A 3D printing platform has been developed for the treatment of nonunion after fracture via the individualized multi-drug-loaded implants combined with dual-gene transfected bone mesenchymal stem cells (BMSCs) coating. A series of in vivo trials were conducted which closely mimicked real clinical conditions. It has been shown that 3D printed polylactic acid (PLA) implants are exceptional carriers for drugs and can be designed with finely tuned physical morphologies and controllable micropore structures. Their favorable biodegradability, cytotoxicity, blood compatibility, subacute toxicity and low sensitization have confirmed their biocompatibility and pharmaceutical properties. Furthermore, we have demonstrated that the individualized multi-drug-loaded plants combined with dual-gene transfection to BMSCs coating exhibits an anti-nonunion efficacy superior to traditional therapy through a series of in vivo tests according to clinical protocols. The proposed 3D printed drug delivery platform can simultaneously realize individual treatment, multi-drug delivery, long-term sustainable drug release, and non-reoperation in anti-nonunion treatment. Our studies enable the utilization of the 3D printing technique in the treatment of nonunion after fracture and guide future clinical trials. The established techniques and principles can also be adapted to the local therapy of other bone defect.

This study built an OA model in rats by monosodium iodoacetate (MIA) injection to determine the effects and mechanism of the voltage-dependent calcium channel subunit alpha-2/delta-1 (CACNA2D1)-calcitonin gene-related protein (CGRP) pathway in osteoarthritis (OA)-induced ongoing pain. CACNA2D1 expression was measured by qPCR assay, western blotting assay, and immunofluorescence. Pain behaviors in rats were assessed with the measurement of thermal paw withdrawal latency (PWL) and mechanical paw withdrawal threshold (PWT). The expression of CACNA2D1, neuropeptide Y (NPY), activating transcription factor 3 (ATF3), CGRP, protein kinase A (PKA), phosphorylated (p)-PKA, adenylyl cyclase (AC), protein kinase C (PKC), p-PKC, phospholipase C (PLC), and mitogen-activated protein kinase (MAPK) signaling pathway proteins were measured, OA rats had higher CACNA2D1 expression than normal rats. Knockdown of CACNA2D1 led to the elevation of the pain threshold of OA rats, and CACNA2D1 over-expression decreased the pain threshold of normal rats. Moreover, CACNA2D1 over-expression inhibited the expression of CGRP, up-regulated the expressions of NPY, ATF3, p-PKA, AC, p-PKC, PLC, p-Jun N-terminal kinase (JNK), and p-p38, and had no significant effect on phosphorylated extracellular signal-regulated kinase (p-ERK) expression in vivo and in vitro. Using this model of MIA-induced OA, we demonstrated that CACNA2D1 might be involved in the process of pain by modulating the CGRP and AC-PKA/PKC/MAPK signaling pathways in the dorsal root ganglion.

A 3D printing technique has been developed which enables the treatment of osteosarcoma via personalized local chemotherapy. A series of in vivo trials were conducted which closely mimicked real clinical chemotherapeutic conditions. We have shown that 3D printed poly L-lactic acid (PLLA) implants are exceptional carriers for anticancer drugs and can be designed with finely tuned physical morphologies and controllable micropore structures. Their favorable biodegradability, in vitro cytotoxicity, in vitro blood compatibility, in vivo subacute toxicity, and in vivo sensitization tests have confirmed their biocompatibility and pharmaceutical properties. Furthermore, we have demonstrated that local chemotherapy with the assistance of the as-prepared PLLA implant exhibits an anti-osteosarcoma efficacy superior to traditional chemotherapy through a series of in vivo anti-osteosarcoma tests according to clinical protocols. The proposed 3D printed drug delivery system can simultaneously realize individual local chemotherapy, multi-drug delivery, long-term sustainable drug release, and non-reoperation in osteosarcoma treatment. Our studies enable the utilization of the 3D printing technique in the treatment of osteosarcomas and guide future clinical trials. The established techniques and principles can also be adapted to the local chemotherapy of other tumors.

AIM:To clarify the role of proteinase-activated receptor 2 (PAR2) in hepatocellular carcinoma, especially in the process of metastasis.METHODS:PAR2 expression levels were assessed by qRT-PCR and immunohistochemistry (IHC) in patient tissues and in hepatocellular carcinoma cell lines SMMC-7721 and HepG2. Cell proliferation and metastasis were assessed both in vitro and in vitro. Immunoblotting was carried out to monitor the levels of mitogen-activated protein kinase (MAPK) and epithelial-mesenchymal transition markers.RESULTS:The prognosis was significantly poorer in patients with high PAR2 levels than in those with low PAR2 levels. Patients with high PAR2 levels had advanced tumor stage (P = 0.001, chi-square test), larger tumor size (P = 0.032, chi-square test), and high microvascular invasion rate (P = 0.037, chi-square test). The proliferation and metastasis ability of SMMC-7721 and HepG2 cells was increased after PAR2 overexpression, while knockdown of PAR2 decreased the proliferation and metastasis ability of SMMC-7721 and HepG2 cells. Knockdown of PAR2 also inhibited hepatocellular carcinoma tumor cell growth and liver metastasis in nude mice. Mechanistically, PAR2 increased the proliferation ability of SMMC-7721 and HepG2 cells via ERK activation. Activated ERK further promoted the epithelial-mesenchymal transition of these cells, which endowed them with enhanced migration and invasion ability.CONCLUSION:These data suggest that PAR2 plays an important role in the proliferation and metastasis of hepatocellular carcinoma. Therefore, targeting PAR2 may present a favorable target for treatment of this malignancy.

Previous studies showed that HCRP1 is decreased in tumor cells compared with normal tissue, and functions as a tumor suppressor. However, its expression pattern and function in human prostate cancer remain unclear. In this study we examined HCRP1 expression in prostate cancer cell lines via western blotting. Thereafter, we performed CCK-8 assay and matrigel invasion assay after cells were transfected with HCRP1 overexpression plasmid or siRNA. We further investigated the possible mechanism involved in HCRP1’s regulation to prostate cancer cell proliferation and invasion. We found that HCRP1 negatively regulates EGFR activity and expression of its downstream proteins. Moreover, we found that HCRP1 is negatively correlated with multi-drug resistant related proteins after cells were treated with paclitaxel, cisplatin or gefitinib, indicating its inhibiting effect of chemotherapy resistance. In summary, our results provided evidence that HCRP1 is a negative regulator in prostate cancer progression, metastasis and multi-drug resistance.

Percutaneous screw insertion for minimally displaced or reducible acetabular fracture using x-ray fluoroscopy and computer-assisted navigation system has been advocated by some authors. The purpose of this study was to compare intraoperative conditions and clinical results between isocentric C-arm 3-dimensional (Iso-C 3D) fluoroscopy and conventional fluoroscopy for percutaneous retrograde screwing of acetabular anterior column fracture.A prospective cohort study was conducted. A total of 22 patients were assigned to 2 different groups: 10 patients in the Iso-C 3D navigation group and 12 patients in the conventional group. The operative time, fluoroscopic time, time of screw insertion, blood loss, and accuracy were analyzed between the 2 groups.There were significant differences in operative time, screw insertion time, fluoroscopy time, and mean blood loss between the 2 groups. Totally 2 of 12 (16.7%) screws were misplaced in the conventional fluoroscopy group, and all 10 screws were in safe zones in the navigation group. Percutaneous screw fixation using the Iso-C 3D computer-assisted navigation system significantly reduced the intraoperative fluoroscopy time and blood loss in percutaneous screwing for acetabular anterior column fracture.The Iso-C 3D computer-assisted navigation system provided a reliable and effective method for percutaneous screw insertion in acetabular anterior column fractures compared to conventional fluoroscopy.

In this study, we developed a novel poly (lactic-co-glycolic acid)-dextran (PLD)-based nanodelivery system to enhance the anticancer potential of cisplatin (CDDP) in osteosarcoma cells. A nanosized CDDP-loaded PLGA-DX nanoparticle (PLD/CDDP) controlled the release rate of CDDP up to 48 h. In vitro cytotoxicity assay showed a superior anticancer effect for PLD/CDDP and with an appreciable cellular uptake via endocytosis-mediated pathways. PLD/CDDP exhibited significant apoptosis of MG63 cancer cells compared to that of free CDDP. Approximately ~25% of cells were in early apoptosis phase after PLD/CDDP treatment comparing to ~15% for free CDDP after 48h incubation. Similarly, PLD/CDDP exhibited ~30% of late apoptosis cells comparing to only ~8% for free drug treatment. PLD/CDDP exhibited significantly higher G2/M phase arrest in MG63 cells than compared to free CDDP with a nearly 2-fold higher arrest in case of PLD/CDDP treated group (~60%). Importantly, PLD/CDDP exhibited a most significant anti-tumor activity with maximum tumor growth inhibition. The superior inhibitory effect was further confirmed by a marked reduction in the number of CD31 stained tumor blood vessels and decrease in the Ki67 staining intensity for PLD/CDDP treated animal group. Overall, CDDP formulations could provide a promising and most effective platform in the treatment of osteosarcoma.

Development of resistance to gemcitabine is a major concern in bladder cancer therapy, and the mechanism remains unclear. Eg5 has been recently identified as an attractive target in cancer chemotherapy, so novel targeted chemotherapy with Eg5 inhibitor is expected to improve the anticancer effect in gemcitabine-resistant bladder cancer. In this research, RT112-Gr cells were 350-fold less sensitive to gemcitabine than the parental cell lines, while KU7-Gr cells were 15-fold less sensitive to gemcitabine than the parental cell lines. Human OneArray Microarray analysis was performed to obtain broad spectrum information about the genes differentially expressed in RT112 and RT112-Gr cells. The anti-proliferative activity of S(MeO)TLC, an Eg5 inhibitor, was analyzed in RT112-Gr cell lines using a cell viability assay. Furthermore, the inhibitory effect was evaluated in vivo using subcutaneous xenograft tumor model. According to the result of Human OneArray GeneChip, RRM1 and RRM2 were up-regulated, while there was no significant change in Eg5. Trypan blue staining confirmed that in S(MeO)TLC and Gemcitabine combining S(MeO)TLC group cell viability were significantly decreased in RT112-Gr cells as compared with other groups. S(MeO)TLC and S(MeO)TLC+gemcitabine groups prominently suppressed tumor growth in comparison with other groups' in vivo. There were no significant differences in S(MeO)TLC and gemcitabine+S(MeO)TLC group in the effect of inhibition of bladder cancer in vivo and in vitro. Our data collectively demonstrated that S(MeO)TLC represents a novel strategy for the treatment of gemcitabine resistant bladder cancer.

The aim of the present investigation was to formulate a docetaxel (DTX) and gemcitabine (GEM) co-loaded PEGylated liposome (DTX/GEM-L) to increase the therapeutic efficacy in osteosarcoma (OS). 2-Hydroxypropyl-gamma-cyclodextrin/DTX inclusion complex was made to increase DTX aqueous solubility. DTX/GEM-L was characterized for morphological shape and size parameters. Release study showed a sustained release pattern for both the drugs. The nanocarriers based combinational drug significantly increased the cytotoxic effect than the free drug combination at the same concentration. The cell cycle analysis showed a predominant G2/M phase arrest for combinational drug. Importantly, more than 20% of cells were in late apoptosis chamber for DTX/GEM-L treatment with significant proportion of cells in the early apoptosis and necrotic phases. The antitumor efficacy was tested in MG63 cancer cell bearing xenograft nude mice. Results showed that DTX/GEM-L significantly reduced the tumor burden comparing to that of free combination cocktail. The PEGylated liposome successfully delivered the anticancer drugs in the osteosarcoma tumor interstitial spaces via EPR effect. DTX/GEM-L showed excellent safety profile along with the remarkable tumor suppression ability. Overall, results suggest that nanocarriers-based delivery system remarkably enhanced the apoptosis and cytotoxicity and increased the potency of combinational drug regimen. (C) 2014 Published by Elsevier B.V.

Ubenimex is a low-molecular-weight dipeptide with the ability to inhibit aminopeptidase N (APN) activity, enhance the function of immunocompetent cells and confer antitumor effects. We sought to characterize the effects of ubenimex on renal cell carcinoma (RCC). The 786-O and OS-RC-2 human RCC cell lines were positive for APN expression and ubenimex decreased APN activity without affecting the expression. Ubenimex suppressed the proliferation of both cell lines in a concentration‑dependent manner, as assessed by curve growth analysis and WST-8 proliferation assay. Wound healing and Matrigel invasion assays demonstrated that the migration and invasion of the RCC cells were also markedly suppressed by ubenimex. Furthermore, ubenimex increased the mortality of both RCC cell lines as determined by the LDH cytotoxicity assay. This affect was accompanied by increased levels of LC3B with no apparent effect on Caspase3; and we observed that autophagy increased significantly after ubenimex treatment in both RCC cell lines by electron microscopy. Moreover, rapamycin enhanced the cytotoxic effect of ubenimex, while 3-methyladenine reversed the effect, indicating that ubenimex cytotoxicity occured through an autophagy-related mechanism. To further assess the potential applicability of ubenimex in the treatment of RCC, we performed immunohistochemistry using tissue microarrays representing 76 RCC patients that underwent radical nephrectomy. The results showed that APN was expressed in most, but not all of the RCC tissues and that the expression was reduced in RCC as compared to the normal kidney tissues, suggesting a potential role for APN in RCC development. Collectively, these results indicated that ubenimex inhibits proliferation, migration and invasion of RCC cells. Ubenimex may induce autophagy, which may be associated with its effect on the growth arrest and the cell death of RCC cells.

The aim of the present study was to provide a theoretical and experimental foundation on the differentiation of stem cells through the induction of multiple genes. The lentiviral vector carrying TGF-β1 and IL-10 genes was transfected to bone marrow-derived mesenchymal stem cells (BMSCs) which differentiated into chondrogenesis. Healthy New Zealand white rabbits, 2-3 months of age were used in the present study. A 6-8 ml of bone marrow was isolated from the iliac and tibial shaft of each rabbit. The BMSCs suspension was aspired following centrifugation of the bone marrow by percoll separating medium. The BMSCs were primarily cultured and subcultured in vitro, then divided into four groups according to the difference of lentivirus vectors: group A, receiving transforming growth factor β1 (TGF‑β1); group B, receiving TGF-β1 and Interleukin-10 (IL-10); group C, empty vector transfection; and group D, receiving no cell growth factor. Fluorescence expression was detected 12 h after transfecting the lentiviral vector carrying the TGF-β1 and IL-10 gene to BMSCs. The transfection efficiency was approximately 70% with a MOI=100 after 96 h. Expression of SOX-9 aggrecan and Type Ⅱ collagen in groups A-E on day 7 and 14 was detected by RT-PCR and western blot analysis. The expression level of three genes expressed in groups A and C were higher compared to the expression in groups B, D and E. The expression level of the three genes expressed in group B was higher compared to the expression in group D. The expression level of three genes expressed in group A and C showed no statistical difference. Cytokines therefore play an important role in cell proliferation and chondrogenic differentiation. TGF-β1 has a synergistic effect in the differentiation. In addition, IL-10 may have a protective role in the restoration of cartilaginous tissue.

Renal cell carcinoma (RCC) is one of the most common malignancies in adults, and there is still no acknowledged biomarker for its diagnosis, prognosis, recurrence monitoring, and treatment stratification. Besides, little is known about the post-translational modification (PTM) of proteins in RCC. Here, we performed quantitative proteomic analysis on 12 matched pairs of clear cell RCC (ccRCC) and adjacent kidney tissues using liquid chromatography-tandem mass spectrometry (nanoLCMS/MS) and Progenesis LC-MS software (label-free) to identify and quantify the dysregulated proteins. A total of 1872 and 1927 proteins were identified in ccRCC and adjacent kidney tissues, respectively. Among these proteins, 1037 proteins were quantified by Progenesis LC-MS, and 213 proteins were identified as dysregulated proteins between ccRCC and adjacent tissues. Pathway analysis using IPA, STRING, and David tools was performed, which demonstrated the enrichment of cancer-related signaling pathways and biological processes such as mitochondrial dysfunction, metabolic pathway, cell death, and acetylation. Dysregulation of two mitochondrial proteins, acetyl-CoA acetyltransferase 1 (ACAT1) and manganese superoxide dismutase (MnSOD) were selected and confirmed by Western blotting and immunohistochemistry assays using another 6 pairs of ccRCC and adjacent tissues. Further mass spectrometry analysis indicated that both ACAT1 and MnSOD had characterized acetylation at lysine residues, which is the first time to identify acetylation of ACAT1 and MnSOD in ccRCC. Collectively, these data revealed a number of dysregulated proteins and signaling pathways by label-free quantitative proteomic approach in RCC, which shed light on potential diagnostic or prognostic biomarkers and therapeutic molecular targets for clinical intervention of RCC.

Eg5 is critical for mitosis and overexpressed in various malignant tumors, which has now been identified as a promising target in cancer therapy. However, the anti-cancer activity of Eg5 inhibitor in renal cell carcinoma (RCC) remains an open issue. In this paper, we evaluated, for the first time, the therapeutic benefit of blocking Eg5 by S-(methoxytrityl)-l-cysteine (S(MeO)TLC) in RCC both in vitro and vivo. The expression of Eg5 was examined in clinical tissue samples and various kidney cell lines, including 293T, 786-0, and OS-RC-2. The anti-proliferative activity of Eg5 inhibitors, (S)-trityl-l-cysteine (STLC) and S(MeO)TLC, was evaluated by a cell viability assay. An apoptosis assay with Hoechst nuclear staining and flow cytometry was applied to investigate the efficacy of the S(MeO)TLC, which is more potent than STLC. Immunofluorescence was used to research the possible mechanism. Furthermore, in vivo studies were performed by using subcutaneous xenograft models, which were used to confirm its role as a potential anti-neoplastic drug. The Eg5 expression was detected in kidney cell lines and RCC tissues, which was low in normal kidney samples. STLC and S(MeO)TLC exhibited their optimal anti-proliferative activity in 72 h, and cells treated with S(MeO)TLC presented characteristic monoastral spindle phenotype in 24 h and apoptotic cells in 48 h. In vivo, S(MeO)TLC effectively suppressed tumor growth in subcutaneous xenograft models. Inhibition of Eg5 represses the proliferation of RCC in vitro and in vivo. All these findings collectively demonstrate that S(MeO)TLC, a potent Eg5 inhibitor, is a promising anti-cancer agent for the treatment of RCC.