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Differential regulation of extracellular matrix components by using different vitamin C derivatives in mono- and co-culture systems Xiaoqing Zhang, Kyle Battiston, Craig A. Simmons, and J. Paul Santerre ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00389 • Publication Date (Web): 18 Oct 2017 Downloaded from http://pubs.acs.org on October 19, 2017

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ACS Biomaterials Science & Engineering

Differential regulation of extracellular matrix components by using different vitamin C derivatives in mono- and co-culture systems Xiaoqing Zhang1, Kyle G. Battiston1, Craig A. Simmons1,2,3, J. Paul Santerre1,3* 1

Translational Biology and Engineering Program, Ted Rogers Centre for Heart Research,

Institute of Biomaterials and Biomedical Engineering, University of Toronto, 661 University Avenue, 14th floor, Toronto, Ontario, Canada M5G 1M1 2

Department of Mechanical and Industrial Engineering, University of Toronto, 5 King's College

Road, Toronto, Ontario, Canada M5S 3G8 3

Faculty of Dentistry, University of Toronto, 124 Edward Street, Toronto, Ontario, Canada M5G

1G6 * Corresponding author: Dr. J. Paul Santerre, Ted Rogers Centre for Heart Research, Institute of Biomaterials and Biomedical Engineering, Faculty of Dentistry, University of Toronto, 661 University Avenue, 14th floor, room 1435, Toronto, ON, Canada M5G 1M1. Phone: 416-946-8158, Fax: 416-979-4760, e-mail: [email protected]

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Abstract Vascular tissue engineering strategies using cell-seeded scaffolds require uniformly distributed vascular cells and sufficient extracellular matrix (ECM) production. However, acquiring sufficient ECM deposition into synthetic biomaterial scaffolds during the in vitro culture period prior to tissue implantation still remains challenging for vascular constructs. Two forms of vitamin C derivatives, ascorbic acid (AA) and sodium ascorbate (SA), are commonly supplemented in cell culture to promote ECM accumulation. However, the literature often refers to AA and SA interchangeably and their differential effects on cell growth and ECM molecule (glycosaminoglycan, collagen, elastin) accumulation have never been reported when used in mono-culture or co-culture systems developed with synthetic three dimensional (3-D) scaffolds. In this study, it was found that 200 µM AA stimulated an increase in cell number while SA (50, 100 and 200 µM) supported more calponin expression (immunostaining) and higher ECM accumulation from vascular smooth muscle cells (VSMCs) after 1 week in the degradable polar hydrophobic ionic polyurethane (D-PHI) scaffold. The influence of AA and SA on ECM deposition was also studied in VSMC-monocyte co-cultures in order to replicate some aspects of a wound healing environment in vitro, and compared to their effects in respective VSMC monocultures after 4 weeks. While 100 µM SA promoted ECM deposition in co-culture, the condition of 100 µM AA + 100 µM SA was more effective towards enhancing ECM accumulation in VSMC mono-culture after 4 weeks. The results demonstrated that AA and SA are not interchangeable and the different effects of AA and/or SA on ECM deposition was both culture system (co-culture vs. mono-culture) and culture period (1 week vs. 4 week) dependent. This study provides further insight into practical vascular tissue engineering strategies when using 3D synthetic biomaterial-based constructs.

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Keywords Tissue engineering, Biodegradable polyurethane, Ascorbic acid, Sodium ascorbate, Vascular smooth muscle cells, Monocytes

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1. Introduction Tissue engineering strategies using cell-seeded scaffolds1 have shown great promise towards the regeneration of functional vascular tissues for the replacement of diseased blood vessels in vivo.2 Prerequisites for functional vascular tissue regeneration include high densities of vascular cells, including vascular smooth muscle cells (VSMCs) distributed uniformly throughout the scaffold, and deposition of sufficient amounts of extracellular matrix (ECM, mainly composed of collagen, elastin and glycosaminoglycans (GAG)) for structural support and to direct cell signalling.3,4 However, it still remains challenging to achieve sufficient ECM accumulation within biomaterial scaffolds for developing functional vascular prostheses prior to implantation.3,5 Strategies to promote ECM accumulation such as growth factor supplementation,6 complex bioreactor systems to provide stimulus7 and retroviral transduction to up-regulate specific ECM gene expression8 have been used with some success. However, some limitations such as potential endotoxin contamination,9 increased risks of culture contamination with more handling steps,10 possible insertional mutagenesis and increased costs9,11 have hindered these approaches. Alternatively, the vitamin C derivatives ascorbic acid (AA)12 and sodium ascorbate (SA)13 have been used as cell culture medium supplements to promote ECM production.14 AA is well known to augment collagen generation within in vitro culture systems,14-16 and SA has been reported to stimulate both elastin and collagen production in human dermal fibroblasts.13 Although AA and SA have similar chemical structures, they have been reported by some groups to have different stability in cell culture medium.13,17,18 AA is highly unstable and can be quickly oxidized to inactive dehydroascorbic acid.17 In comparison with AA, SA can preserve its intact sodium ascorbate form and be transported at a faster rate into the cells through the sodium 1 ACS Paragon Plus Environment

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dependent vitamin C transporters (SVCTs) after they are activated by sodium.13,18 As a result, the faster translocation of SA into the cells (in comparison with AA) would also help reduce the oxidation/inactivation of the ascorbate in the culture medium.13,18 In addition, there are a few studies that have compared AA and SA in fibroblast culture.13,19 It was found that SA (in mM concentration range) was more effective at reducing cell proliferation,19 and SA promoted both collagen and elastin accumulation in fibroblasts while AA up-regulated collagen but not elastin.13 However, another study has reported that AA can induce elastin production in 3-D fibroblast culture, but not 2-D culture.20 Although AA and SA could have different potential to enhance the accumulation of ECM components, they have been often used interchangeably in the literature.20-23 For example, some scientists have referred to "AA" as "ascorbate"20,24,25 and "SA" as "ascorbic acid"26,27 in their papers, which implies that researchers do not fully realize the potential the potential differential effects of AA and SA in their culture systems (e.g. studies assume that AA and SA are identical without further exploration). There do not appear to be any controlled studies that have investigated and compared the effects of both vitamin C derivatives alone or in combination on the deposition of collagen, elastin and GAG within a three dimensional (3-D) biomaterial scaffold (which is more tissue-engineering relevant compared to 2-D) seeded with identical cell sources. Knowledge on the use of AA or SA alone, or combination of both to enhance ECM protein accumulation within a 3-D biomaterial scaffold would be important in the development of vascular constructs and other types of tissues. Therefore, it is desired to systematically compare AA and SA (at different dosages) to determine which of the two (or their combination) may be more effective to enhance ECM accumulation in the same VSMC culture system. This study has specifically focused on VSMCs rather than other vascular cell types (e.g. endothelial cells, fibroblasts) because VSMCs are the predominant cell type that constitute the medial layer of the blood vessel (important for blood pressure regulation, 2 ACS Paragon Plus Environment


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maintenance of vascular homeostasis),28,29 and tissue-engineering mature vascular media has been a great challenge for the biomaterials and tissue regeneration field and a major objective that the lab has striven to achieve in recent years.10,30 Since AA and SA have different stability and transportation kinetics,13,17,18 and they were found to have different effects in fibroblast culture,13,19 it has been hypothesized that there are different effects of AA and SA in VSMCs that would be cultured in 3-D polyurethane biomaterial-based tissue engineering system. Specifically, it has been hypothesized that SA would be more effective at promoting ECM (GAG, collagen and elastin) accumulation and specific VSMC marker expression than AA (due to SA’s faster translocation into the cell interior and reduced oxidation). AA and SA supplementation has also not been explored in the context of co-culture systems, despite the recognized importance of co-culture systems in the vascular tissue engineering field.31 Blood derived monocytes are innate immune cells that play important roles in recruiting tissue-specific cells and guiding wound healing and regeneration of different types of tissues inside the body, including the vascular tissues.32,33 Therefore, there has been a great interest to develop co-culture systems that contain monocytes to more closely mimic the in vivo environment during regeneration and to harness this powerful endogenous cell source for tissue engineering purposes.34,35 In recent years, a degradable polar hydrophobic ionic polyurethane (DPHI) has been shown to support a wound healing phenotype and anti-inflammatory cytokinerelease profile from monocytes, when compared to poly (lactic-co-glycolic acid) (PLGA).36,37 The superior immunomodulatory character of D-PHI in comparison to PLGA is related to the observation that D-PHI does not generate monomeric acid by-products such as lactic acid, which have been shown to contribute to pro-inflammatory responses.38 It was found that monocytes which were co-cultured with VSMCs within a D-PHI scaffold not only showed an anti3 ACS Paragon Plus Environment

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inflammatory phenotype but also improved the distribution of co-cultivated VSMCs into the scaffolds35. Vitamin C derivatives, specifically AA, can inhibit the generation of reactive oxygen species (ROS), regulate the cytokine release profile (e.g. decrease the prolonged release of proinflammatory cytokines IL-6 and TNF-α),39 and protect cells against FAS-mediated apoptosis in monocyte culture.40 Although it seems that vitamin C derivatives could play an important role in co-cultures containing monocytes that are developed for vascular tissue engineering applications, whether supplementation of different vitamin C derivatives (AA or SA) can improve the ECM accumulation within the co-culture systems remains unknown. Therefore, it is also desired to investigate the effects of AA and SA on the accumulation of different ECM components within VSMC-monocyte co-culture systems, and to determine if the effects of the vitamin C derivatives in co-culture would be identical to the effects observed in VSMC mono-culture. Given the above background and the identified needs of the field, the first part of this study focused on better understanding the influence of AA and SA on total cell number, ECM accumulation (collagen, elastin and GAG) and VSMC marker (calponin) expression in VSMC mono-culture on D-PHI, which has been conceived for vascular tissue engineering applications. In addition, the authors investigated the effects of specific AA and SA supplementation conditions on ECM accumulation within VSMC-monocyte co-culture systems. The data was compared to the effects observed in respective VSMC mono-cultures. The findings in this study not only provide significant insight into vascular tissue engineering processes, but could also be applied to the regeneration of other types of tissues using different biomaterial scaffolds, where ECM also plays an important role and its remodeling is influenced by monocytes and their derived macrophages. By investigating the potential differential effects of AA and SA in this defined 3-D culture system for VSMCs (with/without monocytes), this paper also wants to 4 ACS Paragon Plus Environment


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encourage researchers working with other specific cell culture systems to strongly consider the different vitamin C derivatives supplemented, as they might have different effects on cell proliferation and deposition of different ECM components (e.g. GAG, collagen and elastin).

2. Materials and Methods All chemicals were purchased from Sigma-Aldrich unless stated otherwise. 2.1 Fabrication of D-PHI scaffolds D-PHI scaffolds were prepared using previously published protocols.41 Briefly, a divinyl oligomer (DVO) (synthesized with hydroxyethylmethacrylate (HEMA), polyhexamethylene carbonate diol (PCN) and lysine diisocyanate (LDI)) was mixed with methacrylic acid (MAA) and methyl methacrylate (MMA) in a 1:5:15 molar ratio. Benzoyl peroxide (BPO) (0.032 mol mol−1 vinyl group) was added to initiate the polymerization reaction, and polyethylene glycol (PEG) (10 wt.%) and sodium bicarbonate (65 wt.%) were added to the chemical mixture in order to generate a dual macro/micro porous architecture within the D-PHI scaffolds.41 The resulting mixture was stirred for 20 h at room temperature (protected from light) before being packed into Teflon molds and cured in a 110ºC oven for 24 h. After removal from the curing oven, the scaffolds (1 mm thickness D-PHI sheets) were subjected to water Soxhlet extraction for 48 h in order to remove the porogens and un-reacted monomer, resulting in scaffolds with a porosity of 79 ± 3%.41 Disk-shaped scaffolds (6 mm diameter, 1 mm thickness) were cut from the D-PHI sheets with a 6 mm diameter biopsy punch and used in all of the experiments described in this study.

2.2 VSMC and VSMC-monocyte cultures on D-PHI scaffolds

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Human coronary artery smooth muscle cells (Lonza, CC-2583) were cultured in DMEM (supplemented with 10% FBS and 1% penicillin/streptomycin) before being seeded onto D-PHI scaffolds. Passage 7 cells were used in all of the experiments. Monocytes were isolated from human peripheral blood donated by healthy volunteers as described before (University of Toronto ethics approval #22203).37 Blood was collected into EDTA-containing Vacutainers (Becton Dickenson, Toronto, Canada), layered onto Histopaque 1077 and processed by centrifugation and washing to obtain the mononuclear cell fraction. Cells were suspended in RPMI-1640 medium (containing 3 g/L HEPES, 2 g/L sodium bicarbonate, 10% FBS, 1% penicillin/streptomycin, and 0.69 mM L-glutamine) and counted. Previous studies showed that monocytes represented approximately 20% of the counted cell population, with mostly monocytes remaining after performing a medium change at 2 h post-seeding, and with 95% of the cells shown to be monocytes or monocyte-derived macrophages (CD68+) after 72 h in culture, with only 3% CD3 positive T-cells.42 Prior to seeding cells onto D-PHI scaffolds, the scaffolds were placed into 24-well tissue culture polystyrene (TCPS) plates and treated with 70% ethanol for 24 h in a sterile biosafety cabinet and then immersed for another 48 h with sterile filtered DMEM (VSMCs alone) or 50:50 DMEM:RPMI-1640 medium (co-culture of VSMCs and monocytes), with medium change after 24 h incubation for complete removal of residual ethanol. At 30 min before seeding, the medium was aspirated and scaffolds were further dehydrated by removing the absorbed medium in order to maximize the subsequent cell suspension uptake into the scaffolds. VSMC mono-culture (300,000 cells) or VSMC-monocyte co-culture (at a 4:1 ratio: 300,000 VSMCs + 75,000 monocytes) cell suspensions (8.3 µl) were manually pipetted onto a D-PHI scaffold from the top side.43 The seeded scaffolds were placed into a cell culture incubator (37 °C, 5% CO2) for 30 min to allow for initial cell attachment to the biomaterial 6 ACS Paragon Plus Environment


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scaffolds before adding 1 ml of sterile filtered DMEM or 50:50 DMEM:RPMI-1640 medium to the cell culture. Different doses of AA or SA (50, 100, 200 µM, which were commonly used in previous studies and considered to be physiologically relevant44-47) were supplemented to the VSMC mono-culture for one week. The effects of AA vs. SA on GAG, collagen and elastin accumulation within VSMC-seeded D-PHI were studied after the one week culture. In a subsequent study, SA (100 µM) or a combination of AA and SA (100 µM AA + 100 µM SA) were supplemented to both VSMC mono-culture and VSMC-monocyte co-culture for 4 weeks, to investigate the effect of different vitamin C supplementation on ECM accumulation in co-culture and determine if the effects in co-culture would be identical to those in mono-culture. AA and SA were prepared fresh and added to the culture medium everyday with medium change.

2.3 Papain digestion Cell-seeded scaffolds were rinsed twice with DPBS and minced into 8-10 small pieces with a needle before being submerged in papain digestion buffer (35 mM ammonium acetate, 1 mM EDTA, 2 mM DTT, 50 µg/ml papain, pH 6.2), based on a previously published protocol.10 The samples were digested at 65°C for 48 h. To ensure a thorough digestion, the samples were vortexed and centrifuged at the 24 h incubation time point and then placed back into the 65°C water bath for the remainder of the digestion period. Digested samples were stored for further analysis at -80°C. Non-seeded D-PHI scaffolds were digested as negative controls.

2.4 DNA mass quantification Digested samples were thawed on ice and centrifuged for 5 min at 14,000g and 4°C before use for removal of potential D-PHI particulate that might interfere with the analysis. 10 µl papain digest of cell-seeded samples was added to a 96-well plate. The DNA content of each 7 ACS Paragon Plus Environment

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sample was quantified by binding with Hoechst 33258 dye, and comparing with a calf thymus DNA standard of known concentration, as previously described.41

2.5 Sulphated GAG (S-GAG) quantification Similar to the DNA quantification, papain digested samples were thawed on ice and centrifuged at 14,000g (4°C) for 5 min before being analyzed for sulphated GAG content. 20 µl aliquots of papain digestion sample solutions were transferred to a 96-well TCPS plate along with the chondroitin sulfate standard solution of known concentration. An equal volume of dimethylmethylene blue (DMMB) solution (8 mg/500 ml) was then added to all of the sample/standard wells to bind with S-GAG. The absorbance of the plate was measured at 525 nm using a plate reader immediately after the addition of DMMB.10

2.6 Hydroxyproline (OH-Pro) assay for collagen quantification The papain digested sample solutions were also used to quantify their hydroxyproline content, which accounts for about 10% of total collagen in the extracellular matrix.48 Aliquots of papain digest were mixed with equal amount of 6 N HCl in heat-resistant Pyrex glass tubes and the mixture was heated on a test-tube heater at 110°C for 18 h. The acid-digested samples were collected and neutralized with 5.7 N NaOH (same amount as the added 6 N HCl) and transferred to 1.5 ml micro-centrifuge tubes (after being cooled down for 5 min at room temperature following the 110°C incubation period), together with freshly prepared L-hydroxyproline standard solutions. Both the standards and samples were first mixed with 0.05 N chloramine-T (20 min, room temperature), and then with 3.15 N perchloric acid (5 min, room temperature), and finally Ehrlich’s reagent (incubated at 60°C for 20 min to enable color development). The

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sample/standard solutions were pipetted into a 96-well plate, and the absorbance was subsequently read at 560 nm.

2.7 Histological analysis Samples were rinsed with pH 7.4 PBS twice, before being immersed in 4% paraformaldehyde (prepared with pH 7.4 PBS) for 20 min on ice for fixation. Samples were then incubated in 15% and 30% sucrose solution (24 h each) before being processed and embedded in paraffin. Sections of 20 µm thickness were obtained for H&E and picrosirius red staining, as well as all of the immunofluorescence staining. In all of the experiments, a minimum of 3 sections for each cell-seeded D-PHI scaffold were cut and stained for each stain type to ensure that the histological images taken were representative of the complete sample.10

2.8 Immunofluorescence (IF) Paraffin-embedded 20 µm thick sections of cell-seeded D-PHI scaffolds were processed as described in previous protocols.10 Scaffold sections were immunostained for collagen I (rabbit polyclonal to collagen I, Abcam, 1:50 dilution), elastin (rabbit polyclonal to elastin, Abcam, 1:50 dilution), calponin (rabbit monoclonal to calponin, Abcam, 1:50 dilution) and α-SMA (mouse monoclonal to α-SMA, Abcam, 1:50 dilution) for VSMC mono-culture or VSMC-monocyte coculture samples. The scaffold sections were incubated with primary antibody solution (diluted with 0.3v/v% TritonX-100 in PBS solution) overnight at 4°C. The sections were then treated with 10% goat serum (Gibco, 16210064) solution containing 200 times diluted secondary antibodies (AlexaFluor® 568 goat anti-rabbit IgG or AlexaFluor® 488 goat anti-mouse IgG) for 1 hr. Cell nuclei were counter-stained with Hoechst 33342 (1:1000) and the scaffold sections on the glass slides were then mounted with PermaFluor (Thermo Scientific) and imaged with a 9 ACS Paragon Plus Environment

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fluorescence microscope (DM-IRE2, Leica Microsystems, Germany). At least three different sections were imaged for each sample to ensure that the pictures shown were representative. To quantify the IF images, the total collagen I, elastin, calponin, and α-SMA positive areas were calculated and normalized to the total scaffold area using ImageJ, as described previously.10 While both the D-PHI scaffolds and cell nuclei were stained by Hoechst 33342 (1:1000), cells could be easily distinguished from the scaffold itself by looking at the size and circularity of the stain. Cells also tended to infiltrate into the pores of the scaffolds (which appeared black in the images). Cell-seeded scaffold sections treated with only secondary antibodies served as negative controls.

2.9 Fastin elastin assay for total elastin quantification Cell-seeded scaffolds were rinsed twice with DPBS and minced into 8-10 small pieces with a needle before being immersed into 0.25 mM oxalic acid solution and incubated at 100°C for 1 h. After removal of oxalic acid solution containing solubilized elastin, the extraction process was repeated two more times with fresh oxalic acid solution. The extract from the three digestions were pooled together and analyzed using the Fastin elastin kit (Biocolor Life Science) following the manufacturer's instructions.10,49

2.10 Statistical analysis Data analysis was performed with SPSS Statistics 22.0 (SPSS Inc., Chicago, IL) by analysis of variance (ANOVA) using Tukey's test for post-hoc analysis or an independent samples t-test where appropriate. Statistical significance was reported for p

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