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.

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.

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.

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.

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.

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.

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.

[Relationship of iron overload to bone mass density and bone turnover in postmenopausal women with fragility fractures of the hip].

OBJECTIVE: To study relationships between serum ferritin and bone metabolism in patients with hip fragility fractures. METHODS: This cross-sectional study included 76 postmenopausal women with hip fracture from Feburary 2011 to June 2012. The mean age of the women was (73 ± 10) years (range, 55-93 years) and the mean duration of menstruation was (22 ± 10)years (range, 5-50 years). Serum concentrations of ferritin, transferrin, alkaline phosphatase (ALP), amino-terminal extension peptide of type I collagen (P1NP), C-terminal telopeptides of type I collagen (ß-CTX)and femoral and lumbar bone mineral density by dual-energy X-ray absorptiometry were measured. Bone metabolism was compared between normal and elevated ferritin groups with t-test, Pearson linear, partial correlation and multiple regression analysis examined associations between iron- and bone-related markers. RESULTS: Serum ferritin concentration raised to (230 ± 146)µg/L, transferrin concentration reduced to (1.89 ± 0.33)g/L. P1NP concentration raised to (61 ± 32) ng/L when the concentration of serum ALP and ß-CTX were in the normal range. T-scores for bone mineral density in the femoral neck (-2.0 ± 1.1) and lumbar (-2.1 ± 1.2) were below the normal ranges(-1.0-1.0). The subjects were divided into two groups according to serum ferritin concentration, normal group(serum ferritin concentration ≤ 150 µg/L, n = 25) and elevated group(serum ferritin concentration > 150 µg/L, n = 51). Patients of elevated group had lower bone mineral density in femoral neck and lumbar than normal group(t = 3.13,2.89, P < 0.01), and higher P1NP, ß-CTX concentration (t = -2.38, -3.59, P < 0.05) . In partial correlation analysis adjusted for confounders, serum ferritin concentration was correlated negatively with bone mineral density in both femoral neck and lumbar (r = -0.335,-0.295, P < 0.05), and positively with P1NP and ß-CTX (r = 0.467,0.414, P < 0.05), but not correlated with ALP (r = 0.188, P > 0.05). Transferrin concentration tended to be correlated positively with bone mineral density in both femoral neck and lumbar (r = 0.444, 0.262, P < 0.05) and negatively with ALP, P1NP and ß-CTX(r = -0.326,-0.285,-0.278, P < 0.05). CONCLUSIONS: Iron overload has a high prevalence in postmenopausal women with fragility fracture. Increased iron stores, which might lead to bone loss and lower bone mineral density by enhancing the activity of bone turnover, could be an independent factor to take effects on bone metabolism on postmenopausal women.

Outcomes of single-row versus double-row arthroscopic rotator cuff repair: a systematic review and meta-analysis of current evidence.

PURPOSE: Our purpose was to perform a systematic review and meta-analysis of the clinical outcomes of single-row versus double-row repair. METHODS: An electronic search was performed using PubMed, EMBASE, and the Cochrane Library up to September 30, 2012. Studies that met the inclusion and exclusion criteria were assessed for quality of methodology. The primary analysis included Level I evidence from studies examining single-row versus double-row repair. The second meta-analysis and subgroup analysis were performed for evidence Levels I, II, and III. RESULTS: The primary analysis of studies providing 6 Level I randomized controlled trials showed no clinically significant differences in Constant scores, University of California, Los Angeles (UCLA), and American Shoulder and Elbow Surgeons (ASES) scores between double-row and single-row rotator cuff repair. The overall odds ratio (OR) of intact rotator cuff tendon healing was 1.93 in patients treated with double-row versus single-row repair, and the difference was significant. The results of the second meta-analysis including evidence Levels I, II, and III were similar to those of the primary analysis. In the subgroup with tears less than 3 cm, there was no statistically significant difference between the groups with regard to shoulder functional score and structure integrity. A statistically significant benefit of double-row repair in the ASES and UCLA scores was observed in the subgroup with tears greater than 3 cm; however, these differences were not clinically significant. The OR for tendon healing was found to be more favorable for double-row repair than for single-row repair in the subgroup with tears greater than 3 cm. CONCLUSIONS: Double-row repair provides a significantly higher rate of intact tendon healing than does single-row repair, and this advantage was mainly reflected in patients with large or massive tears. However, this benefit did not translate into clinically confirmed functional improvement. Thus, the double-row technique should be used only in carefully selected patients. LEVEL OF EVIDENCE: Level III, systematic review of Levels I, II, and III studies.