Influence of different physical and chemical modifications of titanium surface on its citocompatibility and immunomodulatory properties

Titan i njegove legure se upotrebljavaju kao materijali za dentalne implantate zbog pokazane biokompatibilnosti titana i dobrih fizičkih i mehaničkih svojstva. Korozivna svojstva titana i njegovih legura u kiselim i baznim rastvorima, i u biološkim fluidima nisu zadovoljavajuća što je moguće prevazići modifikacijom njihove površine. Biofunkcionalnost modifikovanih površina titana zavisi od tehnika kojima se sprovode modifikacije. Postoje brojne metode modifikacije površine titana, kao hemijske metode koje zajedno sa naknadnim termičkim tretmanom obezbeđuju formiranje titan-oksidnog sloja ili fizičke metode nanošenja prevlaka hidroksiapatita (HAP). Dentalni implantati mogu dovesti do povećane produkcije citokina i hemokina sa proinflamacijskim delovanjem. Cilj istraživanja: Ispitivanje efekta modifikacije površine titanove legure (Ti6Al4V) hemijskom obradom i naknadnim termalnim tretmanom, i nanošenjem prevlake hidroksiapatita, na biokompatibilnost merenu pomoću određivanja citotoksičnosti in vitro, kao i na imunomodulacijsko delovanje titana in vitro. Materijal i metode: Citotoksičnost je ispitivana in vitro, primenom testova direktnog kontakta uzoraka titanovih legura sa L929 ćelijama ili indirektno, ispitivanjem metaboličke aktivnosti L929 ćelija u prisustvu različitih razblaženja kondicioniranog medijuma (KM). Korišćena je ćelijska linija mišijih fibroblasta (L929). Metabolička aktivnost je merena MTT testom, ćelijska proliferacija je merena testom ugradnje 3[H+]-timidina, dok je produkcija slobodnih radikala kiseonika (ROS) određivana protočnom citofluorimetrijom nakon obeležavanja sa dihloro-dihidro-fluorescin diacetatom (DCFH-DA). Nekroza i apoptoza su merene bojenjem ćelija sa propidijum jodidom, merenjem produkcije laktatdehidrogenaze (LDH), kao i analizom ekspresije gena za mišje kaspaze 3, 8 i 9 metodom lančane reakcije polimeraze u realnom vremenu (Real-Time PCR). Ćelijski rast i morfološka analiza L929 ćelija je procenjivana fazno kontrasno svetlosnom mikroskopijom, dok je vijabilnost određivana pomoću tripan plavog. Imunomodulacijski efekat modifikovanih površina titana je ispitivan na modelu fitohemaglutininom (PHA) aktiviranih humanih mononuklearnih ćelija periferne krvi (PBMNC)...Introduction: Titanium and its alloys are being used as dental implant materials due to its biocompatibility and solid physical and mechanical properties. Corrosion properties of titanium and its alloys in acid and alkaline solutions and biological fluids are not satisfying and can be surpassed by modifying its surface area. Biofunctionality of the modified surface depends on the techniques being used. There are many methods to modify titanium surface such as chemical methods that, together with subsequent thermal treatment, provide the formation of a titanium oxide layer, or physical methods, such as deposition of a hydroxyapatite coatings (HAP). Dental implants can increase production of cytokines and chemokines with proinflammatory properties. Aim: Investigation of the effect of the titanium alloy (Ti6Al4V) surface modification by chemical treatment and subsequent thermal treatment, and deposition of a hydroxyapatite coating, on biocompatibility, assesed by cytotoxicity evaluation in vitro, and on the immunomodulatory action of titanium in vitro. Material and methods: Cytotoxicity was determined by using in vitro test of direct contact of titanium alloy samples with L929 cells or indirectly, by determining metabolic activity of L929 cells in the presence of different solutions of conditioned medium (KM). Mouse fibroblast (L929) cell line was used. Metabolic activity was determined with MTT test, cell proliferation was measured with 3[H+]-thymidine test, while reactive oxygen species (ROS) production was determined by flow cytometry after labeling with dichloro-dihydro-fluorescein diacetate (DCFH-DA). Necrosis and apoptosis were measured with propidium iodide (PI) cell staining, measuring lactate dehydrogenase (LDH) production, and by analyzing gene expression of mice caspases 3, 8 and 9 with Real Time PCR method. Cell growth and morphological analysis of L929 cells were determined with phase contrast light microscopy, while viability was determined with Trypan Blue. Immunomodulating effect of the modified titanium surfaces was measured on phytohemaglutinin (PHA) model of peripheral blood mononuclear cells (PBMNC)..

Association of uPA and PAI-1 tumor levels and 4G/5G variants of PAI-1 gene with disease outcome in luminal HER2-negative node-negative breast cancer patients treated with adjuvant endocrine therapy

Abstract Background The aim of this study was to evaluate the prognostic potential of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI-1) tumor tissue levels and examine the association between these biomarkers and classical prognostic factors in early node-negative luminal breast cancer patients. The clinical value of 4G/5G variants of PAI-1 gene was evaluated. Patients and methods This study involved 81 node-negative, estrogen receptor-positive and/or progesterone receptor-positive and human epidermal growth factor receptor 2-negative operable breast cancer patients who underwent radical surgical resection and received adjuvant endocrine therapy. Determination of uPA and PAI-1 concentrations in the breast cancer tissue extracts was performed using FEMTELLE® uPA/PAI-1 ELISA. An insertion (5G)/deletion (4G) polymorphism at position − 675 of the PAI-1 gene was detected by PCR-RFLP analysis. Results Our research showed that patients with uPA tumor tissue levels higher than 3 ng/mg of protein had significantly reduced disease-free survival (DFS) and overall survival (OS) when compared to patients with uPA tumor tissue levels lower or equal to 3 ng/mg of protein. Patients with PAI-1 tumor tissue levels higher than 14 ng/mg of protein had significantly decreased OS in comparison with patients with PAI-1 tumor tissue levels lower or equal to 14 ng/mg of protein. ROC analysis confirmed the uPA and PAI-1 discriminative potential for the presence/absence of relevant events in these patients and resulted in higher cut-off values (5.65 ng/mg of protein for uPA and 27.10 ng/mg of protein for PAI-1) than standard reference cut-off values for both biomarkers. The prognostic importance of uPA and PAI-1 ROC cut-off values was confirmed by the impact of uPA higher than 5.65 ng/mg of protein and PAI-1 higher than 27.10 ng/mg of protein on poorer DFS, OS and event-free survival (EFS). We observed that patients with dominant allele in PAI-1 genotype (heterozygote and dominant homozygote, − 675 4G/5G and − 675 5G/5G) had significantly increased DFS, OS and EFS when compared with patients with recessive homozygote genotype (− 675 4G/4G). Conclusion Our study indicates that uPA and PAI-1 tumor tissue levels and 4G/5G variants of PAI-1 gene might be of prognostic significance in early node-negative luminal HER2-negative breast cancer patients treated with adjuvant endocrine therapy