Development of Advanced Dressings for the Delivery of Progenitor

Many of these are difficult (if not impossible) to treat with cell suspensions. ... Australia, and was used to label primary amines on the surface of ...
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Development of Advanced Dressings for the Delivery of Progenitor Cells Giles T. S. Kirby,†,‡ Stuart J. Mills,†,‡ Liesbeth Vandenpoel,†,§ Jef Pinxteren,†,§ Anthony Ting,†,∥ Robert D. Short,†,‡ Allison J. Cowin,†,‡ Andrew Michelmore,†,‡,⊥ and Louise E. Smith*,†,‡ †

Cooperative Research Centre for Cell Therapy Manufacturing, North Terrace, Adelaide, South Australia 5000, Australia Future Industries Institute, University of South Australia, Mawson Lakes, South Australia 5095, Australia § ReGenesys BVBA, Bio-Incubator Leuven, Gaston Geenslaan 1, 3001 Heverlee, Belgium ∥ Athersys, Inc., Cleveland, Ohio 44115-2634, United States ⊥ School of Engineering, University of South Australia, Mawson Lakes, South Australia 5095, Australia ‡

S Supporting Information *

ABSTRACT: Culture surfaces that substantially reduce the degree of cell manipulation in the delivery of cell sheets to patients are described. These surfaces support the attachment, culture, and delivery of multipotent adult progenitor cells (MAPC). It was essential that the processes of attachment/ detachment to the surface did not affect cell phenotype nor the function of the cultured cells. Both acid-based and aminebased surface coatings were generated from acrylic acid, propanoic acid, diaminopropane, and heptylamine precursors, respectively. While both functional groups supported cell attachment/detachment, amine coated surfaces gave optimal performance. X-ray photoelectron spectroscopy (XPS) showed that at a primary amine to carbon surface ratio of between 0.01 and 0.02, greater than 90% of attached cells were effectively transferred to a model wound bed. A dependence on primary amine concentration has not previously been reported. After 48 h of culture on the optimized amine surface, PCR, functional, and viability assays showed that MAPC retained their stem cell phenotype, full metabolic activity, and biological function. Consequently, in a proof of concept experiment, it was shown that this amine surface when coated onto a surgical dressing provides an effective and simple technology for the delivery of MAPC to murine dorsal excisional wounds, with MAPC delivery verified histologically. By optimizing for cell delivery using a combination of in vitro and in vivo techniques, we developed an effective surface for the delivery of MAPC in a clinically relevant format. KEYWORDS: surface modification, progenitor cell, cell delivery, cell therapy, chronic wounds

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first described by Jiang et al. in 2002.7 Preclinical assessment of MAPC have shown clear therapeutic benefits in the amelioration of graft versus host disease8 and improving tissue regeneration and function.9−14 MAPC cells can be vastly expanded to the extent that cells isolated from a single donor can provide millions of individual patient doses, with no tissue matching required and the doses can be cryogenically stored for years.15 The future optimum mode of delivery of cell therapies remains unclear. Delivering suspensions of cells is widely reported and is a typical method for cell delivery in vivo.16 For epithelial grafts, including skin, enzymatic detachment of adherent cell sheets is required which adds complexity and may reduce the effectiveness of the cell product. Cell sheet delivery has been largely pioneered by the Okano group17 who have developed cell release surfaces based upon phase change

INTRODUCTION Cell therapies have received a great deal of attention recently as they offer the promise of effective treatment for chronic wounds. Epidermal cell therapies to date have focused almost entirely on autologous expanded cells1,2 with keratinocytes to be the first, reliably, cultured in the laboratory 40 years ago.3 This has translated into the production of thin sheets of cells (2−3 layers thick), known as cultured epithelial autografts (CEA) for use in acute, large surface area burns.4,5 Autografts offer no risk of rejection; however, they take 2−3 weeks for expansion and preparation for grafting.6 Allogeneic grafts are an alternative, and they negate many of the complications associated with autologous approaches. Allogenic products have the major advantage of being available when the patient requires but risks include immune rejection and disease transmission. An allogeneic product promising great potential without the immune risk is the multipotent adult progenitor cell. Multipotent adult progenitor cells (MAPC) are bonemarrow derived nonhematopoietic adherent cells that were © XXXX American Chemical Society

Received: November 16, 2016 Accepted: January 9, 2017 Published: January 9, 2017 A

DOI: 10.1021/acsami.6b14725 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces polymers. These, and allied methods,3,4 still require the detachment of intact sheets and their manipulation without breaking them. However, approaches that avoid enzymatic detachment of cells and allow delivery of complete extracellular matrix are gaining favor.18 Herein, we detail the development of a surface, applied to a robust support, which is tailored for the effective and direct delivery of an adult progenitor cell therapy to the skin. Recent research highlights the advantage of local cell delivery in wound healing, versus systemic delivery via injection. Huang et al. (2015) demonstrated the delivery of mesenchymal stem cells (MSCs) to murine cutaneous wounds within collagen microspheres while maintaining paracrine signaling,19 and Gao et al. (2014) delivered MSCs to diabetic murine cutaneous wounds at concentrations of 3 × 106 cells/ wounds and demonstrated enhanced wound closure rates.20 Plasma polymerization (PP) (also known as glow discharge polymerization) can be used to deposit surfaces from vapors of compounds containing specific chemistries.21 These chemistries can be tailored for cell culture and delivery.22 Radio frequency energy is used to fragment precursor vapor, forming reactive species which deposit onto a collecting substrate.23 These polymers adhere to a wide range of substrates and PP is able to coat objects of complex geometries.24,25 Through careful selection of a precursor, specific surface chemical functionality can be attained.26,27 This technique for the generation of surfaces has been previously utilized for the delivery of autologous keratinocytes,28 melanocytes,29,30 corneal epithelial cells31 and MSCs.22 Plasma polymerization provides a means to deliver cell sheets directly from culture surfaces to the wound site and with a wide range of base materials and formats. This unlocks a new set of approaches/applications. The human body is full of planar membranes such as the skin, esophagus, uterus and bladder. Many of these are difficult (if not impossible) to treat with cell suspensions. Furthermore, cell−cell interactions are important, but as soon as adherent cells are placed into a suspension, this behavior is compromised, potentially effecting function. Direct delivery of cell sheets negates this risk and in some cases may be used to deliver a denser cell population to the target site. However, to maximize the efficacy of the dressing, the surface chemistry must be carefully controlled. If the surface density of chemical functional groups which promote cell attachment is too low the cells will attach in low numbers resulting in delivery of a low number of cells; if the surface density is too high, the cells will easily attach but not release from the surface when placed in contact with the wound site. Plasma polymerization is ideal in this application as the surface chemistry can be controlled though judicious choice of chemical precursor and operating conditions. Here, we describe the testing and development of optimized plasma polymerized surfaces for the delivery of MAPC cell therapy into wounds. Plasma polymers containing different densities of carboxylic acid and amine functionality were produced and tested in vitro for MAPC attachment and delivery to a skin model. Finally, we present an optimized amine-based surface ready for further testing in vivo.

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Sigma-Aldrich and were used following degassing with three freeze thaw cycles. Polymerization was carried out within a parallel plate reactor, as previously described and characterized.32 The reactor consisted of a grounded 30 cm diameter steel cylinder with a height of 25 cm. The electrode was an internal 28 cm diameter plate located approximately 1 cm below the top of the reactor. Radio frequency (rf) power at 13.56 MHz was applied to the top electrode via a Coaxial Power Systems (U.K.) generator (RFG050) with a matching network (AMN150). The chamber was evacuated using a rotary pump to a base pressure of below 1 × 10−4 mbar. Precursor vapors were introduced to the chamber via a needle valve (Chell, U.K.). The initial pressure in the chamber was set at 2 × 10−2 mbar. A deposition time of 20 min was used and the precursor flow was sustained for a further 10 min in order to minimize uptake of atmospheric gases onto radical sites created on the freshly prepared surfaces. Samples were stored in sealed containers for at least 3 days prior to use. X-ray Photoelectron Spectroscopy (XPS). A SPECS SAGE XPS system was used to obtain XP spectra with an Mg Kα radiation source operating at 10 kV and 20 mA. The system included a Phoibos 150 hemispherical analyzer, with an MCD-9 detector. Survey spectra were recorded between 0 and 1000 eV at a pass energy of 100 eV with energy steps of 0.5 eV to determine the elements present on the surface of the plasma polymer films. High-resolution spectra were then recorded for selected peaks using 0.1 eV energy steps at a pass energy of 20 eV. All spectra were corrected for charging effects by setting the aliphatic carbon peak to 285 eV.33 Processing and component fitting of the spectra were performed using CasaXPS (Neal Fairley, U.K.). The C 1s core level spectra of the acidic plasma polymers were peak fitted using 70% Lorentian/30% Gaussian peak shapes with full-width at half-maxima (fwhm) between 1.6 and 1.9. The spectra were corrected for sample charging by setting the hydrocarbon signal to 285 eV and functionalities were fitted based on previously published values:34 alcohol/ether (C−OH/R) at a shift of +1.5 eV; carbonyl (CO) at a shift of +3 eV; carboxylic acid/ester (COOH/R) at +4 eV; and β shifted carbon bonded to carboxylate (C−COOH/R) at +0.7 eV. An example of a peak fitted C 1s is shown in Figure S3 with the corresponding functionalities detailed in Table S1. 4-(Trifluoromethyl)benzaldehyde 98% (TFBA) was sourced from Sigma-Aldrich, Australia, and was used to label primary amines on the surface of the plasma polymer films using the quantitative-elementalanalysis (QEA) method.35 Samples were attached to microscope slides (25 mm × 100 mm), which were then placed in 50 mL centrifuge tubes. A volume of 0.5 mL of TFBA was added to the centrifuge tube, which was then sealed and placed in an oven at 45 °C for 3 h. Samples were immediately analyzed by XPS. By comparing the atomic ratios of TFBA treated (d) and non TFBA treated (u) samples, it was possible to calculate the density of primary amines. Cell Culture. MAPC were obtained from ReGenesys who isolated the cells from bone marrow and cultured under conditions previously described.36 Cryopreserved MAPC cells were thawed and seeded on a primed and fibronectin-coated hollow-fiber cartridge of the Quantum cell expansion system (TerumoBCT, Lakewood). Cell were allowed to expand for 6 days and harvested into a harvest bag using trypsin/ EDTA (Lonza, Verviers). Cells were washed, counted, and cryopreserved in complete medium with 10% DMSO and 10% FBS and thawed immediately prior to seeding onto candidate surfaces. Cells were seeded onto patches at densities of 2.5 × 104 cells/cm2. Cell seeding was constrained within a 10 mm (internal diameter) cell seeding ring and to attain this density, 200 μL of a 105 cells/ml suspension was used. After 24 h in culture, the seeding ring was removed and the cell-laden patch was ready for use. Cells were imaged live using phase contrast microscopy and after fixation using fluorescent microscopy with actin/nuclei staining. This consisted of fixing the cell monolayer in 4% formaldehyde for 10 min, washing with PBS, permeabilizing with 0.2% triton X-100 for 5 min, blocking with 0.5% albumin (from bovine serum) for 30 min, incubation with a mixture of Phalloidin conjugated with Oregon green and DAPI diacetate (both from Life Technologies, Australia) for 45 min, followed by PBS washing. Samples were then mounted using an

EXPERIMENTAL SECTION

Plasma Polymerization. The backing substrate was 0.012 in. thick silicone with a durometer rating of 50 (shore A), obtained from Polymer Systems Technology Ltd. (U.K.). Plasma polymer precursors acrylic acid (AA), propanoic acid (PA), heptylamine (HA), diaminopropane (DAP), and 1,7-octadiene were obtained from B

DOI: 10.1021/acsami.6b14725 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces aqueous mountant and stored in darkness prior to imaging on a Nikon A1R confocal microscope running NIS-Elements AR. mRNA Isolation and Assessment. mRNA and protein was extracted from monolayers of MAPC and donor matched mesenchymal stem cells (MSC) using AllPrep Protein/RNA Isolation Kit according to manufacturer’s protocol (QIAGEN GmBH, Hilden Germany), quantified using the NanoDrop Lite Spectrophotometer (ThermoScientific, Wilmington) and expression of differentiation markers AGT, VCAM1, ANGPTL2, CXCL5, INSC, PTGS1, and ANGPTL4 assessed by qRT-PCR. mRNA expression was quantified as relative normalized expression against ATP5B following iScript cDNA synthesis (BioRad, California) and qPCR on the BioRad CFX Connect using SsoAdvanced SYBR Green Universal Supermix synthesis (BioRad, California). Angiogenic Potency. Stem cells can promote or enhance angiogenesis and neo-vascularization through the secretion of proangiogenic factors.37 Angiogenic potency has been described as a method to test the functional potential of MAPC cells and methods are described fully by Lehman et al. (2012).38 Briefly, growth factorreduced Matrigel (Corning, Lasne, Belgium) was thawed on ice at 4 °C overnight and diluted on ice using ice-cold PBS. Matrigel was distributed into the inner wells of a 24-well tissue culture plate and allowed to solidify for 1 h at 37 °C. The addition of Matrigel was done on ice, and outer wells of the plate were filled with 1 mL of PBS. HUVECs were washed with PBS followed by a brief rinse with 0.25% trypsin/EDTA and then recovered in endothelial cell basal media (EBM) and spun down. Cells were resuspended in EBM and counted by a hemocytometer. HUVECs were added to the MAPC conditioned media at a density of 5.5 × 104 cells/mL/well. Each sample and control was assayed in triplicate. Plates were incubated 18 h at 5% CO 2 and 37 °C to allow for tube formation. Four fields per well were analyzed for a total of 12 fields. Images were taken using a 10× objective. Angiogenesis was scored by counting the number of tubes. Results are expressed as average tubes formed per field ± SEM. T-Cell Inhibition. MAPC are immunoprivledged and demonstrate immunosuppressive qualities on the proliferation of activated T-cell populations.39 This property allows for a quantifiable method to assess immunomodulatory capacity. The assay used to test this directly measures the inhibition of T-cell proliferation in a co-culture model of MAPC and T-cells activated with CD3/CD28 as reported by Jacobs et al. (2012).40 The assay uses CellTrace CFSE (Life Technologies, Gent, Belgium), a well-retained cell-tracing reagent which passively diffused into cells. Every decrease in CFSE signal represents a cell division that can be visualized by flow cytometry. Briefly, MAPC were cultured on amine surfaces and fibronectin coated tissue culture plastic (5.3 cell culture). Peripheral blood mononuclear cells (PBMC) were isolated using gradient centrifugation and frozen down. PBMC were thawed and labeled with 0.2 μM CFSE dye. These labeled PBMC (1 × 105 cells/well) were cocultured with MAPC in a dilution series (1:1− 1:16 MAPC/PBMC) in a round-bottom 96-well plate. PBMC were activated using DynaBeads Human T-cell Activator CD3/CD28 (Life Technologies, Gent, Belgium) in a 1:125 DynaBead/PBMC concentration. Plates were incubated for 6 days at 37 °C, 5% CO2. Cells were harvested (each dilution series performed in triplicate), stained for CD3 and analyzed using flow cytometry. Inhibition was compared with the proliferation of activated PBMC without addition of MAPC. Delivery of MAPC in Vitro. De-epidermized acellular human dermis (DED) was obtained from the European skin bank and prepared to preserve membrane proteins. The glycerol preservative was washed away with multiple PBS washes and the DED was incubated within a 1 M solution of sodium chloride at 37 °C. The epidermis was removed and the dermis was rehydrated and sterility tested for 48 h in antibiotic free cell culture medium at 37 °C. The DED was cut into 15 × 15 mm squares and placed into 6-well plate wells with the papillary side facing upward. Cell-laden patches were carefully placed in contact with the DED and weighted in place to maintain intimate contact. Complete cell culture medium was added and the transfer models were incubated for 24 h, after which the surfaces were separated from the DED and metabolically active cells

were assessed on both the DED and the patch surface. MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Life Technologies, Australia) was used at a concentration of 0.5 mg/mL in PBS and allowed to incubate on the samples for 120 min. The endpoint consisted of imaging and elution/quantification of the stain using 1 mL of acidified isopropanol and measuring the optical density at 540 nm. This procedure was repeated for each PP surface. Delivery of MAPC in Vivo. All experiments were approved by the Women’s and Children’s Health Network Animal Ethics Committee (974/6/2017) following the Australian Code of Practice for the Care and the Use of Animals for Scientific Purposes. Studies were performed using mice with a BALB/c background. Anesthesia was induced by inhalation of isoflurane and oxygen (5% induction at 2 L/ min and 2% maintenance at 500 mL/min). To expose the skin, hair was removed by shaving and then application of hair removal cream (Veet, Reckitt Benckiser, NSW, Australia). Two 6 mm full thickness wounds, one on each side of the midline were created on the dorsum of the mouse using a 6 mm biopsy punch (Stiefel Laboratories, NSW, Australia). MAPC-laden patches at a density of 2.5 × 104 cells/cm2 were applied and held in place with Tegaderm (3M, NSW, Australia) which adhered to the bare skin. Temgesic (buprenorphine 0.05 mg/ kg) was administered intraoperatively to provide analgesia. Patches were removed at day 3, and the mice were euthanized using CO2 asphyxiation (1 L/min) and cervical dislocation. Histology. Histological sections were prepared from wound tissue fixed in 10% buffered formalin and embedded in paraffin. An antibody to human nuclear antigen (ab191181; Abcam, Victoria, Australia) was directly labeled with Dylight 633 probe following manufacturer’s instructions (ThermoFisher, SA, Australia). The 5 μm sections were stained via immunohistochemistry with the labeled human nuclear antigen, at a dilution of 1:20 in phosphate buffered saline, after blocking with 2% fetal bovine serum. Sections were counterstained with DAPI (4′,6-diamidino-2-phenylindole) (1:5000). Control slides were stained with appropriate IgG antibody in isolation and were negative for staining. Images were captured using an Olympus inverted microscope (IX83).

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RESULTS Plasma Deposition and XPS Analysis. In order to obtain plasma polymer surfaces with different densities of functional groups, plasma deposition for the 4 precursors was conducted over a range of input rf powers from 4−20 W. The resulting surfaces were then analyzed by XPS to measure the surface chemistry. The oxygen to carbon ratio of propanoic acid (PA) and acrylic acid (AA) plasma polymers (PPs) varied as a function of applied power; specifically, increased radio frequency (rf) power to the plasma resulted in surfaces with a lower surface elemental concentration of oxygen (see Table 1). Expressed as a ratio of O/C, this trend has previously been reported by O’Toole et al.41 and furthermore the absolute values are relatively consistent with the previous literature. For example, Table 1. Summary of XPS Data for Surfaces Generated from Propanoic Acid (PA) and Acrylic Acid (AA) Precursorsa precursor

rf power (W)

C 1s %

O 1s %

O/C

COOH/R

PA

5 10 15 20

76.9 79.0 79.6 80.0

23.1 21.0 20.4 20.0

0.30 0.27 0.26 0.25

10.8 ± 1.8 6.8 ± 0.7 6.7 ± 0.7 7.7 ± 0.8

AA

5 20

73.5 76.1

26.5 23.9

0.36 0.31

19.2 ± 1.9 13.1 ± 1.3

a Results determined using atomic % and peak fitting. Standard error in elemental analysis is 5% and curve fitting is 10%.

C

DOI: 10.1021/acsami.6b14725 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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°C increased the rate of silicon increase (Figure S5). This indicates that within the experimental window, the amine surfaces remained stable under storage conditions. Thus, candidate patches with a range of functionalities and surface densities was produced for subsequent cell attachment and delivery studies. MAPC Attachment. MAPC cells cultured on candidate patches all exhibited typical morphology comparable to that of MAPC cultured under standard conditions on fibronectin (Figure 1). Following seeding, cells attached to surfaces and elongated to over 50 μm within 5 h. In culture, lamellipodia extended and the cells began to align at higher densities. MAPC cultured on octadiene-coated surfaces were used as a negative control. OD was a good example of an unfavorable surface and this was evidenced by the cells clustering together (Figure 1j) or not attaching at all. Cells seeded on OD surfaces were easily detached from the surface by washing. The metabolic activity of the MAPC on the acid and amine based plasma polymer surfaces was determined using the MTTESTA assay after 48 h of culture (Figure 2). This was compared and normalized to a positive control of MAPC cultured on fibronectin-coated tissue culture plastic. Washing ensured that only attached cells were assessed. All of the PP surfaces (both acid and amine) supported MAPC metabolic levels similar to that of normal culture conditions (Figure 2). Minimal levels of metabolic activity were detected on OD surfaces. MAPC Transfer. MAPC transfer from the patches was tested by applying the cell-laden patch to a piece of acellular human deepidermised dermis (DED). This well reported technique is an effective method for estimating cell transfer into wounds.41 All the patches tested freely detached from the DED following cell transfer, any ripping or adherence of the patch would be highly unfavorable in use in clinical applications. Minimal levels of (cell) metabolic activity were seen on the patches following detachment from the DED and high levels of metabolic activity on the DED. This was taken as a first measure of efficient cell transfer. All of the plasma polymer surfaces capable of MAPC culture were capable of some degree of MAPC delivery but to varying extents. For both acid precursors, the acid-based plasma polymers deposited using lower rf powers permitted high cell transfer (up to 80%) and transfer from the acrylic acid PP was less dependent on rf power than the propanoic acid PP (Figure 3). A proportional relationship between cell transfer and surface chemistry could be postulated. As the O/C ratio increases, so does cell transfer. Heptylamine-based plasma polymer surfaces most effectively delivered MAPC to the wound models, delivering between 80 and 100% of cells. Lower power heptylamine plasma polymers appeared more effective at MAPC delivery but since the surface N/C ratio of the plasma polymer was largely independent of rf power, it was difficult to produce surfaces over a range of N/C to allow for a detailed correlation of cell transfer to surface atomic ratios (Table 2). It was therefore not possible to readily correlate XPS atomic ratios with cell transfer, as it was in the case of the acid based surfaces. It was postulated that the availability of primary amine on the surface may have had an important influence, and consequently this led us to investigate the use of a tagging method (4-(Trifluoromethyl)benzaldehyde) to quantify the surface densities of primary amine. This tagging, shown in Figure 4, confirms that a specific range of primary amine (as generated from heptylamine) were the most effective for the delivery of cells.

plasma deposited PA with 10 W rf power was reported to have an O/C ratio of 0.2841 and herein we have measured it at 0.27. Assuming all oxygen originates from the precursor, in the acid plasma polymers, there was 37−54% oxygen retained based upon the original stoichiometric ratio (O/C) of the precursors of 0.67. The remainder of the oxygen appears to have been lost during the deposition process. Higher levels of carboxylic acid incorporation lead to less stable plasma polymer surfaces in aqueous media.42 The stability of these surfaces was therefore assessed over a period of time using XPS, under both wet and dry conditions. In plasma polymers from each acid precursor, higher levels of carboxylic acid incorporation resulted (upon storage) in higher amounts of silicone detection at day 12 (Figure S4). It was postulated that this silicone had penetrated through from the underlying silicone substrate. This result indicates a lower degree of cross-linking in the PP with more COOH/R. Peak fitting the amine plasma polymer C 1s envelope does not discern between primary and secondary amines.43 This is why primary amines were tagged and quantified using the 4trifluoromethylbenzaldehyde (TFBA) quantitative-elementalanalysis method.36 Both heptylamine (HA) and diaminopropane (DAP) PPs had an N/C ratio that followed the stoichiometric N/C of the precursor but with approximately half of the nitrogen lost during the deposition process (Table 2). Loss of nitrogen is in Table 2. Summary of XPS Data for Surfaces Generated Using Heptylamine (HA) and Diaminopropane (DAP) Precursorsa

a

monomer

rf power (W)

N/C

O/C

NH2/C

HA (N/C 0.14)

5 8 12 16 20

0.05 0.06 0.06 0.07 0.07

0.20 0.14 0.15 0.06 0.12

0.0137 0.0137 0.0098 0.0096 0.0097

± ± ± ± ±

0.002 0.002 0.001 0.001 0.001

DAP (N/C 0.67)

4 8 10 12 15 20

0.32 0.24 0.26 0.31 0.31 0.29

0.11 0.18 0.15 0.10 0.08 0.10

0.0500 0.0251 0.0262 0.0326 0.0277 0.0251

± ± ± ± ± ±

0.005 0.003 0.003 0.003 0.003 0.003

Results determined using atomic % and TFBA tagging.

agreement with previous studies.44−46 It has been suggested that during plasma polymerization, the cleavage of the C−N bond leads to the formation of an •NH2 aminyl radical leading to a pressure increase within the reaction vessel.44 This pressure was observed to increase with increasing rf power (Table S2). TFBA labeling showed that increased rf power also corresponded with a reduction in available primary amine on the HA PP surface (Table 2). Oxygen present in the HA and DAP plasma polymer surfaces is thought to arise from a combination of incorporation of atmospheric oxygen (from exposure to the laboratory atmosphere47) and components migrating from the silicone backing membrane. The relative mobility of the underlying silicone polymer chains, present in the substrate, caused the levels of oxygen and silicon to increase as the surface aged over 12 days. The rate of change under dry conditions was minimal over the storage period and storage under wet conditions at 37 D

DOI: 10.1021/acsami.6b14725 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Figure 1. MAPC morphology imaged using fluorescence microscopy; actin is labeled in green and nuclei in blue. MAPC cultured on candidate surfaces (a−h) have a similar morphology and distribution to the positive fibronectin control (i). Octadiene was used as a negative control, and this shows an unfavorable surface for MAPC culture (j). Scale bars are all 100 μm.

Figure 3. Transfer of MAPC onto DED from surfaces generated with plasma polymers of propanoic acid and acrylic acid. Labels indicate rf power of the PP. n = 3 error bars show standard deviation.

Figure 2. Cell viability of MAPC cells cultured on plasma polymer surfaces for 48 h and normalized to MAPC cultured on fibronectin treated plates (100%). n = 3, error bars show standard deviation.

MAPC Phenotype and Function. A panel of PCR markers was used to monitor changes in the phenotype of MAPC and donor matched MSCs upon culture on ppHA surfaces and control surfaces. MAPC cells cultured on fibronectin-coated tissue culture plastic (positive control) and MAPC cells cultured on an amine based patch within the range 0.01 to 0.02 (NH2/C) were compared. Cells were cultured for 48 h, the maximum time that the cells would be in contact with the patch for anticipated in vivo applications. The results show that MAPC cells cultured on the PP surface had down

This dependence on primary amine has not been previously reported, and we showed that for optimal (near 100%) transfer, the amounts of primary amine available on the heptylamine PPs must be confined to a narrow range from 0.01 to 0.02 (NH2/ C) (Figure 4). Consequently, diaminopropane PP surfaces with their higher levels of primary amine (0.025−0.035 NH2/C) were less effective than heptylamine at delivering cells, with delivery falling below 60%. E

DOI: 10.1021/acsami.6b14725 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Figure 6. Angiogenic potency of MAPC based on their ability to induce HUVEC branching. MAPC on the amine patch had the same angiogenic potential as the MAPC control (*** p < 0.001). Error bars show standard deviation.

Figure 4. Transfer of MAPC onto DED from PP surfaces generated with octadiene (Δ), heptylamine (×), and diaminopropane (◇).

regulation of ANGPTL2, VCAM1, and AGT and upregulation of ANGPTL4, PTGS1, INSC, and CXCL5 (Figure 5). Angiogenic potency of MAPC cells cultured on an amine based patch for 24 h were no different from MAPC cells cultured on fibronectin-coated tissue culture plastic (positive control) (p < 0.001). Since 24 h is the maximum attachment time before application of cell-laden patches in vivo, then it was demonstrated that at the theoretical point of application in vivo, the angiogenic potency of MAPC is at its full potential (Figure 6). MAPC populations cultured on the amine based patch and on fibronectin-coated tissue culture plastic were both able to retain immunogenic potency when assessed with a T cell inhibition assay. Activation of T cell proliferation was inhibited with cells cultured on the amine patch as well as fibronectin coated plastic (42% and 39%, respectively, at a 1:1 MAPC/ PBMC ratio) (Figure 7). Delivery of MAPC in Vivo. Surfaces generated using 5 W heptylamine PPs (an amine patch within an optimized 0.01 to 0.02 NH2/C range) were used to transfer MAPC into wound models in mice. These models consisted of dorsal excisional biopsies. MAPC laden patches (and controls with no cells) were applied to the wounds and kept in place for 3 days. Immunohistochemical (IHC) staining for the presence of

Figure 7. Inhibition of T-cell proliferation from MAPC cultured on amine patch (a) and control MAPC (b). Assay internal controls are shown: UA, unactivated control; AC, activated control. Error bars show standard deviation.

human nuclear antigen in mouse tissue clearly shows the presence of MAPC in the wounds at day 3 and to a lesser

Figure 5. RT-PCR data for MAPC compared with donor-matched MDCs. MSCs expressed ANGPTL2, VCAM1, and AGT (a) while MAPC and MAPC cultured on the heptylamine based patch express higher levels of ANGPTL4, PTGS1, INSC, and CXCL5 (b). F

DOI: 10.1021/acsami.6b14725 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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obtained with amine plasma polymers. The amine surfaces fabricated from a heptylamine precursor were the most effective for MAPC delivery. This surface consistently delivered >95% of the cells seeded yet the amine plasma polymers exhibited a high level of stability. To begin to unravel what drives cell delivery from surfaces, it is important to consider the mechanisms at play. It is strongly indicated that the cells do not interact directly with surface chemistry but in fact with an interfacial layer of adsorbed serum protein.51 To this end we should consider how serum proteins may react with the surface chemistry. The properties of a surface can be defined by surface charge and chemical functionalities.52 These factors are thought to affect the concentration, composition, and conformation of adsorbed proteins on the surface and it is these surface bound proteins to which cells are able to respond.53,54 While characterization of protein adsorption is beyond the scope of this study (and will be the focus of future work), we were able to measure the zeta potential of the amine surfaces Figure S7 which is indicative of surface charge. Measured at pH 5.6 Figure S7a there was a trend in zeta potential correlating with the amount of primary amine measured on the heptylamine-based surfaces fabricated at different rf powers. This supports our assertion that primary amines are present. By measuring the zeta potential across a range of pH values Figure S7b, the isoelectric point was shown to be close to physiological pH (the pH of cell culture conditions). This indicated that while the overall charge may be close to neutral, there is certainly a mixture of positively and negatively charged species with primary amines being one of these components. This combination of charges may favor the adsorption of a protein later conducive with cell delivery.55 While the delivery of viable cells was paramount, it was important that the vehicle/surface did not adversely affect the cells during the process of cell seeding, culture, or delivery. Within our study time (48 h), we did not see any negative effects on metabolic activity, and the expression of mRNA key to MAPC function was unaffected. For an effective MAPCwound treatment it would be important that the dressing would be applied to a wound and shortly thereafter (1−3 days), removed. A relevant in vivo wound model confirmed that the MAPC were delivered successfully in this relatively short time period. Amine-based PP surfaces for cell culture/delivery are less well reported than acid-based ones, this may be due to the difficulties in assessing the amine surface chemistry.35 Amine based surfaces have however been reported as good surfaces for the culture of cells, generally linking cell performance with surface biomolecule adhesion.56 An amine surface with an NH2/C ratio of 0.014 was determined to be effective for both the culture and complete delivery of MAPC cells into wound models. While effective plasma polymer surfaces have previously been described for the culture and delivery of mesenchymal stem cells,56 MAPC cells are a distinctly different cell population that are antigenically distinct. This led to a screening approach highlighting the importance of surfaces tailored for cell types.

extent at day 7 (Figure 8). This demonstrates that the surface had the ability to deliver MAPC to wounds in vivo. It also eludes to the anticipated clearance of MAPC over time.

Figure 8. Human nuclear antigen staining of murine wounds treated with MAPC cell therapy (a and b) and not treated (c and d) at day 3 (a and c) and day 7 (b and d). Red = human nuclear antibody, blue = DAPI. Scale bars indicate 20 μm (a and b) and 100 μm (c and d).

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DISCUSSION A selection of surface coatings were identified that would permit the attachment and subsequent detachment (delivery) of MAPC into a wound environment. Furthermore, the amount of surface primary amine was correlated with the effectiveness of cell delivery to identify optimum surfaces. The premise of cell delivery was to generate surfaces able to achieve the duality of cell attachment and cell release. Consequently, it was important that the surfaces fabricated by plasma polymerization encouraged cell attachment but that this attachment was not so effective that cells would not leave and migrate into wound sites. A balance between adherence and nonadherence was achieved and it was shown that this surface facilitated MAPC culture and maintenance of phenotype. Acid-based surfaces were previously explored for cell sheet delivery but for MAPC the acid-based surfaces were not able to deliver greater than 80% of the seeded cells. The goal was to deliver as close to 100% as possible. Reduced levels of carboxylic acid within PP films have been associated with greater film stability (i.e., greater levels of film cross-linking).42 While this seems an essential prerequisite for a medical device/ cell transfer product, it has been clearly demonstrated that higher levels of carboxylic acid (20%) promote more effective keratinocyte delivery than low (acid) levels.42,48 Furthermore, in line with our premise, high levels of carboxylic acid have been shown to have an antifouling effect and prevent adhesion of fibroblasts.49 It is, therefore, not entirely clear whether better delivery manifests from a slightly less stable surface or less well attached cells (on a less adhesive surface). Our experiments performed with MAPC cells support the trends reported within the existing literature; i.e., high surface carboxylic acid surfaces favor cell delivery. With surface carboxylic acid group density ranging from 6% to 19%, we were able to show an increase in cell delivery. Relevant literature points to cell detachment from plasma surfaces arising from a combination of reduced cell adhesion (less adhesive surfaces) and lower intrinsic surface stability/ cross-linking,50 but this assumption is challenged by the results

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CONCLUSIONS The results herein foreshadow a simple delivery vehicle (surgical dressing with a plasma polymer coating) to deliver dense populations of MAPC to cutaneous wounds, without having to enzymatically detach these cell sheets from a culture vessel. This vehicle could be an important enabler of cell therapy in (real) clinical applications. G

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ACS Applied Materials & Interfaces

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By detailing effective cell delivery from relatively stable amine-based surfaces, we have raised questions regarding the underlying assumptions of how cells are delivered from plasma polymer surfaces. We correlate delivery with an optimal surface primary amine density and while we were able to define an effective surface for the delivery of MAPC, there are still questions regarding mode of action and future research will focus in two very important directions: delivering MAPC to nonhealing wounds and exploring protein−surface interactions on these surfaces.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b14725. Table S1, assignment of anticipated chemical functionalities to binding energies (BE) within C 1s; Table S2, pressure in reaction vessel during plasma polymerization of heptylamine; Figure S3, peak fitted C 1s core levels of PP of acrylic acid (5 W); Figure S4, XPS spectra of amine PP surfaces formed with heptylamine at 5 W detailing Si 2p changes related to aging; Figure S5, XPS spectra of acid PP surfaces fromed with acrylic acid detailing Si 2p changes related to aging; eq S6, methods and equation used to convert measured streaming potential to zeta potential; Figure S7, (a) zeta potential of candidate heptylamine surfaces at pH 5.6 and (b) zeta potential of surfaces generated using heptylamine 5 W plasma polymer (PDF)

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Research Article

ABBREVIATIONS AA, acrylic acid CEA, cultured epithelial autograft DAP, diaminopropane DED, deepidermized acellular human dermis DM, diabetes mellitus EBM, endothelial cell basal media fwhm, full width half-maximum HA, heptylamine MAPC, multipotent adult progenitor cell MSC, mesenchymal stem cell MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide PA, propanoic acid PBMC, peripheral blood mononuclear cell PP, plasma polymer(ization) QEA, quantitative elemental analysis rf, radio frequency TFBA, 4-(trifluoromethyl)benzaldehyde XPS, X-ray photoelectron spectroscopy REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: +61 8 8302 2466. Phone: +61 8 8302 5500. ORCID

Giles T. S. Kirby: 0000-0003-4557-9936 Andrew Michelmore: 0000-0003-3215-8841 Louise E. Smith: 0000-0003-0659-0884 Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare the following competing financial interest(s): A. Ting is an employee of Athersys, L. Vandenpoel and J. Pinxteren are employees of ReGenesys, a subsidiary of Athersys. The remaining authors declare no conflict of interest.

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ACKNOWLEDGMENTS The authors would also like to acknowledge the support of the Cooperative Research Centre for Cell Therapy Manufacturing and the Australian Government’s Cooperative Research Centres Program. This work was performed in part at the South Australian node of the Australian National Fabrication Facility, a company established under the National Collaborative Research Infrastructure Strategy to provide nano- and microfabrication facilities for Australia’s researchers. The authors would also like to thank Dr. Marta Krasowska, of the Future Industries Institute, for training with the Zeta Spin. H

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