. In the present study, we investigated the proliferation and invasion inhibitory effect of apigenin on human OS cells and the possible molecular mechanisms involved. The cell viability of U2OS and MG63 human OS cell lines was detected by MTT assay. Cell cycle progression and invasion were assessed by flow cytometry and the Matrigel Boyden chamber assay, respectively, and the involvement of molecular mechanisms was examined by western blot analysis. We demonstrated that apigenin inhibited proliferation and reduced invasion in human OS cells, and downregulated the expression of ß-catenin in OS cells. Furthermore, the inhibitory effect of apigenin on OS cells was reversed by overexpression of ß-catenin, but enhanced by knockdown of ß-catenin. Collectively, our results showed that apigenin inhibits the tumor growth of OS cells by inactivating Wnt/ß-catenin signaling. Therefore, apigenin is a promising chemotherapeutic agent that may be used in the treatment of human OS.

Injectable and Biodegradable pH-Responsive Hydrogels for Localized and Sustained Treatment of Human Fibrosarcoma.

Injectable hydrogels are an important class of biomaterials, and they have been widely used for controlled drug release. This study evaluated an injectable hydrogel formed in situ system by the reaction of a polyethylene glycol derivative with α,ß-polyaspartylhydrazide for local cancer chemotherapy. This pH-responsive hydrogel was used to realize a sol-gel phase transition, where the gel remained a free-flowing fluid before injection but spontaneously changed into a semisolid hydrogel just after administration. As indicated by scanning electron microscopy images, the hydrogel exhibited a porous three-dimensional microstructure. The prepared hydrogel was biocompatible and biodegradable and could be utilized as a pH-responsive vector for drug delivery. The therapeutic effect of the hydrogel loaded with doxorubicin (DOX) after intratumoral administration in mice with human fibrosarcoma was evaluated. The inhibition of tumor growth was more obvious in the group treated by the DOX-loaded hydrogel, compared to that treated with the free DOX solution. Hence, this hydrogel with good syringeability and high biodegradability, which focuses on local chemotherapy, may enhance the therapeutic effect on human fibrosarcoma.

[Comparative evaluation of the techniques and effects of RetroButton and Interference Screw in anterior cruciate ligament reconstruction].

OBJECTIVE: To evaluate the difference between RetroButton and Interference Screw in Anterior Cruciate Ligament (ACL) Reconstruction. METHODS: Ninety-seven patients with ACL rupture were treated by arthroscopic reconstruction with RetroButton and Interference Screw from June 2008 to December 2011. There were 54 males and 43 females with an average age of 27.5 years (range, 18-53 years). The preoperative magnetic resonance imaging (MRI) of all injured knees revealed the rupture of ACL. The average time from injury to surgery was 13.1 days (range, 6-20 days). All the patients were randomly divided into RetroButton group and Interference Screw group. And all the operations were performed under arthroscopy. Patients were instructed to walk with crutches and weight-bearing 4 weeks after operation. Walk without crutches was allowed 6 to 8 weeks after reconstruction and normal daily activities were gradually resumed. RESULTS: The average follow-up was 54.6 months (range 48-60 months). No adverse biological reactions or infection ever occurred. There was no spontaneous rupture or laxity of graft. The average Lysholm knee score[(56.1 ± 7.9) vs (93.1 ± 6.1); (55.3 ± 8.5) vs (93.2 ± 5.7)], KT-1000 examination [(9.2 ± 1.8) vs (2.1 ± 1.4); (9.5 ± 1.7) vs (2.1 ± 1.5)], International Knee Documentation Committee (IKDC) subjective and objective score [(49.7 ± 5.9) vs (91.7 ± 5.0); (50.2 ± 6.3) vs (91.0 ± 6.1)] were improved significantly in both two groups (all P<0.01), and there was no significant difference between the two groups [(93.1 ± 6.1) vs (93.2 ± 5.7), P>0.05]. CONCLUSIONS: ACL reconstruction using RetroButton and Interference Screw are simple and effective, and can lead to good ligamentous stability and knee function. The two kinds of fixation methods have no significant difference in short and medium term effect. Long-term follow-up should be performed to confirm the durable stability of the knee.

Low-dose X-ray irradiation promotes osteoblast proliferation, differentiation and fracture healing.

Great controversy exists regarding the biologic responses of osteoblasts to X-ray irradiation, and the mechanisms are poorly understood. In this study, the biological effects of low-dose radiation on stimulating osteoblast proliferation, differentiation and fracture healing were identified using in vitro cell culture and in vivo animal studies. First, low-dose (0.5 Gy) X-ray irradiation induced the cell viability and proliferation of MC3T3-E1 cells. However, high-dose (5 Gy) X-ray irradiation inhibited the viability and proliferation of osteoblasts. In addition, dynamic variations in osteoblast differentiation markers, including type I collagen, alkaline phosphatase, Runx2, Osterix and osteocalcin, were observed after both low-dose and high-dose irradiation by Western blot analysis. Second, fracture healing was evaluated via histology and gene expression after single-dose X-ray irradiation, and low-dose X-ray irradiation accelerates fracture healing of closed femoral fractures in rats. In low-dose X-ray irradiated fractures, an increase in proliferating cell nuclear antigen (PCNA)-positive cells, cartilage formation and fracture calluses was observed. In addition, we observed more rapid completion of endochondral and intramembranous ossification, which was accompanied by altered expression of genes involved in bone remodeling and fracture callus mineralization. Although the expression level of several osteoblast differentiation genes was increased in the fracture calluses of high-dose irradiated rats, the callus formation and fracture union were delayed compared with the control and low-dose irradiated fractures. These results reveal beneficial effects of low-dose irradiation, including the stimulation of osteoblast proliferation, differentiation and fracture healing, and highlight its potential translational application in novel therapies against bone-related diseases.

Calcitonin treatment is associated with less severe osteoarthritis and reduced toll-like receptor levels in a rat model.

BACKGROUND: Studies indicate that inflammation promotes progression of osteoarthritis. Cartilage damage is aggravated by the binding of toll-like receptors and endogenous ligands that release large amounts of cytokines and inflammation mediators. Calcitonin can inhibit degeneration of articular cartilage, by inhibiting activation of toll-like receptors and generation of endogenous ligands. To study the effect of calcitonin in the pathogenesis of osteoarthritis and the underlying molecular mechanism, we monitored levels of toll-like receptors during osteoarthritis progression, and after calcitonin injection. METHODS: Male Sprague-Dawley rats were randomly assigned to either a surgery-only or a calcitonin-treatment group (n = 35, each). To induce osteoarthritis, the anterior cruciate ligament and the medial meniscus were cut in the right knees of both groups. Rats in the calcitonin-treatment group received a subcutaneous injection of 15 IU/kg calcitonin once every other day, starting one day post-surgery, until euthanised. Signs of osteoarthritic changes were noted. The amount of collagen II was measured by antibody staining. The amounts of MMP1 and MMP3 in cartilage were measured by use of ELISA. RNA from operated and matched control knee cartilage was extracted to determine expression levels of Col2a1, ACAN, Tlr2, Tlr3, and Tlr4. RESULTS: Signs of osteoarthritis were less severe in rats treated with calcitonin. In the surgery-only group, Tlr2 levels increased early after surgery and then decreased substantially by the latter stages. Tlr3 levels gradually increased and correlated with the severity of osteoarthritis. Tlr4 levels were high but fluctuated over the experimental period. Calcitonin treatment was associated with lower Tlr3 and Tlr4 levels than in the surgery-only group whereas Tlr2 expression was initially lower but increased 28 days after administration of calcitonin. CONCLUSION: Calcitonin treatment may lessen the severity of osteoarthritis in the rat model, perhaps by inhibition of Tlr expression in cartilage.

Bafilomycin A1 inhibits autophagy and induces apoptosis in MG63 osteosarcoma cells.

The purpose of the present study was to investigate the effects of bafilomycin A1 (BafA1) on proliferation, apoptosis and autophagy in MG63 osteosarcoma cells. The growth rate of MG63 cells was determined using a Cell Counting Kit­8 assay. The mitochondrial membrane potential (Δψ) was measured using a fluorescent probe, JC­1, and the inhibition of autophagy and apoptosis was monitored by transmission electron microscopy. In addition, the inhibition of autophagy was monitored by western blot analysis of microtubule­associated protein 1 light chain 3 (LC3), and the ratio of LC3­II/LC3­I protein levels was calculated as an indicator of the extent of autophagy. Furthermore, the expression levels of specific proteins associated with autophagy, including p53, Beclin1 and p62, were detected in cultured MG63 cells by western blotting. It was shown that the viability of MG63 cells was inhibited following the use of BafA1, and an induction in the expression levels of the apoptosis­related protein p53 and the autophagic protein Beclin1 was detected. Furthermore, a collapse in Δψ was observed, together with an induction of apoptotic cell death, following treatment with BafA1. Therefore, following BafA1­mediated inhibition of autophagy, the inhibition of MG63 cell proliferation and induction of apoptosis were observed.

Inhibition of the mammalian target of rapamycin leads to autophagy activation and cell death of MG63 osteosarcoma cells.

It has been well documented that the inhibition of the mammalian target of rapamycin (mTOR) induces autophagy in proliferative cells. Therefore, mTOR inhibitors have been proposed for the treatment of cancer. As autophagy plays significant roles in tumor cell survival, the present study aimed to investigate the contribution of autophagy activation to the antitumor effects of cis-diamminedichloroplatinum (CDDP). An MTT assay was used to determine the cytotoxic effects of rapamycin on MG63 osteosarcoma cells. The cell cycle was assessed using a flow cytometry analysis subsequent to staining the DNA with propidium iodide. The mitochondrial membrane potential (Δψ) was measured using the fluorescent probe, JC-1. Western blot analysis was used to determine the expression of the proteins that are involved in apoptosis and autophagy, including p53, p62, light chain 3 (LC3) and Beclin-1. The viability of the MG63 cells was inhibited following rapamycin or CDDP treatment. The mitochondrial Δψ collapsed following treatment with rapamycin or CDDP. Rapamycin induced cell death and enhanced the effects of the induction of MG63 cell death by CDDP. Western blot analysis detected the induced expression of the p53 and Beclin-1 proteins and the autophagic proteins, LC3 and p62. Rapamycin was observed to induce the death of cancer cells through apoptotic and autophagic mechanisms. Rapamycin may enhance the effects of the activation of autophagy and the induction of apoptosis by CDDP.