AVS2012 Session BI-MoM: Surfaces to Control Cell Response

The development of versatile and robust strategies for the surface modification of multiple classes of materials has proven challenging, with few generalized methods. Many available methods have limitation for widespread use due to the need for specific surface chemistries and/or laborious multistep procedures^[1]. A protocol to deposit brominated plasma polymer (Brpp) thin films on a variety of substrate surfaces (silicon wafers, glass, gold, Teflon) has been developed. These coatings are highly adherent and exhibit good stability in aqueous, biphasic and autoclaving conditions. The Brpp coating was found to be a useful platform for secondary reactions leading to surfaces with specific chemical properties. Following nucleophilic exchange, azide functionalized surfaces were developed and the copper catalysed azide alkyne cycloaddition (CuAAC) reaction^[2]; a paradigm of click chemistry, was successful in immobilizing various acetylenes. A particular highlight is the patterning of cells via selective surface functionalisation of PEG-alkyne using a photomask.^[3] This is the first known example of CuAAC reactions on pp thin films. A detailed physicochemical characterisation study of these films will also be presented.

[1] aH. Lee, S. M. Dellatore, W. M. Miller, P. B. Messersmith, Science 2007, 318, 426-430; bD. Y. Ryu, K. Shin, E. Drockenmuller, C. J. Hawker, T. P. Russell, Science 2005, 308, 236-239.

[2] R. A. Evans, Australian Journal of Chemistry 2007, 60, 384-395.

[3] Chen, R. T.; Marchesan, S.; Evans, R. A.; Styan, K. E.; Such, G. K.; Postma, A.; McLean, K. M.; Muir, B. W*.; Caruso, F., Biomacromolecules 2012, 13, (3), 889-895.

Development of long-term stable surfaces that resist bio-adhesion continues to stimulate the field of biomedical and biological research. While numerous strategies have been developed over the last several decades, the challenge remains in the creation of surfaces that can provide long-term ‘zero’ bio-adhesion from a variety of biological entities that spans lengths scales from biomolecules to cells. Although the physical and chemical properties of the resisting surface itself are important in achieving this ultimate goal, assessing the extent of bio-adhesion must be accompanied by detailed surface analysis via highly sensitive analytical techniques.

We have recently discovered that increasing the temperature alone during the assembly process of poly-l-lysine grafted poly (ethylene glycol) (PLL-g-PEG) results in the formation of highly dense PLL-g-PEG brush coating. The PLL-g-PEG surfaces prepared at various temperatures (20 to 80 °C) have been characterized by X-ray photoelectron spectroscopy (XPS). The PLL-g-PEG surfaces prepared at the ‘standard’ temperature of 20 °C are found to be comparable to the previously reported literatures. Interestingly, the surfaces prepared at 80°C have shown the highest surface grafted density of PLL-g-PEG, with ~ 4 times denser than those prepared at 20 °C.

The degree of cell and protein adhesions on these surfaces has been stringently determined using cell culture and serum/blood adsorption assays combined with XPS and time of flight secondary ion mass spectrometry (ToF-SIMS). The temperature-induced PLL-g-PEG surfaces have achieved ‘zero’ cell adhesions from three different types of mammalian cells for at least 36 days. In addition, XPS and ToF-SIMS analysis have confirmed near-zero protein adsorptions from 10% serum/MEM (at least 36 days), whole undiluted blood (at least 24 hrs) and undiluted serum (at least 24 hrs) with the surfaces being pre-incubated in high ionic strength buffer (2.4 M NaCl for 24 hrs).

Our approach to engineer cellular environments is based on self-organizing spatial positioning of single signaling molecules attached to synthetic extracellular matrices, which offers the highest spatial resolution with respect to the position of single signaling molecules. This approach allows tuning tissue with respect to its most relevant properties, i.e., viscoelasticity, peptide composition, nanotopography and spatial nanopatterning of signaling molecule. Such materials are defined as “nano-digital materials” since they enable the counting of individual signaling molecules, separated by a biologically inert background. Within these materials, the regulation of cellular responses is based on a biologically inert background which does not initiate any cell activation, which is then patterned with specific signaling molecules such as peptide ligands in well defined nanoscopic geometries. This approach is very powerful, since it enables the testing of cellular responses to individual, specific signaling molecules and their spatial ordering. Detailed consideration is also given to the fact that protein clusters such as those found at focal adhesion sites represent, to a large extent, hierarchically-organized cooperativity among various proteins. We found that integrin cluster have a functional packing density which is defined by an integrin-integrin spacing of approximately 68 nanometers. Such critical spacing values vary as matter of transmembrane receptor choice of interest. We have also developed methods which allows the light initiated activation of adhesion processes by switching the chemical composition of the extracellular matrix. This enabled us to identify the frequency of leader cell formation in collective cell migration as a matter of initial cell cluster pattern size and geometry. Moreover, “nano-digital supports” such as those described herein are clearly capable of involvement in such dynamic cellular processes as protein ordering at the cell’s periphery which in turn leads to programming cell responses.

In this study, we focus on a surface initiated Radical Addition-Fragmentation chain Transfer (RAFT) approach and present data demonstrating that dense polymer brushes can be prepared via surface immobilized macro-RAFT agents. The brush nature of the coatings was confirmed using a combination of XPS analysis and direct interaction force measurements with the AFM colloid probe technique. The properties of the coatings could be fine tuned using a variety of parameters such as the RAFT agent surface density, the polymerisation conditions, the monomer feed composition and the conjugation of cell attachment motifs such as cyclic peptides which interact with cell surface integrins. For example, the combination of a low cell adherent, low protein adsorbing polymer brush coating containing a conjugated peptide which interacted with alpha_vbeta₃ integrins resulted in a surface which supported the expansion of hMSCs in a xeno-free, chemically defined, serum replacement media. In addition the expanded cells expressed cell surface markers typical of undifferentiated hMSCs and the expanded cells were able to differentiate along adipogenic, osteogenic and chondrogenic pathways.

Surface properties of implant materials are known to influence biological responses they elicit. However, complex processes operating at the interface remain poorly understood. To get an insight into these processes, we investigated the role played by surface ion equilibrium in defining interactions between an implant material (TiO₂) and components of blood (in this case, platelets), because blood is the first tissue that foreign materials come into contact with when inserted into the body and because platelet response is crucial in defining the implant’s fate.

Titanium is a widely used biomaterial. Its success is in part due the favorable biocompatibility properties conferred by its oxide, TiO₂. We have previously shown that Ca²⁺-TiO₂ interactions affect the distribution of phospholipid phosphatidyl serine (PS) in model lipid membranes prepared on TiO₂. This allowed us to hypothesize that platelet activation will be affected by these interactions as well.

Platelets are anuclear cell fragments circulating in blood. Activated at wound sites, they aggregate and provide a catalytic surface for the formation of a fibrin-based clot that stops the bleeding. Recently, platelets have been recognized to participate in inflammation, wound healing, tissue regeneration, and immune responses. Activation of platelets by foreign surfaces is detrimental to blood-contacting implants but beneficial for osteoimplants. Upon activation, platelets expose on their surface and secrete a number of markers. These include PS, activated form of GPIIb-IIIa, and proteins CD62P and CD63 that are found in the membranes of the intracellular α- and dense granules of quiescent platelets. To assess the state of platelet activation on TiO₂, we assayed for the expression of these markers. In order to isolate a clear cause-and-effect relationship between Ca²⁺-TiO₂ interactions and platelet activation, we focused on purified platelets.

Our main finding is that the platelet activation profile on TiO₂ depends on the presence of Ca²⁺. Furthermore, in the absence of Ca²⁺, the α- and dense granule secretion is differentially regulated on titania. The differential granule secretion by platelets, as regulated by the surface properties, can be applied towards controlled release of molecules from platelets by nanoparticles or implants in drug delivery applications.

Introduction: Strontium (Sr) has been shown to have a beneficial influence on the subsequent remodelling of the bone structure in relation with implant osseointegration. Both decrease of the osteoclast driven bone resorption and enhancement of the osteoblast driven process of bone formation has been shown. Furthermore, Sr has proven to have an anti-inflammatory effect.

Methods: The coatings used in this study were either prepared on Ti implants (rods with diameter = 1.1 mm and length = 6 mm) or on silicon wafers. The Sr containing surface modifications were prepared by co-sputtering in a setup with a pure Ti and a sintered composite target. The samples were characterized using SEM, AFM, XPS and RBS. ICP-AES was used to investigate the amount of Sr released from the samples as a function of time. Human dental pulp stem cell (hDPSC) cultures were used to assess the in vitro cellular response: Cell attachment and proliferation was studied along with the cells ability to mineralize. Quantification of osteogenic expression markers and specific cytokines was performed via RT-PCR. Human blood derived monocyte cultures were carried out to investigate the in vitro differentiation of these into osteoclast-like cells in response to Sr. In vivo experiments were carried by inserting implants into the femur of Wistar rats and evaluation was done by assessing bone-to-implant contact and new bone volume.

Results: The amount of Sr incorporated in the surfaces was found to be between 0 and 8.7 at. %. The Sr release profile showed that the most Sr was released from samples incorporating 5.5 at. % Sr. In relation with the in vitroexperiments, the hDPSC proliferation and mineralization was found to correlate with the surface Sr concentrations. Moreover, the Sr concentration also affected the differentiation of monocytes into osteoclast-like cells. In relation with the in vivo experiment it was found that the incorporation of Sr had a beneficial effect on implant osseointegration, where an increase in direct bone contact and in new bone volume was observed with an increasing Sr release.

Discussion: From the in vitro and in vivo Sr release experiments it was found that a more dense surface structure developed as the Sr concentration were increased. We therefore speculate that the peak in the Sr release around 5.5 at.% can be ascribed to an optimal correlation between the morphology and the amount of incorporated Sr. The results from the in vitro and in vivo models shows that the coating process we have developed for modifying implants is an interesting candidate in relation with shortening the healing period when inserting osseointegrating implants.