Elsevier

Acta Biomaterialia

Volume 8, Issue 4, April 2012, Pages 1627-1638
Acta Biomaterialia

Cellular and transcriptomic analysis of human mesenchymal stem cell response to plasma-activated hydroxyapatite coating

https://doi.org/10.1016/j.actbio.2011.12.014Get rights and content

Abstract

Atmospheric pressure plasma has recently emerged as a technique with a promising future in the medical field. In this work we used the technique as a post-deposition modification process as a means to activate hydroxyapatite (HA) coatings. Contact angle goniometry, optical profilometry, scanning electron microscopy morphology imaging and X-ray photoelectron spectroscopy analysis demonstrate that surface wettability is improved after treatment, without inducing any concomitant damage to the coating. The protein adsorption pattern has been found to be preferable for MSC, and this may result in greater cell attachment and adhesion to plasma-activated HA than to untreated samples. Cell cycle distribution analysis using flow cytometry reveals a faster transition from G1 to S phase, thus leading to a faster cell proliferation rate on plasma-activated HA. This indicates that the improvement in surface wettability independently enhances cell attachment and cell proliferation, which is possibly mediated by FAK phosphorylation. Pathway-specific polymerase chain reaction arrays revealed that wettability has a substantial influence on gene expression during osteogenic differentiation of human MSC. Plasma-activated HA tends to enhance this process by systemically deregulating multiple genes. In addition, the majority of these deregulated genes had been appropriately translated, as confirmed by ELISA protein quantification. Lastly, alizarin red staining showed that plasma-activated HA is capable of improving mineralization for up to 3 weeks of in vitro culture. It was concluded from this study that atmospheric pressure plasma is a potent tool for modifying the biological function of a material without causing thermal damage, such that adhesion molecules and drugs might be deposited on the original coating to improve performance.

Introduction

The term plasma is used to describe a collection of charged particles (an ionized gas) in physicochemical science and also the liquid component of blood in medicine. Both plasma types share a cardinal feature: both are macroscopically neutral and microscopically ionized active media [1]. This article focuses on the type of plasma which is generated by applying energy to a gas resulting in a mixture of ions, electrons and neutral species. A plasma can be classified as thermal or non-thermal, depending on its method of creation. Thermal plasma processes have a long history of industrialization [2], of which a notable application is coating metallic substrates with hydroxyapatite (HA) for orthopedic implantation [3]. Despite its industrial efficiency, thermal plasma deposition results in undesirable changes to thermally sensitive substrates such as HA [4]. Encouragingly, non-thermal plasma techniques have recently extended plasma treatment to living tissue [5], primarily due to its finely tunable effects and selective influence on pathological/physiological tissues [6].

The medical applications of non-thermal plasmas fall into two major types: direct and indirect plasma treatment, differentiated by the amount of charged species applied to the surface of the living tissue. Direct plasma treatment gives off ozone, NO and OH radicals to the surface, with some passing through the living tissue. It is therefore mainly applied in skin sterilization, blood coagulation [7], and assisting wound healing and tissue regeneration [8]. On the other hand, indirect plasma treatment, represented by atmospheric pressure plasma jets (APPJ) [9], delivers reactive species generated between two electrodes to the area of application in the form of a gas flow. Apart from the recently discovered effects of indirect plasma treatment on cell apoptosis [10], necrosis [11] and detachment [12], very little is known regarding its influence on osteogenic lineage cells or bone tissue, let alone its application in surgical repair of hard tissue. In this study, rather than applying APPJ straight to cells, we used an HA coating, which is one of the most commonly used orthopedic biomaterials, as a medium to transfer the effect of APPJ to bone-forming cells. This is possible due to several advantages of APPJ: they are applicable to thermally sensitive materials, they produce uniform treatment across a relatively large area, they do not require a vacuum system [13], and they are applicable to surfaces with micron scale roughness [5].

One well-known approach to surface treatment by atmospheric plasmas is surface activation, after which the surface energy is increased [14]. This has been proven on polymer surfaces after activation by APPJ [15], [16]. Thus we hypothesize that plasma activation of HA coatings may similarly raise the surface energy or wettability. The relationship between surface energy/chemistry and bone-forming cells has been extensively studied and reviewed using various cell lines on both HA-based and non-HA-based materials [17], [18], [19], [20]. A consensus seems to have been reached, at least on the aspect of cell adhesion, which is positively correlated with surface wettability [19], [21]. A systematic study by Lim et al. has revealed that not only osteoblast adhesion but also proliferation rate are strongly correlated with substrate surface wettability [22]. The early effects have been extensively studied and reported in the literature. In contrast, much less is known of the long-term effects of surface energy on cell activities such as differentiation and in vitro mineralization [23], [24], [25].

In this study cultures of undifferentiated and osteogenically differentiated human mesenchymal stem cells (MSC) were used to explore the short- and long-term cell responses to plasma-activated HA coatings, respectively. A further objective of this study was to fully elucidate the cell–surface interaction following plasma activation of HA during osteogenic differentiation, specifically at the transcriptome level. Levels of mRNA are not necessarily directly proportional to the level of expression of the proteins they encode, due to variations in the translation initiation sequence of the mRNA [26]. Therefore, selective protein investigations were performed to bridge the technical quantification of transcriptomes with the physiological relevance of proteomes.

Section snippets

Deposition of HA coatings

The HA coatings used in this study was deposited on grade V Ti–6Al–4V alloy coupons 20 × 20 × 1 mm in size (Lisnabrin Engineering Ltd, Cork, Ireland). The test substrates were polished with 1200 grit size silicon carbide paper. The polished samples were then ultrasonically cleaned with acetone, then methanol, then isopropyl alcohol solution for 5 min to remove any residual particulates. A novel non-thermal coating technique, CoBlast [27], [28], was chosen over the thermal plasma spray procedure, in

Surface analysis of the coatings pre- and post-activation

HA coating wettability was measured using a contact angle goniometer. Three comparisons are demonstrated in Fig. 3A: between control and activated HA, between 1 day and 8 weeks of storage, and among four different liquids. Activating the HA coating significantly enhances wettability, as demonstrated by the dramatic reduction in contact angle from 35° to <5°, irrespective of type of liquid used for the measurement. This is within our expectation, as the energy contained in plasma could be

Conclusions

This study has evaluated the effect of plasma activation of HA on the response of human MSC. After plasma activation the HA liquid contact angle was observed to decrease to <5°. It was found to be stable at this level for the 8 week test period. XPS demonstrated enhanced levels of oxygen and nitrogen in the activated HA coating surface. Quantitative fluid shear studies and qualitative SEM investigations have revealed that the adhesion and attachment of undifferentiated MSC to plasma-activated HA

Acknowledgements

This work was supported by Science Foundation Ireland (Grant No. 08/SRC/I1411). The authors wish to thank EnBIO (Cork, Ireland) for providing technical assistance regarding HA coating deposition.

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