Bangcheng Yang

Characterization of surface oxide films on titanium and adhesion of osteoblast

The relationship between surface characteristics of titanium and initial interactions of titanium-osteoblasts was investigated. Titanium plates were heat-treated in different oxidation atmospheres. The third passage rabbit osteoblasts were cultured on the titanium plates for 24h. After the heat-treatment, the crystal structure of the surface oxide films on titanium was identified using X-ray diffractometer and X-ray photoelectron spectroscopy (XPS). The surface roughness of titanium was measured with a profilometer. The surface energy was obtained by measurement of contact angles and calculation with Owens-Wendt-Kaebles equation. The amount of surface hydroxyl (OH)(s) groups was examined using XPS. The change of binding energy of the some elements on the substrate surface suggested that the interactions between the cells and the titanium involved chemical reactions. The greater surface roughness, higher surface energy and more surface hydroxyl groups resulted in greater numbers of adhered osteoblasts and higher cell activity. Compared to the acidic hydroxyl (OH)(a) groups in (OH)(s) groups and the dispersion component of the total surface energy, the basic hydroxyl (OH)(b) groups and the polar component play more important roles in the osteoblast-titanium interaction.

The order of calcium and phosphate ion deposition on chemically treated titanium surfaces soaked in aqueous solution

The mechanism of apatite deposition on chemically treated Ti surfaces still is being studied. In this study, simulated body fluid, calcium aqueous solution, phosphate aqueous solution, and accelerated calcification solution are used as media to investigate the order of calcium and phosphate ion deposition on chemically treated Ti surfaces. The results of inductively coupled plasma spectra, scanning electron microscopy, and energy dispersive X-ray analysis show that calcium deposition is the prerequisite for phosphate ion deposition.

Collagen nanofilm immobilized on at surfaces by electrodeposition method.

A simple electrodeposition method is presented for the preparing of collagen nanofilms (EAT) on anodic oxidized titanium surfaces (AT). The nanofilms were observed by scanning electron microscopy and atomic force microscopy. Functional TiOx layers with anionic groups of --PO(4), --SO(4) and --OH were investigated on the AT surface by X-ray photoelectron spectroscopy; X-ray diffraction results indicated that the AT surface was composed mainly of anatase and rutile. The bioactive electrodeposited TiOx layers on the AT surface showed lower water contact angles and higher surface energy than pure titanium surfaces (CT) and displayed higher collagen molecule immobilization.

Preparation of a HA/collagen film on a bioactive titanium surface by the electrochemical deposition method*

A hydroxyapatite (HA) film with or without collagen was electrochemically deposited on a bioactivated titanium metal prepared by acid-alkali treatment, so as to improve the biocompatibility of bioactive titanium metals. The cell response of the film was studied with MG63 osteoblasts culture. It was found that the hydroxyapatite formation in the film during the deposition process was inhibited when collagen was added in the electrolyte. More hydroxyapatite with and without collagen could be deposited on the bioactivated titanium than the control titanium metal without treatment, which indicated that the bioactivation process before the electrochemical deposition could accelerate the deposition. The abilities of cell attachment and proliferation were improved by the film especially in the group containing collagen, and the film on the bioactivated metal had higher cell response ability than that on the titanium without treatment. The results indicated that the hydroxyapatite/collagen film could improve the biocompatibility of the bioactive titanium metal surface, and the bioactivation surface modification could further regulate the film and its cell response. It is possible to get a titanium surface with higher bioactivity than the traditional bioactive titanium surface by combining the bioactivation surface modification and electrochemical deposition HA/collagen film.

The controllable lanthanum ion release from Ca-P coating fabricated by laser cladding and its effect on osteoclast precursors

Several studies have suggested that rare earth oxides can improve properties of bioceramic coating, and bone resorption of osteoclast can be inhibited by rare earth ion releasing certain concentration. However, the effects of lanthanum ion (La3+) released from Ca-P coating on osteoclast precursors is not clear. In this work, La2O3-doped gradient bioceramic coatings were fabricated on Ti alloy (Ti-6Al-4V) by laser cladding with mixed powders of CaHPO4·2H2O, CaCO3 and La2O3. And the bioactivity, mechanical properties and the La3+ release from coating were investigated in vitro. Human osteosarcoma cells (MG63) were used as a cell model to evaluate the biocompatibility of coatings. Mouse macrophage RAW264.7 cells were cultured on coatings to study the effect of La3+ release from Ca-P coating on osteoclast precursors. The XRD results reveal that the amount of HA + TCP reaches maximum (2θ = 32-33°) when the content of La2O3 is 0.6 wt%, and the proliferation of MG63 cells is up to highest value, which indicates that compared with other groups, the bioceramic coating with 0.6 wt% La2O3 is of best biocompatibility. Furthermore, the differentiation of RAW264.7 cells into osteoclast could be inhibited by controllable releasing La3+ from Ca-P coating when soaked in SBF, which demonstrates that controllable La3+ release from Ca-P coating is an effective method to prevent osteoclast formation. And a prospective therapy is provided to cure the disease of wear debris in replacement of artificial joint.

AB0003 IL-23R and IL-17A Polymorphisms Correlate with Susceptibility of Ankylosing Spondylitis in Chinese Han Population

Background The association between the IL-23R and IL-17A polymorphisms and ankylosing spondylitis (AS) in a Chinese Han population is still unclear. Objectives The purpose of this study is to detect the association between IL-23R and IL-17A polymorphisms and AS. Methods A case–control study consisting of 486 AS patients and 480 healthy controls was performed. We used the high-resolution melting methods (HRM) to genotype the selected five single nucleotide polymorphisms (SNPs), four of them (rs6693831, rs7517847, rs1884444, rs10889677) on the IL-23R gene and one (rs2275913) on the IL-17A gene. Meanwhile the laboratory indexes (RBC, WBC, PLT, MONO%, LYMPH%, NEUT%, ALB, ALP, ALT, AST, BUN, CHOL, CK, CREA, Cys-C, DBIL, GGT, GLB, GLU, HBDH, HDL-C, IBIL, LDH, LDL-C, TBIL, CRP, C3 and C4) were recorded. Results In this study, patients with genotype CC (p=8.574E-8) and allele C (p=3.206E-31) on SNP rs6693831 (IL-23R) showed decreased risk of AS. The genotype TT (p=4.551E-6) and allele T (p=0.02) on SNP rs1884444 (IL-23R) showed significant lower risk of AS. Individuals carrying the allele A on SNP rs2275913 (IL-17A) showed higher risk of AS [p=0.04, OR (95%CI) = 0.825 (0.690–0.987)]. Significant higher level of CRP was found among AS patients with genotype CT on rs6693831 (χ2=7.633, P=0.030). Conclusions We first demonstrated that rs6693831 and rs1884444 on IL-23R gene and rs2275913 on IL-17A gene are genetic susceptibility factor for AS. We also noticed that patients with CT genotype on rs6693831 have higher CRP level. References Jiang D, Wubuli A, Hu X, Ikramullah S, Maimaiti A, et al. (2015) The variations of IL-23R are associated with susceptibility and severe clinical forms of pulmonary tuberculosis in Chinese Uygurs. BMC Infect Dis 15: 550. Akbal A, Resorlu H, Gokmen F, Savas Y, Zateri C, et al. (2015) The relationship between C-reactive protein rs3091244 polymorphism and ankylosing spondylitis. Int J Rheum Dis. Almodovar R, Rios V, Ocana S, Gobbo M, Casas ML, et al. (2014) Association of biomarkers of inflammation, cartilage and bone turnover with gender, disease activity, radiological damage and sacroiliitis by magnetic resonance imaging in patients with early spondyloarthritis. Clin Rheumatol 33: 237–241. Wright PB, McEntegart A, McCarey D, McInnes IB, Siebert S, et al. (2015) Ankylosing spondylitis patients display altered dendritic cell and T cell populations that implicate pathogenic roles for the IL-23 cytokine axis and intestinal inflammation. Rheumatology (Oxford). Acknowledgement We are grateful to the participating AS patients and their families. This research was sponsored by the National Natural Science Foundation of China (Nos. 81301496, and 81202354). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Disclosure of Interest B. Yang Shareholder of: No, Grant/research support from: No, Consultant for: No, Employee of: No, Paid instructor for: No, Speakers bureau: No, J. Chen: None declared, L. Wang: None declared

THU0365 Light rs1077667 G>A Gene Polymorphism Is Associated with Susceptibility To Ankylosing Spondylitis in A Chinese Han Population

Background Single-nucleotide polymorphisms (SNPs) are single base-pair changes which will lead to the structural changes of mRNA and get involved in the pathogenesis process of ankylosing spondylitis (AS). LIGHT is indicated to be a inflammatory mediator in AS. Meanwhile the SNP rs1077667 G>A, which was located on LIGHT, is associated with the expression of LIGHT. Objectives This study explored the relationship between AS and the SNP rs1077667 G>A in a Han Chinese population. Meanwhile we explored the relationship between the SNP and the laboratory characteristic of AS patients. Methods A case-control study with 497 subjects diagnosed AS and 387 healthy controls to compare their genotype and gene frequencies was delivered. SNP is identified by high-resolution melting methods (HRM). Clinical characteristics of patients with AS and controls were registered at the same time. Results Statistical significance was found in both co-dominant model (GG vs. GA vs. AA) (P=0.000004) and allele [p=4.59E-08, OR (95%CI) = 0.740 (0.663–0.827)] between LIGHT rs1077667 G>A and the risk of AS. However, no significant association was found between the SNP and the laboratory indexes. Conclusions This is the first study to address the association between the LIGHT rs1077667 G>A polymorphisms and AS, and it suggests a potential pathogenic factor for AS. References Smith JA (2015) Update on ankylosing spondylitis: current concepts in pathogenesis. Curr Allergy Asthma Rep 15: 489 Hreggvidsdottir HS, Noordenbos T, Baeten DL (2014) Inflammatory pathways in spondyloarthritis. Mol Immunol 57: 28–37. Wen W, Kato N, Hwang JY, Guo X, Tabara Y, et al. (2016) Genome-wide association studies in East Asians identify new loci for waist-hip ratio and waist circumference. Sci Rep 6: 17958. Acknowledgement We are grateful to the participating AS patients and their families. This research was sponsored by the National Natural Science Foundation of China (Nos. 81301496, and 81202354). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Disclosure of Interest B. Yang Shareholder of: No, Grant/research support from: No, Consultant for: No, Employee of: No, Paid instructor for: No, Speakers bureau: No, L. Wang: None declared

AB0010 Association of Gene Polymorphisms in ETS-1 with Rankl in Rheumatoid Arthritis

Background Rheumatoid arthritis (RA) is a complicated autoimmune disease characterized by progressive destruction of cartilage and bone. Recently, receptor activator of nuclear factor κB ligand (RANKL) have been found to be involved in the differentiation of osteoclasts [1]. Also, the association between the RANKL expression and the pathogenesis of bone-destructive rheumatoid arthritis (RA) has been described in several joints [2]. It indicated that RANKL play a crucial rule in RA. Meanwhile, E26 transformation specific sequence 1 (ETS-1), belonging to the ETS family of transcription factors that regulate the expression of various immune-related genes, was reported to confer susceptibility and development to RA [3]. Moreover, ETS-1 was found to be overexpressed in RA synovial membrane and to be involved in the destructive pathway of RA [4], but it was not clearly defined. Objectives We aimed to identify how RANKL changes in RA and whether polymorphisms in ETS-1 play a role in that changes by describing in Chinese Han population. Methods 170 RA patients and 136 healthy controls were included for this analysis. Clinical information was gathered and disease activity was determined according to the disease activity score for 28 painful/swollen joints (DAS28). The serum level of RANKL were detected by magnetic luminex assays. Four single nucleotide polymorphisms (SNPs) in ETS-1 were genotyped by high resolution melting (HRM) curve method. Results Compared with healthy controls, Level of RANKL in serum of patients was elevated (28.69 (17.24–44.90) versus 14.24 (17.00–20.00), P<0.01). The RA patients with rs73013527 TT genotype had higher RANKL levels (P=0.019 in a dominant model). Furthermore, we found that T allele of rs73013527 was overrepresented in RA group as well (25.7% versus 42.6%, P<0.001). Besides, an association was found between rs73013527 TT genotype and DAS28 (P=0.001). No statistically significant difference was observed in the distribution of other three SNPs (rs10893872, rs4937333 and rs11221332) alleles or genotypes in this study (all P>0.05). Conclusions This study suggests that RANKL increased in RA and the polymorphisms in the ETS-1 region may be associated with the changes of RANKL in RA. The patients with rs73013527 TT genotype may be not prone to RA but have higher RANKL which could lead a sever condiction. However, larger studies, most likely through multicenter collaboration will be needed to fully validate the significance of these findings. References Motiur Rahman M, Takeshita S, Matsuoka K, et al. Proliferation-coupledosteoclast differentiation by RANKL: Cell density as a determinant of osteoclast formation [J]. Bone, 2015, 81:392–399. Liu WW, Xu ZM, Li ZQ, et al. RANKL, OPG and CTR mRNA expression in the temporomandibular joint in rheumatoid arthritis. Exp Ther Med. 2015 Sep;10(3):895–900. Chen L, Huang Z, Yang B, et al. Association of E26 Transformation Specific Sequence 1 Variants with Rheumatoid Arthritis in Chinese Han Population. PLoS One. 2015 Aug 4;10(8):e0134875. Redlich K, Kiener HP, Schett G, Tohidast-Akrad M, Selzer E, Radda I, et al. Overexpression of transcription factor Ets-1 in rheumatoid arthritis synovial membrane: regulation of expression and activation by interleukin-1 and tumor necrosis factor alpha. Arthritis Rheum. 2001;44:266–274. Disclosure of Interest None declared

AB0258 Decreased Phosphostat5 Contributed To Impairment of CD4+CD25+Foxp3+ Tregs in RA Patients

Background Our previous research has demonstrated decreased expression of CD4+CD25+Foxp3+ regulatory T cells (Tregs) enrolled in the pathogenesis of rheumatoid arthritis (RA). Anti-TNF-α mAb therapy could improve Tregs expansion in RA and associate with clinical amelioration of RA patients. STAT5 is reported to have effects on many aspects of immune function, particularly in regulatory T cell development. Objectives In this study, we aim to monitor phosphoSTAT5 expression in Tregs and investigate its role in RA disease. Methods Flowcytometry was employed to detect the phosphorylation level of STAT5 in CD3+CD4+ T cells, CD4+CD25high regulatory T cells and the expression of CD4+CD25+Foxp3+Tregs in peripheral blood of 20 patients with RA disease and 20 age and gender-matched normal controls. STAT5 mRNA expression in peripheral blood mononuclear cells (PBMC) were also monitored of these RA patients and normal controls. Results Phosphorylation level of STAT5 deceased in RA patients both in CD3+CD4+ T cells and in CD4+CD25high regulatory T cells, along with reduction of expression of CD4+CD25+Foxp3+ Tregs. Detection of mRNA also confirmed STAT5 down regulation in Tregs in RA patients. Conclusions Compared with normal controls, the expression of CD4+CD25+Foxp3+Tregs decreased in RA patients. PhosphoSTAT5 reduction in CD3+CD4+ T cells, mainly in CD4+CD25high regulatory T cells contributed to impairment of CD4+CD25+Foxp3+ regulatory T cells in RA. References Niu Q, Cai B, Huang ZC, Shi YY, Wang LL. Disturbed Th17/Treg balance in patients with rheumatoid arthritis. Rheumatol Int. 2012 Sep;32(9):2731–6. Huang Z, Yang B, Shi Y, Cai B, Li Y, Feng W, Fu Y, Luo L, Wang L. Anti-TNF-α therapy improves Treg and suppresses Teff in patients with rheumatoid arthritis. Cell Immunol. 2012 Sep;279(1):25–9. Disclosure of Interest None declared

SAT0020 Association of Hla-Dp/dq and Stat4 Polymorphisms with Ankylosing Spondylitis in A Chinese Population

Background Ankylosing spondylitis (AS) is a highly heritable complex inflammatory arthritis disease. Genetic factors are thought to be crucial in the pathogenesis of ankylosing spondylitis. However, few studies were performed to investigate the relationship between HLA-DP/DQ and STAT4 polymorphisms and AS susceptibility. Objectives To further explore the associations of the four SNPs (HLA-DP rs3077, HLA-DP rs9277535, HLA-DQ rs7453920 and STAT4 rs7574865) with the risk and clinical characteristics of AS in a Chinese Han population. And also to analyze the linkage disequilibrium (LD) in HLA gene and the association between the HLA haplotypes and the susceptibility to AS. Methods 400 patients with AS and 379 age- and sex-matched healthy controls in a Chinese population were included. All the four SNPs were genotyped using polymerase chain reaction high resolution melting (HRM) analysis. Allele, genotype, and haplotype frequencies were compared between AS patients and controls. Besides, stratification analyses of HLA-DP/DQ and STAT4 polymorphisms and risk for AS were also performed. Results No significant difference was observed between AS patients and healthy controls in the allele frequency of rs3077, rs9277535 and rs7574865. However, there was a significant association between the HLA-DQ rs7453920 G/A variant and AS patients, with minor allele A correlated with a reduced risk of AS (allelic frequency, OR=0.44, 95% CI: 0.31–0.62, p=3.0E-06; dominant model, OR =0.42, 95% CI: 0.29–0.62, p=7.0E-06). Moreover, the haplotypes block AAA and GGA in the HLA gene significantly correlated with reduced risk of AS (AAA: OR =0.38, 95%CI: 0.22–0.66, p=0.0004; GGA: OR=0.41, 95% CI: 0.24–0.69, p=0.0006). Conclusions This is the first study demonstrating the relationship between HLA-DQB2 rs7453920 and the risk of AS in a Chinese population. Besides, this study revealed that LD blocks around HLA–DP/DQ are strongly associated with AS susceptibility. This research sheds new light on the significant relationship between the SNPs in the HLA gene and the risk of AS. References Braun, J. & Sieper, J. Ankylosing spondylitis. Lancet. 369, 1379–1390, (2007). Reveille, J. D. Major histocompatibility genes and ankylosing spondylitis. Best practice & research. Clinical rheumatology. 20, 601–609, (2006). Diaz-Pena, R. et al. Fine mapping of a major histocompatibility complex in ankylosing spondylitis: association of the HLA-DPA1 and HLA-DPB1 regions. Arthritis and rheumatism. 63, 3305–3312, (2011). Shen, L. et al. Replication study of STAT4 rs7574865 G/T polymorphism and risk of rheumatoid arthritis in a Chinese population. Gene. 526, 259–264, (2013). Acknowledgement This study was supported by the National Natural Science Foundation of China (No. 81301496, 81202354). Disclosure of Interest X. Liu Shareholder of: No, Grant/research support from: National Natural Science Foundation of China (No. 81301496), Consultant for: No, Employee of: No, Paid instructor for: No, Speakers bureau: No, L. Wang Shareholder of: No, Grant/research support from: National Natural Science Foundation of China (No. 81301496), Consultant for: No, Employee of: No, Paid instructor for: No, Speakers bureau: No, B. Yang Shareholder of: No, Grant/research support from: National Natural Science Foundation of China (No. 81301496), Consultant for: No, Employee of: No, Paid instructor for: No, Speakers bureau: No