Kaempferol attenuates particle-induced osteogenic impairment by regulating ER stress via the IRE1α-XBP1s pathway.

Periprosthetic osteolysis and subsequent aseptic loosening are the primary causes of failure following total joint arthroplasty. Wear particle-induced osteogenic impairment is recognized as an important contributing factor in the development of osteolysis, with endoplasmic reticulum (ER) stress emerging as a pivotal underlying mechanism. Hence, searching for potential therapeutic targets and agents capable of modulating ER stress in osteoblasts is crucial for preventing aseptic loosening. Kaempferol (KAE), a natural flavonol compound, has shown promising osteoprotective effects and anti-ER stress properties in diverse diseases. However, the influence of KAE on ER stress-mediated osteogenic impairment induced by wear particles remains unclear. In this study, we observed that KAE effectively relieved TiAl6V4 particles-induced osteolysis by improving osteogenesis in a mouse calvarial model. Furthermore, we demonstrated that KAE could attenuate ER stress-mediated apoptosis in osteoblasts exposed to TiAl6V4 particles, both in vitro and in vivo. Mechanistically, our results revealed that KAE mitigated ER stress-mediated apoptosis by upregulating the IRE1α-XBP1s pathway while concurrently partially inhibiting the IRE1α-regulated RIDD and JNK activation. Collectively, our findings suggest that KAE is a prospective therapeutic agent for treating wear particle-induced osteolysis and highlight the IRE1α-XBP1s pathway as a potential therapeutic target for preventing aseptic loosening.

Gene Expression in Synovium of Rotator Cuff Tear Patients Determined by RNA Sequencing.

Rotator cuff tear (RCT) is a common shoulder disorder related to pain and dysfunction. However, the pathological mechanism of RCT remains unclear. Thus, this study aims to investigate the molecular events in RCT synovium and identify possible target genes and pathways as determined by RNA sequencing (RNA-Seq). The synovial tissue was biopsied from 3 patients with RCT (RCT group) and 3 patients with shoulder instability (Control group) during arthroscopic surgery. Then, differentially expressed (DE) mRNAs, long non-coding RNAs (lncRNAs) and micro RNAs (miRNAs) were comprehensively profiled by RNA-Seq. Gene ontology (GO) enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, and competing endogenous RNA (ceRNA) network analysis were performed to identify the potential functions of these DE genes. 447 mRNAs, 103 lncRNAs and 15 miRNAs were identified differentially expressed. The DE mRNAs were highlighted in inflammatory pathway including up-regulated T cell costimulation, positive regulation of T cell activation, and T cell receptor signaling. Down-regulated fatty acid degradation pathway and 5'-AMP-activated protein kinase (AMPK) signaling in RCT group are also enriched. Validation assay showed that the expression of pro-inflammatory molecules including IL21R, CCR5, TNFSF11, and MMP11 was significantly increased in RCT group compared with Control group. CeRNA analysis further revealed lncRNA-miRNA-mRNA regulatory networks involving IL21R and TNFSF11 in RCT. Activated synovial inflammation is the remarkable event of RCT. Importantly, increased T cell activation and disordered fatty acid metabolism signaling might play a significant role. ceRNA networks involving IL21R and TNFSF11 identified could potentially control the progression of RCT. In conclusion, our findings could provide new evidence for the molecular mechanisms of RCT and might identify new therapeutic targets.

Biomechanical evaluation of reconstruction of the posterior complex in restorative laminoplasty with miniplates.

OBJECTIVE: To evaluate the biomechanical effects of different miniplates on restorative laminoplasty. METHODS: Assembled restorative laminoplasty models were developed based on 3D printed L4 lamina. Based on different internal fixations, the research was divided into H-shaped miniplates (HSMs) group, two-hole miniplates (THMs) group, and L-shaped miniplates (LSMs) group. The static and dynamic compression tests were analyzed to investigate the biomechanical effects of different internal fixations in restorative laminoplasty, until the failure and fracture of miniplates, or the collapse of miniplates. The static compression tests adopted the speed control mode, and the dynamic fatigue compression tests adopted the load control mode. RESULTS: The "door close" and the collapse of lamina occurred in THMs group and LSMs group, and plate break occurred in LSMs group. However, these phenomenon was absent in HSMs group, and only plate crack around a screw and looseness of a screw tail cap were found in HSMs group. The sustainable yield load of HSMs group was greater than that of THMs group and LSMs group (P < 0.05). No significant difference in yielding-displacement was found between HSMs group and LSMs group (P > 0.05), while both were much less than that of THMs (P < 0.05). Moreover, the compressive stiffness and the axial displacement under the same mechanical load were arranged as follows: HSMs group > LSMs group > THMs group (P < 0.05). The results of dynamic compression test revealed that the peak load of HSMs group could reached 873 N and was 95% of the average yield load of the static compression, and was better than that in THMs group and LSMs group (P < 0.05). Besides, according to the fatigue life-peak load diagram, the ultimate load of HSMs group was more than twice that of THMs group or LSMs group. CONCLUSIONS: The mechanical strength of H-shaped miniplates was superior to two-hole miniplates and L-shaped miniplates in maintaining spinal canal enlargement and spinal stability, and was more excellent in fatigue stability and ultimate load.

Lonicerin alleviates the progression of experimental rheumatoid arthritis by downregulating M1 macrophages through the NF-κB signaling pathway.

The present study aimed to evaluate anti-rheumatoid arthritis (RA) effect of Lonicerin (LON), a safe compound with anti-inflammatory and immunomodulatory properties. Nevertheless, the exact role of LON in RA remains elusive. In this test, the anti-RA effect of LON was evaluated in collagen-induced arthritis (CIA) mouse model. Relevant parameters were measured during the experiment; ankle tissue and serum were collected at the end of the experiment for radiology, histopathology, and inflammation analysis. ELISA, qRT-PCR, immunofluorescence, and western blot were used to explore the effect of LON on the polarization of macrophages and related signal pathways. It was discovered that LON treatment attenuated the disease progression of CIA mice with lower paw swelling, clinical score, mobility, and inflammatory response. LON treatment significantly decreased M1 marker levels in CIA mice and LPS/IFN-γ-induced RAW264.7 cells, while slightly increasing M2 marker levels in CIA mice and IL-4-induced RAW264.7 cells. Mechanistically, LON attenuated the activation of the NF-κB signaling pathway, which contributes to M1 macrophage polarization and inflammasome activation. In addition, LON inhibited NLRP3 inflammasome activation in M1 macrophages, thereby reducing inflammation by inhibiting IL-1ß and IL-18 release. These results indicated that LON might exert anti-RA effects by regulating the polarization of M1/M2 macrophage, especially by inhibiting macrophage polarization toward M1.

All-inside technique versus in situ transtendon repair for Ellman â ¢ partial articular supraspinatus tendon avulsion.

BACKGROUND: The current surgical techniques for repairing Ellman Ⅲ partial articular supraspinatus tendon avulsion (PASTA) is mainly tear completion followed by repair and in situ transtendon repair, and both techniques have been proven to have good clinical effects. In situ transtendon repair is more widely used because of its high performance in preserving the integrity of the bursal side supraspinatus tendon. However, there is still some scope for improvement. Our purpose was to compare the clinical outcomes of the all-inside repair technique and in situ transtendon repair for Ellman III PASTA. METHODS: A retrospective comparative study was conducted on 56 patients who suffered from Ellman Ⅲ PASTA and underwent rotator cuff repair; 28 patients were treated with the all-inside technique (group A), and 28 patients were treated with the transtendinous technique (group B). All patients were followed up for at least 2 years. The visual analog scale (VAS), Constant, and American Shoulder and Elbow Surgeons (ASES) scores were used to evaluate the patient's shoulder joint function before surgery, 1 month and 3 months after surgery, and at the last time of follow-up. RESULTS: Group A showed superiority in pain and functional improvement 1 month after the operation: the VAS score, 2.8 ± 0.3 in group A vs. 4.7 ± 0.4 in group B (P = .042); Constant score, 73 ± 5 in group A vs. 60 ± 6 in group B (P = .038); and ASES score, 75 ± 5 in group A vs. 58 ± 7 in group B (P = .043), whereas there was no statistical difference in group B. However, 3 months after surgery and at the last follow-up, the VAS, Constant, and ASES scores in both groups were significantly improved (P < .01), and there was no significant statistical difference between the groups (P > .05). Magnetic resonance imaging showed that the repaired rotator cuff tendon-bone healed well; at the last follow-up, all patients were in good function, the pain was completely relieved, and no revision was performed in both groups. CONCLUSION: Arthroscopic repair of Ellman Ⅲ PASTA provided satisfactory functional improvements and pain relief regardless of the all-inside and in situ transtendon repair techniques. However, the all-inside repair technique is more beneficial due to its dual function in preserving the intact bursa and avoidance of uneven tension of the articular side, which is advantageous to early postoperative rehabilitation.

Chronic Thrombocytopenia and In-Hospital Outcomes After Primary Total Hip and Knee Arthroplasty.

BACKGROUND: There is a lack of data on the influence of chronic thrombocytopenia (cTCP) on clinical outcomes following primary total hip arthroplasty (THA) and total knee arthroplasty (TKA). Limited studies mainly focused on postoperative heparin-induced TCP from single centers with small sample sizes. This study aims to describe the characteristics, trend, and outcomes of cTCP in patients undergoing THA and TKA from a nationally reprehensive perspective. METHODS: We identified THA and TKA patients with and without cTCP from the 2005 to 2015 Nationwide Inpatient Sample. Annual percent changes were calculated to reflect cTCP trends. Multivariable regression and propensity score analyses were conducted to investigate the association of cTCP and mortality, preoperative complications, cost as well as length of stay. RESULTS: In total, 578,278 and 1,237,331 patients underwent primary THA and TKA, respectively. Proportion of cTCP annually increased by 6.95% in THA and 6.66% in TKA. Patients with cTCP were associated with higher risk of medical (odds ratio [OR] 2.00, 95% confidence interval [CI] 1.89-2.11) and surgical complications (OR 2.72, 95% CI 2.55-2.90) in THA, and higher risk of mortality (OR 1.68, 95% CI 1.22-2.31), medical (OR 1.94, 95% CI 1.85-2.03) and surgical complications (OR 2.55, 95% CI 2.38-2.73) in TKA. Additionally, higher cost and longer length of stay were observed in patients with cTCP for both surgical procedures. CONCLUSION: Patients with cTCP had higher risk of mortality for TKA, more perioperative complications for both TKA and THA. Further studies are warranted to improve the preoperative management and to prevent worse outcomes associated with cTCP.

Survival outcomes of patients with brain metastasis of osteosarcoma can be improved by aggressive multi-disciplinary interventions including chemotherapy.

BACKGROUND/OBJECTIVE: Brain metastasis in osteosarcoma (BMO) is rare and its clinical characteristics are often buried among studies on brain metastasis of bone and soft tissue sarcomas. The aim of the present study was to summarize the incidence, clinical characteristics, treatment and outcomes of patients with BMO. METHODS: This retrospective study included 7 patients with BMO who received treatment in our center between 2005 and 2019. The clinical medical records of the 7 patients, together with data of 70 BMO patients published in 33 articles and retrieved by means of PubMed and Medline, were analyzed, retrospectively. RESULTS: Data analysis of the 97 BMO patients showed a high correlation between the interval from the primary diagnosis to BMO occurrence and the interval from the primary diagnosis to prior metastases. Multivariate analysis showed that chemotherapy, radiotherapy and surgery were three main factors affecting the overall survival of BMO patients (HR = 0.427; HR = 0.372; HR = 0.296). Surgery combined with chemotherapy or radiotherapy offered a better overall survival than surgery alone. CONCLUSION: Patients with BMO may obtain survival benefits from regular neuroimaging and early aggressive multi-disciplinary interventions including surgical resection, postoperative radiotherapy and chemotherapy. SYNOPSIS: This is a retrospective study describing the characteristics of metastasic intervals, locations, clinical features and prognosis in 97 patients with brain metastasis of osteosarcoma (BMO). Multivariate analysis showed that chemotherapy was effective as surgery and radiotherapy for the treatment of BMO. Our findings emphasize the importance of regular neuroimaging and early aggressive multi-disciplinary interventions including surgical resection, postoperative radiotherapy and chemotherapy.

miR-181c-5p mediates simulated microgravity-induced impaired osteoblast proliferation by promoting cell cycle arrested in the G2 phase.

Impaired osteoblast proliferation plays fundamental roles in microgravity-induced bone loss, and cell cycle imbalance may result in abnormal osteoblast proliferation. However, whether microgravity exerts an influence on the cell cycle in osteoblasts or what mechanisms may underlie such an effect remains to be fully elucidated. Herein, we confirmed that simulated microgravity inhibits osteoblast proliferation. Then, we investigated the effect of mechanical unloading on the osteoblast cell cycle and found that simulated microgravity arrested the osteoblast cell cycle in the G2 phase. In addition, our data showed that cell cycle arrest in osteoblasts from simulated microgravity was mainly because of decreased cyclin B1 expression. Furthermore, miR-181c-5p directly inhibited cyclin B1 protein translation by binding to a target site in the 3'UTR. Lastly, we demonstrated that inhibition of miR-181c-5p partially counteracted cell cycle arrest and decreased the osteoblast proliferation induced by simulated microgravity. In conclusion, our study demonstrates that simulated microgravity inhibits cell proliferation and induces cell cycle arrest in the G2 phase in primary mouse osteoblasts partially through the miR-181c-5p/cyclin B1 pathway. This work may provide a novel mechanism of microgravity-induced detrimental effects on osteoblasts and offer a new avenue to further investigate bone loss induced by mechanical unloading.

Hydrogen treatment reduces tendon adhesion and inflammatory response.

A rat model of tendon repair was established to investigate the effects of hydrogen water on tendon adhesion reduction. Thirty-six Sprague Dawley rats were randomly divided into a normal saline (NS) group and a hydrogen water (HS) group according to the processing reagents after a tendon repairing operation. Pre- and postoperative superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione (GSH) in subjects' serum were observed. Skin fibroblasts were grouped into an NS group, H2 O2 group, H2 group, and H2 O2 H2 group. Expressions of Nrf2, CATA, and γ-GCS were also tested by Western blot analysis. 8-OHdG, GSH, MDA, and SOD of the cells were analyzed by the enzyme-linked immunosorbent assay method. The postoperative SOD activity and GSH contents were significantly reduced (P < 0.05), whereas the postoperative MDA level was significantly increased (P < 0.05). Similarly, the postoperative HS group showed significantly higher SOD activity and GSH contents (P < 0.05) but lower MDA (P < 0.05) compared with the postoperative NS group. MDA and 8-OHdG were significantly decreased in hydrogen-rich medium, while SOD and GSH were increased. The expression of Nrf2, CATA, and γ-GCS in antioxidant system were reduced after H2 O2 processing, which were restored after the application of hydrogen-rich medium. Hydrogen water can reduce tendon adhesion after tendon repairing and prohibit excessive inflammatory response, which could be associated with the activation of the Nrf2 pathway.

Simulated microgravity reduces intracellular-free calcium concentration by inhibiting calcium channels in primary mouse osteoblasts.

Calcium homeostasis in osteoblasts plays fundamental roles in the physiology and pathology of bone tissue. Various types of mechanical stimuli promote osteogenesis and increase bone formation elicit increases in intracellular-free calcium concentration in osteoblasts. However, whether microgravity, a condition of mechanical unloading, exerts an influence on intracellular-free calcium concentration in osteoblasts or what mechanisms may underlie such an effect are unclear. Herein, we show that simulated microgravity reduces intracellular-free calcium concentration in primary mouse osteoblasts. In addition, simulated microgravity substantially suppresses the activities of L-type voltage-sensitive calcium channels, which selectively allow calcium to cross the plasma membrane from the extracellular space. Moreover, the functional expression of ryanodine receptors and inositol 1,4,5-trisphosphate receptors, which mediate the release of calcium from intracellular storage, decreased under simulated microgravity conditions. These results suggest that simulated microgravity substantially reduces intracellular-free calcium concentration through inhibition of calcium channels in primary mouse osteoblasts. Our study may provide a novel mechanism for microgravity-induced detrimental effects in osteoblasts, offering a new avenue to further investigate bone loss induced by mechanical unloading.

microRNA-7 inhibition protects human osteoblasts from dexamethasone via activation of epidermal growth factor receptor signaling.

Sustained dexamethasone (Dex) treatment could induce secondary osteoporosis, osteonecrosis, or even bone fractures. Dex can induce potent cytotoxicity in cultured human osteoblasts. The aim of this study was to test the potential role of microRNA-7 (miR-7), which targets the epidermal growth factor receptor (EGFR), in Dex-treated human osteoblasts. In OB-6, hFOB1.19, and primary human osteoblasts, miR-7 depletion by a lentiviral antagomiR-7 construct (LV-antagomiR-7) increased EGFR expression and downstream Akt activation, protecting cells from Dex-induced viability reduction, cell death, and apoptosis. In contrast, forced overexpression of miR-7 by a lentiviral miR-7 construct (LV-miR-7) inhibited EGFR expression and Akt activation, potentiating Dex-induced cytotoxicity in OB-6, hFOB1.19, and primary human osteoblasts. EGFR is the primary target of miR-7 in human osteoblasts. Luciferase activity of the EGFR 3-untranslated region was enhanced by LV-antagomiR-7, but decreased by LV-miR-7 in OB-6 cells. Further, LV-antagomiR-7-induced osteoblast cytoprotection against Dex was abolished by the EGFR inhibitors AG1478 and PD153035. Moreover, neither LV-antagomiR-7 nor LV-miR-7 was functional in EGFR-KO OB-6 cells. We also show that miR-7 is upregulated in the necrotic femoral head tissues of Dex-administered patients, correlating with EGFR downregulation. Together, we conclude that miR-7 inhibition protects human osteoblasts from Dex via activation of EGFR signaling.

miR-338-5p Regulates the Viability, Proliferation, Apoptosis and Migration of Rheumatoid Arthritis Fibroblast-Like Synoviocytes by Targeting NFAT5.

BACKGROUND/AIMS: MicroRNAs (miRNAs) have been reported to be involved in Rheumatoid arthritis (RA) pathogenesis and prognosis. However, little is known about the disease mechanism in RA. Here, we aim to investigate the potential association between miR-338-5p and NFAT5 in RA. METHODS: Aberrant expression of miR-338-5p in RA tissues and rheumatoid arthritis fibroblast-like synoviocytes (RAFLSs) compared to the normal were determined by RT-qPCR. Cell viability was determined using the CCK-8 assay, and cell apoptosis was analyzed via Annexin V-FITC/PI double staining and was detected using flow cytometry. The targeted relationship was determined by TargetScan database and dual luciferase reporter gene assay. RESULTS: Upregulation of miR-338-5p facilitated the proliferation, migration, invasion and induced G0/G1 arrest of RAFLSs while miR-338-5p inhibitor functioned oppositely. Nuclear factor of activated T-cells 5 (NFAT5) was confirmed as a downstream target of miR-338-5p which expression was directly suppressed by miR-338-5p. Overexpression of NFAT5 attenuated the proliferation and metastasis of RAFLSs and those changes could be rescued by co-transfection of miR-338-5p. CONCLUSION: miR-338-5p promotes RAFLS's viability and proliferation, migration by targeting NFAT5, suggesting a novel therapeutic strategy for RA.

Long Non-Coding RNA MALAT1 Protects Human Osteoblasts from Dexamethasone-Induced Injury via Activation of PPM1E-AMPK Signaling.

BACKGROUND/AIMS: Dexamethasone (Dex) induces injuries to human osteoblasts. In this study, we tested the potential role of the long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (Lnc-MALAT1) in this process. MATERIALS: Two established human osteoblastic cell lines (OB-6 and hFOB1.19) and primary human osteoblasts were treated with Dex. Lnc-MALAT1 expression was analyzed by quantitative real-time polymerase chain reaction assay. Cell viability, apoptosis, and death were tested by the MTT assay, histone-DNA assay, and trypan blue staining assay, respectively. AMP-activated protein kinase (AMPK) signaling was evaluated by western blotting and AMPK activity assay. RESULTS: Lnc-MALAT1 expression was downregulated by Dex treatment in the established osteoblastic cell lines (OB-6 and hFOB1.19) and primary human osteoblasts. The level of Lnc-MALAT1 was decreased in the necrotic femoral head tissues of Dex-administered patients. In osteoblastic cells and primary human osteoblasts, forced overexpression of Lnc-MALAT1 using a lentiviral vector (LV-MALAT1) inhibited Dex-induced cell viability reduction, cell death, and apoptosis. Conversely, transfection with Lnc-MALAT1 small interfering RNA aggravated Dex-induced cytotoxicity. Transfection with LV-MALAT1 downregulated Ppm1e (protein phosphatase, Mg2+/ Mn2+-dependent 1e) expression to activate AMPK signaling. Treatment of osteoblasts with AMPKα1 short hairpin RNA or dominant negative mutation (T172A) abolished LV-MALAT1-induced protection against Dex-induced cytotoxicity. Furthermore, LV-MALAT1 induced an increase in nicotinamide adenine dinucleotide phosphate activity and activation of Nrf2 signaling. Dex-induced reactive oxygen species production was significantly attenuated by LV-MALAT1 transfection in osteoblastic cells and primary osteoblasts. CONCLUSION: Lnc-MALAT1 protects human osteoblasts from Dex-induced injuries, possibly via activation of Ppm1e-AMPK signaling.

Effects of Transplanting Bone Marrow Stromal Cells Transfected with CXCL13 on Fracture Healing of Diabetic Rats.

BACKGROUND/AIMS: Diabetic fracture have poor treatment and serious complications. Therefore, how to treat diabetic fracture is receiving increasing attention. This study aimed to investigate the effects of transplanting CXCL13-stimulated bone marrow stromal cells (BMSCs) on the fracture healing in diabetic rats. METHODS: In vitro, RT-PCR was employed to examine the expression of CXCL13 in BMSCs in high glucose environment. MTT assay and apoptosis assay were utilized to determine the effects of CXCL13 overexpression on the proliferation and apoptosis of BMSCs respectively. ALP staining was applied to detect the ALP activity. In vivo, CXCL13-stimulated BMSCs were transplanted into the fracture sites of diabetic rats. At the 1st week, 2nd weeks, 4th week and 6th week after the operation, bone mineral density (BMD) and callus area measurement, ELISA detection, and HE staining were performed to evaluate the fracture healing. RESULTS: Low BMD and less area of callus in diabetic rats showed that the recovery after fracture was worse in diabetic rats than in non-diabetic rats. Meanwhile, the expression of CXCL13 in serum was lower in diabetic rats than in non-diabetic rats. Overexpression of CXCL13 promoted the proliferation of BMSCs in vitro high glucose environment. After BMSCs transfected with CXCL13 being transplanted into the fracture sites of diabetic rats, it was found that the fracture healing was enhanced and ALP expression in serum became higher. HE staining results further verified the effects of transplantation of BMSCs transfected with CXCL13 on fracture healing in diabetic rats. CONCLUSION: These finding indicated that CXCL13 may play a critical role in the process of fracture healing, which could provide a deeper insight into molecular targets for the fracture healing in diabetic people.

LAG-3 Represents a Marker of CD4+ T Cells with Regulatory Activity in Patients with Bone Fracture.

The lymphocyte activation gene 3 (LAG-3) is a CD4 homolog with binding affinity to MHC class II molecules. It is thought that LAG-3 exerts a bimodal function, such that co-ligation of LAG-3 and CD3 could deliver an inhibitory signal in conventional T cells, whereas, on regulatory T cells, LAG-3 expression could promote their inhibitory function. In this study, we investigated the role of LAG-3 expression on CD4+ T cells in patients with long bone fracture. We found that LAG-3+ cells represented approximately 13% of peripheral blood CD4+ T cells on average. Compared to LAG-3- CD4+ T cells, LAG-3+ CD4+ T cells presented significantly higher Foxp3 and CTLA-4 expression. Directly ex vivo or with TCR stimulation, LAG-3+ CD4+ T cells expressed significantly higher levels of IL-10 and TGF-ß than LAG-3- CD4+ T cells. Interestingly, blocking the LAG-3-MHC class II interaction actually increased the IL-10 expression by LAG-3+ CD4+ T cells. The frequency of LAG-3+ CD4+ T cell was positively correlated with restoration of healthy bone function in long bone fracture patients. These results together suggested that LAG-3 is a marker of CD4+ T cells with regulatory function; at the same time, LAG-3 might have limited the full suppressive potential of Treg cells.

Downregulation of regulatory T cell function in patients with delayed fracture healing.

Bone fracture healing is a multistage regenerative process that requires the collaboration of various cell types, with approximately 5%-10% of fractures not healing properly. Accumulating evidence suggests that dysregulations in the immune system are associated with defective healing. In a cohort of 30 bone fracture patients between 50 and 62 years of age, 8 patients displayed delayed healing. Compared to the 22 normal healing patients, these 8 delayed healing patients presented significantly lower frequencies of CD4+ CD25hi Foxp3+ canonical regulatory T cells immediately following bone fracture and early on during the healing process. The CD4+ CD25+/hi T cells from delayed healing patients also presented reduced capacity to express transforming growth factor beta (TGF-ß), and presented reduced surface expression levels of inhibitory molecules, including CTLA-4 and Lag-3, compared to CD4+ CD25+/hi T cells from normal healing patients. Moreover, CD4+ CD25+/hi T cells from delayed healing patients were less potent in the suppression of CD4+ CD25- autologous conventional T cell proliferation, and presented reduced expansion capacity in response to interleukin (IL)-2 stimulation. Overall, our results demonstrated multiple reductions in regulatory T cell function in delayed healing patients that could produce long-lasting consequences in the bone fracture healing process.

Endotoxin Contributes to Artificial Loosening of Prostheses Induced by Titanium Particles.

BACKGROUND Aseptic loosening of orthopedic implants caused by wear particles is a major cause of joint replacement failure. However, the mechanism of aseptic loosening has not yet been defined. The present study explored whether endotoxin adherent on the titanium (Ti) particles contributes to aseptic loosening. MATERIAL AND METHODS Limulus amebocyte lysate detection was conducted to detect the levels of endotoxin adhered to the Ti particles. A mouse air pouches model was established and mice were divided into 4 groups and injected with phosphate-buffered saline (PBS) or Ti particles suspensions (0.1, 1, 10 mg/mL), following detection of the number of macrophages and the level of endotoxin. Scanning electron microscopy (SEM) was used to characterize the microstructures of Ti particles adhered with endotoxin. RESULTS In vitro experiments showed that the level of endotoxin adhered to the Ti particles was significantly increased after adding LPS back to these "endotoxin-free" particles. In vivo experiments showed that Ti particles injection significantly increased the number of macrophages and the level of endotoxin. CONCLUSIONS In conclusion, these results suggest that adherent endotoxin may play an important role in aseptic loosening induced by Ti particles.

FK506 Attenuates the Inflammation in Rat Spinal Cord Injury by Inhibiting the Activation of NF-κB in Microglia Cells.

FK-506 (Tacrolimus) is a very commonly used immunomodulatory agent that plays important roles in modulating the calcium-dependent phosphoserine-phosphothreonine protein phosphatase calcineurin and thus inhibits calcineurin-mediated secondary neuronal damage. The biological function of FK-506 in the spinal cord has not been fully elucidated. To clarify the anti-inflammatory action of FK-506 in spinal cord injury (SCI), we performed an acute spinal cord contusion injury model in adult rats and hypoxia-treated primary spinal cord microglia cultures. This work studied the activation of NF-κB and proinflammatory cytokine (TNF-a, IL-1b, and IL-6) expression. ELISA and q-PCR analysis revealed that TNF-a, IL-1b, and IL-6 levels significantly increased 3 days after spinal cord contusion and decreased after 14 days, accompanied by the increased activation of NF-κB. This increase was reversed by an FK-506 treatment. Double immunofluorescence labeling suggested that NF-κB activation was especially prominent in microglia. Immunohistochemistry confirmed no alteration in the number of microglia. Moreover, the results in hypoxia-treated primary spinal cord microglia confirmed the effect of FK-506 on TNF-a, IL-1b, and IL-6 expression and NF-κB activation. These findings suggest that FK-506 may be involved in microglial activation after SCI.

Arachidonic acid causes hidden blood loss-like red blood cell damage through oxidative stress reactions.

BACKGROUND: Hidden blood loss (HBL) often occurs in the prosthetic replacement for joint, but the mechanism is still not clear. MATERIALS AND METHODS: This study tried to establish an animal model of HBL by injecting arachidonic acid (AA) into the Sprague-Dawley rats. Different concentrations of AA were injected into the tail veins of the rats, and blood samples were collected before and after administration at 24, 48, and 72 h. A complete blood count was obtained by to find the hemoglobin (Hb) and red blood cell (RBC) count changes. The glutathione peroxidase (GSH-PX) and total superoxide dismutase (T-SOD) activities and hydrogen peroxide (H2O2) levels were detected. The morphological changes of erythrocyte were observed under a polarizing microscope. The absorbance values of the blood samples were tested to determine the presence of ferryl Hb. RESULTS: HBL occurred in the experimental groups when the concentration of AA reached 10 mmol/L; Hb and RBC values decreased sharply at 24- and 48-h postinjection. This was followed by reduced activities of GSH-PX and T-SOD and decreased levels of H2O2. Moreover, the pathologic changes of red cell morphology mainly presented as pleomorphic RBC morphology, including cell rupture. The absorbance values of the blood samples were in accordance with ferryl Hb features. RBC and Hb values were relatively stable at 72 h. The GSH-PX and T-SOD activities and H2O2 levels gradually increased up to a balanced state. CONCLUSIONS: The study concluded that high concentrations of AA can induce oxidative stress reactions in the body, causing acute injury of RBCs, which is closely related to HBL.

Regulatory B cell is critical in bone union process through suppressing proinflammatory cytokines and stimulating Foxp3 in Treg cells.

Bone fractures may result in delayed union (DU) or non-union (NU) in some patients. Evidence suggests that the skewing of the immune system toward the proinflammatory type is a contributing factor. Because B cells were previously found to infiltrate the fracture healing site at abundant levels, we examined the regulatory B cells (Bregs) in DU/NU patients. In bone fracture patients with normal healing, the frequency of interleukin (IL)-10-expressing B cells was significantly upregulated in the early healing process (6 weeks post-surgery) and was downregulated later on (18 weeks post-surgery), whereas in DU/NU patients, the early upregulation of IL-10-expressing B cells was missing. The majority of IL-10-expressing B cells were concentrated in the IgM+ CD27+ fraction in both controls and patients. IgM+ CD27+ B cells effectively suppressed interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), and IL-2 expression from CD4+ T cells, as well as IFN-γ and TNF-α expression from CD8+ T cells. The IgM+ CD27+ B cell-mediated suppression was restricted to the sample from the early healing time point in controls, as the IgM+ CD27+ B cells from normal healing patients later on or from DU/NU patients did not present significant regulatory function. In addition, culturing of CD4+ CD25+ Tregs with IgM+ CD27+ B cells from controls at early healing time point resulted in higher Foxp3 expression, a function absent in controls at later time point, or in DU/NU patients. In conclusion, our results support a role of B cell-mediated regulation early during the bone healing process.