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Chitosan/Chondroitin Sulfate Membranes Produced by Polyelectrolyte Complexation for Cartilage Engineering Mariana N. Rodrigues, Mariana B Oliveira, Rui R. Costa, and João F. Mano Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b00399 • Publication Date (Web): 18 May 2016 Downloaded from http://pubs.acs.org on May 19, 2016

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Chitosan/Chondroitin Sulfate Membranes Produced by Polyelectrolyte Complexation for Cartilage Engineering Mariana N. Rodrigues†,‡, Mariana B. Oliveira†,‡, Rui R. Costa†,‡, João F. Mano†,‡,* †3B’s Research Group – Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence of Tissue Engineering and Regenerative Medicine, Avepark – Parque de Ciência e Tecnologia, Zona Industrial da Gandra, 4805-017 Barco GMR, Portugal. ‡ICVS/3B’s, PT Government Associated Laboratory, Braga/Guimarães, Portugal. KEYWORDS. Polyelectrolyte complexes; Saloplastics; Biomaterials; Articular cartilage; Tissue engineering.

ABSTRACT. Membranes made of chitosan (CHT) and chondroitin sulfate (CS) are herein presented using a polyelectrolyte complexation sedimentation/evaporation method. The membranes present high roughness and heterogeneous morphology induced by salt crystals. Exposing the membranes to different salt concentrations induces saloplastic behavior, as shown by an increasing water absorption and decreasing stiffness while exposed to increasing

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concentrations of salt. Establishing contact between two parts of a cut membrane leads to their self-adhesion and partial recovery of their tensile mechanical properties. The membranes sustain the adhesion of ATDC5 pre-chondrocyte cells, inducing their rearrangement in cellular aggregates typical of chondrogenesis, and the expression of cartilage markers. Impregnated TGF-β3 remains loaded after 14 days of incubation, releasing only 1.2% of its total loaded mass. CHT/CS polyelectrolyte membranes are here shown as suitable candidates for the biomedical field, namely for cartilage regeneration.

INTRODUCTION Membranes are widely used in biomedical applications as wound healing patches, permselective barriers for hemodialysis and advanced cell culture substrates.1,2 A wide plethora of techniques can be employed to fabricate membranes, such as film casting and hot-melt extrusion.3 The most commonly used techniques usually rely on the use of organic solvents or plasticizers, which may have a negative impact on cell viability. Techniques that are based on “Green” concepts discarding the use of aggressive ingredients, minimizing energy consumption and potentially increasing biocompatibility are in great demand in conventional medicine, and cutting-edge applications such as the engineering of native biological tissues. The compaction of polyelectrolyte complexes (PECs) has been recently introduced to fabricate hydrogels and films aimed at soft tissue regeneration. PECs are synthesized via electrostatic interactions between positively and negatively charged polyelectrolytes, driven by an entropic release of counterions and water molecules,4-6 and is governed by Equation 1:

Pol + A− (aq ) + Pol − B + (aq ) ↔ Pol + Pol − ( s ) + A− (aq ) + B + (aq )


(1)

2

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where Pol+ and Pol– represent the polycation and polyanion, respectively and A– and B+ stand for monovalent salt counterions (like Na+ and Cl–). Although PECs can be easily prepared, they are difficult to process into complex structures due to their brittleness and water insolubility.7 For this reason, PECs have been mostly limited to develop simple devices, such as drugs carriers,8 biospecific sorbents,9 hydrogels,10 and ultrathin films.11 More recently, Schlenoff et al. showed that PECs could be centrifuged5 or extruded12 resulting in compact three-dimensional aggregates and sheets, yielding compact polyelectrolyte complexes (CoPECs) that behave as tough macroscopic hydrogels. Our group showed recently that CoPEC membranes could also be obtained following a sedimentation/evaporation compaction method.13 CoPECs can be considered hydrogels at high electrostatic cross-link densities. Their ionic bonds can be broken at high ionic strengths and replaced by polymer/counterion pairs – accompanied by an increase of water mass – but are be remade at low ionic strengths. The plasticizing effect of salt over CoPECs led these structures to be called saloplastics. Salt doping has proved to influence the bulk mechanical properties of CoPECs, exhibiting moduli ranging from a few kPa to a few MPa.12,14 This is a relevant range to heal or replace soft tissues, since many soft tissues behave as hydrogels, with similar mechanical properties, viscoelasticity and transport properties.15 Articular cartilage tends to be under constant mechanical stress since it covers the head of the joints. Due to avascularity and consequent low access to nutrients or circulating cells16, cartilage ability to heal is limited, leading to the formation of fibrocartilage, a functionally and biomechanically inferior tissue compared to the original cartilage.16,17 Moreover, articular cartilage is a complex tissue with a gradient of properties along its composition – superficial, middle, deep and calcified zones.18 Importantly, the proportion of different types of collagens in


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each part of articular cartilage is different. In the bone-cartilage interface, the content of collagen type I decreases from the bone site to the cartilage, while it is accompanied by an increase in collagen type II. Collagen type X is restricted to the cartilage calcified area, characterized by the presence of hypertrophic chondrocytes.19 The controlled interplay between different chondrocytes is crucial to obtain a fully functional cartilage/bone-cartilage interface tissue. The infiltration of mesenchymal stem cells or osteogenic progenitor cells through the calcified hypertrophic zone must also be avoided, so that vascularization or presence of osteoblasts in the cartilage structure are not promoted.20,21 In this work, we explore the synthesis of CoPEC membranes via a sedimentation/evaporation compaction following the complexation of chitosan (CHT) and chondroitin sulfate (CS). We aimed at developing a system targeting cartilage regeneration. CHT and CS are among the most used polysaccharides for cartilage tissue engineering. CHT is a natural polycationic polymer showing immunological, antibacterial and wound healing activities.22,23 Moreover, CHT shares structural similarities with many GAGs present in articular cartilage24. CS is a linear anionic polysaccharide present in cartilage, bone and connective tissue25. It is a component of aggrecan, the major glycosaminoglycan found in the proteoglycans of articular cartilage.26 CS has numerous biological properties including anti-inflammatory activity, water and nutrient absorption, improved wound healing, and biological activity at cellular level that may help to restore arthritic joint function.27 Moreover, CS has shown chondrogenic activity while present in hydrogel mixtures.28 The combination of CHT and CS is considered of great potential since it can enhance hydrophilicity, biological compatibility, mechanical strength and can also help in chondrogenesis, producing

good

support

structures

for cartilage

tissue engineering

applications.29


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The CoPEC membranes produced herein will be characterized for the extent of their saloplastic behavior and degradation in vitro. Moreover, PECs were previously proposed to produce nanoparticles for drug delivery purposes.30 In this regard, the CHT/CS membranes produced were also assessed for their potential to be used as growth-factor carriers. Their effect on a pre-chondrocyte cell line proliferation, morphological arrangement and collagens expression will allow assessing their potential for cartilage defects treatment.

EXPERIMENTAL SECTION Materials. CHT of medium molecular weight (MW=190,000–310,000 Da, 75–85% degree of deacetylation, viscosity 200–800 cP), CS A sodium salt from bovine trachea – lyophilized powder (MW=50,000–100,000 Da), and lysozyme from chicken egg white (dialyzed, lyophilized, powder, ≈100000 units/mg) were purchased from Sigma-Aldrich. CHT was purified by a series of filtration and precipitation steps in water and ethanol, as described by Signini and Filho,31 followed by freeze-drying. Recombinant human TGF-β3 was purchased from Prepotech Inc. (Rocky Hill, NJ, USA), reconstituted in 5–10 mM of citric acid up to a concentration of 1.0 mg/mL. All other chemical agents including sodium chloride (NaCl), sodium hydroxide (NaOH) and acetic acid were of reagent grade and were used as received. Zeta (ζ)-potential measurements. A 1 mg/mL CHT solution in 1% (v/v) acetic acid and a 1 mg/mL CS solution were prepared in 0.5 M NaCl, in distilled water. Both solutions were adjusted to different pHs (4.0, 4.5, 5.0 and 5.5) using 2 M NaOH and 1% (v/v) acetic acid. The zeta (ζ)-potentials were determined using a Nano-ZS from Malvern (UK). The optimal pH value to carry out the experiments was the condition that the best electrostatic equilibrium (charge ratio of 1:1).


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CoPEC membranes production. Membranes were produced as depicted in Figure 1. The solutions used to determine the ζ-potential were hereafter used for the production of the membranes. The pH of both solutions was adjusted to 5.5, as determined experimentally to provide a charge ratio close to 1:1. 200 mL of CHT and 200 mL of CS were heated to 37 °C. The CHT solution was added slowly to the CS solution, being left to stir vigorously for 30 min, at 37 ºC, 1400 rpm. The complexes were then left to precipitate for 1h. After sedimentation, excess supernatant was carefully removed and the remaining suspension was centrifuged for 15 min at 500g. The pellet was resuspended in 30 mL of supernatant, equally distributed by 4 petri dishes (55 mm in diameter), which were left to dry in an incubator at 37 ºC for 24h. The resulting dry membranes were stored at room temperature. The described conditions lead to the synthesis of 4 individual membranes.

Figure 1. Schematic representation of the production of CHT/CS polyelectrolyte complexes membranes.


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CHT membranes production. CHT powder was dissolved to a concentration of 2% (w/v) in 0.2 M aqueous acetic acid. The solution was cast into a petri dish and dried at room temperature for 4 days. The membranes were neutralized by soaking them in a 4% (v/v) NaOH for 10 min, followed by washing with PBS and distilled water until the pH of the latter reaches a value of 7. Scanning electron microscopy (SEM). The membranes were washed with distilled water for 24h and cut into pieces of 1×1 cm2 each. They were then dehydrated in increasing series of ethanol (20%, 50%, 70%, 90%, 95% and 100%), twice for 15 min, and later dried using a critical point drier Autosamdri-815 Series A (Tousimis, USA). Shortly, a sample saturated with ethanol is placed in the specimen chamber and filled with CO2 at 0 ºC (±5 ºC) and pressure ranging from 1072 to 1350 psi. The drying of the sample starts after the temperature is raised to 31 ºC and the pressure returns to atmospheric pressure, after which the sample can be retrieved. Supercritical drying was preferred over conventional freeze-drying, since the former preserves the structure of the samples and the latter is often associated with specimen structural collapse.32,33 The dry samples were coated with a gold sputter and observed using a JSM-6010LV scanning electron microscope (JEOL, Japan) operated at 10 kV accelerating voltage. For a cross-section observation, the membranes were immersed in liquid nitrogen and ruptured. Energy dispersive spectroscopy (EDS). This analysis was used to determine the presence of residual NaCl after washing the membranes for 24h with ultrapure water. The samples used and the preparation mode were the same as the ones used for SEM observation. The samples were analyzed using EDS with a Leica Cambridge S360 scanning electron microscope. Atomic force microscopy (AFM). The membranes were dried using the same procedure as described for SEM analysis. Samples with 1×1 cm2 were studied using a L018W46 Dimension Icon AFM equipment with a ScanAsyst Air cantilever (Bruker, France), at a spring constant of


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6 N/m. The topographies were imaged with 512×512 pixels at a line rate of 1 Hz. The upper part of the membranes, i.e. the face that did not touch the Petri dish during the drying stage, was analyzed. The obtained images had a resolution of 5×5 µm2 and were analyzed using the NanoScope analysis software. At least three acquisitions were made with roughness parameters analysis. Uniaxial tensile testing. Different samples were first washed with ultrapure water to remove residual salt crystals resulting from the synthesis process. They were then immersed in one of various aqueous solutions for 24h: ultrapure water, PBS, 0.15 M NaCl, 0.5 M NaCl, 1 M NaCl and 2.5 M NaCl. The membranes were tested in wet conditions using the INSTRON 5543 universal mechanical testing equipment (Instron Int. Ltd, High Wycombe, UK) with a load cell of 1 kN. The membranes were cut in 5 mm width and 2 cm length stripes, fixing them between two grips positioned 1 cm in the length axis. The thickness of the samples was determined using a Mitutoyo thickness measurement tool. The crosshead speed was 1 mm/min and for each condition the specimens were loaded until rupture. At least 5 specimens were used. Dynamic mechanical analysis (DMA). Samples previously washed in ultrapure water for 24h were immersed in different concentrations of NaCl, to verify the effect of salt in the mechanical properties of the membranes produced. They were immersed in one of 6 solutions: ultrapure water, PBS, 0.15 M NaCl; 0.5 M NaCl; 1 M NaCl and 2.5 M NaCl for 24h. After that period these were cut in stripes the same length as for uniaxial mechanical tests, and the thickness of each membrane was measured using Mitutoyo thickness measurement tool. DMA tensile tests were performed using the Tritec 2000B equipment (Triton Technology, UK). The measurements were carried out at 37 ºC, with the samples immersed in each solution in a Teflon bath, with an increasing frequency ranging from 0.1 to 10 Hz. Five samples per each condition were used.


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Water uptake. The water uptake ability of the membranes was measured by soaking them in 6 different solutions with different concentrations of salt (ultrapure water, PBS, 0.15 M NaCl, 0.5 M NaCl, 1 M NaCl and 2.5 M NaCl). The swollen membranes were removed at predetermined time points (5 min, 15 min, 30 min, 1h, 3h, 5h, 7h, 24h and 48h). After removing the excess of water with a filter paper, the membranes were weighed in an analytical balance (Denver Instrument, Germany). The water uptake was calculated as described in Equation 2:

Water uptake (%) =

Ww − Wd × 100 Wd

(2)

where Ww and Wd are the weights of the swollen and dried membranes, respectively. For each time point and for each condition, 3 samples were weighed and the calculations were made with the average of the 3 measurements. Enzymatic degradation. Dry membranes were first weight and then placed at 37 °C in three different solutions: (i) PBS with NaN3 (pH=7.4), (ii) PBS with NaN3 and 13 µg/mL lysozyme and (iii) PBS with NaN3 and 130 µg/mL lysozyme. In the enzyme-containing solutions, the pH was adjusted to 6.2, to maximize the enzyme’s activity. PBS and enzymatic solutions where changed every 3 days. The membranes were retrieved at predetermined time-points (1, 3, 7, 14 and 21 days) and washed with distilled water to remove any remaining salts. The membranes were then dried in an incubator at 37 °C, to be weighed afterwards. The percentage of weight loss was calculated as shown in Equation 3:

Weight loss (%) =

Wi − W f Wi

× 100


(3)

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where Wi and Wf are the weights of the initial dry membrane and after incubation in PBS or enzymatic solution, respectively. For each time-point, 3 samples were weighed and the calculations were made with the average of the 3 measurements. Self-Adhesion test. The membranes were cut longitudinally in stripes with the same dimensions as the samples used for mechanical assays. Two pieces of a cut membrane were placed in contact and pressure was applied by using a weight of 5 kg for about 1 min. They were then immersed in a solution of 2.5 M NaCl and stored in an incubator at 37 ºC for 24h. After this step, they were tested using the universal mechanical testing equipment INSTRON 5543 (Instron Int. Ltd, High Wycombe, UK) with a load cell of 1 kN operating in tensile mode. The crosshead speed of 1 mm/min was applied until rupture. At least 3 specimens were tested. Uptake and release quantification of TGF-β3. The dry membranes were first sterilized using UV light for 20–30 min on each side of the membranes. Each CHT/CS membrane was immersed in 1 mL of a 150 ng/mL TGF-β3 solution in PBS under sterile conditions; the experiment was performed in quadriplicate. For the determination of TGF-β3 uptake by the membranes, after 24h of immersion in the solutions, the membranes were removed and rinsed with PBS. The quantification of TGF-β3 in the remaining solution was determined using the OmniKine Human TGFBeta3 ELISA Kit. The uptake of TGF-β3 was calculated as shown in Equation 4:

TGF _ β 3 uptake (%) =

Wi − W f Wi

× 100

(4)

where Wi is the mass of TGF-β3 measured before the beginning of the experiment and Wf is the measured mass after overnight incubation at room temperature.


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For the TGF-β3 release quantification, the membranes were immersed in 1 mL of sterile PBS. For every time-point – 2h, 4h, 6h, 8h, 24h, 48h, 7 days and 14 days – 300 µL of PBS was removed from the tubes containing each sample, to assess the TGF-β3 release with time. The same volume of fresh PBS was replenished to the tube in each time point. The samples were kept in a bath at 37 ºC and 60 rpm. Enzyme-linked immunosorbent assays (ELISA) were performed according to the manufacturer’s instructions. The plates were read on a Synergie HT microplate ELISA reader (BioTek, USA) at 450 nm, and the values were adjusted to the calibration curve to assess the TGF-β3 content. ATDC5 cell seeding. Four different biomaterial substrates were used to study cell response. Pure CHT membranes and tissue culture polystyrene (TCPS) dishes were used as controls. Moreover cell tests were also done in CHT/CS membranes loaded with TGF-β3 (after immersion in 150 ng/ml solution, as previously described), and without TGF-β3. CHT/CS membranes were washed for 24h to remove the salt and were left to dry and cut into pieces of 1×1 cm2 each. For sterilization of all the substrates UV light was applied for 20–30 min on each side of the membranes. The pre-chondrocyte ATDC5 cell line was obtained from ECACC (UK). The cells were expanded in flasks using Dulbecco’s modified Eagle’s medium nutrient mixture F-12 (DMEM/Ham’s F12; Invitrogen) supplemented with 10% (v/v) fetal bovine serum (FBS, Invitrogen) and 1% antibiotic/antimicotic (Gibco). The cells, maintained at 37 °C in a humidified CO2 (5%) atmosphere, were dissociated with 0.25% trypsin-EDTA (Sigma), centrifuged at 1200 rpm for 5 min and resuspended in culture medium prior to cell seeding. Three samples per material per culturing time (7, 14 and 21 days) were studied, for ALP and dsDNA quantification. For immunolabelling, 5 samples were used per time point. A total of 5×104 cells were seeded in each membrane in a total volume of 100 µL of cell culture medium. Samples were then placed


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into the incubator for 2h. After that time, more cell culture medium was added to each sample, up to a volume of 600 μL, and the plates were placed into the incubator until the established time periods. Immunocytochemistry. The expression of collagens type I, II and X was assessed by immunocytochemistry for solvent casted CHT membranes, CHT/CS membranes, CHT/CS membranes loaded with TGF-β3 and TCPS. The time points studied were 7, 14 and 21 days of cell culture in DMEM/F12 medium. Samples were washed with PBS, permeabilized with 1% Triton X-100 (Sigma, USA) and blocked with a 1% BSA/PBS solution. Cells were incubated for 1h at room temperature with the primary antibodies. The samples were divided in half: one part was incubated with rabbit anti-mouse IgG collagen type I (abcam ab292; 1:100 dilution) and a mouse IgG antibody with multiple reactivity for collagen type II (Millipore MAB1330; 1:100 dilution); the other half of the samples was incubated with rabbit anti-mouse IgG collagen type X (abcam ab58632; 1:100 dilution). All antibody dilutions were performed in 1% BSA/PBS. After incubation, cells were washed three times with 1% BSA/PBS and incubated with the respective secondary antibodies (goat anti-mouse IgG labeled with Alexa Fluor® 488 (Invitrogen, USA), or goat anti-rabbit IgG labeled with Alexa Fluor® 594, both diluted at 1:200 in 1% BSA/PBS). Samples were visualized and images were acquired using a reflected light microscope (Axio Imager Z1m, Zeiss). All images were acquired with the same exposure settings. Five replicas per condition were analyzed for the fluorescence intensity corresponding to each antibody using ZEN software (Zeiss). Such values were then normalized by the intensity quantified for nuclei staining in each image. All images from the same biomaterial conditions were treated for background removal using the same image analysis settings, for every time point.


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dsDNA quantification. Double stranded DNA (dsDNA) content was quantified using the PicoGreen dsDNA kit (Gibco, USA). After each culturing time point (7, 14 and 21 days), the samples were washed using sterile PBS twice and then placed into 1.5 mL tubes, after which 1 mL of ultrapure water was added. The tubes were then placed in a 37 ºC bath for 1h. The tubes were then placed in a -80 ºC freezer for at least 24h before use. At the time of use, quantification standard solution of dsDNA (0, 0.2, 0.5 and 1 mg/mL) was prepared to obtain a standard curve and all the solutions were prepared according to manufacturer’s instructions. Triplicates were made from each sample. Fluorescence emission was read at 528/20 nm in a SYNERGY HT microplate (BIO-TEK, USA), and concentration of dsDNA was correlated with calibration curve. ALP quantification. The quantification of alkaline phosphatase (ALP) activity was performed after a culturing period of time (7, 14 and 21 days). The activity of this marker is evaluated using p-nitrophenol assay. p-nitrophenylphosphate, which is colorless, is hydrolyzed by ALP at pH 10.5 at 37 ºC to form free p-nitrophenol, which is yellow in color. This reaction is stopped using sodium hydroxide (NaOH). ALP activity assays were performed on cell lysates. All reagents were purchased from Sigma. All samples were prepared in triplicate and compared against pnitrophenol standards. The absorbance was read at 405 nm on a microplate reader (Synergy HT, USA) to determine the enzyme concentration per mL of cell lysate. Statistical analysis. Statistical analysis was performed using GraphPad Prism version 6.01 for Windows (GraphPad Software, USA). The results were presented as mean±standard deviation (SD). Statistical analysis was performed using the one-way ANOVA test followed by Tukey’s multiple comparisons. For the DNA, ALP and collagens quantification the two-way ANOVA


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was performed. Statistical significance was set to p-values lower than 0.05 (symbols: * for p

Chondroitin Sulfate Membranes ... - ACS Publications

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