- Email: [email protected]
Uniform cell colonization of porous 3-D scaffolds achieved using radial control of surface chemistry
Acta Biomaterialia 7 (2011) 3336–3344
Contents lists available at ScienceDirect
Acta Biomaterialia journal homepage: www.elsevier.com/locate/actabiomat
Uniform cell colonization of porous 3-D scaffolds achieved using radial control of surface chemistry Francesca Intranuovo a, Daniel Howard b, Lisa J. White b, Ramneek K. Johal c, Amir M. Ghaemmaghami c, Pietro Favia a, Steven M. Howdle d, Kevin M. Shakesheff b, Morgan R. Alexander e,⇑ a
Department of Chemistry, University of Bari, 70126 Bari, Italy Tissue Engineering Group, School of Pharmacy, University of Nottingham, Nottingham NG7 2RD, UK Allergy Research Group, School of Molecular Medical Sciences and Respiratory Biomedical Research Unit, University of Nottingham, Nottingham NG7 2RD, UK d School of Chemistry, University of Nottingham, Nottingham NG7 2RD, UK e Laboratory of Biophysics and Surface Analysis, School of Pharmacy, University of Nottingham, Nottingham NG7 2RD, UK b c
a r t i c l e
i n f o
Article history: Received 2 February 2011 Received in revised form 11 May 2011 Accepted 18 May 2011 Available online 24 May 2011 Keywords: Tissue engineering Porous scaffold Plasma polymerization Allyl amine Cell distribution
a b s t r a c t Uniform cellular distribution is a prerequisite to forming tissue within porous scaffolds, but the seeding process often results in preferential adhesion of cells at the periphery. We develop a vapour phase coating strategy which is readily applicable to any porous solid to provide a uniform cellular distribution. Plasma polymerized allyl amine (ppAAm) is used to form a thin nitrogen-containing coating throughout porous three-dimensional (3-D) poly(D,L-lactic acid) scaffolds. Subsequent controlled deposition of a hydrocarbon plasma polymerized hexane (ppHex) allows control of the fibroblast penetration into these porous 3-D objects. In order to optimize the coating conditions, a planar pinhole model of plasma penetration into pores is developed to rapidly measure deposit penetration using picolitre water contact angle measurement. Sufficiently good control over the plasma deposition within the porous scaffold is achieved using this approach to superimpose a relatively cell-repellent ppHex coating at the scaffold periphery onto the ppAAm-coated core, with a chemical gradient between the two. This 3-D chemical gradient encourages 3T3 fibroblast cells to adhere homogeneously from the periphery to the centre, when balanced by the tortuousity of the pore structure, which cells experience when passing from the surrounding medium to the centre. Ó 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction In the field of tissue engineering, biodegradable polymer scaffolds represent an important strategy for supporting cells in three dimensions. The interconnected structure of porous objects is designed to provide a shape into which cells penetrate, adhere, proliferate and generate their own extracellular matrix and allow vascularization prior to degradation of the scaffold. Cell attachment and adhesion are essential prerequisites to forming functional tissues using this approach [1]. A challenge when using preformed scaffolds is to achieve uniform cellular colonization of the macroporous structure, since adhesion to the periphery is often favoured [2]. In order to achieve uniform cell colonization, the production of three-dimensional (3-D) scaffolds allows the control of a high degree of open and interconnected porosity, a large surface area, appropriate mechanical strength and dimensional stability, and ⇑ Corresponding author. Tel.: +44 (0) 115 951 5119; fax: +44 (0) 115 951 5102. E-mail address: [email protected] (M.R. Alexander).
ready control over the shape, biodegradability and biocompatibility [3]. Polyesters represent attractive polymers in biomedical applications, with a range of scaffold production techniques used to process these materials to obtain appropriate porous structures. Here, porous poly(D,L-lactic acid) (PDLLA) scaffolds were prepared by means of a supercritical carbon dioxide (scCO2) method [4–10]. The scCO2 method is advantageous because organic solvents are not used, CO2 is non-toxic, it does not require a leaching process to eliminate the porogen and the process can be used to distribute sensitive bioactive biomolecules such as growth factors for extended release. Cell adhesion resulting from the commonly used agitated culture approach is often restricted to the peripheries of the scaffold, because of the tortuous path the cells must navigate to reach the interior [11]. The cellular attachment and activity are influenced by the surface properties of scaffolds. Chemical [12], mechanical [13] and topographical [14] stimuli have been shown to affect cell attachment, proliferation, differentiation and other cellular functions [15]. There is increasing interest from researchers in the surface modification of polymer scaffolds, with the aim of controlling
1742-7061/$ - see front matter Ó 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.actbio.2011.05.020
F. Intranuovo et al. / Acta Biomaterialia 7 (2011) 3336–3344
surface properties such as hydrophilicity, chemical composition and topography [16]. Surface modification of scaffolds by applying biomimetic peptides, such as arginine-glycine-aspartic acid, has been used to promote the MC3T3-E1 pre-osteoblast cell attachment [17]. Pre-treatment with tricalcium phosphate under alkaline conditions has been utilized to increase the degradation rate and improve mechanical properties of scaffolds, thereby increasing bone formation [18]. Another method utilized gelatin spheres with a smooth surface, prepared by non-surfactant emulsification, solvent extraction and freeze-drying, as a porogen in the fabrication of PLLA scaffolds, with the result of improving MC3T3-E1 osteoblast cell adhesion and proliferation [19]. With respect to these surface modification strategies, plasma polymer deposition is an effective and versatile tool to modify surface chemistry by coating, which is able to create a range of different chemistries in a controlled way on solid surfaces with minimal influence on the bulk morphological or mechanical properties. Plasma coatings are free of voids and pinholes and exhibit good adhesion to the substrate. Furthermore, plasma processes are dry techniques that do not require solvents, and can occur at relatively low (ambient) temperatures. Many studies have been devoted to the plasma surface modification of materials, intended for use in biomedical applications [20]. Plasma treatments and plasma polymer deposition, which results in surfaces that contain amine, carboxy, hydroxy and aldehyde groups, have been the most used plasma processes in the biomedical field. Amine-containing surfaces have been prepared mainly by NH3, N2 or N2/H2 plasma treatments or plasma polymerization of alkyl amine monomers. The plasma surface modification treatment approach suffers from the short-lived nature of the treatment, leading to relaxation of the modified surface into the polymer bulk [20]. With plasma polymer deposition, relatively stable coatings with a high density of nitrogen-containing groups compared to plasma-treated substrates can be obtained over a broad range of plasma parameters. Indeed, it is possible to tune the chemical composition of material surfaces and in turn increase their cell affinity, e.g. coatings from allyl amine to produce amines and other nitrogen moieties [21,22]. The application of plasma processes to macroscopic porous objects is a challenge and only a few papers have reported success, e.g. NH3 grafting [11] and allyl amine polymer deposition [23,24]. The challenge is to achieve deposition on the inner surfaces of the scaffolds and thereby control the surface chemistry, although there is much work focusing on treatment of the external surfaces of scaffolds [25–31]. Modification of the surface chemistry throughout a PDLLA scaffold has previously been performed by depositing a plasmapolymerized allyl amine (ppAAm) film, and compared with allyl amine plasma grafting. The nitrogen concentration at the exterior and interior scaffold surfaces was demonstrated to be greater for the plasma deposits than for the grafted surfaces, suggesting in the latter case a limitation by diffusion of depositing species into the scaffold pores [32]. An increase in the adherent cell number, although in a non-uniform distribution, was achieved with the ppAAm-coated scaffolds. A later study utilized a subsequent deposition of plasma-polymerized hexane (ppHex) film to obtain a peripheral ‘‘sheath’’ region that dampened cell adhesion at the perimeter of the object and encouraged cells to move towards the core [24]. Here, we propose a new process for ppAAm/ppHex deposition that improves the deposit penetration and consequently betters the homogeneity of the 3T3 fibroblast cell distribution inside the scaffolds to the point where it is uniform. Since this is a coating technique, the approach is readily applicable to any porous scaffold materials, and this study of fibroblast cells on PDLLA serves as a proof of concept example.
3337
2. Materials and methods 2.1. Scaffold preparation PDLLA (Mw 57 kDa; Purac, Gorinchem, the Netherlands) scaffolds were produced by scCO2 processing [4]. Powdered PDLLA (150 ± 3 mg) was placed in a Teflon mould (12 wells, each with a diameter and height of 10 mm, as previously shown [33]). The mould was then placed into a 60 ml clamp-sealed stainless-steel high-pressure autoclave. The autoclave was equipped with a pressure transducer and a heating jacket with a CAL 3300 temperature controller. A high-pressure PM101 pump (New Ways of Analytics) was used to load CO2 into the autoclave. The vessel was heated to an external temperature of 43 °C, then pressurized to a pressure of 23 MPa over 20 min of filling time. The polymer/CO2 mixture was maintained at this constant pressure for a soaking time of 60 min, after which the vessel was depressurized to ambient pressure over a 60 min venting time [7]. The three stages of the process (filling, soaking and venting) were controlled by a backpressure regulator under computer control. On release of the pressure, pores were formed in the polymer by the nucleation and escape of CO2 [34]. Porous scaffolds with a layer of nonporous skin and a size of approximately 10 mm diameter and 5 mm height were generated by this procedure. The nonporous skin of the scaffolds was removed by cutting with a scalpel blade. 2.2. Surface modification by plasma deposition Plasma polymer deposition was carried out in a T-shaped borosilicate glass chamber with stainless steel endplates (shown schematically in Fig. 1). The plasma discharge was initiated with two external, capacitively coupled copper band ring-electrodes, connected to a 13.56 MHz radiofrequency power source (Coaxial Power System Ltd.). The impedance was matched manually so that the reflected power was <1 W. The gas pressure in the chamber was monitored via a Pirani gauge (Kurt J. Lesker) and controlled with needle valves (LV10 K Leak Valve, BOC Edwards). Samples were placed on a metal tray (18.5 12.5 cm2) in the centre of the chamber. A quartz crystal sensor (STM-100/MF, Sycon Instruments) was positioned inside the chamber to monitor the deposition rate and the thickness of the coating. Prior to deposition, the samples were cleaned by an oxygen etching plasma (20 W, 40 Pa) for 5 min. PDLLA scaffolds were plasma modified by allyl amine (AAm) depositions in continuous and pulsing power modes (Table 1), where the average power is defined as the total input power tON/(tON + tOFF), with a fixed tOFF of 10 ms. In continuous mode, the power was set at 20 W, while the pressure was varied in the 7–107 Pa range. In pulsing mode, the total input power (20, 50 and 70 W) and the tON (1 and 2 ms) were varied, with fixed pressure (40 Pa). Two hexane (Hex) depositions were tested at different deposition times, 1 and 4 min, equivalent to 5 and 20 nm depositions respectively, on an unshielded substrate. Before cell culture and surface analyses, samples were stored in a cool dry place. 2.3. Water contact angle The penetration of ppAAm coatings at different plasma conditions was studied using a model pore with which ready assessment of the deposit penetration could be performed. This comprised (Fig. 2) a metal mask with a hole (0.7 mm diameter), held above a ppHex (20 W, power in continuous mode; 40 Pa, pressure; 5 min deposition time) coated glass coverslip at a 0.11 mm distance, provided by an aluminium ring. The analysis of the coverslip
3338
F. Intranuovo et al. / Acta Biomaterialia 7 (2011) 3336–3344
Fig. 1. Schematic of the plasma chamber.
Table 1 Plasma depositing conditions included in the study of the effect of plasma parameters on polymer penetration into a model pore and corresponding WCA values (n = 5). Process
Monomer
Pressure (Pa)
Total power (W)
Average power (W)
tON (ms)
Power mode
Deposition rate (nm min 1)
Time (min)
WCA (°)
ppAAm (7 Pa) ppAAm (20 Pa) ppAAm (40 Pa) ppAAm (80 Pa) ppAAm (107 Pa) p-ppAAm (1.8 W) p-ppAAm (3.3 W) p-ppAAm (4.5 W) p-ppAAm (6.4 W) p-ppAAm (8.3 W) p-ppAAm (11.7 W) ppHex (1 min) ppHex (4 min) ppHex (5 min)
AAm AAm AAm AAm AAm AAm AAm AAm AAm AAm AAm
7 20 40 80 107 40 40 40 40 40 40
20 20 20 20 20 20 20 50 70 50 70
/ / / / / 1.8 3.3 4.5 6.4 8.3 11.7
/ / / / / 1 2 1 1 2 2
Continuous Continuous Continuous Continuous Continuous Pulsing Pulsing Pulsing Pulsing Pulsing Pulsing
5 10 20 25 17 2 3 5 4 6 7
20 10 5 4 6 50 33 20 25 17 14
49 ± 3 49 ± 1 59 ± 1 51 ± 1 50 ± 2 53 ± 1 55 ± 1 54 ± 3 51 ± 2 53 ± 1 57 ± 2
Hex Hex Hex
40 40 40
20 20 20
/ / /
/ / /
Continuous Continuous Continuous
5 5 5
1 4 5
92 ± 1 91 ± 2 91 ± 1
Allyl amine coatings at different pressures in continuous power mode (ppAAm), different average powers in pulsed power mode (p-ppAAm) and hexane coatings at different deposition times in continuous power mode (ppHex) were plasma deposited.
Fig. 2. Schematic of the experimental setup used to study the plasma polymer gradients.
F. Intranuovo et al. / Acta Biomaterialia 7 (2011) 3336–3344
was able to determine the penetration of the depositing species through the hole and along the channel formed between the mask and the substrate [35]. The water contact angle (WCA) measurements on gradient samples were taken every 0.2 mm over a 4 mm total diameter, using a DSA100 instrument (Krüss). This allowed the deposition of picolitre-sized droplets on the sample surface. The dispensed drop volume was 110 pl, corresponding to a base diameter of approximately 75 lm for a WCA of 90°. The sample was moved via an automated stage. The WCA measurements were made radially from the centre of the sample to the edge. Videos of the evaporating droplets were captured in 18 ms intervals, for a duration of 1 s. The WCA was determined from the first stable image of the water droplet, between a tangent and a circular curve fit, intersecting with a straight line [36]. 2.4. Chemical analysis X-ray photoelectron spectroscopy (XPS) was carried out using a Kratos Axis Ultra spectrometer, employing a monochromatic Al Ka X-ray source (hm = 1486.6 eV), hybrid (magnetic/electrostatic) optics, hemispherical analyser, a multi-channel plate and a delay line detector with a collection angle of 30° and a take-off angle of 90°. The X-ray gun power was set to 100 W. All spectra were recorded using an aperture slot of 300 700 lm2, with a pass energy of 80 eV for survey scans and 20 eV for high-resolution core level scans. Spectra were recorded using the Kratos VISION II software and processed using the CASA XPS™ software package. Charge compensation (Kratos AXIS Nova charge neutralization system: a coaxial low energy electron source within the field of the magnetic lens) was used during the experiments, and samples were earthed via the stage using a standard BNC connector. Binding energies were charge corrected by setting the C1s aliphatic carbon signal from adventitious carbon contamination to 285.0 eV [37]. The C1s curve fits were carried out by restricting the full width at half maximum of all components to that of the C–C/C–H component (C1) at 285.0 eV. The other components had fixed energies at 287.1 eV (C(@O)OC, C2) and 289.1 eV (C(@O)O, C3) for the PDLLA samples, according to its chemical structure and the literature [37]. When samples were plasma treated, two other contributions were considered, at 286.0 eV (C–N/C–O, C4) and 288.0 eV (N–C@O, C5), respectively. In order to analyse the chemical composition inside the 3-D scaffolds, sections parallel and perpendicular to their surface were made using a scalpel blade. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) analysis was carried out on a SIMS IV time-of-flight instrument (ION-TOF GmbH) equipped with a bismuth liquid metal ion gun and a single-stage reflectron analyser. The instrument was typically operated at a primary ion energy of 25 kV, a pulsed target current of 1.3 pA and a post-acceleration of 10 kV. Charges induced on the surface by the positively charged ion beam were compensated with a flux of low energy electrons (20 eV). Chemical images were obtained by rastering the stage under the pulsed primary ion beam, using a field of 0.5 mm2. All analyses were carried out below the static limit of 1012 ions per cm2 for both polarities combined. The acquisition of full raw datasets allowed for the retrospective construction of spectra from the imaged areas. Positive spectra were normalized to the total ion intensity for comparison between samples. 2.5. Cell culture Untreated and plasma modified PDLLA scaffolds were sterilized and pre-wetted using phosphate-buffered saline (PBS) and 5 antibiotic/antimycotic solution (500 U ml 1 penicillin, 0.5 mg ml 1
3339
streptomycin, 1.25 lg ml 1 amphotercin B). After being rinsed with PBS, the scaffolds were soaked in cell culture medium in either a 6-well plate or a Falcon tube in a cell culture incubator for 24 h. The scaffolds were then placed in a non-tissue culture treated 6-well plate and seeded with 3T3 fibroblasts (1 106 cells per scaffold), for culture times of 48 and 96 h. After 24 h the medium was changed to remove any unattached cells. 2.6. Cell viability assays Cell viability was measured using an MTS assay (CellTiter 96 AQueous One solution, Promega Corporation). The scaffolds were incubated with a 20% MTS (3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) solution in cell culture medium for 3 h, at 37 °C in a 5% CO2 incubator. At the end of the incubation period, 100 ll of MTS solution and medium was transferred to a 96-well plate and the absorbance of the dye was measured at 490 nm, using an Optima FLUOstarH plate reader. Statistical analysis was performed by a paired Student’s t-test. To determine the viability of adherent or non-adherent cells, the LIVE/DEAD viability/cytotoxicity test was used. The LIVE/DEAD working solution was composed of PBS/ethidium homodimer-1 with calcein AM (LIVE/DEAD Viability/Cytotoxicity Assay Kit, Molecular Probes). Live cells were distinguished by the presence of intracellular esterase activity, determined by the enzymatic conversion of the non-fluorescent calcein AM to the green fluorescent calcein. Ethidium homodimer-1 is a high-affinity, red-fluorescent nucleic acid stain, able to pass through the compromised membranes of dead cells, producing a red fluorescence. After seeding cells, the LIVE/DEAD working solution was added to cover the scaffolds and incubated for 40 min, at room temperature. The scaffolds were then washed with PBS solution, cut in the middle and fixed with DABCO UV mountant–working solution (9:1 ratio of glycerol:DABCO/PBS) to retard photobleaching of the dyes. A confocal microscope (Zeiss 710, ex/em 495/515 nm Calcein: ex/em 495/ 635 nm EB) was used to visualize the area of interest in the scaffold sections. 2.7. X-ray microcomputed tomography of scaffolds After certain incubation times (48 and 96 h), the culture medium was removed and the scaffolds were washed in PBS. The cells were then fixed to the scaffolds by treating with paraformaldehyde/10% neutral buffered formalin for 30 min. The scaffolds were extensively rinsed first with PBS and then with distilled water, before staining with osmium tetroxide (1% solution) for 1 h. They were then rinsed in distilled water and dried for 2–3 days for X-ray microcomputed tomography (lCT) analysis. A high-resolution Skyscan lCT system (Skyscan 1174, Skyscan, Aartselaar, Belgium) was used to produce reconstructed 3-D images of cell distribution in the scaffolds. The scanner was set to a voltage of 50 kV and a current of 800 lA, and the samples were scanned at 11.9 lm voxel resolution. Two hundred slices were taken from the central section of each sample (3 mm thickness) and analysed for osmium tetroxide-stained cells by comparing the radiodense area. Three-dimensional images were obtained by using SkyScan 1174 NRecon and then SkyScan 1174 CTAn software to produce a 3-D volumetric space from the reconstructed 2-D slices (cross-sections). Morphometric parameters, such as porosity and pore size, were then calculated in the 3-D volumetric space or in two dimensions, from approximately 3 mm cross-sectional slices through the centre of the scaffolds. The area (mm2) occupied by cells in each slice was converted to a volume fraction.
3340
F. Intranuovo et al. / Acta Biomaterialia 7 (2011) 3336–3344
3. Results The theory describing plasma polymer deposition into pores is developing, but as yet it is not possible to predict how deposition into such structures is influenced by a particular plasma parameter [38]. A number of plasma conditions could influence the deposition of plasma polymers; thus, to determine the optimal parameters, a model pore was devised to enable ready empirical assessment of the effect of variables, such as input power, pulse/continuous power modes and monomer pressure on the deposit penetration down the pore. This allowed the penetrative nature of the plasma polymer deposition process to be assessed for a large range of plasma conditions (Table 1) on a flat surface (glass coverslip) behind an aperture. This acts as the pore opening, while the plate containing the hole and the glass coverslip act as the pore walls. Recently, a few other strategies have been proposed to create model pores, e.g. by designing microstructured specimens using the micromoulding technique [39], or by preparing polyolefin plastomer masks, equipped with a channel frame, that mimic the scaffold pore structure [38]. Glass coverslips were plasma coated with a uniform layer of hydrophobic ppHex thin film, on which was superimposed a radial gradient in the deposit of hydrophilic ppAAm through the pinhole mask. The wettability gradient was demonstrated by WCA mapping (Fig. S1) [12]. This gradient arose from the diffusion of the AAm depositing species under the mask, resulted in the deposition of a coating with a thickness decreasing from the region closest to the plasma through the mask hole to the areas beneath the mask. The AAm plasma condition, identified as producing the lowest penetration, was a pulsed deposition with an average power of 3.3 W (deposition time = 33 min), having a WCA value of 55 ± 1° (n = 5, Table 1) and the highest penetration at an average pulsed power of 11.7 W (deposition time = 14 min), having a WCA value of 57 ± 2° (n = 5, Table 1).
3.1. Characterization of plasma-coated PDLLA scaffolds PDLLA scaffolds of 10 mm diameter and 5 mm thickness were formed using supercritical CO2 processing. lCT was used to analyse the morphology of the scaffolds, from which the average porosity was measured to be 70 ± 1%, with an average pore size of 234 ± 15 lm. An O2 plasma was applied immediately before the AAm plasma deposition to encourage plasma polymer adhesion. The AAm plasmas identified as providing the lowest and highest deposit penetrations, pulsed ppAAm (p-ppAAm) at 3.3 and 11.7 W respectively, were subsequently struck in the chamber. After plasma polymer deposition, the scaffold pore size, assessed from the 3-D lCT images, was found to increase slightly compared to the untreated scaffolds, with unchanged porosity (Table 2). The slight increase in pore size is likely to be due to removal of PDLLA as a result of the initial O2 etching process before the p-ppAAm deposition. The penetration of p-ppAAm deposits was studied using XPS, by mechanical sectioning of the scaffold. The surface nitrogen concentration was used to quantify the presence of p-ppAAm coating. In Fig. 3, it can be seen from the nitrogen concentration that a gradient in p-ppAAm thickness was achieved
Table 2 Average porosity and pore size of untreated and p-ppAAm-coated scaffolds, assessed from the 3-D lCT analysis (the standard deviation is reported from analysis of five different scaffolds before and five after the p-ppAAm deposition).
Untreated p-ppAAm
Average porosity (%)
Average pore size (lm)
70 ± 1 71 ± 1
234 ± 15 265 ± 41
Fig. 3. XPS determined nitrogen surface concentration (atomic percent) along the diameter of a PDLLA scaffold sectioned at a depth of 2 mm. The pulsed allyl amine plasma was struck at a pressure of 40 Pa. (N), p-ppAAm at power = 3.3 W (33 min deposition time); (d), p-ppAAm at power = 11.7 W (14 min deposition time).
from the periphery to the centre of the disc. The penetration of p-ppAAm coating into the scaffold section was greater for the one deposited at 11.7 W compared to that at 3.3 W. This was consistent with the predictions made from water contact angle measurements on the pore model, which indicated that the shorter exposure of the AAm plasma at 11.7 W resulted in better deposit penetration. ToF-SIMS analysis was carried out to provide a high lateral resolution characterization of the distribution of the p-ppAAm coating on PDLLA in the centre of the scaffold, where the XPS determined that the nitrogen concentration was lowest (3–5 at.%). In all analyses, a uniform p-ppAAm layer was observed (Fig. S2). In order to counteract the tendency of cells to adhere to the outside of the scaffold, after the deposition of the p-ppAAm coating with the most penetrating plasma condition (at 11.7 W), a thin layer of the cell-attachment-resistant ppHex chemistry was deposited [35] (ppHex/p-ppAAm). This scaffold was subject to a hexane plasma for 4 min (power in continuous mode = 20 W, pressure = 40 Pa). To complement the analysis of the deposit distribution across the diameter of the scaffold, a mechanical section along the cylindrical axis was undertaken to provide the distribution through the thickness of the scaffold. The XPS analysis of a 3 mm vertical section (5 mm total scaffold thickness), shown in Fig. 4, indicates a deposition thickness gradient consistent with plasma species penetration from the top to the bottom. The nitrogen content of the p-ppAAm-coated samples (triangles) decreased from the top to the bottom of the scaffold. For the ppHex/p-ppAAm coating (squares), the nitrogen concentration was lower than the p-ppAAm-coated scaffold, indicating that the overcoat of ppHex markedly attenuated the contribution of the nitrogen at the periphery of the object. 3.2. Cell response to plasma polymer-coated scaffolds An in vitro cell culture experiment was performed using 3T3 fibroblast cells to assess and compare the cell viability and penetration in the scaffolds for the following samples: untreated, p-ppAAm and ppHex/p-ppAAm-coated scaffolds. To probe the survival of the fibroblast cell population attached to the whole scaffold, the retention of their metabolic and/or
F. Intranuovo et al. / Acta Biomaterialia 7 (2011) 3336–3344
Fig. 4. XPS determined nitrogen concentration along the mechanical section perpendicular to the PDLLA scaffold top surface. A pulsed 40 Pa allyl amine plasma with and without a ppHex overcoat were deposited. (N), p-ppAAm (11.7 W power in pulsing mode; 40 Pa pressure; 14 min deposition time); (j), ppHex/p-ppAAm (Hex deposition: 20 W power in continuous mode; 40 Pa pressure; 4 min deposition time).
proliferative ability was measured by MTS assay. The mitochondrial activity of cells in the scaffolds after 48 h of culture time is presented in Fig. 5. The mitochondrial activity of the p-ppAAmcoated scaffolds increased relative to the untreated scaffolds, and deposition became higher with the ppHex/p-ppAAm film. This was most likely due to a larger number of cells initially attaching. The ppHex/p-ppAAm-coated scaffolds were additionally evaluated at 96 h cell culture, which indicated that the cell number had increased. The MTS value (p < 0.01) further increased over the 48 h cell culture, with a cell proliferation improvement of 18%.
3341
The global distribution of 3T3 fibroblasts through the scaffold was determined with lCT, using osmium tetroxide staining to provide sufficient X-ray absorption contrast, to distinguish the cells from the PDLLA. The lCT images obtained after 48 h culture time are presented in Fig. 6. For each sample, images representing the top and side views of the scaffolds are displayed. Each represents a summation of the cells in a 3 mm thick section from the centre of the section viewed. A clear difference in cell distribution was observed between the untreated (Fig. 6A) and plasma-treated scaffolds (Fig. 6B–D). There was a considerable increase in the overall loading of cells in the p-ppAAm (11.7 W)-coated scaffolds, which were determined to have the best ppAAm penetration, as represented by the cell area intersecting the viewing plane in 3 mm slices through the centre of the scaffolds (718 ± 79 mm2; Fig. 6B), compared to untreated scaffolds (176 ± 23 mm2; Fig. 6A). The increase in the area occupied by cells as a result of the ppHex/pppAAm coating was greater still (828 ± 63 mm2), as seen in Fig. 6C. A further 2 days of cell culture was employed to explore the development of the cell distribution in the ppHex/p-ppAAmcoated scaffolds. After 96 h of culture (Fig. 6D), the 3T3 fibroblasts were more homogeneously distributed than the equivalent 48 h cultured sample. The cell area (903 ± 9 mm2) reached the greatest value and the cells were spread evenly (top to bottom) throughout the porous scaffold structure. The cell distribution and area shown by lCT data are in agreement with the metabolic activity levels determined with the MTS assay. To quantify the cell distribution within the images shown in Fig. 6, the cell coverage of the scaffolds was calculated for the core and for the outer sheath area and presented in Fig. 7. This illustrates that these cells in the untreated PDLLA scaffold were concentrated within the sheath region, with a total cell volume fraction of only 1% reaching the core, compared to 4% in the sheath (Fig. 7). For the plasma polymer-treated samples, an increase in the cell content for both core and sheath was noted. The p-ppAAm-coated scaffold exhibited a cell volume fraction of 7% in the core and 12% in the sheath, which represents an overall increase compared to the uncoated samples. For the samples with a ppHex layer (ppHex/p-ppAAm), a further increased penetration into the core, to give a volume fraction of 11% in the core and 13% in the sheath, was noted. Increasing the culture time to 96 h further promoted the scaffold colonization, with the highest value volume fraction occupied by cells in both core (12.5%) and sheath (14%) regions. At culture times of 48 and 96 h, evaluation of the cell viability inside the scaffolds was performed using a LIVE/DEAD assay. Confocal images from cells located in the centre of the scaffolds accessed by mechanical sectioning indicated that live cells (green) lined the struts and pores of the scaffolds, as shown in the representative images presented in Fig. 8A–C (untreated, p-ppAAm and ppHex/p-ppAAm-coated scaffold sections after 48 h cell culture, respectively). In Fig. 8D (ppHex/p-ppAAm-coated scaffold section, after 96 h cell culture), long dendrites were observed extending from the cells, which could aid in cell migration and monitoring of the scaffold environment. This branched morphology has been previously noted on 3-D electrospun poly(D,L-lactide-coL-lactide) matrices [40,41], as the typical shape of migrating fibroblasts in 3-D fibrous scaffolds or matrices. Very few dead cells (red) were observed for all scaffolds.
4. Discussion 4.1. Cell response to scaffolds Fig. 5. MTS activity of 3T3 fibroblasts cultured for 48 and 96 h in untreated and plasma-modified scaffolds: p-ppAAm (11.7 W power in pulsing mode; 40 Pa pressure; 14 min deposition time); ppHex/p-ppAAm (Hex deposition: 20 W power in continuous mode; 40 Pa pressure; 4 min deposition time). For each scaffold type, the standard deviation is reported from analysis of three different scaffolds.
The attachment level and distribution of 3T3 fibroblasts on untreated scaffolds in vitro was poor (Figs. 6 and 7). An improvement in cell adhesion and distribution was achieved after the p-ppAAm
3342
F. Intranuovo et al. / Acta Biomaterialia 7 (2011) 3336–3344
Fig. 6. Cumulative 3T3 cell area in approximately 3 mm slices through the centre of the scaffolds, within the core and the sheath regions (half the total scaffold area) of untreated and plasma-coated scaffolds.
without improving the cell area in the total scaffold [24]. In the present study, the ppHex/p-ppAAm coating had the effect of improving the fibroblast cell colonization of the interior, as shown in Fig. 6, with the increase in cell loading in the core approaching that of the sheath scaffold regions (Fig. 7). Furthermore, by increasing the cell culture time from 48 to 96 h, both the total cell loading and proliferation increased to the point where the scaffold was virtually uniformly populated, when viewed from the top and the side.
4.2. Plasma polymers
Fig. 7. lCT top and side images of scaffolds cultured with 3T3 fibroblasts for 48 h: (A) untreated; (B) p-ppAAm, 11.7 W power in pulsing mode, 40 Pa pressure, 14 min deposition time; (C) ppHex/p-ppAAm, Hex deposition: 20 W power in continuous mode, 40 Pa pressure, 4 min deposition time; and for 96 h: (D) ppHex/p-ppAAmcoated scaffolds. The pictures show lCT images from approximately 3 mm slices through the centre of the scaffolds.
coating, consistent with other similar studies with ppAAm-coated surfaces [42,43]; however, with an optimized coating regime developed using a pinhole model, a uniform cell distribution was obtained. The improved cell response on the p-ppAAm surface has been rationalized as the presence of nitrogen-containing groups modifying the protein adsorption from the serum in the medium, thereby improving cell adhesion [32]. The p-ppAAm-coated scaffolds showed a greater area occupied by cells in the total scaffold with respect to untreated scaffolds, with a greater proportion penetrating to the core (Fig. 6). In a previous study [24], continuous-wave ppAAm deposition did not achieve any great improvement in cell area, which was approximately 0.3 mm2 and was higher in the outer area than in the inner area. When a ppHex coating was applied, the fibroblast area was increased and more uniform compared to the ppAAm-coated scaffolds. This allowed an increase in the cell area in the core, but
There has been much debate about which components of the plasma dominate the deposition process and in turn influence the deposition rate and polymer chemistry (see e.g. Ref. [44]). In the gas phase of allyl amine plasma, mass spectral measurements have found evidence of reactions between cations and intact neutral allyl amine monomers or radicals, which contrast with the more traditional view of the dominant radical–radical and radical–neutral reactions [22,45,46]. Swindells et al. [47] proposed another alternative of negative ion-neutral polymerization mechanism from acrylic acid vapours, demonstrating that negative ions may also contribute to plasma polymer formation. The plasma is expected to be excluded from the porous body of the scaffolds, based on previous investigations on plasma polymer deposition into channels [38]. Modification of the chemistry of the internal surfaces of 3-D porous scaffolds by plasma polymer deposition would therefore be controlled by diffusion of the depositforming species from the plasma phase down the pores and the deposition of these species on the walls. This process leads to radial gradients of plasma polymer from the periphery to the centre of the scaffolds. Radicals are known to be sufficiently long lived to make this journey, as are some charged species which have been found to be surprisingly long lived by mass spectrometry of pulsed plasma polymer systems [42]. To readily empirically assess coating deposition inside the porous structure, we used a pinhole as a model pore and investigated the effect of plasma parameters such as monomer pressure, power and pulse mode on penetration therein. We used simple picolitre WCA measurements, which were a quick and inexpensive means of mapping penetration into this model pore that was compatible with an iterative search of the optimal deposition parameters. XPS and ToF-SIMS provide a fuller characterization, but the throughput and turnaround from sample fabrication to result is much lower, preventing iterative studies.
F. Intranuovo et al. / Acta Biomaterialia 7 (2011) 3336–3344
3343
Fig. 8. Confocal images (n = 3) of 3T3 fibroblast cells, labelled with the LIVE/DEAD working solution on the sections from the centre of untreated (48 h, A); p-ppAAm (48 h, B); ppHex/p-ppAAm (48 h, C); and ppHex/p-ppAAm (96 h, D) coated scaffolds. Live cells were distinguished by enzymatic conversion of the non-fluorescent calcein AM to the green fluorescent calcein. Dead cells were determined by the red-fluorescent ethidium homodimer-1 stain.
The optimal polymer deposit penetration condition was found to be at a high pulsed power. The higher power pulsed plasma is likely to give better ppAAm penetration than pulsed and continuous-wave plasmas at lower power because of the greater monomer fragmentation resulting in a higher density of ions. This is consistent with previous deposition studies on 2-D surfaces [43,45,48]. The higher concentration of depositing species outside the pores would result in greater penetration through the pores towards the centre of the scaffold, although the many other factors that change when the power is modified, e.g. the plasma sheath, make this proposal difficult to verify. XPS analysis on horizontal and perpendicular scaffold mechanical sections allowed quantification of the p-ppAAm penetration using the nitrogen elemental signal. Across the diameter of a scaffold section, the nitrogen content decreased from the edges to the core (Fig. 3), consistent with a previous study [32]. A similar dependence on the distance from the edge of the scaffold was seen through the thickness (Fig. 4). This marked difference between the external and internal regions of the scaffold relates to the diffusion of depositing species from the excluded plasma phase. The chemistry of the p-ppAAm, indicated by the N1s and C1s core levels, does not appear to vary with depth, being consistent with the assignment of the chemistry to amines, imines and nitrile groups in previous studies [45,48,49]. After the p-ppAAm deposition, the scaffolds were plasma coated for a short while in a hexane plasma. This has been shown to result in a ppHex film at the periphery [32]. Thus, gradients of surface chemistry within the porous structure, from a peripheral hydrocarbon to an internal nitrogen-containing film, were produced in order to encourage the cell colonization in the scaffold core. By tuning the ppHex deposition time, a good control of cell
adhesion and penetration into the scaffolds was achieved on the optimized p-ppAAm coating (Figs. 6 and 7). Fast and uniform cell colonization was achieved with the fibroblasts used in this study. This chemical surface modification strategy is an attractive alternative to complex methods for forcing cell loading inside the scaffolds. This simple and effective plasma-generated chemical gradient could be easily applied to any porous scaffold material. Plasma polymer coatings have previously been shown to be a powerful strategy for controlling the attachment and function of a wide range of cell types, indicating that this can be applied beyond the model of fibroblast cell type used here. 5. Conclusions An empirical study of the dependence of deposit penetration down a model pore on plasma parameters has enabled optimal plasma conditions to be identified. A radial chemical gradient has been achieved from a cell-repulsive ppHex film on the top to a cell-adhesive p-ppAAm coating throughout the scaffold. By controlling the allyl amine and hexane plasma polymerization mechanisms and parameters, a uniform fibroblast cell colonization throughout porous scaffolds was successfully achieved. This indicates the potential of this coating technique to provide a uniform distribution of many cell types in porous 3-D scaffolds made of any material. Acknowledgements We thank Paul Roach and Mathieu Lanniel for support with plasma deposition experiments and Yvonne Reinwald for scaffold
3344
F. Intranuovo et al. / Acta Biomaterialia 7 (2011) 3336–3344
preparation. We further thank David J. Scurr, Susan Dodson and Emily Smith for help with ToF-SIMS, lCT and XPS measurements, respectively. Appendix A. Figures with essential colour discrimination Certain figures in this article, particularly Figures 1, 2, 6, and 8, are difficult to interpret in black and white. The full colour images can be found in the on-line version, at doi:10.1016/ j.actbio.2011.05.020. Appendix B. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.actbio.2011.05.020. References [1] Nerem RM. The challenge of imitating nature. In: Lanza R, Langer R, Vacanti J, editors. Principles of tissue engineering. Burlington, MA: Elsevier Academic Press; 2007. p. 7–14. [2] Sengers BG, Taylor M, Please CP, Oreffo ROC. Computational modelling of cell spreading and tissue regeneration in porous scaffolds. Biomaterials 2007;28:1926–40. [3] Khang G, Kim MS, Lee HB. Introduction. In: Khang G, Kim MS, Lee HB, editors. Manuals in biomedical research. A manual for biomaterials/scaffold fabrication technology, vol. 4. Singapore: World Scientific Publishing Co. Pte. Ltd.; 2007. p. 1–12. [4] Barry JJA, Silva MMCG, Popov VK, Shakesheff KM, Howdle SM. Supercritical carbon dioxide: putting the fizz into biomaterials. Phil Trans R Soc A 2006;364:249–61. [5] Hile DD, Amirpour ML, Akgerman A, Pishko MV. Active growth factor delivery from poly(D,L-lactide-coglycolide) foams prepared in supercritical CO2. J Control Release 2000;66:177–85. [6] Singh L, Kumara V, Ratner BD. Generation of porous microcellular 85/15 poly (DL-lactide-co-glycolide) foams for biomedical applications. Biomaterials 2004;25:2611–7. [7] Tai H, Mather ML, Howard D, Wang W, White LJ, Crowe JA, et al. Control of pore size and structure of tissue engineering scaffolds produced by supercritical fluid processing. Eur Cell Mater 2007;14:64–77. [8] Pini R, Storti G, Mazzotti M, Tai H, Shakesheff KM, Howdle SM. Sorption and swelling of poly(DL-lactic acid) and poly(lactic-co-glycolic acid) in supercritical CO2: an experimental and modeling study. J Polym Sci Part B: Polym Phys 2008;46:483–96. [9] Duarte ARC, Mano JF, Reis RL. Preparation of starch-based scaffolds for tissue engineering by supercritical immersion precipitation. J Supercrit Fluids 2009;49:279–85. [10] Zhu XH, Lee LY, Jackson JSH, Tong YW, Wang CH. Characterization of porous poly(D, L-lactic-co-glycolic acid) sponges fabricated by supercritical CO2 gasfoaming method as a scaffold for three-dimensional growth of Hep3B cells. Biotechnol Bioeng 2008;100:998–1009. [11] Wan Y, Tu C, Yang J, Bei J, Wang S. Influences of ammonia plasma treatment on modifying depth and degradation of poly(L-lactide) scaffolds. Biomaterials 2006;27:2699–704. [12] DeForest CA, Polizzotti BD, Anseth KS. Sequential click reactions for synthesizing and patterning three-dimensional cell microenvironments. Nat Mater 2009;8:659–64. [13] Engler AJ, Sen S, Sweeney HL, Discher DE. Matrix elasticity directs stem cell lineage specification. Cell 2006;126:677–89. [14] Dalby MJ, Gadegaard N, Tare R, Andar A, Riehle MO, Herzyk P, et al. The control of human mesenchymal cell differentiation using nanoscale symmetry and disorder. Nat Mater 2007;6:997–1003. [15] Cukierman E, Pankov R, Stevens DR, Yamada KM. Taking cell-matrix adhesions to the third dimension. Science 2001;294:1708–12. [16] Moroni L, Habibovic P, Mooney DJ, van Blitterswijk CA. Functional tissue engineering through biofunctional macromolecules and surface design. MRS Bull 2011;35:584–90. [17] Lan PX, Lee JW, Seol Y-J, Cho D-W. Development of 3D PPF/DEF scaffolds using micro-stereolithography and surface modification. J Mater Sci: Mater Med 2009;20:271–9. [18] Yeo A, Wong WJ, Teoh S-H. Surface modification of PCL-TCP scaffolds in rabbit calvaria defects: evaluation of scaffold degradation profile, mechanical properties and bone healing patterns. J Biomed Mater Res A 2010;93:1358–67. [19] Liu X, Won Y, Ma PX. Porogen-induced surface modification of nano-fibrous poly(L-lactic acid) scaffolds for tissue engineering. Biomaterials 2006;27:3980–7. [20] Siow K, Britcher L, Kumar S, Griesser H. Plasma methods for the generation of chemically reactive surfaces for biomolecule immobilisation and cell colonisation – a review. Plasma Process Polym 2006;3:392–418.
[21] Fally F, Doneux C, Riga J, Verbist JJ. Quantification of the functional groups present at the surface of plasma polymers deposited from propylamine, allylamine, and propargylamine. J Appl Polym Sci 1995;56:567–614. [22] Shard AG, Whittle JD, Beck AJ, Brookes PN, Bullett NA, Talib RA, et al. A NEXAFS examination of unsaturation in plasma polymers of allylamine and propylamine. J Phys Chem B 2004;108:12472–80. [23] Roach P, Howard D, Barry JR, Shakesheff KM, Howdle SM, Alexander MR. Chemical modification of porous scaffolds using plasma polymers. In: Berthiaume F, Morgan J, editors. 3D Tissue Engineering. Boston: Artech House; 2010. p. 1–20. [24] Barry JJA, Howard D, Shakesheff KM, Howdle SM, Alexander MR. Using a coresheath distribution of surface chemistry through 3D tissue engineering scaffolds to control cell ingress. Adv Mater 2006;18:1406–10. [25] Djordjevic I, Britcher LG, Kumar S. Morphological and surface compositional changes in poly(lactide-co-glycolide) tissue engineering scaffolds upon radio frequency glow discharge plasma treatment. Appl Surf Sci 2008;254:1929–35. [26] Safinia L, Datan N, Höhse M, Mantalarisa A, Bismarck A. Towards a methodology for the effective surface modification of porous polymer scaffolds. Biomaterials 2005;26:7537–47. [27] Yang J, Shi G, Bei J, Wang S, Cao Y, Shang Q, et al. Fabrication and surface modification of macroporous poly(L-lactic acid) and poly(L-lactic-co-glycolic acid) (70/30) cell scaffolds for human skin fibroblast cell culture. J Biomed Mater Res 2002;62:438–46. [28] Hu Y, Winn SR, Krajbich I, Hollinger JO. Porous polymer scaffolds surfacemodified with arginine-glycine-aspartic acid enhance bone cell attachment and differentiation in vitro. J Biomed Mater Res 2003;64A:583–90. [29] Chim H, Ong JL, Schantz JT, Hutmacher DW, Agrawal CM. Efficacy of glow discharge gas plasma treatment as a surface modification process for threedimensional poly (D, L-lactide) scaffolds. J Biomed Mater Res 2003;65A:327–35. [30] Armentano I, Ciapetti G, Pennacchi M, Dottori M, Devescovi V, Granchi D, et al. Role of PLLA plasma surface modification in the interaction with human marrow stromal cells. J Appl Polym Sci 2009;114:3602–11. [31] Yildirim ED, Pappas D, Güçeri S, Sun W. Enhanced cellular functions on polycaprolactone tissue scaffolds by O2 plasma surface modification. Plasma Process Polym 2011;8:256–67. [32] Barry JJA, Silva MMCG, Shakesheff KM, Howdle SM, Alexander MR. Using plasma deposits to promote cell population of the porous interior or threedimensional poly(D, L-lactic acid) tissue-engineering scaffolds. Adv Funct Mater 2005;15:1134–40. [33] Quirk RA, France RM, Shakesheff KM, Howdle SM. Supercritical fluid technologies and tissue engineering scaffolds. Curr Opin Solid State Mater Sci 2004;8:313–21. [34] Woods HM, Silva MMCG, Nouvel C, Shakesheff KM, Howdle SM. Materials processing in supercritical carbon dioxide: surfactants, polymers and biomaterials. J Mater Chem 2004;14:1663–78. [35] Zelzer M, Majani R, Bradley JW, Rose FRAJ, Davies MC, Alexander MR. Investigation of cell-surface interactions using chemical gradients formed from plasma polymers. Biomaterials 2008;29:172–84. [36] Taylor M, Urquhart AJ, Zelzer M, Davies MC, Alexander MR. Picoliter water contact angle measurement on polymers. Langmuir 2007;23:6875–8. [37] Moulder J, Stickle WF, Sobol PE, Bomben KD, Chastian J. Handbook of X-ray Photoelectron Spectroscopy. Eden Prairie: Perkin-Elmer; 1992. [38] Zelzer M, Scurr D, Abdullah B, Urquhart AJ, Gadegaard N, Bradley JW, et al. Influence of the plasma sheath on plasma polymer deposition in advance of a mask and down pores. J Phys Chem B 2009;113:8487–94. [39] Besch W, Lippold O, Reinecke H, Schröder K, Ohl A. A test specimen for investigation of plasma modification of interior surfaces in polymeric threedimensional microstructures. Surf Coat Tech 2003;174–175:112–7. [40] Kluger PJ, Wyrwa R, Weisser J, Maierle J, Votteler M, Rode C, et al. Electrospun poly(D/L-lactide-co-L-lactide) hybrid matrix: a novel scaffold material for soft tissue engineering. J Mater Sci: Mater Med 2010;21(9):2665–71. [41] Grinnell F. Fibroblast mechanics in three-dimensional collagen matrices. J Bodywork Mov Ther 2008;12:191–3. [42] Swindells I, Voronin SA, Bryant PM, Alexander MR, Bradley JW. Temporal evolution of an electron-free afterglow in the pulsed plasma polymerisation of acrylic acid. J Phys Chem B 2008;112:3938–47. [43] Harsch A, Calderon J, Timmons RB, Gross GW. Pulsed plasma deposition of allylamine on polysiloxane: a stable surface for neuronal cell adhesion. J Neurosci Methods 2000;98:135–44. [44] Short RD, Steel DA. Testing the hypothesis: comments on plasma polymerisation of acrylic acid revisited. Plasma Process Polym 2010;7:366–70. [45] Choukourov A, Biederman H, Slavinska D, Hanley L, Grinevich A, Boldyryeva H, et al. Mechanistic studies of plasma polymerization of allylamine. J Phys Chem B 2005;109:23086–95. [46] Beck AJ, Candan S, Short RD, Goodyear A, Braithwaite NSJ. The role of ions in the plasma polymerization of allylamine. J Phys Chem B 2001;105:5730–6. [47] Swindells I, Voronin SA, Fotea C, Alexander MR, Bradley JW. Detection of negative molecular ions in acrylic acid plasma: some implications for polymerization mechanisms. J Phys Chem B 2007;111:8720–2. [48] Hamerli P, Weigel T, Groth T, Paul D. Surface properties of and cell adhesion onto allylamine-plasma coated poly(ethylene terephthalate) membranes. Biomaterials 2003;24:3989–99. [49] Denis L, Cossement D, Godfroid T, Renaux F, Bittencourt C, Snyders R, et al. Synthesis of allylamine plasma polymer films: correlation between plasma diagnostic and film characteristics. Plasma Process Polym 2009;6:199–208.
Contents lists available at ScienceDirect
Acta Biomaterialia journal homepage: www.elsevier.com/locate/actabiomat
Uniform cell colonization of porous 3-D scaffolds achieved using radial control of surface chemistry Francesca Intranuovo a, Daniel Howard b, Lisa J. White b, Ramneek K. Johal c, Amir M. Ghaemmaghami c, Pietro Favia a, Steven M. Howdle d, Kevin M. Shakesheff b, Morgan R. Alexander e,⇑ a
Department of Chemistry, University of Bari, 70126 Bari, Italy Tissue Engineering Group, School of Pharmacy, University of Nottingham, Nottingham NG7 2RD, UK Allergy Research Group, School of Molecular Medical Sciences and Respiratory Biomedical Research Unit, University of Nottingham, Nottingham NG7 2RD, UK d School of Chemistry, University of Nottingham, Nottingham NG7 2RD, UK e Laboratory of Biophysics and Surface Analysis, School of Pharmacy, University of Nottingham, Nottingham NG7 2RD, UK b c
a r t i c l e
i n f o
Article history: Received 2 February 2011 Received in revised form 11 May 2011 Accepted 18 May 2011 Available online 24 May 2011 Keywords: Tissue engineering Porous scaffold Plasma polymerization Allyl amine Cell distribution
a b s t r a c t Uniform cellular distribution is a prerequisite to forming tissue within porous scaffolds, but the seeding process often results in preferential adhesion of cells at the periphery. We develop a vapour phase coating strategy which is readily applicable to any porous solid to provide a uniform cellular distribution. Plasma polymerized allyl amine (ppAAm) is used to form a thin nitrogen-containing coating throughout porous three-dimensional (3-D) poly(D,L-lactic acid) scaffolds. Subsequent controlled deposition of a hydrocarbon plasma polymerized hexane (ppHex) allows control of the fibroblast penetration into these porous 3-D objects. In order to optimize the coating conditions, a planar pinhole model of plasma penetration into pores is developed to rapidly measure deposit penetration using picolitre water contact angle measurement. Sufficiently good control over the plasma deposition within the porous scaffold is achieved using this approach to superimpose a relatively cell-repellent ppHex coating at the scaffold periphery onto the ppAAm-coated core, with a chemical gradient between the two. This 3-D chemical gradient encourages 3T3 fibroblast cells to adhere homogeneously from the periphery to the centre, when balanced by the tortuousity of the pore structure, which cells experience when passing from the surrounding medium to the centre. Ó 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction In the field of tissue engineering, biodegradable polymer scaffolds represent an important strategy for supporting cells in three dimensions. The interconnected structure of porous objects is designed to provide a shape into which cells penetrate, adhere, proliferate and generate their own extracellular matrix and allow vascularization prior to degradation of the scaffold. Cell attachment and adhesion are essential prerequisites to forming functional tissues using this approach [1]. A challenge when using preformed scaffolds is to achieve uniform cellular colonization of the macroporous structure, since adhesion to the periphery is often favoured [2]. In order to achieve uniform cell colonization, the production of three-dimensional (3-D) scaffolds allows the control of a high degree of open and interconnected porosity, a large surface area, appropriate mechanical strength and dimensional stability, and ⇑ Corresponding author. Tel.: +44 (0) 115 951 5119; fax: +44 (0) 115 951 5102. E-mail address: [email protected] (M.R. Alexander).
ready control over the shape, biodegradability and biocompatibility [3]. Polyesters represent attractive polymers in biomedical applications, with a range of scaffold production techniques used to process these materials to obtain appropriate porous structures. Here, porous poly(D,L-lactic acid) (PDLLA) scaffolds were prepared by means of a supercritical carbon dioxide (scCO2) method [4–10]. The scCO2 method is advantageous because organic solvents are not used, CO2 is non-toxic, it does not require a leaching process to eliminate the porogen and the process can be used to distribute sensitive bioactive biomolecules such as growth factors for extended release. Cell adhesion resulting from the commonly used agitated culture approach is often restricted to the peripheries of the scaffold, because of the tortuous path the cells must navigate to reach the interior [11]. The cellular attachment and activity are influenced by the surface properties of scaffolds. Chemical [12], mechanical [13] and topographical [14] stimuli have been shown to affect cell attachment, proliferation, differentiation and other cellular functions [15]. There is increasing interest from researchers in the surface modification of polymer scaffolds, with the aim of controlling
1742-7061/$ - see front matter Ó 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.actbio.2011.05.020
F. Intranuovo et al. / Acta Biomaterialia 7 (2011) 3336–3344
surface properties such as hydrophilicity, chemical composition and topography [16]. Surface modification of scaffolds by applying biomimetic peptides, such as arginine-glycine-aspartic acid, has been used to promote the MC3T3-E1 pre-osteoblast cell attachment [17]. Pre-treatment with tricalcium phosphate under alkaline conditions has been utilized to increase the degradation rate and improve mechanical properties of scaffolds, thereby increasing bone formation [18]. Another method utilized gelatin spheres with a smooth surface, prepared by non-surfactant emulsification, solvent extraction and freeze-drying, as a porogen in the fabrication of PLLA scaffolds, with the result of improving MC3T3-E1 osteoblast cell adhesion and proliferation [19]. With respect to these surface modification strategies, plasma polymer deposition is an effective and versatile tool to modify surface chemistry by coating, which is able to create a range of different chemistries in a controlled way on solid surfaces with minimal influence on the bulk morphological or mechanical properties. Plasma coatings are free of voids and pinholes and exhibit good adhesion to the substrate. Furthermore, plasma processes are dry techniques that do not require solvents, and can occur at relatively low (ambient) temperatures. Many studies have been devoted to the plasma surface modification of materials, intended for use in biomedical applications [20]. Plasma treatments and plasma polymer deposition, which results in surfaces that contain amine, carboxy, hydroxy and aldehyde groups, have been the most used plasma processes in the biomedical field. Amine-containing surfaces have been prepared mainly by NH3, N2 or N2/H2 plasma treatments or plasma polymerization of alkyl amine monomers. The plasma surface modification treatment approach suffers from the short-lived nature of the treatment, leading to relaxation of the modified surface into the polymer bulk [20]. With plasma polymer deposition, relatively stable coatings with a high density of nitrogen-containing groups compared to plasma-treated substrates can be obtained over a broad range of plasma parameters. Indeed, it is possible to tune the chemical composition of material surfaces and in turn increase their cell affinity, e.g. coatings from allyl amine to produce amines and other nitrogen moieties [21,22]. The application of plasma processes to macroscopic porous objects is a challenge and only a few papers have reported success, e.g. NH3 grafting [11] and allyl amine polymer deposition [23,24]. The challenge is to achieve deposition on the inner surfaces of the scaffolds and thereby control the surface chemistry, although there is much work focusing on treatment of the external surfaces of scaffolds [25–31]. Modification of the surface chemistry throughout a PDLLA scaffold has previously been performed by depositing a plasmapolymerized allyl amine (ppAAm) film, and compared with allyl amine plasma grafting. The nitrogen concentration at the exterior and interior scaffold surfaces was demonstrated to be greater for the plasma deposits than for the grafted surfaces, suggesting in the latter case a limitation by diffusion of depositing species into the scaffold pores [32]. An increase in the adherent cell number, although in a non-uniform distribution, was achieved with the ppAAm-coated scaffolds. A later study utilized a subsequent deposition of plasma-polymerized hexane (ppHex) film to obtain a peripheral ‘‘sheath’’ region that dampened cell adhesion at the perimeter of the object and encouraged cells to move towards the core [24]. Here, we propose a new process for ppAAm/ppHex deposition that improves the deposit penetration and consequently betters the homogeneity of the 3T3 fibroblast cell distribution inside the scaffolds to the point where it is uniform. Since this is a coating technique, the approach is readily applicable to any porous scaffold materials, and this study of fibroblast cells on PDLLA serves as a proof of concept example.
3337
2. Materials and methods 2.1. Scaffold preparation PDLLA (Mw 57 kDa; Purac, Gorinchem, the Netherlands) scaffolds were produced by scCO2 processing [4]. Powdered PDLLA (150 ± 3 mg) was placed in a Teflon mould (12 wells, each with a diameter and height of 10 mm, as previously shown [33]). The mould was then placed into a 60 ml clamp-sealed stainless-steel high-pressure autoclave. The autoclave was equipped with a pressure transducer and a heating jacket with a CAL 3300 temperature controller. A high-pressure PM101 pump (New Ways of Analytics) was used to load CO2 into the autoclave. The vessel was heated to an external temperature of 43 °C, then pressurized to a pressure of 23 MPa over 20 min of filling time. The polymer/CO2 mixture was maintained at this constant pressure for a soaking time of 60 min, after which the vessel was depressurized to ambient pressure over a 60 min venting time [7]. The three stages of the process (filling, soaking and venting) were controlled by a backpressure regulator under computer control. On release of the pressure, pores were formed in the polymer by the nucleation and escape of CO2 [34]. Porous scaffolds with a layer of nonporous skin and a size of approximately 10 mm diameter and 5 mm height were generated by this procedure. The nonporous skin of the scaffolds was removed by cutting with a scalpel blade. 2.2. Surface modification by plasma deposition Plasma polymer deposition was carried out in a T-shaped borosilicate glass chamber with stainless steel endplates (shown schematically in Fig. 1). The plasma discharge was initiated with two external, capacitively coupled copper band ring-electrodes, connected to a 13.56 MHz radiofrequency power source (Coaxial Power System Ltd.). The impedance was matched manually so that the reflected power was <1 W. The gas pressure in the chamber was monitored via a Pirani gauge (Kurt J. Lesker) and controlled with needle valves (LV10 K Leak Valve, BOC Edwards). Samples were placed on a metal tray (18.5 12.5 cm2) in the centre of the chamber. A quartz crystal sensor (STM-100/MF, Sycon Instruments) was positioned inside the chamber to monitor the deposition rate and the thickness of the coating. Prior to deposition, the samples were cleaned by an oxygen etching plasma (20 W, 40 Pa) for 5 min. PDLLA scaffolds were plasma modified by allyl amine (AAm) depositions in continuous and pulsing power modes (Table 1), where the average power is defined as the total input power tON/(tON + tOFF), with a fixed tOFF of 10 ms. In continuous mode, the power was set at 20 W, while the pressure was varied in the 7–107 Pa range. In pulsing mode, the total input power (20, 50 and 70 W) and the tON (1 and 2 ms) were varied, with fixed pressure (40 Pa). Two hexane (Hex) depositions were tested at different deposition times, 1 and 4 min, equivalent to 5 and 20 nm depositions respectively, on an unshielded substrate. Before cell culture and surface analyses, samples were stored in a cool dry place. 2.3. Water contact angle The penetration of ppAAm coatings at different plasma conditions was studied using a model pore with which ready assessment of the deposit penetration could be performed. This comprised (Fig. 2) a metal mask with a hole (0.7 mm diameter), held above a ppHex (20 W, power in continuous mode; 40 Pa, pressure; 5 min deposition time) coated glass coverslip at a 0.11 mm distance, provided by an aluminium ring. The analysis of the coverslip
3338
F. Intranuovo et al. / Acta Biomaterialia 7 (2011) 3336–3344
Fig. 1. Schematic of the plasma chamber.
Table 1 Plasma depositing conditions included in the study of the effect of plasma parameters on polymer penetration into a model pore and corresponding WCA values (n = 5). Process
Monomer
Pressure (Pa)
Total power (W)
Average power (W)
tON (ms)
Power mode
Deposition rate (nm min 1)
Time (min)
WCA (°)
ppAAm (7 Pa) ppAAm (20 Pa) ppAAm (40 Pa) ppAAm (80 Pa) ppAAm (107 Pa) p-ppAAm (1.8 W) p-ppAAm (3.3 W) p-ppAAm (4.5 W) p-ppAAm (6.4 W) p-ppAAm (8.3 W) p-ppAAm (11.7 W) ppHex (1 min) ppHex (4 min) ppHex (5 min)
AAm AAm AAm AAm AAm AAm AAm AAm AAm AAm AAm
7 20 40 80 107 40 40 40 40 40 40
20 20 20 20 20 20 20 50 70 50 70
/ / / / / 1.8 3.3 4.5 6.4 8.3 11.7
/ / / / / 1 2 1 1 2 2
Continuous Continuous Continuous Continuous Continuous Pulsing Pulsing Pulsing Pulsing Pulsing Pulsing
5 10 20 25 17 2 3 5 4 6 7
20 10 5 4 6 50 33 20 25 17 14
49 ± 3 49 ± 1 59 ± 1 51 ± 1 50 ± 2 53 ± 1 55 ± 1 54 ± 3 51 ± 2 53 ± 1 57 ± 2
Hex Hex Hex
40 40 40
20 20 20
/ / /
/ / /
Continuous Continuous Continuous
5 5 5
1 4 5
92 ± 1 91 ± 2 91 ± 1
Allyl amine coatings at different pressures in continuous power mode (ppAAm), different average powers in pulsed power mode (p-ppAAm) and hexane coatings at different deposition times in continuous power mode (ppHex) were plasma deposited.
Fig. 2. Schematic of the experimental setup used to study the plasma polymer gradients.
F. Intranuovo et al. / Acta Biomaterialia 7 (2011) 3336–3344
was able to determine the penetration of the depositing species through the hole and along the channel formed between the mask and the substrate [35]. The water contact angle (WCA) measurements on gradient samples were taken every 0.2 mm over a 4 mm total diameter, using a DSA100 instrument (Krüss). This allowed the deposition of picolitre-sized droplets on the sample surface. The dispensed drop volume was 110 pl, corresponding to a base diameter of approximately 75 lm for a WCA of 90°. The sample was moved via an automated stage. The WCA measurements were made radially from the centre of the sample to the edge. Videos of the evaporating droplets were captured in 18 ms intervals, for a duration of 1 s. The WCA was determined from the first stable image of the water droplet, between a tangent and a circular curve fit, intersecting with a straight line [36]. 2.4. Chemical analysis X-ray photoelectron spectroscopy (XPS) was carried out using a Kratos Axis Ultra spectrometer, employing a monochromatic Al Ka X-ray source (hm = 1486.6 eV), hybrid (magnetic/electrostatic) optics, hemispherical analyser, a multi-channel plate and a delay line detector with a collection angle of 30° and a take-off angle of 90°. The X-ray gun power was set to 100 W. All spectra were recorded using an aperture slot of 300 700 lm2, with a pass energy of 80 eV for survey scans and 20 eV for high-resolution core level scans. Spectra were recorded using the Kratos VISION II software and processed using the CASA XPS™ software package. Charge compensation (Kratos AXIS Nova charge neutralization system: a coaxial low energy electron source within the field of the magnetic lens) was used during the experiments, and samples were earthed via the stage using a standard BNC connector. Binding energies were charge corrected by setting the C1s aliphatic carbon signal from adventitious carbon contamination to 285.0 eV [37]. The C1s curve fits were carried out by restricting the full width at half maximum of all components to that of the C–C/C–H component (C1) at 285.0 eV. The other components had fixed energies at 287.1 eV (C(@O)OC, C2) and 289.1 eV (C(@O)O, C3) for the PDLLA samples, according to its chemical structure and the literature [37]. When samples were plasma treated, two other contributions were considered, at 286.0 eV (C–N/C–O, C4) and 288.0 eV (N–C@O, C5), respectively. In order to analyse the chemical composition inside the 3-D scaffolds, sections parallel and perpendicular to their surface were made using a scalpel blade. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) analysis was carried out on a SIMS IV time-of-flight instrument (ION-TOF GmbH) equipped with a bismuth liquid metal ion gun and a single-stage reflectron analyser. The instrument was typically operated at a primary ion energy of 25 kV, a pulsed target current of 1.3 pA and a post-acceleration of 10 kV. Charges induced on the surface by the positively charged ion beam were compensated with a flux of low energy electrons (20 eV). Chemical images were obtained by rastering the stage under the pulsed primary ion beam, using a field of 0.5 mm2. All analyses were carried out below the static limit of 1012 ions per cm2 for both polarities combined. The acquisition of full raw datasets allowed for the retrospective construction of spectra from the imaged areas. Positive spectra were normalized to the total ion intensity for comparison between samples. 2.5. Cell culture Untreated and plasma modified PDLLA scaffolds were sterilized and pre-wetted using phosphate-buffered saline (PBS) and 5 antibiotic/antimycotic solution (500 U ml 1 penicillin, 0.5 mg ml 1
3339
streptomycin, 1.25 lg ml 1 amphotercin B). After being rinsed with PBS, the scaffolds were soaked in cell culture medium in either a 6-well plate or a Falcon tube in a cell culture incubator for 24 h. The scaffolds were then placed in a non-tissue culture treated 6-well plate and seeded with 3T3 fibroblasts (1 106 cells per scaffold), for culture times of 48 and 96 h. After 24 h the medium was changed to remove any unattached cells. 2.6. Cell viability assays Cell viability was measured using an MTS assay (CellTiter 96 AQueous One solution, Promega Corporation). The scaffolds were incubated with a 20% MTS (3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) solution in cell culture medium for 3 h, at 37 °C in a 5% CO2 incubator. At the end of the incubation period, 100 ll of MTS solution and medium was transferred to a 96-well plate and the absorbance of the dye was measured at 490 nm, using an Optima FLUOstarH plate reader. Statistical analysis was performed by a paired Student’s t-test. To determine the viability of adherent or non-adherent cells, the LIVE/DEAD viability/cytotoxicity test was used. The LIVE/DEAD working solution was composed of PBS/ethidium homodimer-1 with calcein AM (LIVE/DEAD Viability/Cytotoxicity Assay Kit, Molecular Probes). Live cells were distinguished by the presence of intracellular esterase activity, determined by the enzymatic conversion of the non-fluorescent calcein AM to the green fluorescent calcein. Ethidium homodimer-1 is a high-affinity, red-fluorescent nucleic acid stain, able to pass through the compromised membranes of dead cells, producing a red fluorescence. After seeding cells, the LIVE/DEAD working solution was added to cover the scaffolds and incubated for 40 min, at room temperature. The scaffolds were then washed with PBS solution, cut in the middle and fixed with DABCO UV mountant–working solution (9:1 ratio of glycerol:DABCO/PBS) to retard photobleaching of the dyes. A confocal microscope (Zeiss 710, ex/em 495/515 nm Calcein: ex/em 495/ 635 nm EB) was used to visualize the area of interest in the scaffold sections. 2.7. X-ray microcomputed tomography of scaffolds After certain incubation times (48 and 96 h), the culture medium was removed and the scaffolds were washed in PBS. The cells were then fixed to the scaffolds by treating with paraformaldehyde/10% neutral buffered formalin for 30 min. The scaffolds were extensively rinsed first with PBS and then with distilled water, before staining with osmium tetroxide (1% solution) for 1 h. They were then rinsed in distilled water and dried for 2–3 days for X-ray microcomputed tomography (lCT) analysis. A high-resolution Skyscan lCT system (Skyscan 1174, Skyscan, Aartselaar, Belgium) was used to produce reconstructed 3-D images of cell distribution in the scaffolds. The scanner was set to a voltage of 50 kV and a current of 800 lA, and the samples were scanned at 11.9 lm voxel resolution. Two hundred slices were taken from the central section of each sample (3 mm thickness) and analysed for osmium tetroxide-stained cells by comparing the radiodense area. Three-dimensional images were obtained by using SkyScan 1174 NRecon and then SkyScan 1174 CTAn software to produce a 3-D volumetric space from the reconstructed 2-D slices (cross-sections). Morphometric parameters, such as porosity and pore size, were then calculated in the 3-D volumetric space or in two dimensions, from approximately 3 mm cross-sectional slices through the centre of the scaffolds. The area (mm2) occupied by cells in each slice was converted to a volume fraction.
3340
F. Intranuovo et al. / Acta Biomaterialia 7 (2011) 3336–3344
3. Results The theory describing plasma polymer deposition into pores is developing, but as yet it is not possible to predict how deposition into such structures is influenced by a particular plasma parameter [38]. A number of plasma conditions could influence the deposition of plasma polymers; thus, to determine the optimal parameters, a model pore was devised to enable ready empirical assessment of the effect of variables, such as input power, pulse/continuous power modes and monomer pressure on the deposit penetration down the pore. This allowed the penetrative nature of the plasma polymer deposition process to be assessed for a large range of plasma conditions (Table 1) on a flat surface (glass coverslip) behind an aperture. This acts as the pore opening, while the plate containing the hole and the glass coverslip act as the pore walls. Recently, a few other strategies have been proposed to create model pores, e.g. by designing microstructured specimens using the micromoulding technique [39], or by preparing polyolefin plastomer masks, equipped with a channel frame, that mimic the scaffold pore structure [38]. Glass coverslips were plasma coated with a uniform layer of hydrophobic ppHex thin film, on which was superimposed a radial gradient in the deposit of hydrophilic ppAAm through the pinhole mask. The wettability gradient was demonstrated by WCA mapping (Fig. S1) [12]. This gradient arose from the diffusion of the AAm depositing species under the mask, resulted in the deposition of a coating with a thickness decreasing from the region closest to the plasma through the mask hole to the areas beneath the mask. The AAm plasma condition, identified as producing the lowest penetration, was a pulsed deposition with an average power of 3.3 W (deposition time = 33 min), having a WCA value of 55 ± 1° (n = 5, Table 1) and the highest penetration at an average pulsed power of 11.7 W (deposition time = 14 min), having a WCA value of 57 ± 2° (n = 5, Table 1).
3.1. Characterization of plasma-coated PDLLA scaffolds PDLLA scaffolds of 10 mm diameter and 5 mm thickness were formed using supercritical CO2 processing. lCT was used to analyse the morphology of the scaffolds, from which the average porosity was measured to be 70 ± 1%, with an average pore size of 234 ± 15 lm. An O2 plasma was applied immediately before the AAm plasma deposition to encourage plasma polymer adhesion. The AAm plasmas identified as providing the lowest and highest deposit penetrations, pulsed ppAAm (p-ppAAm) at 3.3 and 11.7 W respectively, were subsequently struck in the chamber. After plasma polymer deposition, the scaffold pore size, assessed from the 3-D lCT images, was found to increase slightly compared to the untreated scaffolds, with unchanged porosity (Table 2). The slight increase in pore size is likely to be due to removal of PDLLA as a result of the initial O2 etching process before the p-ppAAm deposition. The penetration of p-ppAAm deposits was studied using XPS, by mechanical sectioning of the scaffold. The surface nitrogen concentration was used to quantify the presence of p-ppAAm coating. In Fig. 3, it can be seen from the nitrogen concentration that a gradient in p-ppAAm thickness was achieved
Table 2 Average porosity and pore size of untreated and p-ppAAm-coated scaffolds, assessed from the 3-D lCT analysis (the standard deviation is reported from analysis of five different scaffolds before and five after the p-ppAAm deposition).
Untreated p-ppAAm
Average porosity (%)
Average pore size (lm)
70 ± 1 71 ± 1
234 ± 15 265 ± 41
Fig. 3. XPS determined nitrogen surface concentration (atomic percent) along the diameter of a PDLLA scaffold sectioned at a depth of 2 mm. The pulsed allyl amine plasma was struck at a pressure of 40 Pa. (N), p-ppAAm at power = 3.3 W (33 min deposition time); (d), p-ppAAm at power = 11.7 W (14 min deposition time).
from the periphery to the centre of the disc. The penetration of p-ppAAm coating into the scaffold section was greater for the one deposited at 11.7 W compared to that at 3.3 W. This was consistent with the predictions made from water contact angle measurements on the pore model, which indicated that the shorter exposure of the AAm plasma at 11.7 W resulted in better deposit penetration. ToF-SIMS analysis was carried out to provide a high lateral resolution characterization of the distribution of the p-ppAAm coating on PDLLA in the centre of the scaffold, where the XPS determined that the nitrogen concentration was lowest (3–5 at.%). In all analyses, a uniform p-ppAAm layer was observed (Fig. S2). In order to counteract the tendency of cells to adhere to the outside of the scaffold, after the deposition of the p-ppAAm coating with the most penetrating plasma condition (at 11.7 W), a thin layer of the cell-attachment-resistant ppHex chemistry was deposited [35] (ppHex/p-ppAAm). This scaffold was subject to a hexane plasma for 4 min (power in continuous mode = 20 W, pressure = 40 Pa). To complement the analysis of the deposit distribution across the diameter of the scaffold, a mechanical section along the cylindrical axis was undertaken to provide the distribution through the thickness of the scaffold. The XPS analysis of a 3 mm vertical section (5 mm total scaffold thickness), shown in Fig. 4, indicates a deposition thickness gradient consistent with plasma species penetration from the top to the bottom. The nitrogen content of the p-ppAAm-coated samples (triangles) decreased from the top to the bottom of the scaffold. For the ppHex/p-ppAAm coating (squares), the nitrogen concentration was lower than the p-ppAAm-coated scaffold, indicating that the overcoat of ppHex markedly attenuated the contribution of the nitrogen at the periphery of the object. 3.2. Cell response to plasma polymer-coated scaffolds An in vitro cell culture experiment was performed using 3T3 fibroblast cells to assess and compare the cell viability and penetration in the scaffolds for the following samples: untreated, p-ppAAm and ppHex/p-ppAAm-coated scaffolds. To probe the survival of the fibroblast cell population attached to the whole scaffold, the retention of their metabolic and/or
F. Intranuovo et al. / Acta Biomaterialia 7 (2011) 3336–3344
Fig. 4. XPS determined nitrogen concentration along the mechanical section perpendicular to the PDLLA scaffold top surface. A pulsed 40 Pa allyl amine plasma with and without a ppHex overcoat were deposited. (N), p-ppAAm (11.7 W power in pulsing mode; 40 Pa pressure; 14 min deposition time); (j), ppHex/p-ppAAm (Hex deposition: 20 W power in continuous mode; 40 Pa pressure; 4 min deposition time).
proliferative ability was measured by MTS assay. The mitochondrial activity of cells in the scaffolds after 48 h of culture time is presented in Fig. 5. The mitochondrial activity of the p-ppAAmcoated scaffolds increased relative to the untreated scaffolds, and deposition became higher with the ppHex/p-ppAAm film. This was most likely due to a larger number of cells initially attaching. The ppHex/p-ppAAm-coated scaffolds were additionally evaluated at 96 h cell culture, which indicated that the cell number had increased. The MTS value (p < 0.01) further increased over the 48 h cell culture, with a cell proliferation improvement of 18%.
3341
The global distribution of 3T3 fibroblasts through the scaffold was determined with lCT, using osmium tetroxide staining to provide sufficient X-ray absorption contrast, to distinguish the cells from the PDLLA. The lCT images obtained after 48 h culture time are presented in Fig. 6. For each sample, images representing the top and side views of the scaffolds are displayed. Each represents a summation of the cells in a 3 mm thick section from the centre of the section viewed. A clear difference in cell distribution was observed between the untreated (Fig. 6A) and plasma-treated scaffolds (Fig. 6B–D). There was a considerable increase in the overall loading of cells in the p-ppAAm (11.7 W)-coated scaffolds, which were determined to have the best ppAAm penetration, as represented by the cell area intersecting the viewing plane in 3 mm slices through the centre of the scaffolds (718 ± 79 mm2; Fig. 6B), compared to untreated scaffolds (176 ± 23 mm2; Fig. 6A). The increase in the area occupied by cells as a result of the ppHex/pppAAm coating was greater still (828 ± 63 mm2), as seen in Fig. 6C. A further 2 days of cell culture was employed to explore the development of the cell distribution in the ppHex/p-ppAAmcoated scaffolds. After 96 h of culture (Fig. 6D), the 3T3 fibroblasts were more homogeneously distributed than the equivalent 48 h cultured sample. The cell area (903 ± 9 mm2) reached the greatest value and the cells were spread evenly (top to bottom) throughout the porous scaffold structure. The cell distribution and area shown by lCT data are in agreement with the metabolic activity levels determined with the MTS assay. To quantify the cell distribution within the images shown in Fig. 6, the cell coverage of the scaffolds was calculated for the core and for the outer sheath area and presented in Fig. 7. This illustrates that these cells in the untreated PDLLA scaffold were concentrated within the sheath region, with a total cell volume fraction of only 1% reaching the core, compared to 4% in the sheath (Fig. 7). For the plasma polymer-treated samples, an increase in the cell content for both core and sheath was noted. The p-ppAAm-coated scaffold exhibited a cell volume fraction of 7% in the core and 12% in the sheath, which represents an overall increase compared to the uncoated samples. For the samples with a ppHex layer (ppHex/p-ppAAm), a further increased penetration into the core, to give a volume fraction of 11% in the core and 13% in the sheath, was noted. Increasing the culture time to 96 h further promoted the scaffold colonization, with the highest value volume fraction occupied by cells in both core (12.5%) and sheath (14%) regions. At culture times of 48 and 96 h, evaluation of the cell viability inside the scaffolds was performed using a LIVE/DEAD assay. Confocal images from cells located in the centre of the scaffolds accessed by mechanical sectioning indicated that live cells (green) lined the struts and pores of the scaffolds, as shown in the representative images presented in Fig. 8A–C (untreated, p-ppAAm and ppHex/p-ppAAm-coated scaffold sections after 48 h cell culture, respectively). In Fig. 8D (ppHex/p-ppAAm-coated scaffold section, after 96 h cell culture), long dendrites were observed extending from the cells, which could aid in cell migration and monitoring of the scaffold environment. This branched morphology has been previously noted on 3-D electrospun poly(D,L-lactide-coL-lactide) matrices [40,41], as the typical shape of migrating fibroblasts in 3-D fibrous scaffolds or matrices. Very few dead cells (red) were observed for all scaffolds.
4. Discussion 4.1. Cell response to scaffolds Fig. 5. MTS activity of 3T3 fibroblasts cultured for 48 and 96 h in untreated and plasma-modified scaffolds: p-ppAAm (11.7 W power in pulsing mode; 40 Pa pressure; 14 min deposition time); ppHex/p-ppAAm (Hex deposition: 20 W power in continuous mode; 40 Pa pressure; 4 min deposition time). For each scaffold type, the standard deviation is reported from analysis of three different scaffolds.
The attachment level and distribution of 3T3 fibroblasts on untreated scaffolds in vitro was poor (Figs. 6 and 7). An improvement in cell adhesion and distribution was achieved after the p-ppAAm
3342
F. Intranuovo et al. / Acta Biomaterialia 7 (2011) 3336–3344
Fig. 6. Cumulative 3T3 cell area in approximately 3 mm slices through the centre of the scaffolds, within the core and the sheath regions (half the total scaffold area) of untreated and plasma-coated scaffolds.
without improving the cell area in the total scaffold [24]. In the present study, the ppHex/p-ppAAm coating had the effect of improving the fibroblast cell colonization of the interior, as shown in Fig. 6, with the increase in cell loading in the core approaching that of the sheath scaffold regions (Fig. 7). Furthermore, by increasing the cell culture time from 48 to 96 h, both the total cell loading and proliferation increased to the point where the scaffold was virtually uniformly populated, when viewed from the top and the side.
4.2. Plasma polymers
Fig. 7. lCT top and side images of scaffolds cultured with 3T3 fibroblasts for 48 h: (A) untreated; (B) p-ppAAm, 11.7 W power in pulsing mode, 40 Pa pressure, 14 min deposition time; (C) ppHex/p-ppAAm, Hex deposition: 20 W power in continuous mode, 40 Pa pressure, 4 min deposition time; and for 96 h: (D) ppHex/p-ppAAmcoated scaffolds. The pictures show lCT images from approximately 3 mm slices through the centre of the scaffolds.
coating, consistent with other similar studies with ppAAm-coated surfaces [42,43]; however, with an optimized coating regime developed using a pinhole model, a uniform cell distribution was obtained. The improved cell response on the p-ppAAm surface has been rationalized as the presence of nitrogen-containing groups modifying the protein adsorption from the serum in the medium, thereby improving cell adhesion [32]. The p-ppAAm-coated scaffolds showed a greater area occupied by cells in the total scaffold with respect to untreated scaffolds, with a greater proportion penetrating to the core (Fig. 6). In a previous study [24], continuous-wave ppAAm deposition did not achieve any great improvement in cell area, which was approximately 0.3 mm2 and was higher in the outer area than in the inner area. When a ppHex coating was applied, the fibroblast area was increased and more uniform compared to the ppAAm-coated scaffolds. This allowed an increase in the cell area in the core, but
There has been much debate about which components of the plasma dominate the deposition process and in turn influence the deposition rate and polymer chemistry (see e.g. Ref. [44]). In the gas phase of allyl amine plasma, mass spectral measurements have found evidence of reactions between cations and intact neutral allyl amine monomers or radicals, which contrast with the more traditional view of the dominant radical–radical and radical–neutral reactions [22,45,46]. Swindells et al. [47] proposed another alternative of negative ion-neutral polymerization mechanism from acrylic acid vapours, demonstrating that negative ions may also contribute to plasma polymer formation. The plasma is expected to be excluded from the porous body of the scaffolds, based on previous investigations on plasma polymer deposition into channels [38]. Modification of the chemistry of the internal surfaces of 3-D porous scaffolds by plasma polymer deposition would therefore be controlled by diffusion of the depositforming species from the plasma phase down the pores and the deposition of these species on the walls. This process leads to radial gradients of plasma polymer from the periphery to the centre of the scaffolds. Radicals are known to be sufficiently long lived to make this journey, as are some charged species which have been found to be surprisingly long lived by mass spectrometry of pulsed plasma polymer systems [42]. To readily empirically assess coating deposition inside the porous structure, we used a pinhole as a model pore and investigated the effect of plasma parameters such as monomer pressure, power and pulse mode on penetration therein. We used simple picolitre WCA measurements, which were a quick and inexpensive means of mapping penetration into this model pore that was compatible with an iterative search of the optimal deposition parameters. XPS and ToF-SIMS provide a fuller characterization, but the throughput and turnaround from sample fabrication to result is much lower, preventing iterative studies.
F. Intranuovo et al. / Acta Biomaterialia 7 (2011) 3336–3344
3343
Fig. 8. Confocal images (n = 3) of 3T3 fibroblast cells, labelled with the LIVE/DEAD working solution on the sections from the centre of untreated (48 h, A); p-ppAAm (48 h, B); ppHex/p-ppAAm (48 h, C); and ppHex/p-ppAAm (96 h, D) coated scaffolds. Live cells were distinguished by enzymatic conversion of the non-fluorescent calcein AM to the green fluorescent calcein. Dead cells were determined by the red-fluorescent ethidium homodimer-1 stain.
The optimal polymer deposit penetration condition was found to be at a high pulsed power. The higher power pulsed plasma is likely to give better ppAAm penetration than pulsed and continuous-wave plasmas at lower power because of the greater monomer fragmentation resulting in a higher density of ions. This is consistent with previous deposition studies on 2-D surfaces [43,45,48]. The higher concentration of depositing species outside the pores would result in greater penetration through the pores towards the centre of the scaffold, although the many other factors that change when the power is modified, e.g. the plasma sheath, make this proposal difficult to verify. XPS analysis on horizontal and perpendicular scaffold mechanical sections allowed quantification of the p-ppAAm penetration using the nitrogen elemental signal. Across the diameter of a scaffold section, the nitrogen content decreased from the edges to the core (Fig. 3), consistent with a previous study [32]. A similar dependence on the distance from the edge of the scaffold was seen through the thickness (Fig. 4). This marked difference between the external and internal regions of the scaffold relates to the diffusion of depositing species from the excluded plasma phase. The chemistry of the p-ppAAm, indicated by the N1s and C1s core levels, does not appear to vary with depth, being consistent with the assignment of the chemistry to amines, imines and nitrile groups in previous studies [45,48,49]. After the p-ppAAm deposition, the scaffolds were plasma coated for a short while in a hexane plasma. This has been shown to result in a ppHex film at the periphery [32]. Thus, gradients of surface chemistry within the porous structure, from a peripheral hydrocarbon to an internal nitrogen-containing film, were produced in order to encourage the cell colonization in the scaffold core. By tuning the ppHex deposition time, a good control of cell
adhesion and penetration into the scaffolds was achieved on the optimized p-ppAAm coating (Figs. 6 and 7). Fast and uniform cell colonization was achieved with the fibroblasts used in this study. This chemical surface modification strategy is an attractive alternative to complex methods for forcing cell loading inside the scaffolds. This simple and effective plasma-generated chemical gradient could be easily applied to any porous scaffold material. Plasma polymer coatings have previously been shown to be a powerful strategy for controlling the attachment and function of a wide range of cell types, indicating that this can be applied beyond the model of fibroblast cell type used here. 5. Conclusions An empirical study of the dependence of deposit penetration down a model pore on plasma parameters has enabled optimal plasma conditions to be identified. A radial chemical gradient has been achieved from a cell-repulsive ppHex film on the top to a cell-adhesive p-ppAAm coating throughout the scaffold. By controlling the allyl amine and hexane plasma polymerization mechanisms and parameters, a uniform fibroblast cell colonization throughout porous scaffolds was successfully achieved. This indicates the potential of this coating technique to provide a uniform distribution of many cell types in porous 3-D scaffolds made of any material. Acknowledgements We thank Paul Roach and Mathieu Lanniel for support with plasma deposition experiments and Yvonne Reinwald for scaffold
3344
F. Intranuovo et al. / Acta Biomaterialia 7 (2011) 3336–3344
preparation. We further thank David J. Scurr, Susan Dodson and Emily Smith for help with ToF-SIMS, lCT and XPS measurements, respectively. Appendix A. Figures with essential colour discrimination Certain figures in this article, particularly Figures 1, 2, 6, and 8, are difficult to interpret in black and white. The full colour images can be found in the on-line version, at doi:10.1016/ j.actbio.2011.05.020. Appendix B. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.actbio.2011.05.020. References [1] Nerem RM. The challenge of imitating nature. In: Lanza R, Langer R, Vacanti J, editors. Principles of tissue engineering. Burlington, MA: Elsevier Academic Press; 2007. p. 7–14. [2] Sengers BG, Taylor M, Please CP, Oreffo ROC. Computational modelling of cell spreading and tissue regeneration in porous scaffolds. Biomaterials 2007;28:1926–40. [3] Khang G, Kim MS, Lee HB. Introduction. In: Khang G, Kim MS, Lee HB, editors. Manuals in biomedical research. A manual for biomaterials/scaffold fabrication technology, vol. 4. Singapore: World Scientific Publishing Co. Pte. Ltd.; 2007. p. 1–12. [4] Barry JJA, Silva MMCG, Popov VK, Shakesheff KM, Howdle SM. Supercritical carbon dioxide: putting the fizz into biomaterials. Phil Trans R Soc A 2006;364:249–61. [5] Hile DD, Amirpour ML, Akgerman A, Pishko MV. Active growth factor delivery from poly(D,L-lactide-coglycolide) foams prepared in supercritical CO2. J Control Release 2000;66:177–85. [6] Singh L, Kumara V, Ratner BD. Generation of porous microcellular 85/15 poly (DL-lactide-co-glycolide) foams for biomedical applications. Biomaterials 2004;25:2611–7. [7] Tai H, Mather ML, Howard D, Wang W, White LJ, Crowe JA, et al. Control of pore size and structure of tissue engineering scaffolds produced by supercritical fluid processing. Eur Cell Mater 2007;14:64–77. [8] Pini R, Storti G, Mazzotti M, Tai H, Shakesheff KM, Howdle SM. Sorption and swelling of poly(DL-lactic acid) and poly(lactic-co-glycolic acid) in supercritical CO2: an experimental and modeling study. J Polym Sci Part B: Polym Phys 2008;46:483–96. [9] Duarte ARC, Mano JF, Reis RL. Preparation of starch-based scaffolds for tissue engineering by supercritical immersion precipitation. J Supercrit Fluids 2009;49:279–85. [10] Zhu XH, Lee LY, Jackson JSH, Tong YW, Wang CH. Characterization of porous poly(D, L-lactic-co-glycolic acid) sponges fabricated by supercritical CO2 gasfoaming method as a scaffold for three-dimensional growth of Hep3B cells. Biotechnol Bioeng 2008;100:998–1009. [11] Wan Y, Tu C, Yang J, Bei J, Wang S. Influences of ammonia plasma treatment on modifying depth and degradation of poly(L-lactide) scaffolds. Biomaterials 2006;27:2699–704. [12] DeForest CA, Polizzotti BD, Anseth KS. Sequential click reactions for synthesizing and patterning three-dimensional cell microenvironments. Nat Mater 2009;8:659–64. [13] Engler AJ, Sen S, Sweeney HL, Discher DE. Matrix elasticity directs stem cell lineage specification. Cell 2006;126:677–89. [14] Dalby MJ, Gadegaard N, Tare R, Andar A, Riehle MO, Herzyk P, et al. The control of human mesenchymal cell differentiation using nanoscale symmetry and disorder. Nat Mater 2007;6:997–1003. [15] Cukierman E, Pankov R, Stevens DR, Yamada KM. Taking cell-matrix adhesions to the third dimension. Science 2001;294:1708–12. [16] Moroni L, Habibovic P, Mooney DJ, van Blitterswijk CA. Functional tissue engineering through biofunctional macromolecules and surface design. MRS Bull 2011;35:584–90. [17] Lan PX, Lee JW, Seol Y-J, Cho D-W. Development of 3D PPF/DEF scaffolds using micro-stereolithography and surface modification. J Mater Sci: Mater Med 2009;20:271–9. [18] Yeo A, Wong WJ, Teoh S-H. Surface modification of PCL-TCP scaffolds in rabbit calvaria defects: evaluation of scaffold degradation profile, mechanical properties and bone healing patterns. J Biomed Mater Res A 2010;93:1358–67. [19] Liu X, Won Y, Ma PX. Porogen-induced surface modification of nano-fibrous poly(L-lactic acid) scaffolds for tissue engineering. Biomaterials 2006;27:3980–7. [20] Siow K, Britcher L, Kumar S, Griesser H. Plasma methods for the generation of chemically reactive surfaces for biomolecule immobilisation and cell colonisation – a review. Plasma Process Polym 2006;3:392–418.
[21] Fally F, Doneux C, Riga J, Verbist JJ. Quantification of the functional groups present at the surface of plasma polymers deposited from propylamine, allylamine, and propargylamine. J Appl Polym Sci 1995;56:567–614. [22] Shard AG, Whittle JD, Beck AJ, Brookes PN, Bullett NA, Talib RA, et al. A NEXAFS examination of unsaturation in plasma polymers of allylamine and propylamine. J Phys Chem B 2004;108:12472–80. [23] Roach P, Howard D, Barry JR, Shakesheff KM, Howdle SM, Alexander MR. Chemical modification of porous scaffolds using plasma polymers. In: Berthiaume F, Morgan J, editors. 3D Tissue Engineering. Boston: Artech House; 2010. p. 1–20. [24] Barry JJA, Howard D, Shakesheff KM, Howdle SM, Alexander MR. Using a coresheath distribution of surface chemistry through 3D tissue engineering scaffolds to control cell ingress. Adv Mater 2006;18:1406–10. [25] Djordjevic I, Britcher LG, Kumar S. Morphological and surface compositional changes in poly(lactide-co-glycolide) tissue engineering scaffolds upon radio frequency glow discharge plasma treatment. Appl Surf Sci 2008;254:1929–35. [26] Safinia L, Datan N, Höhse M, Mantalarisa A, Bismarck A. Towards a methodology for the effective surface modification of porous polymer scaffolds. Biomaterials 2005;26:7537–47. [27] Yang J, Shi G, Bei J, Wang S, Cao Y, Shang Q, et al. Fabrication and surface modification of macroporous poly(L-lactic acid) and poly(L-lactic-co-glycolic acid) (70/30) cell scaffolds for human skin fibroblast cell culture. J Biomed Mater Res 2002;62:438–46. [28] Hu Y, Winn SR, Krajbich I, Hollinger JO. Porous polymer scaffolds surfacemodified with arginine-glycine-aspartic acid enhance bone cell attachment and differentiation in vitro. J Biomed Mater Res 2003;64A:583–90. [29] Chim H, Ong JL, Schantz JT, Hutmacher DW, Agrawal CM. Efficacy of glow discharge gas plasma treatment as a surface modification process for threedimensional poly (D, L-lactide) scaffolds. J Biomed Mater Res 2003;65A:327–35. [30] Armentano I, Ciapetti G, Pennacchi M, Dottori M, Devescovi V, Granchi D, et al. Role of PLLA plasma surface modification in the interaction with human marrow stromal cells. J Appl Polym Sci 2009;114:3602–11. [31] Yildirim ED, Pappas D, Güçeri S, Sun W. Enhanced cellular functions on polycaprolactone tissue scaffolds by O2 plasma surface modification. Plasma Process Polym 2011;8:256–67. [32] Barry JJA, Silva MMCG, Shakesheff KM, Howdle SM, Alexander MR. Using plasma deposits to promote cell population of the porous interior or threedimensional poly(D, L-lactic acid) tissue-engineering scaffolds. Adv Funct Mater 2005;15:1134–40. [33] Quirk RA, France RM, Shakesheff KM, Howdle SM. Supercritical fluid technologies and tissue engineering scaffolds. Curr Opin Solid State Mater Sci 2004;8:313–21. [34] Woods HM, Silva MMCG, Nouvel C, Shakesheff KM, Howdle SM. Materials processing in supercritical carbon dioxide: surfactants, polymers and biomaterials. J Mater Chem 2004;14:1663–78. [35] Zelzer M, Majani R, Bradley JW, Rose FRAJ, Davies MC, Alexander MR. Investigation of cell-surface interactions using chemical gradients formed from plasma polymers. Biomaterials 2008;29:172–84. [36] Taylor M, Urquhart AJ, Zelzer M, Davies MC, Alexander MR. Picoliter water contact angle measurement on polymers. Langmuir 2007;23:6875–8. [37] Moulder J, Stickle WF, Sobol PE, Bomben KD, Chastian J. Handbook of X-ray Photoelectron Spectroscopy. Eden Prairie: Perkin-Elmer; 1992. [38] Zelzer M, Scurr D, Abdullah B, Urquhart AJ, Gadegaard N, Bradley JW, et al. Influence of the plasma sheath on plasma polymer deposition in advance of a mask and down pores. J Phys Chem B 2009;113:8487–94. [39] Besch W, Lippold O, Reinecke H, Schröder K, Ohl A. A test specimen for investigation of plasma modification of interior surfaces in polymeric threedimensional microstructures. Surf Coat Tech 2003;174–175:112–7. [40] Kluger PJ, Wyrwa R, Weisser J, Maierle J, Votteler M, Rode C, et al. Electrospun poly(D/L-lactide-co-L-lactide) hybrid matrix: a novel scaffold material for soft tissue engineering. J Mater Sci: Mater Med 2010;21(9):2665–71. [41] Grinnell F. Fibroblast mechanics in three-dimensional collagen matrices. J Bodywork Mov Ther 2008;12:191–3. [42] Swindells I, Voronin SA, Bryant PM, Alexander MR, Bradley JW. Temporal evolution of an electron-free afterglow in the pulsed plasma polymerisation of acrylic acid. J Phys Chem B 2008;112:3938–47. [43] Harsch A, Calderon J, Timmons RB, Gross GW. Pulsed plasma deposition of allylamine on polysiloxane: a stable surface for neuronal cell adhesion. J Neurosci Methods 2000;98:135–44. [44] Short RD, Steel DA. Testing the hypothesis: comments on plasma polymerisation of acrylic acid revisited. Plasma Process Polym 2010;7:366–70. [45] Choukourov A, Biederman H, Slavinska D, Hanley L, Grinevich A, Boldyryeva H, et al. Mechanistic studies of plasma polymerization of allylamine. J Phys Chem B 2005;109:23086–95. [46] Beck AJ, Candan S, Short RD, Goodyear A, Braithwaite NSJ. The role of ions in the plasma polymerization of allylamine. J Phys Chem B 2001;105:5730–6. [47] Swindells I, Voronin SA, Fotea C, Alexander MR, Bradley JW. Detection of negative molecular ions in acrylic acid plasma: some implications for polymerization mechanisms. J Phys Chem B 2007;111:8720–2. [48] Hamerli P, Weigel T, Groth T, Paul D. Surface properties of and cell adhesion onto allylamine-plasma coated poly(ethylene terephthalate) membranes. Biomaterials 2003;24:3989–99. [49] Denis L, Cossement D, Godfroid T, Renaux F, Bittencourt C, Snyders R, et al. Synthesis of allylamine plasma polymer films: correlation between plasma diagnostic and film characteristics. Plasma Process Polym 2009;6:199–208.