Cenk Aktas

Femtosecond laser treatment of 316L improves its surface nanoroughness and carbon content and promotes osseointegration: An in vitro evaluation.

Cell-material surface interaction plays a critical role in osseointegration of prosthetic implants used in orthopedic surgeries and dentistry. Different technical approaches exist to improve surface properties of such implants either by coating or by modification of their topography. Femtosecond laser treatment was used in this study to generate microspotted lines separated by 75, 125, or 175μm wide nanostructured interlines on stainless steel (316L) plates. The hydrophobicity and carbon content of the metallic surface were improved simultaneously through this method. In vitro testing of the laser treated plates revealed a significant improvement in adhesion of human endothelial cells and human bone marrow mesenchymal stem cells (hBM MSCs), the cells involved in microvessel and bone formation, respectively, and a significant decrease in fibroblast adhesion, which is implicated in osteolysis and aseptic loosening of prostheses. The hBM MSCs showed an increased bone formation rate on the laser treated plates under osteogenic conditions; the highest mineral deposition was obtained on the surface with 125μm interline distance (292±18mg/cm(2) vs. 228±43mg/cm(2) on untreated surface). Further in vivo testing of these laser treated surfaces in the native prosthetic implant niche would give a real insight into their effectiveness in improving osseointegration and their potential use in clinical applications.

Differential Behavior of Fibroblasts and Epithelial Cells on Structured Implant Abutment Materials: A Comparison of Materials and Surface Topographies

PURPOSE The aim of this study was to compare the proliferation and attachment behavior of fibroblasts and epithelial cells on differently structured abutment materials. MATERIALS AND METHODS Three different surface topographies were prepared on zirconia and titanium alloy specimens and defined as follows: machined (as delivered without further surface modification), smooth (polished), and rough (sandblasted). Energy-dispersive X-ray spectroscopy, topographical analysis, and water contact angle measurements were used to analyze the surface properties. Fibroblasts (HGF1) and epithelial cells (HNEpC) grown on the specimens were investigated 24 hours and 72 hours after seeding and counted using fluorescence imaging. To investigate adhesion, the abundance and arrangement of the focal adhesion protein vinculin were evaluated by immunocytochemistry. RESULTS Similar surface topographies were created on both materials. Fibroblasts exhibited significant higher proliferation rates on comparable surface topographies of zirconia compared with the titanium alloy. The proliferation of fibroblasts and epithelial cells was optimal on different substrate/topography combinations. Cell spreading was generally higher on polished and machined surfaces than on sandblasted surfaces. Rough surfaces provided favorable properties in terms of cellular adhesion of fibroblasts but not of epithelial cells. CONCLUSIONS Our data support complex soft tissue cell-substrate interactions: the fibroblast and epithelial cell response is influenced by both the material and surface topography.

Reduced myofibroblast differentiation on femtosecond laser treated 316LS stainless steel

In-stent restenosis is a common complication after stent surgery which leads to a dangerous wall narrowing of a blood vessel. Laser assisted patterning is one of the effective methods to modify the stent surface to control cell-surface interactions which play a major role in the restenosis. In this current study, 316 LS stainless steel substrates are structured by focusing a femtosecond laser beam down to a spot size of 50 μm. By altering the laser induced spot density three distinct surfaces (low density (LD), medium density (MD) and high density (HD)) were prepared. While such surfaces are composed of primary microstructures, due to fast melting and re-solidification by ultra-short laser pulses, nanofeatures are also observed as secondary structures. Following a detailed surface characterization (chemical and physical properties of the surface), we used a well-established co-culture assay of human microvascular endothelial cells and human fibroblasts to check the cell compatibility of the prepared surfaces. The surfaces were analyzed in terms of cell adherence, proliferation, cell morphology and the differentiation of the fibroblast into the myofibroblast, which is a process indicating a general fibrotic shift within a certain tissue. It is observed that myofibroblast proliferation decreases significantly on laser treated samples in comparison to non-treated ones. On the other hand endothelial cell proliferation is not affected by the surface topography which is composed of micro- and nanostructures. Such surfaces may be used to modify stent surfaces for prevention or at least reduction of restenosis.

Improved Endothelialisation on Nanostructured Surfaces

Abstract . Topography plays a major role on surface-cell interaction beside the surface chemistry. We investigated the effect of the nanotopography on vascular cell adhesion and proliferation in order to improve endothelialisation for restenosis treatment. In this context, Al2O3 nanowires (NWs) composed of a stable Al2O3 shell and an Al core were synthesized by chemical vapour deposition (CVD) of the molecular precursor (tBuOAlH2)2. After the detailed material characterization, human umbilical vein endothelial cells (HUVEC) and human umbilical vein smooth muscle cells (HUVSMC) were seeded and cultivated on these surfaces. Our preliminary results showed that there is a preference of HUVEC adhesion on NWs in comparison to that of HUVSMC. The control of the cell–surface interaction by the topography may represent a key issue for the future stent material design.

Nano Calcium Phosphate Powder Production through Chemical Agitation from Atlantic Deer Cowrie Shells (Cypraea cervus Linnaeus)

The process is a simple chemical method and aims to produce nano-structured calcium phosphate powders from natural sources, for biomedical applications. For this purpose, Atlantic Deer Cowrie (ADC) shells (Cypraea cervus Linnaeus, 1771) were collected from a local gift store in Istanbul. The empty shells were cleaned and crushed then were ball milled and sieved under 100µm. The raw powders were suspended on a hotplate stirrer for a simple chemical agitation. The temperature was kept at 80°C for 15 min. and then appropriate amount of H3PO4 was added by titration into the prepared solution to form calcium phosphate precursors. The solution was stirred on a hotplate for 8 hours then dried at 100°C for 24 hours. Afterwards the resulting dried sediments were collected and heat treated between 400-800°C for 4 hours, dependent on the required specific calcium phosphate phase. X-ray diffraction (XRD) analysis and scanning electron microscopy (SEM) were carried out for identifying various hydroxyapatite (HA), tricalcium phosphate (TCP) and other calcium phosphate phases. Various particle sizes ranging from nano to micron, are obtained depending on the chemistry used and the processing technique applied during the production. A range of calcium phosphate phases can be obtained from ADC shells, by using a simple and economic conversion method. Proper cleaning methods developed and appropriate preparation techniques will enable us to use these nano calcium phosphate powders in orthopedic and dental applications.

Nano-Bioceramic Production via Mechano-Chemical Conversion (Ultrasonication)

The production of nano-calcium phosphate powders, such as HA (hydroxyapatite), from synthetic chemicals can be expensive and time consuming. The skeleton or shells of sea creatures (e.g. sea urchins, shells, corals) could be an alternative source of materials to produce very fine and even nano-structured calcium phosphate biomaterial powders. Ηydrothermal conversion under very high pressures or methods such as hot-plating (chemical) or ultrasonication (mechano-chemical), have been proposed to transform naturally derived CaCO3, e.g. aragonite, into apatite based materials. The aim of the present work was to prepare inexpensive nano-sized HA and TCP bioceramics powders from a local sea snail shells as a possible raw material for HA/TCP bioceramics. Empty shells of a local sea snail (Nassarius hinia reticulatus) from Marmara Sea, Turkey were collected from a beach near Istanbul. The collected shells were ground to a particle size <75µm. Thermal analyses (DTA/TGA) were performed to determine the exact CaCO3 content and thermal behavior. The raw powder was suspended in an aqueous media which was placed in an ultrasonic bath. The temperature was set at 80°C for 15min. Then, an equivalent (to CaO content) amount of H3PO4 was added drop by drop very gently into the solution. The reaction continued for 8h, following which the liquid component was evaporated off in an incubator at 100°C for 24h. The dried sediment was collected and heat treated at two different temperatures, 400 and 800°C. The morphology of the powders produced was examined using SEM. The crystalline phases were indentified using X-ray analysis. X-ray diffractograms indicated the presence of two calcium phosphate phases, namely HA and whitlockite. SEM observations showed that the powder produced comprised nano-sized particles. FTIR results also indicated the presence of HA and whitlockite structures. The experimental results suggest that Nassariushinia reticulatus shells could be an alternative source for the production of various mono or biphasic calcium phosphates. In this study, local sea snail shells were successfully converted to HA and whitlockite with a simple mechano-chemical (ultrasonic) conversion method without the use of complex hydrothermal methods.

Al2O3 micro- and nanostructures affect vascular cell response

In-stent restenosis (ISR) is one of the most common and serious complications observed after stent implantation. ISR is characterized by the inordinate proliferation of smooth muscle cells (SMC) that leads to narrowing of the blood vessels. To achieve a healthy endothelium, it is critical to selectively enhance the growth of endothelial cells (EC) while suppressing the growth of smooth muscle cells, which is still a major challenge and yet to be achieved. In this study, novel surfaces have been developed to support the selective growth of endothelial cells. Micro- and nanostructured Al2O3 surfaces with unique topographical features were fabricated and tested. Surface characterization and cellular response of endothelial cells (HUVEC) as well as smooth muscle cells (HUVSMC) has been investigated at cellular and molecular levels. A topography driven selective cell response of ECs over SMCs was demonstrated successfully. This selective response of ECs was also analyzed at protein levels in order to understand the basic mechanism.

Surface modification by plasma etching impairs early vascularization and tissue incorporation of porous polyethylene (Medpor(®) ) implants.

Porous polyethylene (Medpor®) is commonly used in craniofacial reconstructive surgery. Rapid vascularization and tissue incorporation are crucial for the prevention of migration, extrusion, and infection of the biomaterial. Therefore, we analyzed whether surface modification by plasma etching may improve the early tissue response to Medpor®. Medpor® samples were treated in a plasma chamber at low (20 W; LE-PE) and high energy levels (40 W; HE-PE). The samples and non-treated controls were implanted into mouse dorsal skinfold chambers to analyze angiogenesis, inflammation, and granulation tissue formation over 14 days using intravital fluorescence microscopy, histology, and immunohistochemistry. Scanning electron microscopy (SEM) analyses revealed that elevating energy levels of plasma etching progressively increase the oxygen surface content and surface roughness of Medpor®. This did not affect the leukocytic response to the implants. However, LE-PE and HE-PE samples exhibited an impaired vascularization. This was associated with a reduced formation of a collagen-rich granulation tissue at the implantation site. Additional in vitro experiments showed a reduced cell attachment on plasma-etched Medpor®. Thus, plasma etching may not be recommended to improve the clinical outcome of reconstructive interventions using Medpor®. However, it may be beneficial for temporarily implanted polyethylene-based biomedical devices for which tissue incorporation is undesirable.

Ultra‐Rapid Growth of Biphasic Nanowires in Micro‐ and Hypergravity

Aluminium/aluminium oxide wires form under microgravity, earth conditions, and hypergravity in different forms. While under 0.04 G the biphasic wires are predominantly linear, they form bundles of wires of high curvature at 1 G and 1.8 G. The absence (0.04 G) and presence (1 G, 1.8 G) of gradients are reflected by the agglomeration and growth direction of the nanowires.

Recombinant Phage Coated 1D Al2O3 Nanostructures for Controlling the Adhesion and Proliferation of Endothelial Cells

A novel synthesis of a nanostructured cell adhesive surface is investigated for future stent developments. One-dimensional (1D) Al2O3 nanostructures were prepared by chemical vapor deposition of a single source precursor. Afterwards, recombinant filamentous bacteriophages which display a short binding motif with a cell adhesive peptide (RGD) on p3 and p8 proteins were immobilized on these 1D Al2O3 nanostructures by a simple dip-coating process to study the cellular response of human endothelial EA hy.926. While the cell density decreased on as-deposited 1D Al2O3 nanostructures, we observed enhanced cell proliferation and cell-cell interaction on recombinant phage overcoated 1D Al2O3 nanostructures. The recombinant phage overcoating also supports an isotropic cell spreading rather than elongated cell morphology as we observed on as-deposited Al2O3 1D nanostructures.