AVS2014 Session SM+AS+BI+PS-ThA: Plasma Processing of Biomemetic Materials

Non equilibrium plasmas have many advantages over wet chemistry approaches: they are highly eco-friendly, have high potentialities in developing surfaces with peculiar characteristics, are capable to be part of in-line material processing and most importantly, can be applied to any material. Consequently, it has opened many new opportunities for investigation of surface modification in various fields like tissue and organ regeneration and biosensing. In this talk, we shall give a brief review on the recent developments of plasma processing of porous materials. We shall describe our experience on non-equilibrium plasmas to modify materials of biomedical interest like: scaffolds for tissue engineering and 3D carbon nanotubes carpets for bio-sensing. This research is aimed to gain new insights on the potentialities of plasma processing of biomedical materials. This work is encouraged by a deep characterization of material’s surface and investigation of the material/ bio-environment interface.

Poly(N-isopropyl acrylamide) (pNIPAM) undergoes a conformation change in a physiologically relevant temperature range: it is relatively hydrophobic above its lower critical solution temperature (LCST, ~32oC), and mammalian cells are easily cultured on pNIPAM-grafted surfaces. When the temperature is lowered below the LCST, the polymer’s chains rapidly hydrate, and cells detach as intact sheets capable of being used to engineer tissues (“cell sheet engineering”). This behavior has led to a great deal of interest from the bioengineering community, resulting in a variety of film deposition methods, substrate storage techniques, and cell release methods. Unfortunately, this has also resulted in widely varying responses (e.g., % of cells released, biocompatibility and stability of surfaces, etc.) from the resulting cell sheets. In this work, we present a comprehensive comparison of the surface chemistry, biocompatibility, and effect on reversible cell adhesion that results from pNIPAM substrates fabricated using the most common polymerization (free radical and plasma polymerization) and deposition (spin coating and plasma polymerization) techniques. The relative biocompatibility of different mammalian cells (e.g., endothelial, epithelial, smooth muscle, and fibroblasts) was evaluated using appropriate cytotoxicity tests (MTS, Live/Dead, plating efficiency). The pNIPAM-coated surfaces were evaluated for their thermoresponse and surface chemistry using X-ray photoelectron spectroscopy and goniometry. We find that plasma polymerized NIPAM substrates (ppNIPAM) are more stable under a variety of storage conditions prior to their use. Furthermore, when used for cell culture, ppNIPAM films exhibit no cytotoxicity toward any of the cell types tested and yield excellent cell detachment (~85%), which is an important consideration for their ultimate use in engineered tissues.

Despite major research efforts in the field of biomaterials, rejection, severe immune responses, scar tissue and poor integration continue to seriously limit the performance of today’s implantable biomedical devices. Implantable biomaterials that interact with their host via an interfacial layer of active biomolecules to direct a desired cellular response to the implant would represent a major leap forward. Another, perhaps equally revolutionary, development that is on the biomedical horizon is the introduction of cost-effective microarrays for fast, highly multiplexed screening for biomarkers on cell membranes and in a variety of analyte solutions.

Both of these advances will rely on the availability of methods to strongly attach biomolecules to surfaces whilst retaining their biological activity. Radicals embedded in nanoscale carbon rich surface layers by energetic ion bombardment can covalently immobilize bioactive proteins [Proc. Nat. Acad. Sci 108(35) pp.14405-14410 (2011)] onto the surfaces of a wide range of materials, including polymers, metals, semiconductors and ceramics . This new approach delivers the strength and stability of covalent coupling without the need for chemical linker molecules and multi-step wet chemistry. Immobilization occurs in a single step directly from solution and the hydrophilic nature of the surface ensures that the bioactive 3D shapes of the protein molecules are minimally disturbed.

This presentation will describe recently developed approaches that use energetic ions extracted from plasma to facilitate simple, one-step covalent surface immobilization of bioactive molecules. A kinetic theory model of the biomolecule immobilization process via reactions with long-lived, mobile, surface-embedded radicals and supporting experimental data will be presented. Progress on applications of this technology to create antibody microarrays for highly multiplexed, simple analysis of cell surface markers and to engineer bioactive surfaces for implantable biomedical devices will be reviewed.

Three-dimensional (3D) bioresorbable polymeric materials, such as porous scaffolds made of poly(ε-caprolactone) (PCL), have desirable bulk properties for tissue engineering, wound healing, and controlled-release drug delivery applications. However, the surface properties (e.g., chemical functionality and wettability) are often undesirable for certain biomedical applications. Therefore, the ability to fabricate 3D materials with ideal bulk properties and customizable surface properties is a critical aspect of biomaterial development. Here, we demonstrate the deposition of conformal films throughout the 3D porous scaffold network using plasma enhanced chemical vapor deposition (PECVD). Resulting film properties can be tailored by using different precursor species. Octofluoropropane (C₃F₈) and hexafluoropropylene oxide (HFPO) precursors were chosen as model hydrophobic film PECVD systems, whereas a copolymerization system consisting of allylamine/allyl alcohol (allylNH/allylOH) precursors was chosen as a model hydrophilic, nitrogen containing PECVD system. To ensure the efficiency and reproducibility of the treatments, both the exterior and interior of the plasma treated scaffolds were characterized using contact angle goniometry, X-ray photoelectron spectroscopy (XPS), and scanning electron microscopy (SEM) to assess changes in wettability, chemical functionality, and scaffold architecture in comparison to untreated scaffolds. C₃F₈ and HFPO PECVD on scaffolds resulted in fluorocarbon films on the exterior of the scaffold, and the extent of deposition throughout the scaffold’s 3D structure was controlled by treatment time. The nitrogen content of the allylNH/allylOH films was tailored by changing the feed gas composition of the copolymerized films. After surface modifications, modified PCL scaffold surface interactions with cells and bacteria were assessed to confirm the relevance of these coatings for the biomedical field. We also explored the effect of different plasma treatments on cell adhesion/proliferation using both human dermal fibroblasts and endothelial cells, bacterial attachment, and biofilm formation using Escherichia coli.

Wound healing is a multi-billion dollar drain on healthcare systems around the work. This is particularly true in developed countries as they deal with aging populations and conditions such as vascular disease and diabetes. More than 30% of the costs associated with treating diabetes can be attributed to management of chronic wounds. Dressings for the clinical management of wounds are constantly evolving to provide antimicrobial environments and optimal gas exchange, pH and hydration to facilitate wound healing. Ideally, the next generation of wound dressings will also provide a favourable surface for cell attachment, proliferation and migration to further promote the healing process. A number of approaches have been developed for healing chronic wounds, many of which involve culturing of explanted cells, or donor cells, and returning them to the wound site. In this paper, we have used plasma polymerisation to develop surfaces which influence the migration rate of primary cells (keratinocytes, fibroblasts and endothelial cells). A pro-migratory surface will enable cell transport into the wound bed. Earlier workers have concentrated on cell attachment as a key measurement of clinical potential, but we have observed that cell mobility exhibits a preference for different surface chemistry to attachment, and this preference depends on cell type. We show how plasma polymerization can be used to produce surfaces with controllable chemistry, and explore the effect of changing surface chemistry on the migration rate of primary fibroblasts and keratinocytes in vitro. We also investigate the effect of these surfaces on wound closure rate using an in-vitro wounding model based on an engineered skin composite. We also explore the application of plasma polymerized pro-migratory surfaces to electrospun scaffolds for use with deeper wounds.