Haptotatic Plasma Polymerized Surfaces for Rapid Tissue

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Haptotatic plasma polymerized surfaces for rapid tissue regeneration and wound healing. Louise E. Smith, Christian Bryant, Marta Krasowska, Allison J. Cowin, Jason D. Whittle, Sheila MacNeil, and Robert D. Short ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b11320 • Publication Date (Web): 07 Nov 2016 Downloaded from http://pubs.acs.org on November 8, 2016

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ACS Applied Materials & Interfaces

Haptotatic Plasma Polymerized Surfaces for Rapid Tissue Regeneration and Wound Healing Louise E. Smith1,2*, Christian Bryant1, Marta Krasowska2, Allison J. Cowin1,2, Jason D. Whittle1,3, Sheila MacNeil4 and Robert D. Short1,2 1 Wound Management Innovation Cooperative Research Centre, Brisbane, 4059, QLD, Australia 2 Future Industries Institute University of South Australia, Adelaide, 5095, SA, Australia, 3 School of Engineering, University of South Australia, Adelaide, 5095, SA, Australia, 4 Kroto Research Institute, University of Sheffield, Sheffield S3 7HQ, South Yorkshire, UK

KEYWORDS Surface Modification; Plasma Polymerization; Wound healing; Cell Migration, Haptotaxis

ABSTRACT Skin has a remarkable capacity for regeneration; however, with an ever aging population there is a growing burden to the healthcare system from chronic wounds. Novel therapies are required to address the problems associated with non-healing chronic wounds. Novel wound dressings that can encourage increased reepithelialization could help to reduce the burden of chronic wounds. A suite of chemically defined surfaces have been produced using plasma polymerization and the ability of these surfaces to support the growth of primary human skin cellshas been assessed. Additionally, the ability of these surfaces to modulate cell migration and morphology has also been investigated. Keratinocytes and endothelial cells were extremely sensitive to surface chemistry showing increased viability, 1 ACS Paragon Plus Environment


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and migration with an increased number of carboxylic acid functional groups. Fibroblasts proved to be more tolerant to changes in surface chemistry, however these cells migrated fastest over amine functionalized surfaces. The novel combination of comprehensive chemical characterization coupled with the focus on cell migration provides a unique insight into how a materials physico-chemical properties affect cell migration.

1. Introduction Chronic wounds are an area of growing concern given our aging population. In these wounds poor vasculature, chronic inflammation and intermittent bacterial infection all combine to prevent the normal wound healing programme occurring. In superficial burns where there is extensive skin loss but no underlying pathology cells at the margin of the wound and in the epidermal inclusions in the dermis need to migrate rapidly to restore the skin barrier layer. In the treatment of both burns and chronic wounds there is a need for new biomaterial strategies that will stimulate patients cells to engage in the healing process 1. For many wound products, xenobiotic materials are often used to enhance the surfaces of synthetic (polymeric) materials. The base materials of wound dressings are usually polyesters, polysiloxanes or polyurethanes 2-4, which themselves, because of low surface energy do not support cell attachment and migration. These materials are selected based upon their pre-existing FDA approvals, bulk properties, and their availability 2-4. However, they are far from ideal for direct contact with cells, biological tissues and/or fluids. For example, Biobrane® (Smith and Nephew) which promotes cell migration over superficial burns, comprises a silicone membrane bonded to a nylon mesh. Functionality, to guide cellular migration, is derived from a coating of dermal porcine peptides 5. Dermagraft® (Organogenesis, Inc) a 3-D scaffold, used in chronic wounds and deep burns, comprises a

2 ACS Paragon Plus Environment

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polyglactin mesh that is conditioned by neonatal foreskin fibroblasts to promote cell migration 5-6. Cellular migration over biological substrates is particularly relevant in wound healing 7-8. In vitro studies of keratinocyte migration are usually performed on collagen surfaces using serum-free defined media, containing little, > 0.25mM calcium, whilst physiological calcium is approximately 1.2mM. This simple reduction in media Ca2+ levels results in a doubling of the keratinocyte migration rate from 30 µm per hour (µm/h) to 60 µm/h 9. Migration of NIH 3T3 mouse fibroblasts, over collagen I coated substrates versus collagen IV-coated substrates also show a measurable difference in migration rates of 11 µm/h versus 9 µm/h. 3T3 fibroblasts have been shown to migrate at rates of up to 42 µm/h over tissue culture plastic, with the rate of migration highly dependent on seeding density 10. Whilst there is an abundance of data on the migration of 3T3 fibroblasts, finding comparative data on keratinocyte and endothelial cell migration remains limited. Whilst extracellular matrix materials (ECM) support the migration of skin cells, significant advantages exist for employing entirely xenobiotic-free materials based upon new surface coating chemistries; these include, minimising the transmission of infective agents and reducing the complexity, variability and cost of manufacture in healthcare products. In this context it is important to recognise that cells in contact with polymeric materials respond to different surface properties including: chemical functional groups 11-12, surface topography 1314

, stiffness 15-16 and combinations of the above.

The effects of surface chemical groups have been extensively studied in cell culture both using cell lines 17-18 and primary cells 19-23. These studies have focused primarily on cell attachment, proliferation, and the maintenance of cell phenotype and/or cell differentiation 23. However, in the development of the next generation of tissue repair and wound healing



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products, a key feature will also include the ability to promote cell migration by tailoring the physio-chemical properties of the surface i.e. haptotaxis. Plasma polymerization 24 is highly suited to improve the surface characteristics of base polymeric materials for applications in tissue repair and wound healing products. Importantly, plasma polymerization applies new chemistry to the surface without affecting the bulk properties or topography of the material. Plasma polymerization reproducibly produces nanometre thick coatings of defined chemistry on most substrates from planar 2-D surfaces 25 and simple 3-D objects such as micro-titre plates 26-27 to complex 3-D tissue engineering scaffolds 28-31, and fabrics and fibres 32. As in conventional polymerization, the functionality of the polymer can be controlled through the choice of monomer and the density of functional groups in the final material through co-polymerization of a functional compound with a diluent co-monomer. In this study, plasma polymerization and copolymerization were used to prepare a suite of polymeric plasma polymer (PP) surfaces (from four organic monomers namely acrylic acid (ppAAc), allyl amine (ppAAm), allyl alcohol (ppAAOH) and copolymers with the diluent hydrocarbon 1,7, octadiene, (ppOD) 18. In previous studies of cell attachment to plasma polymerized coatings, the surface analysis has been restricted to x-ray photoelectron spectroscopy (XPS). However, this technique only provides chemical data on dry samples. Therefore in this study, the chemistries of plasma copolymer surfaces were confirmed using XPS, but the analysis was extended to surface wettability, zeta potential determination, as well as surface topography measurements. All of these data were collected for wet PP substrates. These additional techniques provide a far more complete physico-chemical surface analysis. Primary human skin cells, keratinocytes, fibroblasts and microvascular dermal endothelial cells, were cultured on these plasma polymerized surfaces and measurements of viability, morphology and migration determined.


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These studies have allowed us to identify, candidate haptotactic surfaces that support migration of skin cells for subsequent preclinical development. 2. Results 2.1 Characterization of surface chemistry Plasma polymer surfaces were characterised using XPS to confirm elemental composition of the presence of the desired functional groups (Table 1). Tissue culture polystyrene (TCP) was used as a control and therefore XPS data from this material are also included. AAc plasma polymers (ppAAc) contain approximately 20% COOH/R (carboxyl or ester) functionality. The peak fit for the high resolution XPS C1s linescan is shown in Figure S1. In this peak fit, we can be reasonably confident to assign the 289.2 eV peak to carboxylic acid (as opposed to ester) because of the relatively low corresponding C-OH/R (alcohol/ether) signal at 8.4%. However, when the proportion of the acrylic acid monomer in the plasma was decreased (≤ 75%) with the addition of octadiene, a marked drop off in the COOH/R signal was observed. The same trend in functional group retention (COH/R) was seen in the plasma polymers produced from AAOH vapour (ppAAOH), where the amount of alcohol/ether functionality decreased as the amount of AAOH in the plasma decreased. This can be assumed to be mostly alcohol, based upon previous labelling studies 33. The N:C ratio decreased in a similar manner in allyl amine plasma polymers (ppAAm) with the addition of octadiene, but due to the complication relating to the number of potential functional groups present in the amine-containing films, it is difficult to quantify the amount of amine functionality, therefore the N:C ratio is used as a surrogate. Recent work investigating the retention of amine groups in plasma polymer films (produced under similar



conditions, and in the same plasma reactor) suggests that a large proportion of the original primary amines are retained within the ppAAm films 34. Further XPS data, widescan spectra and C1s coreline spectra for 100% ppAAc, ppAAOH and ppAAm, along with TCP are shown in Supporting Information (S1).

Table 1. Chemical composition of plasma polymers assessed by XPS: elemental N:C ratio calculated from survey spectra; percent C-OH/R, C=O, and C-OOH/R functionality calculated from peaking fitting of high resolution C1s scans. Elemental ratios and peak fitted data show the effect of dilution of OD in the polymerization of the three functionalized monomers (AAc, AAm and AAOH). Data are expressed as the mean of 3 batches of plasma polymers. Shaded sections represent surface functionalities of interest for the monomers and blends used. In no sample was there any evidence of the Al substrate, indicating that all PPs were at least 10 nm thick, the accepted sampling depth of XPS.

Acrylic Acid

% functional monomer in gas flow

100%

75%

50%

25%

C1s

71.6

82.8

87.2

90.2

O1s N1s CO/R CO COO/R

28.4

17.2

12.7

9.7

8.4 4.337 19.9

8.4 1.524 8.5

7.5 0.764 4.2

5.5 0.731 1.5

C1s

78.3

87.8

89.5

93.8

O1s N1s

7.1 14.6

4.9 7.3

6.0 4.5

4.1 2.1

N:C Allyl amine

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CO/R CO COO/R N:C

0.19

0.09

0.05

0.02

C1s

84.9

90.2

93.3

95.2

O1s N1s CO/R CO COO/R N:C

15.0

9.8

6.7

4.8

18.1 4.509 1.9

12.1 2.594 0.7

9.1 1.690 0.4

7.0 1.067 0.1

C1s

93.5

77.7

O1s N1s CO/R CO COO/R N:C

6.4 0.1 4.6 2.695 0.1 0.0

21.2 1.1 14.6 2.148 11.2 0.0

TCP

Octadiene

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Allyl alcohol

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TCP can be highly variable with respect to surface chemistry, and more specifically it is prone to ageing post- plasma. 35-36. Therefore, in employing TCP as the control across all experiments the surface O/C and functional group composition at the time was used. The XPS of TCP revealed a highly-oxidized surface with an O/C of (0.27) and a good proportion of carbon in a highly oxidised environment (COOH/R). 2.2 Film Thickness and Roughness The PP surfaces were analyzed by AFM for thickness (in air and in PBS), swelling/dissolution [%] and roughness (RMS). These data are presented in Figure 1. Selected AFM images are presented in the supporting information (Figure S2).



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Figure 1: - A) Atomic force microscope measurements of film thickness both dry and wet, data expressed as mean ± SEM (n=5). Most films showed some degree of swelling when immersed in PBS. The exceptions being the 100% ppAAc films which showed significant dissolution. B) RMS roughness measurements of dry and hydrated plasma polymer films. All films whether dry or wet were smooth with a RMS roughness of less than 1 nm.

From Figure 1, it can be seen that all monomers were fast depositing monomers (i.e. they provided thick coatings in the dry state, which is consistent with our previous findings 37. Acrylic acid deposited at a rate of 1.3 nm/min with the rate increasing slightly as the concentration of octadiene in the plasma was increase. This was reflected by an increase in the starting pressure of the system needed to maintain the 4 sccm flowrate. Allylamine deposited at a rate of 1 nm/min, again the deposition rate increased with increase amounts of octadiene in the plasma. Allyl alcohol initially deposited at a rate of 2 nm/min, however in this case the introduction of octadiene into the plasma depressed the deposition rate.


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Octadiene itself deposited at a rate of 1.6 nm/min. The films were also smooth, RMS

Haptotatic Plasma Polymerized Surfaces for Rapid Tissue

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