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    Calcified and mechanically debilitated three-dimensional hydrogel environment induces hypertrophic trend in chondrocytes
    (Sage Publications Ltd, 2016) Celik, Ekin; Bayram, Cem; Akcapinar, Rumeysa; Turk, Mustafa; Denkbas, Emir Baki
    Currently, the main focus on tissue engineering strategies is to mimic the extracellular matrix of the related tissues. Many studies accomplished to build tissue scaffolds to act as the natural surroundings of the specific interest, which can be established to behave like either healthy or unhealthy tissues. The latter one of these conditions is a quite new approach and crucial for the design of three-dimensional in vitro disease models. This study investigates the potential of a composite scaffold consisting hydroxyapatite-integrated fluorenyl-9-methoxycarbonyl diphenylalanine hydrogels by focusing on the optimization of this hybrid scaffold for the development of an in vitro model of degenerative cartilage. Cell growth, chondrocyte proliferation, extracellular matrix production, hypertrophy marker monitoring, scaffold mechanical properties, and morphological analysis were evaluated. Fluorenyl-9-methoxycarbonyl diphenylalanine dipeptides were dissolved in null cell culture media and pH decreased sequentially to compel peptides to self-organize into fibrous hydrogel scaffolds. Nano-hydroxyapatite crystals were incorporated into fluorenyl-9-methoxycarbonyl diphenylalanine hydrogels during the gelation to investigate the effect on chondrocytes. It is observed that hydroxyapatite incorporation into peptide hydrogels significantly increased the alkaline phosphatase activity and assymetrical cell divisions, which is appraised as an outcome of chondrocyte hypertrophy. It is concluded that chondrocytes develop a hypertrophic potential when they are cultured in a media with nano-hydroxyapatites in a three-dimensional cell culture matrix mimicking the extracellular matrix conditions of degenerative cartilage.
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    Dual delivery of platelet-derived growth factor and bone morphogenetic factor-6 on titanium surface to enhance the early period of implant osseointegration
    (WILEY, 2020) Keceli, H. Gencay; Bayram, Cem; Celik, Ekin; Ercan, Nuray; Demirbilek, Murat; Nohutcu, Rahime Meral
    Objective To test the surface properties and in vitro effects of a new sequential release system on MC3T3-E1 cells for improved osseointegration. Background BMP6-loaded anodized titanium coated with PDGF containing silk fibroin (SF) may improve osseointegration. Methods Titanium surfaces were electrochemically anodized, and SF layer was covered via electrospinning. Five experimental groups (unanodized Ti (Ti), anodized Ti (AnTi), anodized + BMP6-loaded Ti (AnTi-BMP6), anodized + BMP6 loaded + silk fibroin-coated Ti (AnTi-BMP6-SF), and anodized + BMP6-loaded + silk fibroin with PDGF-coated Ti (AnTi-BMP6-PDGF-SF)) were tested. After SEM characterization, contact angle analysis, and FTIR analysis, the amount of released PDGF and BMP6 was detected using ELISA. Cell proliferation (XTT), mineralization, and gene expression (RUNX2andALPL) were also evaluated. Results After successful anodization and loading of PDGF and BMP6, contact angle measurements showed hydrophobicity for TiO(2)and hydrophilicity for protein-adsorbed surfaces. In FTIR, protein-containing surfaces exhibited amide-I, amide-II, and amide-III bands at 1600 cm(-1)-1700 cm(-1), 1520 cm(-1)-1540 cm(-1), and 1220 cm(-1)-1300 cm(-1)spectrum levels with a significant peak in BMP6- and/or SF-loaded groups at 1100 cm(-1). PDGF release and BMP6 release were delayed, and relatively slower release was detected in SF-coated surfaces. Higher MC3T3-E1 proliferation and mineralization and lower gene expression ofRUNX2andALPLwere detected in AnTi-BMP6-PDGF-SF toward day 28. Conclusion The new system revealed a high potential for an improved early osseointegration period by means of a better factor release curve and contribution to the osteoblastic cell proliferation, mineralization, and associated gene expression.
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    The effect of calcium chloride concentration on alginate/Fmoc-diphenylalanine hydrogel networks
    (Elsevier Science Bv, 2016) Celik, Ekin; Bayram, Cem; Akcapinar, Rumeysaa; Turk, Mustafa; Denkbas, Emir Baki
    Peptide based hydrogels gained a vast interest in the tissue engineering studies thanks to great superiorities such as biocompatibility, supramolecular organization without any need of additional crosslinker, injectability and tunable nature. Fmoc-diphenylalanine (FmocFF) is one of the earliest and widely used example of these small molecule gelators that have been utilized in biomedical studies. However, Fmoc-peptides are not feasible for long term use due to low stability and weak mechanical properties at neutral pH. In this study, Fmoc-FF dipeptides were mechanically enhanced by incorporation of alginate, a biocompatible and absorbable polysaccharide. The binary hydrogel is obtained via molecular self-assembly of FmocFF dipeptide in alginate solution followed by ionic crosslinldng of alginate moieties with varying concentrations of calcium chloride. Hydrogel characterization was evaluated in terms of morphology, viscoelastic moduli and diffusional phenomena and the structures were tested as 3D scaffolds for bovine chondrocytes. In vitro evaluation of scaffolds lasted up to 14 days and cell viability, sulphated glycosaminoglycan (sGAG) levels, collagen type II synthesis were determined. Our results showed that alginate incorporation into FmocFF hydrogels leads to better mechanical properties and higher stability with good biocompatibility. (C) 2016 Elsevier B.V. All rights reserved.
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    Novel layer-by-layer self-assembled peptide nanocarriers for siRNA delivery
    (Royal Soc Chemistry, 2017) Bozdogan, Betul; Akbal, Oznur; Celik, Ekin; Turk, Mustafa; Denkbas, Emir Baki
    All complex and functional structures of nature consist of simple building blocks that are thermodynamically balanced and self-assembled at the molecular level. Production of functional bio-nanomaterials with molecular self-assembly mechanisms, based on a bottom-up approach, has become increasingly important in recent years. In this study, a biodegradable and biocompatible siRNA nanocarrier system, consisting of diphenylalaninamide (FFA) based nanoparticles, was developed for silencing of HER2, a gene known to be overexpressed in breast cancer. FFA contains an amide functional group that has a dipolar nature with zero net charge. Here we report an original approach to functionalizing peptide nanoparticles based on layer-by-layer polyelectrolyte deposition (LbL PD) technique. The resulting well-defined FFA nanoparticles (FFANPs) were coated with polycationic poly-Llysine (PLL) by cation-dipole interaction, giving rise to a net positive surface charge. The PLL coating improved the physical stability of FFANPs at physiological pH and temperature. The cationized FFANP was then interacted with the polyanionic siRNA, forming an FFANP-PLL/siRNA complex. Nanoparticles were then interacted with PLL one more time, to create a third layer that can prevent degradation of the siRNA by nucleases and achieve effective delivery of the siRNA into the cytoplasm. These original FFANP-PLL/siRNA/PLL were optimized to achieve efficient in vitro gene silencing. Overall, this study shows that FFANP-PLL/siRNA/PLL are promising gene carriers for gene silencing therapies.