Human Infrapatellar Fat Pad Mesenchymal Stem Cell-derived Extracellular Vesicles Purified by Anion Exchange Chromatography Suppress Osteoarthritis Progression in a Mouse Model.

BACKGROUND: Extracellular vesicles derived from mesenchymal stem cells (MSCs) show great promise in treating osteoarthritis (OA). However, studies from the perspective of clinical feasibility that consider an accessible cell source and a scalable preparation method for MSC-extracellular vesicles are lacking. QUESTIONS/PURPOSES: (1) Does an infrapatellar fat pad obtained from patients undergoing TKA provide a suitable source to provide MSC-extracellular vesicles purified by anion exchange chromatography? Using an in vivo mouse model for OA in the knee, (2) how does injection of the infrapatellar fat pad-derived MSC-extracellular vesicles alter gait, cartilage structure and composition, protein expression (Type II collagen, MMP13, and ADAMTS5), subchondral bone remodeling and osteophytes, and synovial inflammation? METHODS: The infrapatellar fat pad was collected from three patients (all female; 62, 74, 77 years) during TKA for infrapatellar fat pad-derived MSC culturing. Patients with infection, rheumatic arthritis, and age > 80 years were excluded. MSC-extracellular vesicles were purified by anion exchange chromatography. For the animal study, we used 30 male C57BL/6 mice aged 10 weeks, divided into six groups. MSC-extracellular vesicles were injected weekly into the joint of an OA mouse model during ACL transection (ACLT). To answer our first research question, we characterized MSCs based on their proliferative potential, differentiation capacity, and surface antigen expression, and we characterized MSC-extracellular vesicles by size, morphology, protein marker expression, and miRNA profile. To answer our second research question, we evaluated the effects of MSC-extracellular vesicles in the OA mouse model with quantitative gait analysis (mean pressure, footprint area, stride length, and propulsion time), histology (Osteoarthritis Research Society International Score based on histologic analysis [0 = normal to 24 = very severe degeneration]), immunohistochemistry staining of joint sections (protein expression of Type II collagen, MMP13, and ADAMTS5), and micro-CT of subchondral bone (BV/TV and Tb.Pf) and osteophyte formation. We also examined the mechanism of action of MSC-extracellular vesicles by immunofluorescent staining of the synovium membrane (number of M1 and M2 macrophage cells) and by analyzing their influence on the expression of inflammatory factors (relative mRNA level and protein expression of IL-1ß, IL-6, and TNF-α) in lipopolysaccharide-induced macrophages. RESULTS: Infrapatellar fat pads obtained from patients undergoing TKA provide a suitable cell source for producing MSC-extracellular vesicles, and anion exchange chromatography is applicable for isolating MSC-extracellular vesicles. Cultured MSCs were spindle-shaped, proliferative at Passage 4 (doubling time of 42.75 ± 1.35 hours), had trilineage differentiation capacity, positively expressed stem cell surface markers (CD44, CD73, CD90, and CD105), and negatively expressed hematopoietic markers (CD34 and CD45). MSC-extracellular vesicles purified by anion exchange chromatography had diameters between 30 and 200 nm and a typical cup shape, positively expressed exosomal marker proteins (CD63, CD81, CD9, Alix, and TSG101), and carried plentiful miRNA. Compared with the ACLT group, the ACLT + extracellular vesicle group showed alleviation of pain 8 weeks after the injection, indicated by increased area (0.67 ± 0.15 cm 2 versus 0.20 ± 0.03 cm 2 , -0.05 [95% confidence interval -0.09 to -0.01]; p = 0.01) and stride length (5.08 ± 0.53 cm versus 6.20 ± 0.33 cm, -1.12 [95% CI -1.86 to -0.37]; p = 0.005) and decreased propulsion time (0.22 ± 0.06 s versus 0.11 ± 0.04 s, 0.11 [95% CI 0.03 to 0.19]; p = 0.007) in the affected hindlimb. Compared with the ACLT group, the ACLT + extracellular vesicles group had lower Osteoarthritis Research Society International scores after 4 weeks (8.80 ± 2.28 versus 4.80 ± 2.28, 4.00 [95% CI 0.68 to 7.32]; p = 0.02) and 8 weeks (16.00 ± 3.16 versus 9.60 ± 2.51, 6.40 [95% CI 2.14 to 10.66]; p = 0.005). In the ACLT + extracellular vesicles group, there was more-severe OA at 8 weeks than at 4 weeks (9.60 ± 2.51 versus 4.80 ± 2.28, 4.80 [95% CI 0.82 to 8.78]; p = 0.02), indicating MSC-extracellular vesicles could only delay but not fully suppress OA progression. Compared with the ACLT group, the injection of MSC-extracellular vesicles increased Type II collagen expression, decreased MMP13 expression, and decreased ADAMTS5 expression at 4 and 8 weeks. Compared with the ACLT group, MSC-extracellular vesicle injection alleviated osteophyte formation at 8 weeks and inhibited bone loss at 4 weeks. MSC-extracellular vesicle injection suppressed inflammation; the ACLT + extracellular vesicles group had fewer M1 type macrophages than the ACLT group. Compared with lipopolysaccharide-treated cells, MSC-extracellular vesicles reduced mRNA expression and inhibited IL-1ß, IL-6, and TNF-α in cells. CONCLUSION: Using an OA mouse model, we found that infrapatellar fat pad-derived MSC-extracellular vesicles could delay OA progression via alleviating pain and suppressing cartilage degeneration, osteophyte formation, and synovial inflammation. The autologous origin of extracellular vesicles and scalable purification method make our strategy potentially viable for clinical translation. CLINICAL RELEVANCE: Infrapatellar fat pad-derived MSC-extracellular vesicles isolated by anion exchange chromatography can suppress OA progression in a mouse model. Further studies with large-animal models, larger animal groups, and subsequent clinical trials are necessary to confirm the feasibility of this technique for clinical OA treatment.

Axial Compressive Loading Attenuates Early Osteoarthritis by Reducing Subchondral Bone Remodeling.

BACKGROUND: Mechanical loading and alendronate (ALN) can be used as noninvasive physical therapy methods for osteoarthritis (OA). However, the timing and efficacy for treatments are unknown. PURPOSE: To determine whether the timing of mechanical loading and ALN influences the pathobiological changes of OA. STUDY DESIGN: Controlled laboratory study. METHODS: Mice with OA induced by anterior cruciate ligament transection were subjected to early (1-3 weeks) or late (5-7 weeks) axial compressive dynamic load or intraperitoneal injection of ALN. Changes in gait were analyzed using gait analysis system, pathobiological changes in subchondral bone, cartilage, osteophyte, and synovitis were assessed using micro-computed tomography, tartrate-resistant acid phosphatase staining, pathologic section staining, and immunohistochemistry at 1, 2, 4, and 8 weeks. RESULTS: At 1, 2, and 4 weeks, the OA limb had lower mean footprint pressure intensity, lower bone volume per tissue volume (BV/TV) in the subchondral bone, and more osteoclasts. At 4 weeks, the early loading, ALN, and load + ALN treatments induced less cartilage destruction, with a corresponding reduction in Osteoarthritis Research Society International score and increased hyaline cartilage thickness. The treatments also resulted in fewer osteoclasts and higher BV/TV and bone mineral density of subchondral bone and suppressed inflammation and interleukin 1ß- and tumor necrosis factor α-positive cells in synovium. At 8 weeks, early loading or load + ALN improved the mean footprint pressure intensity and knee flexion. At 8 weeks, early load + ALN had a synergistic effect on protecting hyaline cartilage and proteoglycans. Footprint pressure intensity and cartilage destruction were worse in late loading limbs, and no differences in BV/TV, bone mineral density, osteophyte formation, and synovium inflammation were found between the late load, ALN, and load + ALN groups and the anterior cruciate ligament transection group. CONCLUSION: Dynamic axial mechanical loading or ALN in the early stages of knee trauma protected against OA by suppressing subchondral bone remodeling. However, late loading promoted cartilage degeneration in advanced OA, indicating that reduced loading should be performed in the late stages of OA to avoid the acceleration of OA. CLINICAL RELEVANCE: Early low-level functional exercise or antiosteoporotic drugs could clearly slow or prevent the progression of early OA. For patients with mild to severe OA, loading reduction via brace protection or maintenance of joint stability via early ligament reconstruction surgery may ameliorate OA exacerbation.

The effect of femoral component valgus/varus angle on the mid-term efficacy of unicondylar knee arthroplasty.

OBJECTIVE: To investigate the effect of femoral component valgus/varus angle (FCVA) on the mid-term outcome after unicondylar knee arthroplasty. METHODS: Patients who underwent unicompartmental knee replacement in the Department of Bone and Joint Surgery, were retrospectively analyzed. According to the postoperative femoral prosthesis internal and external rotation angle, patients were divided into six groups the postoperative femoral prosthesis internal and external rotation angle: standard group, mildly abnormal group. The patients were followed up for 12 months, and complications were recorded during the follow-up period. The Range of motion (ROM), visual analog scale (VAS), and Knee society score (KSS) were compared between the six groups. RESULTS: One hundred twenty-four patients with 124 knees were included in this study. There were no statistical differences in age, gender, body mass index, preoperative ROM, or preoperative VAS, KSS-C, and KSS-F scores among the six groups. Comparison of postoperative outcomes and efficacy grades showed that: (1) the differences in ROM grades, as well as VAS, KSS-C, and KSS-F efficacy at 12 months postoperatively were statistically significant in all six groups by rank sum test; and patients in the normal group had better ROM grades and KSS-F grades than those in the mild and severe abnormality groups; (2) During the follow-up, no patient records had other serious complications such as periprosthetic infection, prosthetic loosening, periprosthetic fracture, and spacer dislocation. CONCLUSION: Controlling the femoral prosthetic internal rotation angle between 6° and 0° in unicompartmental knee replacements can lead to better mid-term outcomes for patients.

Tracking tools of extracellular vesicles for biomedical research.

Imaging of extracellular vesicles (EVs) will facilitate a better understanding of their biological functions and their potential as therapeutics and drug delivery vehicles. In order to clarify EV-mediated cellular communication in vitro and to track the bio-distribution of EV in vivo, various strategies have been developed to label and image EVs. In this review, we summarized recent advances in the tracking of EVs, demonstrating the methods for labeling and imaging of EVs, in which the labeling methods include direct and indirect labeling and the imaging modalities include fluorescent imaging, bioluminescent imaging, nuclear imaging, and nanoparticle-assisted imaging. These techniques help us better understand the mechanism of uptake, the bio-distribution, and the function of EVs. More importantly, we can evaluate the pharmacokinetic properties of EVs, which will help promote their further clinical application.

A 3D printed mimetic composite for the treatment of growth plate injuries in a rabbit model.

Growth plate injuries affecting the pediatric population may cause unwanted bony repair tissue that leads to abnormal bone elongation. Clinical treatment involves bony bar resection and implantation of an interpositional material, but success is limited and the bony bar often reforms. No treatment attempts to regenerate the growth plate cartilage. Herein we develop a 3D printed growth plate mimetic composite as a potential regenerative medicine approach with the goal of preventing limb length discrepancies and inducing cartilage regeneration. A poly(ethylene glycol)-based resin was used with digital light processing to 3D print a mechanical support structure infilled with a soft cartilage-mimetic hydrogel containing chondrogenic cues. Our biomimetic composite has similar mechanical properties to native rabbit growth plate and induced chondrogenic differentiation of rabbit mesenchymal stromal cells in vitro. We evaluated its efficacy as a regenerative interpositional material applied after bony bar resection in a rabbit model of growth plate injury. Radiographic imaging was used to monitor limb length and tibial plateau angle, microcomputed tomography assessed bone morphology, and histology characterized the repair tissue that formed. Our 3D printed growth plate mimetic composite resulted in improved tibial lengthening compared to an untreated control, cartilage-mimetic hydrogel only condition, and a fat graft. However, in vivo the 3D printed growth plate mimetic composite did not show cartilage regeneration within the construct histologically. Nevertheless, this study demonstrates the feasibility of a 3D printed biomimetic composite to improve limb lengthening, a key functional outcome, supporting its further investigation as a treatment for growth plate injuries.

Unexpected intradural disc herniation instead of space-occupying tumor at L3-L4 level: a case report and literature review.

BACKGROUND: Lumbar disc herniation (LDH) is a common disease, with a conventional treatment method, as well as well-established surgical procedure, when necessary. However, some rare cases of LDH, such as intradural disc herniation (IDH), accounting for a very small proportion (approximately 0.3%) of all LDH cases, could lead to intra-operation or post-operation complications, which requires a more circumspect pre-operational radiology analysis and overall management. Herein, we reported a case with L3-L4 IDH identified by pathological examination. Recent studies on PubMed were reviewed to summarize the unique characteristics of IDH, as well as diagnosis and treatments. Case introduction: A 69-year-old male was admitted to our department due to complaints of chronic low back pain for the past one month, along with radiating pain along the left lower hip and posterolateral left lower extremity. Disk herniation and space-occupying mass inside the canal at the L3-L4 level were confirmed by both lumbar CT and MRI. In surgery, after resection of the disc at L3-L4, further exploration revealed unsatisfactory volume of disk tissue and local eminence posterior to ventral dura, which emphasized the need for preoperatively identifying the mass inside the spinal canal. The tumor-like mass was found inside the dura. Finally, transforaminal lumbar interbody fusion (TLIF) was performed followed by resection of the mass. However, the histology examination showed a disc-like fibrocartilage tissue. The symptoms were immensely improved after the operation. CONCLUSION: IDH has a low incidence and is sporadically reported. Misdiagnosis is very common preoperatively as well as intraoperatively. IDH usually develops more rapidly compared with intradural tumors. Adhesion between dura mater and posterior longitudinal ligament may play a critical role in the disease onset.

Clinical effect of using MultiLoc® nails to treat four-part proximal humeral fractures.

OBJECTIVE: This study was performed to evaluate the clinical effect of MultiLoc® nails (DePuy Synthes, Raynham, MA, USA) on the treatment of four-part proximal humeral fractures (PHFs). METHODS: From January 2014 to January 2018, 32 patients with four-part PHFs were treated with intramedullary MultiLoc® nails in our hospital. The operation time, bleeding volume, postoperative X-ray findings, and fracture healing status were recorded and analyzed. At the end of follow-up, the clinical outcome was evaluated based on the visual analog scale (VAS) score, American Shoulder and Elbow Surgeons (ASES) shoulder score, Constant-Murley score (CMS), and occurrence of any complications. RESULTS: Among all patients, the average operation time was 124.5 minutes (range, 91-152 minutes), the average amount of bleeding was 90 mL (range, 55-150 mL), and the fracture healing rate was 100%. At the end of follow-up, the mean VAS score was 1.6 ± 0.4, mean ASES score was 84.4 ± 6.3, and mean CMS was 70.3 ± 6.1; no serious complications had occurred; and the patients exhibited good recovery of shoulder function. CONCLUSIONS: MultiLoc nails® can be applied to the treatment of four-part PHFs. This surgical fixation method has no obvious complications and helps to restore shoulder function.

The treatment of complex intra-articular distal radius fractures with turning radius and distal volaris radius plate fixation.

BACKGROUND: Although distal radius fractures (DRFs) are clinically common, intra-articular DRFs accompanied by dorsally displaced free fragments are much less so. At present, it is very difficult to fix and stabilize the intra-articular distal radius fractures accompanying dorsally displaced free fragments with a plate. Our aim was to investigate the clinical effect of DRFs with distally displaced dorsal free mass treated with distal volaris radius (DVR) combined with turning of the radius via the distal palmar approach. METHODS: From 2015 to 2019, 25 patients with intra-articular distal radius fractures associated with dorsally displaced free fragments were selected and treated with distal volaris radius (DVR) combined with turning of the radius via the distal palmar approach. This study involved 14 males and 11 females, with an average age of 34.5 years (ranging from 21 to 50 years). The mean follow-up period was 16.5 months (ranging from 12 to 22 months). The dorsal displacement of the free fragments was analyzed by X-ray and three-dimensional computed tomography, allowing characterization of postoperative recovery effects by radial height, volar tilt and radial inclination. For the follow-up, we evaluated effects of the surgery by analyzing range of motion (ROM); Modified Mayo Wrist Score (MMWS); and Disabilities of Arm, Shoulder and Hand (DASH) score. Postoperative wound recovery and complications were also monitored to evaluate the clinical therapeutic effects of the surgical procedures. RESULTS: X-ray showed that all patients showed reduced fractures, well-healed wounds and recovered function with no obvious complications. Based on the follow-up, patients had a mean radial height of 10.5 mm (ranging from 8.1 to 12.6 mm), mean MMWS of 78.8° (ranging from 61° to 90°), mean DASH score of 16.25 (ranging from 11 to 21), mean ROM for volar flexion of 76.5° (ranging from 62° to 81°), mean ROM for dorsiflexion of 77.1° (ranging from 59 to 83) and mean VAS score of 1.4 (ranging from 1 to 3). CONCLUSION: Treatment of the intra-articular distal radius fractures accompanying dorsally displaced free fragments with turning of the radius and the DVR plate system via the distal palmar approach is effective and has no obvious complications.

The design of an "H" joystick for closed reduction and its application in segmental and comminuted femoral shaft fractures: an innovative technique.

BACKGROUND: Closed reduction and locked intramedullary nailing has become a common surgical method in the treatment of femoral shaft fractures. Overlap and rotation displacements can usually be corrected through the use of an orthopedic traction table. However, lateral displacement and angulation persist. METHODS: In this paper, we describe a joystick that can be used in the closed reduction of a fracture. It can correct lateral displacement and angulation, and has the advantage of multi-direction reduction. The device described in this paper includes two parallel horizontal joysticks, one vertical main joystick and four assistant rods. Moreover, there are many specific spacing holes in the two parallel horizontal joysticks and a groove structure in the vertical main joystick. When the main "H" joystick is pressed, it can adjust lateral displacements and angulation because of the lever principle. The distance between parallel horizontal joysticks and assistant rods can be adjusted to the fracture position and body mass index of different patients. RESULTS: The study participants consisted of 11 males and 5 females with a mean age of 31.0 years. All participants had good closed reduction and achieved bony union without any complications such as infection, nerve injury, non-union, malunion, and limb length discrepancy. By using an "H" joystick, closed femoral shaft fracture reduction and locked intramedullary nailing becomes simpler and faster. CONCLUSION: Based on the use of this instrument, we can easily and conveniently obtain the correct reduction situation, which leads to better surgical results. This device can be applied in the reduction of clinical femoral fractures and gradually extended to the reduction of other fractures.

A novel reduction device for the minimally invasive treatment of femoral shaft fractures.

OBJECTIVE: In this study, a new type of reduction device for femoral shaft fractures was developed and designed. The reduction procedure was also standardized and is expected to be useful in clinical practice. METHODS: A bone traction retractor that consisted of a special traction needle, a resistant sleeve, a crossbar and an arc-adjusting bar was designed. Forty-eight patients (32 males and 16 females, mean age 33.21±7.03 years old) with femoral shaft fractures treated in our hospital from January 2016 to December 2017 were selected. According to the AO classification, there were 15 patients with type A, 24 patients with type B and 9 patients with type C fractures. All patients were treated with transverse bone traction for closed reduction of femoral shaft fractures and femoral reconstruction with intramedullary nails for final fixation. The injured side, preoperative delay time, reduction and operative times, operative blood loss, drilling frequency, number of open reduction cases, hospitalization days, fracture healing time, postoperative HSS function score and complications were recorded. RESULTS: All 48 patients were treated with transverse bone traction using our novel device to obtain reduction. The average time needed for reduction was 19.98±4.66 min. The operating time was 60-100 min, with an average of 78.65±16.81 min, and the average intraoperative blood loss was 131.91±30.22 ml. Open reduction was performed in 8 patients: 1 patient in the experimental group and 7 patients in the control group. The average hospitalization days was 7.78±2.81 days, the fracture healing time was 10 to 15 weeks, with an average of 12.44±2.63 weeks, and the postoperative HSS score was 80-95 points, with an average of 86.52±6.03 points. None of the patients had coxa vara, nonunion, internal fixation failure, infection, nerve injury, limb length discrepancy or other complications. CONCLUSION: In this study, the transverse bone traction reduction technique and the design of a proprietary reduction device system were proposed, with high clinical application. The transverse bone traction reduction technique has the advantages of simple operation, reliable reduction and limited intraoperative fluoroscopy in the minimally invasive treatment of femoral shaft fractures.

MiR-33b-3p promotes chondrocyte proliferation and inhibits chondrocyte apoptosis and cartilage ECM degradation by targeting DNMT3A in osteoarthritis.

Osteoarthritis (OA) is a common and frequently-occurring disease in middle-aged and older people. A growing number of studies have shown that microRNAs (miRNAs) are involved in the development of OA. However, the role and mechanism of miR-33b-3p in OA remain ill-defined. The levels of miR-33b-3p and DNA methyltransferase 3A (DNMT3A) mRNA were determined by quantitative real-time polymerase chain reaction (qRT-PCR). The levels of DNMT3A protein, matrix metalloprotein 13 (MMP-13), a disintegrin and metalloproteinase with thrombospondin motif-5 (ADAMTS-5), collagen II, aggrecan, cleaved Caspase-3, B-cell lymphoma-2 (Bcl-2) and BCL2-Associated X (Bax) were measured by Western blot assay. Cell proliferation and cell apoptosis were assessed by Cell Counting Kit-8 (CCK-8) assay and flow cytometry analysis, respectively. The targeting relationship between miR-33b-3p and DNMT3A was verified by dual-luciferase reporter assay. The expression of miR-33b-3p was decreased and the expression of DNMT3A was increased in OA cartilage tissues and IL-1ß-induced chondrocytes. There was an inverse correlation between miR-33b-3p and DNMT3A in OA cartilage tissues. MiR-33b-3p overexpression or DNMT3A knockdown inhibited extracellular matrix (ECM) degradation and cell apoptosis and promoted cell proliferation in IL-1ß-induced chondrocytes. Moreover, DNMT3A was confirmed to be a direct target of miR-33b-3p. Upregulation of DNMT3A weakened the effects of miR-33b-3p overexpression on cartilage ECM degradation, cell proliferation and apoptosis in IL-1ß-activated chondrocytes. MiR-33b-3p overexpression suppressed cartilage ECM degradation and cell apoptosis, and promoted cell proliferation by directly targeting DNMT3A in IL-1ß-stimulated chondrocytes.

Rabbit Model of Physeal Injury for the Evaluation of Regenerative Medicine Approaches.

Physeal injuries can lead to bony repair tissue formation, known as a bony bar. This can result in growth arrest or angular deformity, which is devastating for children who have not yet reached their full height. Current clinical treatment involves resecting the bony bar and replacing it with a fat graft to prevent further bone formation and growth disturbance, but these treatments frequently fail to do so and require additional interventions. Novel treatments that could prevent bone formation but also regenerate the injured physeal cartilage and restore normal bone elongation are warranted. To test the efficacy of these treatments, animal models that emulate human physeal injury are necessary. The rabbit model of physeal injury quickly establishes a bony bar, which can then be resected to test new treatments. Although numerous rabbit models have been reported, they vary in terms of size and location of the injury, tools used to create the injury, and methods to assess the repair tissue, making comparisons between studies difficult. The study presented here provides a detailed method to create a rabbit model of proximal tibia physeal injury using a two-stage procedure. The first procedure involves unilateral removal of 25% of the physis in a 6-week-old New Zealand white rabbit. This consistently leads to a bony bar, significant limb length discrepancy, and angular deformity within 3 weeks. The second surgical procedure involves bony bar resection and treatment. In this study, we tested the implantation of a fat graft and a photopolymerizable hydrogel as a proof of concept that injectable materials could be delivered into this type of injury. At 8 weeks post-treatment, we measured limb length, tibial angle, and performed imaging and histology of the repair tissue. By providing a detailed, easy to reproduce methodology to perform the physeal injury and test novel treatments after bony bar resection, comparisons between studies can be made and facilitate translation of promising therapies toward clinical use. Impact Statement This study provides details to create a rabbit model of physeal injury that can facilitate comparisons between studies and test novel regenerative medicine approaches. Furthermore, this model mimics the human, clinical situation that requires a bony bar resection followed by treatment. In addition, identification of a suitable treatment can be seen in the correction of the growth deformity, allowing this model to facilitate the development of novel physeal cartilage regenerative medicine approaches.

Successful treatment for acute prosthetic joint infection due to MRSA and Candida albicans: a case report and literature review.

Methicillin-resistant Staphylococcus aureus (MRSA) and Candidal prosthetic joint infections (PJIs) are very rare, and the optimal management for these patients is still unknown. A 54-year-old man with traumatic arthritis due to previous electric injury successfully retained the implant despite the successive infection with MRSA and Candida albicans after total knee arthroplasty (TKA). Continuous lavage with vancomycin was used to control MRSA infection and repeated local washout plus oral swallow with voriconazole tablet were administered to eradicate C. albicans. Additional three reported cases were identified by the criteria of selecting patients with concomitant and/or successive MRSA and Candidal PJIs. Different methods were applied with variable outcomes. Therefore, several risk factors such as intra-articular corticosteroid injection, high frequency of door openings in the operating room, excessive blood loss and allogeneic red blood cell transfusions should be avoided. Debridement, antibiotics and implant retention (DAIR) can be an alternative in dedicated patients to control acute MRSA and Candidal PJIs. Particularly, repeated intra-articular washout with susceptible drugs and a prolonged duration of oral antibiotics was essential for microbial control.

KLF15 regulates in vitro chondrogenic differentiation of human mesenchymal stem cells by targeting SOX9.

Mesenchymal stem cells (MSCs) are multipotent stromal cells residing in the bone marrow. MSCs have the potential to differentiate into adipocytes, chondrocytes, and other types of cells. However, the mechanism underlying MSC differentiation is still not fully understood. Here we aimed to investigate the function of the Kruppel-like factor (KLF) transcriptional factor family in regulating chondrogenic differentiation from human MSCs. Among the KLF family members, KLF15 was activated during different models of chondrogenic differentiation in a time-dependent manner. Lentivirus-mediated knockdown of KLF15 in MSCs repressed chondrogenic differentiation whereas KLF15 overexpression facilitated chondrogenic differentiation. KLF15 promoted the chondrogenic differentiation of human MSCs by activating the expression of SOX9, which is critically involved in KLF15 function during chondrogenic differentiation. Our mechanism study demonstrated that KLF15 bound the promoter of SOX9 and promoted the activation of the SOX9 promoter. Taken together, our findings show that KLF15 promotes chondrogenic differentiation of human MSCs by activating SOX9.

Overexpression of microRNA-210 promotes chondrocyte proliferation and extracellular matrix deposition by targeting HIF-3α in osteoarthritis.

The present study aimed to determine the effect of microRNA (miR)­210 on osteoarthritis (OA). The expression levels of miR­210, type I and X collagen (COL1A1 and COL10A1) and matrix metallopeptidase 13 (MMP13) in OA and normal chondrocytes were determined using reverse transcription­quantitative polymerase chain reaction analysis. The OA chondrocytes were transfected with an miRNA precursor for miR­210 or a negative control. After 3, 7, 14 and 21 days, the expression levels of miR­210 were examined, the proliferation of the OA chondrocytes were determined using an XTT assay and the protein levels of Ki67 and HIF­3α were analyzed by Western blotting. After 21 days, the mRNA and protein levels of COL1A1, COL10A1 and MMP13 were analyzed. Th present study demonstrated that the expression levels of miR­210 and COL1A1 were lower, and the expression levels of COL10A1 and MMP13 were higher in the OA chondrocytes, compared with the levels of expression in the normal chondrocytes. Overexpression of miR­210 significantly promoted the proliferation of OA chondrocytes and induced the protein expression of Ki67. In addition, miR­210 overexpression markedly increased the expression of COL1A1 expression, but decreased the expression levels of COL10A1 and MMP13. A luciferase reporter assay confirmed the direct interaction between miR­210 and hypoxia­inducible factor (HIF)­3α. miR­210 did not alter the mRNA expression of HIF­3α, however, it suppressed the protein expression of HIF­3α. Additionally, HIF­3α knockdown significantly promoted OA chondrocyte proliferation and increased the mRNA levels of COL1A1, whereas it decreased the mRNA levels of COL10A1 and MMP13. The results of the present study suggested that miR­210 may be a negative regulator of the progression of OA, which increases chondrocyte proliferation and prompts extracellular matrix deposition by directly targeting HIF­3α.

Two-stage therapeutic utility of ectopically formed bone tissue in skeletal muscle induced by adeno-associated virus containing bone morphogenetic protein-4 gene.

BACKGROUND: The major disadvantage of using a stem cell-based bone morphogenetic protein-4 (BMP4) gene therapy for skull defect is the overgrowth of generated bone tissue in situ. In the present study, to overcome bony overgrowth of stem cell based-gene therapy, a new strategy of two-stage bone tissue engineering by an adeno-associated virus containing BMP4 gene (AAV-BMP4) gene therapy was used. METHODS: AAV-BMP4 was purposely implanted in the skeletal muscle of mice to generate ectopic bone tissues during the first stage. Next, the newly formed ectopic bone tissues were harvested and then transplanted to repair the mouse skull defect during the second stage. RESULTS: The results showed that skeletal muscle implantation of AAV-BMP4 yielded a large amount of new bone tissues. The ectopic bone tissues can be harvested as a bone graft and can successfully repair the mouse skull defect without any bony overgrowth in situ. CONCLUSION: The results indicate that the bone tissues purposely generated by AAV-BMP4 in the skeletal muscle may be a new alternative of bone grafting for clinical purposes.

The case for primary salivary rhabdomyosarcoma.

Rhabdomyosarcomas of the parotid and submandibular glands have the histological appearance of a skeletal muscle tumor yet can be found in tissue with no striated muscular elements. We examine the potential cell-of-origin for rhabdomyosarcoma and whether salivary tumors represent primary malignancy or metastasis. We have previously established genetically engineered mouse models of rhabdomyosarcoma. In these mice, rhabdomyosarcoma is only induced when a Pax3:Foxo1 fusion oncogene is activated with concurrent loss of p53 function (for alveolar rhabdomyosarcoma) or loss of p53 function alone (for embryonal rhabdomyosarcoma) using Cre-lox technology. These mutations are only activated under the control of promoters specific for selected cell lineages, previously thought to be myogenesis-restricted. RT-PCR and immunohistochemistry for lineage-specific promoter gene products reveal these promoters are active in wild-type mouse salivary gland. Given that mouse rhabdomyosarcoma frequently originates in the salivary glands and these myogenic-related promoters are normally expressed in salivary tissue, a high likelihood exists that the salivary gland contains a cell-of-origin of this muscle-related cancer.

Celecoxib Combined with Diacerein Effectively Alleviates Osteoarthritis in Rats via Regulating JNK and p38MAPK Signaling Pathways.

Osteoarthritis (OA) has long been a difficult to overcome joint disease for medical workers. However, there is still a lack of effective treatments for OA. In the present study, we aimed to evaluate the treatment effect of celecoxib (CLX) combined with diacerein (DC) on OA and delineate the underlying molecular mechanism. The OA model was established by using rats, and OA rats were treated with either CLX alone, DC alone, and CLX combined with DC. The results showed that, as compared with a single treatment of CLX or DC, CLX combined with DC markedly attenuated OA and inhibited the levels of inflammatory mediators interleukin-1ß and nitric oxide, improved bone cartilage metabolism, and suppressed chondrocyte apoptosis. Most importantly, CLX combined with DC significantly inactivated the c-Jun N-terminal kinases (JNK) signaling pathway by the inhibition of MEKK1 and MKK7, as detected by Western blot analysis. Furthermore, the protein expression of downstream genes of JNK, including activating-transcription factor (Atf-2), matrix metalloproteinase-13 (MMP-13), and cyclooxygenase (COX-2), were also significantly inhibited by CLX combined with DC as compared with single treatments. Furthermore, CLX combined with DC also effectively inhibits p38 mitogen-activated protein kinase and nuclear factor-κB signaling pathways. Taken together, our study suggests that CLX combined with DC has satisfactory treatment effects on OA via a stronger inhibitory effect on inflammatory signaling pathway.

Effect and mechanism of miRNA to osteosarcoma cell.

MicroRNA has proved to be low expression in many tumor cells. In addition, it was also proved that as a kind of cancer suppressor gene, miR-199a-3p in miRNA can affect the growth and invasion ability of tumor cells. This paper aims to discuss the effect of miRNA to osteosarcoma cell. It used synthetic mature miR-199a-3p sequence simulants to transfect osteosarcoma cell and took negative contrast sequence (NC mimics) transfection cell as negative contrast. After transfection, qRT-PCR was applied to detect the expression quantity of miR-199a-3p in every group. Western blot method was applied to detect the expression level of MCL-(1) protein and shear situation of PARP in groups of cells. Flow cytometry was used for detecting apoptosis rate of cells and the experimental result was made a statistical analysis. The result shows that in cells from experimental group of transfection miR-199a-3p sequence simulants, expression quantity of mi-R-199a-3p significantly increased while MCL⁻¹ protein expression decreased compared to control group. In addition, shear level of PARP protein and apoptosis rate of cells increased. The differences all had statistical significance (P<0.05). It was concluded that miR-199a-3p can effectively promote the apoptosis rate of osteosarcoma cells.

IL-4 receptor blockade abrogates satellite cell: rhabdomyosarcoma fusion and prevents tumor establishment.

Tumor cells of the muscle-related cancer alveolar rhabdomyosarcoma (aRMS) have dysregulated terminal myogenic differentiation that is characterized by continuous proliferation, decreased capacity to express markers of terminal differentiation, and inability of tumor cells to fuse to one another in the manner seen for normal myoblasts. Whether aRMS tumor cells can fuse with normal myogenic progenitors such as skeletal muscle stem cells (satellite cells) or myoblasts is unknown, as is the biological effect of fusion events if the phenomenon occurs. To study this possibility, we isolated primary satellite cells harboring a lacZ Cre-LoxP reporter gene for coculture with murine aRMS primary tumor cells expressing Cre. Results of in vitro and in vivo experiments demonstrated tumor cell-muscle cell progenitor fusion events as well as accelerated rates of tumor establishment and progression when satellite cells and derived muscle progenitors were coinjected with tumor cells in an orthotopic allograft model. Interleukin 4 receptor (IL-4R) blocking antibody treatment reversed fusion events in vitro and blocked tumor initiation and progression in vivo. Taken together, this study supports a potential role of tumor cell-host cell fusion and the strong therapeutic potential of IL-4R blockade to prevent the establishment of RMS tumors at new anatomical sites.