PVD and CVD processes for deposition of thin coatings – Coating of polymer materials and deposition of diamond-like carbon Wolfgang Waldhauser JOANNEUM RESEARCH Forschungs-GmbH, MATERIALS – Functional Surfaces, Austria Thin-film coatings play a prominent role in the manufacturing of tribological and mechanical stressed components, decorative objects, components for electronics and optics, biocompatible implants, etc.. Physical Vapor Deposition (PVD) and Chemical Vapor Deposition (CVD) are two most common types of thin-film coating techniques. PVD coatings involve atom-by-atom, molecule-by-molecule, or ion deposition of various materials on solid substrates in vacuum systems. Main basics of PVD are the evaporation or sputtering of a solid or liquid target material, the transport of this material in gas or plasma state towards the substrate and the deposition of a thin film on its surface. While thermal evaporation lacks on the low energy of the evaporated vapor and, thus, provides low energy during film nucleation and growth for adhesion and densification, evaporation by pulsed laser beams (pulsed laser deposition) overcomes this problem. Magnetron sputtering possesses about one magnitude lower energy in plasma, but allows high-rate layer deposition on large areas. Contrasting to the PVD coating in the "line of sight", the CVD can coat all surfaces of the substrate. It’s based on the thermal or plasma activated decomposition (PACVD) of precursor gases. Plasma-Assisted CVD coating processes can be performed at lower temperature than CVD techniques. Recently, the trend of using temperature-sensitive materials like polymers demands new low-temperature coating techniques for protective surface finishing as well as for functionalization of the surfaces. However, till now there is a lack of industrially-scaled low-temperature vacuum coating techniques (<50 °C). Pulsed laser deposition and magnetron sputtering with ion beam pretreatment step could overcome these problems connected with thin film adhesion and porous structures, where traditional coating processes struggle when being used at low or room temperature coating. Crucial aspect is the interface formation in room-temperature coating, which will be shown for pulsed laser deposited (PLD) coating on polymer substrates. A titanium film on polyurethane was chosen as model system for transmission electron microscopy and X-ray photoelectron spectroscopy investigations. Pseudodiffusion interfaces were found due to the high-energetic particle bombardment during PLD coating. Additionally, changes of the polyurethane chemical binding are evident, like the transition from C=O to C–O–R binding, in which titanium atoms could act as new binding partners to the O species. Diamond-like carbon (DLC) is an amorphous, metastable form of carbon in sp2 and sp3 hybridisation. Since DLC films have superior properties with a unique combination of high hardness, good thermal conductivity, low friction coefficients, excellent wear resistance, ultra-smoothness and chemical inertness, the films have found a wide range of applications. DLC films are used for example as protective coatings on magnetic hard discs and on engineering components as wear resistant coatings, or as reported in recent years, as biocompatible or barrier coatings. Structure-properties correlation of DLC coatings prepared by employing different coating techniques will be shown and discussed.

This talk will conclude results from surface topography formation studies of hard metal and ceramic films on soft polymers, deposited by PLD and magnetron sputtering and results from plasmapolymerization of carbon and silicon based plasma-polymer coatings on the inner surfaces of tubes. Functionalization of surfaces is an important task for nanotechnology to add specially designed physico-chemical properties to materials, e.g. physical or chemical vapour deposited (PVD, CVD) films. Besides chemical modification of surfaces, physical adaptations gain increasing interest, from which topography formation is one highly interesting aspect. Topography of coating can evolve from both the thin film growth itself and mechanical interactions with the substrate. The thin film growth follows structure zone models, which were developed in the 1970 for crystalline coatings and conclude influences of diffusion processes, triggered by temperature of the substrate (initiates bulk and surface diffusion) and particle energy from plasma (initiates nearly only surface diffusion). Growth of columns and cones starting from nuclei (e.g. at the substrate surface) forms on the surface a dome-shaped topography. Mechanical interaction between the surface region of the substrate and the growing thin film are due to film stresses evolving from lattice distortion during growth by binding of atoms on wrong positions. This phenomenon – also known as wrinkling – requires high adhesion at the film-substrate interface and leads to a wave-like surface, whereby their wavelength is increasing step-wise during film growth. The ratio of elasticity of the film and the substrate is one major influence to the wave formation, leading to effective surface structuring of soft polymer materials. The modification of inner surfaces of tubes becomes increasingly important in many applications (e.g. fluid transport). However, long tubes require special plasma techniques for surface functionalization on the nanometer scale without damaging the bulk. This work focuses on pulsed DC glow discharges in inert argon, reactive oxygen, nitrogen and air plasma as well as polymerizing acetylene and hexamethyldisiloxane plasma revealing strong influence on the wetting behavior dependent on the total duration of the plasma discharge (plasma on-time). While the investigated polymers polyamide, polycarbonate, polyethylene terephtalate, and thermoplastic polyurethane react very differently on the inert and reactive etching (activation) conditions a plasma-assisted deposition of polymer films (polymer-like carbon with decreasing cross-linking at lower plasma intensity and > 50% hydrogen content) on these surfaces suppresses all substrate influences.

The choice of an appropriate scaffold - in terms of three-dimensional structure and of chemical and physical properties - is crucial. The structure of the scaffold is controlled by the technology applied in scaffold fabrication. Many techniques have been developed to achieve the production of porous scaffolds, as for example particulate leaching, gas foaming, phase inversion, etc.[1-4] In addition to the mentioned strategies, in recent years electrospinning (ES) technology has attracted growing interest. Indeed, ES enables the production of porous scaffolds, with interconnected pores, composed of polymeric micro- or nanofibers that mimic the extra-cellular matrix (ECM) topology. [5-7] In fact, in the most common configuration of the instrumental apparatus, fibers are collected in the form of randomly oriented non-woven mats, reminiscent of the collagen fiber distribution in the ECM. Recent instrumental implementation involves ad hoc modification of both spinning devices and fiber collectors, leading to a broad choice of fiber layering options (isoorientation, specific patterning, etc.).[8] Moreover, careful tuning of the processing parameters allows fabrication of nanofibers with desired diameter. Micro/nano-architecture of the scaffold (i.e. fiber size and orientation) are expected to influence cell behavior and to play a crucial role in engineered tissue fabrication. [9]

A wide variety of materials have been used to produce scaffolds. They are mostly polymers, sometimes associated with inorganic substances, especially in the realization of scaffolds for bone tissue engineering. Polymeric materials include: natural polymers, such as polysaccharides (hyaluronic acid, agarose, alginate, etc.), proteins (collagen, gelatin, silk fibroin, etc.) and synthetic polymers, such as poly(lactide-co-glycolide) copolymers, polycaprolactone, poly-hydroxyalkanoates. The most well know member of PHA family is the polyester poly[(R)-3-hydroxybutyrate] which is produced and stored by many prokaryotic organisms as carbon and energy source [10], unfortunately it is highly crystalline. In order to decrease this undesirable feature it can be blended with biomimetic poly[(R,S)-3-hydroxybutyrate] (aPHB) which is an amorphous synthetic analogue which can be obtained, among other ways, via ring-opening polymerization (ROP) of β-butyrolactone [11-14].

Some basis information regarding electrospinning and its application in fabrication of artificial scaffolds will be discussed.

References:

[1] Agrawal, C. M.; Ray, R. B.Journal of Biomedical Materials Research 2001, 55, 141.

[2] Hutmacher, D. W. Biomaterials 2000, 21, 2529.

[3] Ma, P. X. Materials Today 2004, 7, 30.

[4] Salgado, A. J.; Coutinho, O. P.; Reis, R. L. Macromolecular Bioscience 2004, 4, 743.

[5] Chew, S. Y.; Wen, Y.; Dzenis, Y.; Leong, K. W. Current Pharmaceutical design 2006, 12, 4751.

[6] Lannutti, J.; Reneker, D.; Ma, T.; Tomasko, D.; Farson, D. Materials Science and Engineering C 2007, 27, 504.

[7] Pham, Q. P.; Sharma, U.; Mikos, A. G. Tissue Engineering 2006, 12, 1197.

[8] Teo, W.-E.; Ramakrishna, S. Nanotechnology 2006, 17, R89.

[9] Stevens, M. M.; George, J. H. Science 2005, 310, 1135.

[10] Doi, Y. Microbial Polyesters; VCH Publishers: Weinheim, 1990.

[11] Rieth, L. R.; Moore, D. R.; Lobkovsky; E. B.; Coates G. W. J.Am.Chem.Soc. 2002, 124, 15239.

[12] Jedliński, Z.; Kurcok, P.; Kowalczuk, M.; Kasperczyk, J. Makromol. Chem. 1986, 187, 1651.

[13] Abe, H.; Matsubara, I.; Doi, Y.; Hori, Y.; Yamaguchi, A. Macromolecules 1994, 27, 6018.

[14] Kurcok, P.; Śmiga, M.; Jedliński, Z. J.Polym. Sci. Polym. Chem. 2002, 40, 2184.

Microparticles (MPs) are small membrane vesicles released from activated or apoptotic cells. MPs circulating in blood plasma are 0.1 to 1 µm in diameter and express surface antigens (platelets, endothelial cells, leukocytes and red blood cells). Increase in the number of plasma MPs has been found in various pathological conditions such as thrombosis, inflammation and cancer. A vast majority of circulating MPs are shed from platelets (PDMPs – platelet derived microparticles), as a result of their activation upon high shear stress or adhesion. PDMPs have procoagulant properties, for they are rich in aminophospholipids and tissue factor. Therefore MPs released at the site of platelet activation (vascular wall injury, inflammation or artificial surfaces) serve as platforms for thrombin generation both in situ and in remote organs via their circulation.

There are two main methods of detecting plasma MPs: (1) enzyme-linked immunosorbent assay (ELISA), and (2) flow cytometry. The majority of ELISA assays are designed to detect MPs specific antigens (immunological method) or their procoagulant activity (functional method). In the functional ELISA, diluted citrated plasma is incubated in the microplate well coated with phosphatidylserine (PS) binding protein (Annexin-V). The procoagulant activity of solid-phase captured MPs is quantified upon addition of coagulation factors, prothrombin and color thrombin substrate (Figure 1). MPs concentration is calculated from calibration curve generated with serial dilutions of platelet derived MPs with known procoagulant activity. The advantage of functional ELISA is the ability to measure the thrombotic properties of MPs contained in plasma, thus reflecting their major biological activity.

Figure 1. Schematic representation of functional ELISA detecting plasma MPs (see details in text).

The second group of methods is based on flow cytometry, a laboratory technique measuring different physical and immunological properties of single particles (sells or MPs), as they flow through the laser beam. Plasma samples are stained with fluorochrome conjugated Annexin-V or monoclonal antibodies detecting antigens expressed on the surface of PDMPs (e.g. CD41a, CD61, CD62P) and analyzed by flow cytometry (Figure 2).

Figure 2. Flow cytometric detection of platelet derived MPs in plasma. A. Physical properties (size [FSC] vs. granularity [SSC]) of isolated plasma MPs (left) and fixed/lysed whole blood cells (right). B. Characteristic surface markers of platelet derived MPs.

The major advantage of flow cytometry is the capability of multiple staining to determine the cellular origins of MPs, their phospholipid content, and quantity in the sample. The main disadvantage of flow cytometry is that objects lower than 0.4 µm cannot be counted precisely. In both techniques fresh or frozen citrate plasma is used as an analytical material. Some methods (e.g. Annexin-V binding) require preparation of concentrated MPs from plasma aliquots, by repeated centrifugation and buffer washes. However, MPs could be detected and phenotyped directly from citrated plasma sample. Direct methods are characterized by better reproducibility, and do not require time-consuming pre-analytical steps.

As evidenced by in vitro studies, MPs are released efficiently from platelets activated under high shear stress on foreign surfaces (Figure 3). Additionally, increased amount of circulating MPs is also observed during the course of cardiopulmonary bypass and hemodialysis. Nevertheless, the relation between the consumption of platelets and generation of MPs during exposure of blood in vivo to a foreign materials has not been determined so far. Little is also known if measuring of circulating MPs in such a setting, could constitute a new biomarker of platelet activation and haemocompatibility.

Figure 3. Generation of platelet MPs following arterial shear stress in vitro (left). Tested materials: polystyrene (PS) and polyurethane (PU) are characterized by substantial increase of MPs measured by functional ELISA as phosphatydylserine concentration. Under static conditions (STAT) or with adenosine diphosphorane (ADP), MPs level is within physiological range. Several artificial coatings varies with their hemobiocompatibility either by consumption of platelets or by generation of platelet MPs. A reliable ranking of modified titanium (Ti), silicon (Si) and diamond like carbon (DLC) is presented.

The concept of the design is to reconstitute the structure of the natural vascular tissue on the artificial material scaffold. The appropriate scaffold, cells and the signal to stimulate the cells for the efficient growth in the biomaterial design would greatly reduce the invasiveness after implantation. The consequences of the inadequate nutrient supply to implanted cells are the major challenges to successful scaffold design [1]. The novel solution proposed by the Brendan et al. considers the collagen base vascular bed [1]. The authors confirmed the properties, which are generally known, of endothelium cells (EC) that the healthy endothelium acts as a barrier between blood and the underlying bio material as well as actively inhibits thrombosis. They have proposed the functional co-culture human umbilical vein endothelial cells (HUVEC) and umbilical vein smooth muscle cells (UVSMC). Regarding tissue analogues, it is crucial to point out the necessity to full fill all the regulatory mechanisms.

The presented work was focused on the similar aspects of the novel biomaterial design. Following relatively old, but still valid elaboration submitted by R. O. Chynes [2], we propose to incorporate fibronectin, as a main component of the extracellular matrix, introduced under the endothelium. The ECM is not just a static scaffold, it is a dynamic, information-rich source of inputs for cells [2]. The idea of the basal lamina reconstruction is based on the three aspects, porous scaffold, adsorbed fibronectin (FN) as a endothelium bed and adsorbed collagen on the other side of the porous material under a muscle cell (Fig. 1). FN is a prominent constituent of ECM around and beneath many cells, and FN-rich matrices provide substrates for cell adhesion and migration during development, wound healing, and other situations, as well as affecting many cellular functions including proliferation, survival, and differentiation. Collagen is a group of naturally occurring proteins. Collagen constitutes 1% to 2% of muscle tissue.

Acknowledgments

               This research was supported financially by the multiyear strategic project “Polish Artificial Heart” No: 2/0-PW/PO1-PBZ-MNiSW/2007 and the project CardioBioMat MNT Era-Net-MNT/15/2009 “Nanostructural materials for implants and cardiovascular biomedical devices”.

Literature

1.)  B. M. Leung  and M. V. Sefton; A Modular Tissue Engineering Construct Containing Smooth Muscle Cells and Endothelial Cells; Annals of Biomedical Engineering, Vol. 35, No. 12, December  (2007) pp. 2039–2049

 2.)  RichardO.Hynes; The dynamic dialogue between cells and matrices: Implications of

fibronectin’s elasticity; Proc. Natl. Acad. Sci. USA Vol. 96, pp. 2588–2590, March (1999)

Wear resistant coatings are frequently used to protect and enhanced life time of industrial components working under high and constant wear load. Wear of hard coatings directly depends on applied load, sliding speed, contact geometry and humidity. Cracks initiation and propagation are often responsible for wear. The presence of cracks at columnar boundaries are formed during deposition process (micro- cracks are formed because of high residual stress) and are enhanced during wear. Coatings delamination can be caused by several factors: (i) fragility of coatings which are characterized by columnar microstructure; (ii) the weak coating/ substrate adhesion caused by the presence of oxide layer on the substrate. Titanium nitride is the standard material among transition metal nitrides used as a hard coatings in industry [1,2]. Modification of this material are intended to achieve better tribological properties. The second hard material which is of a high interest for coatings is amorphous carbon (a:C-H) (known as a DLC- diamont like carbon) [3]. On one side it is characterized by high  hardness on the other side by high elastic modulus. Amorphous carbon is biologically neutral which gives it additional advantage for application. Recently composite or multilayer coatings are of special interest. The soft phase is in combination with hard one. In the case of composite coatings hard, ceramic phase is set in soft, metallic matrix. In the case of multilayer systems hard, ceramic layers are deposited in sequence with soft, metallic layers [4- 6]. The risk of sudden coating delamination under applied load is reduced. Multilayer Ti/TiN or Ti/a:C-H systems display an improved fracture resistance as compared to homogenous TiN [7] or a:C-H. The wear of multilayer coatings can be much more predictable. Cracks in ceramic layers can be stopped by plastically deformed metallic layers. The Ti layer would allow extensive plastic deformation at crack tip, deflects cracks. The current work is focused on description of wear mechanisms of single (ceramic) layers as well as multilayer coatings by transmission electron microscopy. Ceramic, single layer coatings wear treated as a reference materials for multilayer systems. Different wear settings were applied to present several wear stages form initiation till completely destruction of coating. 

References:

[1]   R.F.Bunshah: Handbook of Hard Coatings. ISBN 0-8155-1438-7, NP New Jersey USA (2001)

[2]   D.S. Rickerby, A.Matthews. Advanced Surface Coatings; Handbook of Surface Engineering. Chapman and Hall N.Y. USA (1991)

[3]   B.K.Gupta, Bharat Bushan: Micromechanical properties of amorphous carbon coatings deposited by different deposition techniques. Thin Solid Films 270(1995)391- 398

[4]   K.Ichijo, H.Hasegawa, T.Suzuki: Microstructures of (Ti,Cr,Al,Si)N films synthesized by cathodic arc method. Surface & Coatings Technology, 201 (2007) 5477–5480

[5]   P.C.Jindal, A.T.Santhanam, U.Schleinkofer, A.F.Shuster: Performance of PVD TiN, TiCN, and TiAlN coated cemented carbide tools in turning. International Journal of Refractory Metals and Hard Materials, 17 (1999) 163-170

[6]   Li Chen, S.Q. Wang, S.Z. Zhou, Jia Li, Y.Z. Zhang: Microstructure and mechanical properties of Ti(C,N) and TiN/Ti(C,N) multilayer PVD coatings. International Journal of Refractory Metals and Hard Materials, 26 (2008) 456–460

[7]   Ł.Major, J.Morgiel, J.Lackner, M.J.Szczerba, M.Kot, B.Major: Microstructure Design and Tribological Properties of Cr/CrN and TiN/CrN Multilayer Films. Advanced Engineering Materials 10(2008)617-621

Hydroxyapatite bioceramics are widely used is therapy of bone defects. Recent research of hydroxyapatite based bioceramics focuses on development of materials of increasingly biomimetic character. This feature is expected to provide the material with enhanced biocompatibility. Hydroxyapatite derived from porcine bones, being material of biological origin, is excellent candidate for new bioceramic materials intended for treatment of bone defects.

In presented work stability and biocompatibility dense and microporous bioceramics based on porcine origin hydroxyapatite were investigated under in vitro conditions. Stability investigations  involved incubation of hydroxyapatite bioceramics in Simulated Body Fluid for 7 and 14 days at 37°C. In the course of biocompatibility investigations colonies of human osteosarcoma (CAL-72 cells) were grown on the surface of selected hydroxyapatite bioceramics for 12 days. As reference materials served bioceramics prepared from stoichiometric synthetic hydroxyapatites.

The research showed that bioceramics based on hydroxyapatite of porcine origin are stable under conditions simulating the inside of human body. Results of biocompatibility investigations confirmed the assumption that biomimetic character of hydroxyapatite derived from pig bones will yield more favorable reaction of biological material towards the ceramic sample compared to ceramics based on stoichiometric synthetic hydroxyapatites.

The main objectives of the work are polymer tubes with modified internal surface towards blood cells biocompatibility. New blood contacting material for forced external and internal blood circulation is under the elaboration. The internal surfaces of the tube like devices were covered with anti thrombogenic coatings or biomimeticly behaved to vessel surface, which means to influence on the endothelial cells to cover. Carbon, titanium and biopolymer based materials are considered for deposition. In the frame of the performed work, cooperation between several European institutions dealing with materials science, biology, biomechanics and medicine is performed.

Material characterization and analysis was done with atomic force microscope (AFM) and differential interference contrast microscope (DIC). Biokinetic interactions were measured by means of cone platelet analyzer adapted in this case. Results were observed with confocal microscope and compared with flow cytometry. One of the most common ways to study cellular characteristics, like activated platelets, uses fluorescent molecules such as fluorophore-labeled antibodies. In the work we used antybody CD 45 and CD 62P coniugated with Phycoerithrin and fluoresceine FITC, respectively. In these experiments, the labeled antibody is added to the cell sample. Then, it binds to a specific molecule on the cell surface or inside the cell.              Average roughness, amount of platelets and aggregates remained on the surface and in blood sample after bio-kinetic test were major parameters taken into consideration.

Acknowledgments

This research was supported financially by the project CardioBioMat MNT Era-Net-MNT/15/2009 “Nanostructural materials for implants and cardiovascular biomedical devices”.

Our sincere thanks to Mateusz Więcaszek from Department of Physics of Nanostructures and Nanotechnology , Institute of Physics, Jagiellonian University for Atomic Force Microscopy Measurements.

Increasingly, disorders of organs and tissues included in the cardiovascular system require the treatment involving implantation of prothesis which can support or replace damaged part of the organ. Polimeric biomaterials offer potentially high constructional possibilities . There are a lot of stringent restrictions on physico-chemical properties of material and it’s biocompatibility, compliance with which is a prerequisite for the proper functioning of the implant in the host environment. After implantation of biologically incompatible material, significant changes in immunologic function may occur. Host response to the presence of incompatible a material may involve the reaction at the cellular level, including activation of selected cell populations. This can be manifested by an increase in intracellular calcium ion concentration and change in activity of cell surface’s receptors, such as: CD3, CD4, CD8, CD14, CD16, CD19, CD40, CD45, CD86. In addition to this, biologically incompatible material may potentially induce apoptosis of peripheral T and B cells.

Therefore, the purpose of the work is to assess the biocompatibility of polymer materials through the analysis of lymphocyte activation in in vitro conditions.

The study will be performed with the use of microscopy and flow cytometry techniques. Fluo-3 dye will be used in order to detect changes of intracellular Ca²⁺ concentration.  Corresponding primary antibodies labeled with fluorochromes will be used to evaluate the activity of extracellular receptors of T and B cells,. In turn, detection of apoptotic cells will be performed with the use of  Annexin V / Propidium iodide apoptosis detection kit.

Acknowledgments

This research was supported financially by the project CardioBioMat MNT Era-Net-MNT/15/2009 “Nanostructural materials for implants and cardiovascular biomedical devices”.

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