Detrimental alteration of mesenchymal stem cells by an articular inflammatory microenvironment results in deterioration of osteoarthritis.

BACKGROUND: Articular injection of mesenchymal stem cells (MSCs) has been applied to treat knee osteoarthritis (kOA), but its clinical outcomes are controversial. This study investigated whether an articular inflammatory microenvironment (AIM) impacts MSC-based therapy in a rat model of kOA. METHODS: The biological change of MSCs and the functional change of MSCs on chondrocytes were evaluated under AIM. The key mediator and mechanism for the AIM impact on MSC therapy were explored via gain- and loss-of-function approaches. RESULTS: The results showed that MSCs exerted potent anti-kOA effects in vivo and in vitro, but that this therapy become chondrodestructive if a chronic AIM was present. Mechanistically, the overexpression of MMP13 in the injected MSCs via a MAPKs-AP1 signaling axis was revealed as the underlying mechanism for the detriment outcome. CONCLUSIONS: This study thus clarifies recent clinical findings while also suggesting a means to overcome any detrimental effects of MSC-based therapy while improving its efficacy.

Fuzi decoction ameliorates pain and cartilage degeneration of osteoarthritic rats through PI3K-Akt signaling pathway and its clinical retrospective evidence.

BACKGROUND: Osteoarthritis (OA) is a difficult disease but the clinic lacks effective therapy. As a classic formula of traditional Chinese medicine (TCM), Fuzi decoction (FZD) has been clinically applied for treating OA-related syndromes, but its anti-OA efficacy and mechanism remain unclear. PURPOSE: To experimentally and clinically determine the anti-OA efficacy of FZD and clarify the underlying mechanism. METHODS: UPLC/MS/MS was applied to identify the main components of FZD. A monoiodoacetate (MIA)-induced OA rat model was employed to evaluate the in vivo efficacy of FZD against OA, by using pain behavior assessment, histopathological observation, and immunohistochemical analysis. Primary rat chondrocytes were isolated to determine the in vitro effects of FZD by using cell viability assay, wound healing assay, and real-time PCR (qPCR) analysis on anabolic/catabolic mRNA expressions. RNA sequencing (RNA-seq) and network pharmacology analysis were conducted and the overlapping data were used to predict the mechanism of FZD, followed by verification with qPCR and Western blot assays. Finally, a retrospective analysis was performed to confirm FZD's efficacy and safety in OA patients. RESULTS: The UPLC/MS/MS result showed that FZD contained atractylenolide I, benzoylhypaconitine, benzoylmesaconitine, benzoylaconitine, hypaconitine, mesaconitine, aconitine, lobetyolin, paeoniflorin, and pachymic acid. The in vivo data showed that FZD restored the cartilage degeneration in MIA-induced OA rats by ameliorating pain behavior parameters, recovering histopathological alterations, benefitting cartilage anabolism (up-regulating Col2 expression), and suppressing catabolism (down-regulating MMP13 and Col10 expressions). The in vitro data showed that FZD increased cell viability and wound healing capacity of chondrocytes, and restored the altered expressions of anabolic and catabolic genes of chondrocytes. The overlapping results of RNA-seq and network pharmacology analysis suggested that PI3K/Akt signaling mediated the anti-OA mechanism of FZD, which was verified by qPCR and Western blot experiments. Clinically, the anti-OA efficacy and safety of FZD were confirmed by the retrospective analysis on OA patients. CONCLUSION: The scientific innovation of this study was the determination of anti-OA efficacy of FZD by experimental and clinical evidence and the discovery of its mechanism by integrated RNA-seq, network pharmacology, and molecular experiments, which suggests FZD as a promising TCM agency for OA treatment.

Mirabilite with Ice Pack after Total Knee Arthroplasty: A Randomized Controlled Trial Study.

BACKGROUND: Total knee arthroplasty (TKA) is a well-established procedure for end-stage arthritis of the knee with complications such as swelling and pain. The aim of this study is to estimate the effect of mirabilite with ice pack versus ice pack in relieving pain, swelling, range of motion (ROM), and serum CRP level on patients after TKA. METHODS: Eighty patients undergoing primary unilateral TKA were randomly assigned to two groups (MIP group and WIP group). We used VAS to measure knee pain at 24 h, 48 h, and 72 h after the surgery, respectively. Knee swelling degree was evaluated by measuring the girth of the leg at the center of the patella and 10 cm above and below it at the same frequency. The active ROM of the knee was measured by using a universal goniometer of plastic material at 72 h postoperatively. The serum CRP level was also measured at 72 h postoperatively. RESULTS: The MIP group showed statistically significant lower knee girth at 48 h (p < 0.05) and 72 h (p < 0.05) postoperatively and VAS score at 72 h (p=0.018) postoperatively after TKA than the WIP group. The ROM of the MIP group was significantly wider than the WIP group (p=0.024). The CRP level (p=0.036) and length of stay (LOS) (p=0.037) of the MIP group were significantly lower than the WIP group. CONCLUSION: Mirabilite with ice pack after TKA showed superiority in relieving pain, reducing swelling, and improving ROM than ice pack only.

Growth factors-based beneficial effects of platelet lysate on umbilical cord-derived stem cells and their synergistic use in osteoarthritis treatment.

Poor viability of mesenchymal stem cells (MSCs) at the transplanted site often hinders the efficacy of MSCs-based therapy. Platelet lysate (PL) contains rich amounts of growth factors, which benefits cell growth. This study aimed to explore how human PL benefits umbilical cord-derived MSCs (huc-MSCs), and whether they have synergistic potential in osteoarthritis (OA) treatment. As quality control, flow cytometry and specific staining were performed to identify huc-MSCs, and ELISA was used to quantify growth factors in PL. CCK-8 and flow cytometry assays were performed to evaluate the effects of PL on the cell viability and cell cycle progression of huc-MSCs. Wound healing and transwell assays were conducted to assess the migration of huc-MSCs. RNA sequencing, real time PCR, and Western blot assays were conducted to explore the growth factors-based mechanism of PL. The in vitro results showed that PL significantly promoted the proliferation, cell cycle, and migration of huc-MSCs by upregulating relevant genes/proteins and activating beclin1-dependent autophagy via the AMPK/mTOR signaling pathway. The main growth factors (PDGF-AA, IGF-1, TGF-ß, EGF, and FGF) contributed to the effects of PL in varying degrees. The in vivo data showed that combined PL and huc-MSCs exerted significant synergistic effect against OA. The overall study determined the beneficial effects and mechanism of PL on huc-MSCs and indicated PL as an adjuvant for huc-MSCs in treating OA. This is the first report on the growth factors-based mechanism of PL on huc-MSCs and their synergistic application. It provides novel knowledge of PL's roles and offers a promising strategy for stem cell-based OA therapy by combining PL and huc-MSCs.

Theaflavin Induces Apoptosis of A375 Human Melanoma Cells and Inhibits Tumor Growth in Xenograft Zebrafishes Through P53- and JNK-Related Mechanism.

Theaflavin (TF) is a major active pigment and polyphenol of tea, possessing anti-cancer activities. However, little is known about its activity and mechanism on melanoma cells. To fill this gap, we conducted in vitro experiments (cell viability assay, morphology observation, DAPI staining, and flow cytometry) and in vivo experiment by using a xenograft model of larval zebrafishes. Real-time PCR (qPCR) and Western blot (WB) analyses were conducted to explore the mechanism of TF. The in vitro data showed that TF exerted significant anti-proliferative and pro-apoptotic effects on A375 cells in a concentration-dependent manner. In vivo, TF significantly inhibited A375 tumor growth in larval zebrafishes at 0.67 and 2.0 µg/ml (1.3 to 3.9 µM). qPCR and WB data showed that TF significantly activated the P53 pathway-related proteins (ATM, CHK1/2, P53, and CASP8/3) and the JNK pathway-related proteins (ASK1, JNK, and C-JUN) through phosphorylation and cleavage, followed by activation of pro-apoptotic molecules (PARP, BAX, BIM, PUMA, and P53). In sum, TF possessed cytotoxic pro-apoptotic and tumor-inhibitory effects on A375 cells through activations of P53 and JNK pathways. This is the first report on TF regarding its effects and mechanism on A375 cells, making it a promising candidate of natural products for clinical treatment of melanoma.

Expression Profile Analysis of Differentially Expressed Circular RNAs in Steroid-Induced Osteonecrosis of the Femoral Head.

BACKGROUND: A growing number of studies have suggested that circular RNAs (circRNAs) serve as potential diagnostic biomarkers in many diseases. However, the role of circRNAs in steroid-induced osteonecrosis of the femoral head (SONFH) has not been reported. METHODS: Secondary sequencing was performed to profile circRNA expression in peripheral blood samples from three SONFH patients and three healthy individuals. We confirmed our preliminary findings by qRT-PCR. Bioinformatics analysis was conducted to predict their functions. RESULTS: The result showed 345 dysregulated circRNAs. qRT-PCR of eight selected circRNAs preliminarily confirmed the results, which were consistent with RNA sequencing. Bioinformatics analyses were performed to predict the functions of circRNAs to target the genes of miRNAs and the networks of circRNA-miRNA-mRNA interactions. CONCLUSIONS: This study provides a new and fundamental circRNA profile of SONFH and a theoretical basis for further studies on the functions of circRNAs in SONFH.

MiR-495 targeting dvl-2 represses the inflammatory response of ankylosing spondylitis.

Ankylosing spondylitis (AS) is a type of rheumatic inflammatory disease. miRNAs participate in the process of regulating inflammatory response and bone differentiation. Herein, we aimed to test the effect of miR-495 on AS. The serum and tissues were obtained from traumatic fracture (health) and AS patients. The human fibroblast-like synovial (HFLS) cells were extracted from AS tissues. The contents of inflammatory factors and dishevelled 2 (DVL-2) were examined using enzyme-linked immunosorbent assay (ELISA). The ossification factors were detected by immunohistochemistry assay. Osteoclast was assessed by tartaric acid acid phosphatase (TRAP) assay. The cell viability and luciferase activity were measured using cell counting kit-8 (CCK-8) and dual-luciferase reporter system. The levels of factors were evaluated using quantitative real-time PCR (qRT-PCR) and western blotting. DVL-2 was a target gene for miR-495, according to the MicroRNA.org website and luciferase activity assay. The expressions of miR-495 and DVL-2 were negative corrected in AS. miR-495 and si-DVL-2 did not affect the cell viability. miR-495 and si-DVL-2 obviously inhibited inflammatory response by down-regulating tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) and IL-6 levels, and facilitated bone differentiation by up-regulating osteoprotegerin (OPG) and receptor activator for nuclear factor-κB ligand (RANKL) levels in HFLS cells. Besides, miR-495 and si-DVL-2 increased the expression of wnt3a, runt-related transcription factor 2 (RUNX-2) and ß-catenin and reduced the phosphorylation of ß-catenin. Collectively, miR-495 depressed inflammatory response and promoted bone differentiation of HFLS cells, and this was accompanied by mediating wnt/ß-catenin/Runx-2 pathway by targeting DVL-2.

Chondroprotective effects of platelet lysate towards monoiodoacetate-induced arthritis by suppression of TNF-α-induced activation of NF-ĸB pathway in chondrocytes.

Platelet lysate (PL) contains a cocktail of growth factors that actively participates in cartilage repair. This study was designed to determine the effect and mechanism of PL on osteoarthritis (OA). An arthritis model was established to mimic human OA by intra-articular injection of monoiodoacetate (MIA) to Sprague Dawley (SD) rats. The model was weekly treated with PL by intra-articular injection. Thermal withdrawal latency, mechanical withdrawal threshold, and treadmill gait were tested for pain behavior observation. Histopathological and immunohistochemical analyses were conducted for evaluating cartilage degradation. Real time PCRs and Western blots were conducted to elucidate the mechanism of PL on primary chondrocytes. Results showed that, in vivo, PL significantly attenuated pain symptoms and exerted chondrocyte-protective and extracellular matrix (ECM)-modifying effect on the arthritic cartilage in a dose-dependent manner. The in situ expressions of type II Collagen (Col2) and matrix metalloproteinase 13 (Mmp13) in the arthritic cartilage was abnormal and was restored by PL. In vitro, PL significantly restored tumor necrosis factor α (TNF-α)-suppressed anabolic gene expression (Col2 and aggrecan) and TNF-α-increased catabolic gene expression (Col10, Mmp13, Adamts5, and Adamts9) in chondrocytes. The effects were mediated by TNF-α downstream signaling, including inhibition of NF-κB and c-Jun activities. This study provides certain knowledge of anti-OA effect and TNF signaling-related mechanism of PL, placing it as a promising and alternative option for OA therapy in the future.

Elevated expression of matrix metalloproteinase-3 in human osteosarcoma and its association with tumor metastasis.

PURPOSE: Matrix metalloproteinase-3 (MMP-3) is one of the several MMPs that is associated with malignant tumors of breast, colon, cervix and lung, where its expression has been correlated with tumor invasion and metastasis. However, the role of MMP-3 in metastasis of osteosarcoma has not yet been explored. METHODS: MMP-3 expression in 15 primary and metastatic osteosarcomas with case-matched adjacent non-tumor tissue was assessed by immunohistochemistry and quantitative RT-PCR. Further, MMP-3 mRNA and protein levels were also determined in osteoblast and osteosarcoma cell lines. Additionally, migration and invasion assays were performed in MMP-3 knockdown cells. RESULTS: MMP-3 was expressed in 86.6% (13/15) of the osteosarcoma patients and its expression was significantly higher in metastatic tumors as compared to the primary osteosarcoma tumor tissues. Furthermore, osteosarcoma cell lines showed higher MMP-3 expression as compared to osteoblast cell lines. siRNA-mediated MMP-3 knockdown in osteosarcoma cell lines significantly inhibited their migration and invasion properties. CONCLUSION: Our results demonstrated that MMP-3 expression is deregulated in osteosarcomas and this potentially contributes to metastasis and might be a promising marker for the prognosis and therapy of metastatic osteosarcoma.

Elevated expression of matrix metalloproteinase-3 in human osteosarcoma and its association with tumor metastasis.

PURPOSE: Matrix metalloproteinase-3 (MMP-3) is one of the several MMPs that is associated with malignant tumors of breast, colon, cervix and lung, where its expression has been correlated with tumor invasion and metastasis. However, the role of MMP-3 in metastasis of osteosarcoma has not yet been explored. METHODS: MMP-3 expression in 15 primary and metastatic osteosarcomas with case-matched adjacent non-tumor tissue was assessed by immunohistochemistry and quantitative RT-PCR. Further, MMP-3 mRNA and protein levels were also determined in osteoblast and osteosarcoma cell lines. Additionally, migration and invasion assays were performed in MMP-3 knockdown cells. RESULTS: MMP-3 was expressed in 86.6% (13/15) of the osteosarcoma patients and its expression was significantly higher in metastatic tumors as compared to the primary osteosarcoma tumor tissues. Furthermore, osteosarcoma cell lines showed higher MMP-3 expression as compared to osteoblast cell lines. siRNA mediated MMP-3 knockdown in osteosarcoma cell lines significantly inhibited their migration and invasion properties. CONCLUSION: Our results demonstrated that MMP-3 expression is deregulated in osteosarcomas and this potentially contributes to metastasis and might be a promising marker for the prognosis and therapy of metastatic osteosarcoma.

Programmed cell death 2 functions as a tumor suppressor in osteosarcoma.

OBJECTIVES: To investigate the role of programmed cell death 2 (PDCD2) in osteosarcoma (OS), along with correlations between PDCD2 and CD4(+)/CD8(+). METHODS: Sprague-Dawley (SD) rats were randomly assigned to control group and OS group. The OS group rats were subjected to induce models of OS by transplantation with UMR106 cells. Peripheral blood was collected to test the percentages of the CD4(+) and CD8(+) cell subsets using flow cytometry (FCM). Western blotting was performed to determine the PDCD2 protein level. The correlations between PDCD2 and CD4(+)/CD8(+) were analyzed by Pearson correlation coefficient. Besides, specific small interfering RNAs (siRNA) against PDCD2 and nonspecific (NS)-siRNA were transfected into UMR106 cells. Cell viability and invasive ability were determined after transfection. RESULTS: CD4(+) cells percentages were significantly decreased in the OS group, while CD8(+) cells were significantly increased (P < 0.05). The PDCD2 protein levels were markedly lower than that in the control group (P < 0.05). Additionally, PDCD2 was positively correlated with CD4(+) (R(2) = 0.66, P < 0.05), but was negatively correlated with CD8(+) (R(2) = -0.94, P < 0.05). Moreover, the cell viability and invasion ability were significantly higher than that in the control group and the NS siRNA group after transfection with PDCD2 siRNA (P < 0.05). CONCLUSION: These results suggest that PDCD2 is involved in the pathogenesis of OS, and PDCD2 may play an important role in tumor suppression. These mechanisms might be related to immune response induced by CD4(+) and CD8(+) T cells.

Serum bone-specific alkaline phosphatase as a biomarker for osseous metastases in patients with malignant carcinomas: a systematic review and meta-analysis.

OBJECTIVE: Bone metastasis was common in patients with malignant tumors. The purpose of this study was to investigate the serum bone-specific alkaline phosphatase (B-ALP) as a biomarker in the diagnosis of osseous metastases in patients with cancers. METHODS: We searched the databases of Pubmed, Cochrane Library, Medline, CNKI and Wanfang to screen the relevant articles about the serum B-ALP detection in the diagnosis of osseous metastases in patients with malignant carcinomas. The pooled sensitivity, specificity, summary receiver operating characteristic (SROC) curve were calculated by STATA12.0 software. RESULTS: Nineteen trials with 3 268 subjects were finally included in this study. The mean level of serum B-ALP was 41.50 ± 26.61 µg/L (216.90 ± 139.00U/L) in patients with osseous metastases and 14.49 ± 5.52 µg/L (103.30 ± 39.44 U/L) in patients without osseous metastases. The serum level of B-ALP was significant higher in the osseous metastases group than that in the control group (P < 0.05); The pooled sensitivity and specificity for diagnosis of osseous metastases were 0.74 with its 95% confidence interval (95% CI) of 0.62-0.83 and 0.80 (95% CI: 0.67-0.89), respectively. The area under the SRCO was 0.86 (95% CI: 0.83-0.89). CONCLUSION: Serum B-ALP can be a promising biomarker for detection of osseous metastases in patients with cancers.