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Hyaluronic Acid/Chitosan Coacervate-Based Scaffolds Ozge Karabiyik Acar, A. Basak Kayitmazer, and Gamze Torun Kose Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00047 • Publication Date (Web): 19 Mar 2018 Downloaded from http://pubs.acs.org on March 20, 2018

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Hyaluronic Acid/Chitosan Coacervate-Based Scaffolds Ozge Karabiyik Acar&, A. Basak Kayitmazer♯*, Gamze Torun Kose&* &

Department of Genetics and Bioengineering, Yeditepe University, Istanbul, Turkey ♯

Department of Chemistry, Bogazici University, Bebek 34342, Istanbul, Turkey

ABSTRACT: Chitosan-chloride (CHI) and sodium hyaluronate (HA), two semi-flexible biopolymers, self-assemble to form non-stoichiometric coacervates. The effect of counterions was briefly investigated by preparing HA/CHI coacervates in either CaCl2 or NaCl solutions to find only a small difference in their tendency to coacervate. Higher water content in coacervates within CaCl2 was attributed to the chaotropic nature of Ca+2 ions. This effect was also evidenced with smaller pore sizes for coacervates in NaCl. Besides, for coacervation of chitosan-glutamate (CHI-G) with HA, dynamic light scattering at different charge ratios indicated a wider coacervation region for the HA/CHI-G pair than the HA/CHI. This was attributed to the chaotropic and “soft” ion nature of glutamate compared to chloride as a counterion of chitosan. Positive zeta potential values for both coacervate suspensions were explained by the contribution of charge mismatch; chain semi-flexibility; intra- and intercomplex disproportionation. Finally, HA/CHI coacervates were used to encapsulate bone marrow stem cells. While cell viabilities in HA/CHI coacervates were remarkable up to 21 days, their well-spread morphology has proved that HA/CHI coacervates are promising scaffolds for cartilage tissue engineering.

KEYWORDS: Complex coacervation; stem cell encapsulation; counterions; Hofmeister series, polyelectrolytes

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INTRODUCTION Organ and tissue failure have been great challenges in medicine. Cartilage damage is potentially serious and very common - once damaged, cartilage has low natural repair capacity because of being avascular and aneural.1 The damage can result in severe pain, knee swelling and restrictions in mobility. There are several medical2 and clinical approaches3-5 to repair the damaged tissue; however, current treatments have various shortcomings.6 Cartilage tissue engineering is a promising approach to overcome those limitations. Engineering cartilage tissue is a revolutionizing approach, where scientific principles are applied to the synthesis of living tissues using relevant cells attached to biodegradable scaffolds stimulated with various factors.7,8 Ideally, a scaffold should be biocompatible and biodegradable with non-toxic by-products; it should also have a porous structure.9-11 For cartilage tissue regeneration, there are additional important features required; i.e. (i) usage of biologically active 3D environment to support cell-material interaction,12 (ii) encapsulation potential to maintain cell viability and differentiation, (iii) viscoelastic properties to fill the gaps in cartilage-damaged areas, and (iv) high water content as in natural extracellular matrix (ECM).13 In cartilage repair and regeneration, chondrocytes are the most frequently used cell type because they are native cells; however, there is a limitation about reaching sufficient cell number after their isolation.14 At this point, stem cells become important alternative cell sources due to their ease of isolation and their retention of multi-lineage potential during expansion. Mesenchymal stem cells (MSCs) are multipotent cells that have the potential to differentiate into mesodermal originated cells. Bone marrow-derived mesenchymal stem cells (BMSCs) are a promising source of MSCs; they also have chondrogenic differentiation potential and high in vitro proliferation capacity, making them suitable for cartilage repair.15 Encapsulation of cells within hydrogel-based 3D biomaterials is a frequently used-technique for cartilage tissue regeneration. In particular, polysaccharides such as hyaluronic acid (HA) and chitosan ACS Paragon Plus Environment

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hybrids were observed to form biomimetic hydrogels for the survival of cells.16,17 Mechanical and rheological analysis of gels prepared using HA and CHI derivatives were comprehensively studied in the literature.18-22 Rheology and dynamic mechanical analysis of the scaffolds were directly related to the preparation method, crosslinking degree, molecular weight and concentration of the polymer.23-25 Park et al.,19 investigated the compressive modulus of the methacrylated glycol chitosan (MeGC)/HA composite hydrogels and showed enhancement of modulus (modulus of 10 kPa to 25 kPa) in accordance with the molecular weight of the HA. However, encapsulating cells within hydrogels has limitations: (i) use of organic solvents, initiators or stabilizers during the preparation may present cytotoxicity.26 (ii) formation of thick constructs (pore size limitation) may prohibit the transfer of oxygen and nutrient.27 (iii) rate and extent of degradation of biomaterials, which affect the mechanical strength of the scaffold, should be optimized for maintaining the viability of the cells.28 Still, there are some encouraging results regarding usage of HA for encapsulation.29,30 For example, a very recent paper described preparation of HA-coated poly(lactic-co-glycolic acid) (PLGA) scaffolds as a microenvironment to seed and encapsulate cancer cells within microfluidic devices.30 In that study, higher cell viability and proliferation were possible when scaffolds included higher amounts of HA-coating. Complex coacervation is a self-assembly phenomenon based upon liquid-liquid phase separation between oppositely charged macro-ions (e.g. micelles, proteins, dendrimers, polyelectrolytes, polypeptides, etc) in an aqueous solution.31,32 This separation occurs due to entropic gain through the release of counterions in solution, which follows electrostatic interaction between the oppositely charged macroions. Phase transition of complex coacervation depends on various parameters such as temperature, pH, ionic strength, molecular weight and charge density of polyelectrolytes and mixing stoichiometry.33,34 There is a narrow range for observation of coacervation which is determined by optimization of these physicochemical parameters for every oppositely charged macro-ion pair. Meanwhile, for physical gels containing HA, Collins and Birkinshaw35 have also pointed to the



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importance of complete dissolution of polymers chains, which can take over 24 hours and result as a better extension of HA chains for association with other macromolecules. Coacervation is extensively employed in the fields of pharmaceuticals,36 foods,37 and cosmetics.38 More recently, promising applications of coacervates for tissue engineering have been reported as a consequence of their desirable properties: (i) coacervates can be prepared in physiological conditions, (ii) high water content and heterogenous structure in coacervates that are tunable by physicochemical variables allow easier transport of nutrients and waste from cells encapsulated in coacervate scaffolds compared to hydrogels,39 (iii) coacervate phase has a low interfacial tension, which is a required condition for wet tissue adhesives,40 (iv) ability of coacervates to recover after pre-shearing (its selfhealing property)41 allows them to be moldable to the shape of a tissue defect, (v) shear-thinning behavior42 of coacervates enables their injection without invasive surgery. As a result, coacervation has been successfully utilized as scaffold or coating material on implants to enhance seeding efficiency and viability,43 in vitro proliferation,44 and healing efficiency,45 osteogenic differentiation46 of cells. Moreover, recent investigations have focused on the adhesive features of complex coacervates to attain a tissue adhesive to be usable under wet conditions (e.g. water, blood)47 for bone grafting,48 sealing of orthopaedic wounds,49 and regeneration of craniofacial fractures.50 As an encapsulation platform, coacervation enhances therapeutic potential of growth factors by enabling a sustained release profile and on-site delivery. Epidermal growth factor, fibroblast growth factor-2 and stromal cell-derived factor (SDF)-1α were all encapsulated in coacervates of heparin/poly(ethylene argininylaspartate diglyceride) for purposes of wound healing,51,52 the remodeling of cell-free vascular grafts,53 and the promotion of angiogenesis.54 Coacervates can be prepared with stimuli-responsive properties that change with ionic strength, pH or temperature. Lawrence et al. showed that coacervation of poly(allylamine hydrochloride) (PAH) with pyrophosphate (PPi) or tripolyphosphate (TPP) established an underwater adhesive with reversible attachment ability depending on pH and ionic strength.55 Shimada et al. reported that changes in


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temperature led to liquid-liquid phase separation of poly(allylamine-co-allylurea), where the morphology of seeded cells was switched from monolayer to spheroid culture.56 In another system, hyperbranched poly(β-amino esters) synthesized from 5-amino-1-penthanol and trimethylolpropane ethoxylate triacrylate were shown to form coacervates above 33°C. By increasing the degrees of protonation of this polymer at low pH and 37°C, this positively charged polymer could bind more strongly to negatively charged cell membrane.57 Protein-loaded poly(glutamic acid)/poly(lysine) coacervates were demonstrated to release their cargo by decreasing the polypeptide molecular weight and/or pH.58 The aim of this work was to develop porous biodegradable scaffolds using the complex coacervation technique. The 3D coacervates were prepared using oppositely charged weak polyelectrolytes, i.e. sodium hyaluronate (HA) were mixed with either chitosan-chloride (CHI) or chitosan-glutamate (CHIG) in salts of different ion types and valences (Na+ and Ca2+). In literature, salt valence and type have been shown to affect complex coacervation.59-62 However, in this study, the main goal was to accomplish scaffolds at the conditions suitable for cell growth using NaCl as monovalent or CaCl2 as divalent salts. Furthermore, we explored the effect of chitosan salts (chitosan-glutamate and chitosanchloride) in attaining coacervates that are the most suitable for cell encapsulation. At the same degrees of acetylation and pH, we compared binding affinity of HA to CHI and CHI-G, where the latter was highlighted as water-soluble even at neutral pH.63 The present work primarily focuses on in vitro characterization of the HA/CHI coacervate-based scaffolds. First, optimal preparation conditions for HA/CHI coacervation were determined using light microscopy, dynamic light scattering, and zeta potential experiments. Microstructure of coacervates was examined with scanning electron microscopy to estimate the porosity available for cell encapsulation. The expression of relevant cell surface markers was analyzed for BMSCs which were isolated from rat bone tissue. Finally, cell morphology and viability assays were performed to evaluate the potential of the coacervates as ideal scaffolds under in vitro conditions for tissue engineering purposes.



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EXPERIMENTAL SECTION Materials. Sodium hyaluronate (HA, Pharma Grade 150), chitosan-chloride (CHI, PROTASAN UP CL 213) with 83% degree of deacetylation (DD), and chitosan-glutamate (CHI-G, PROTASAN UP G 213) with also 83% DD were purchased from Novamatrix (Sandvika, Norway). CHI has an average molecular weight (MW) of 260 kDa as reported by the manufacturer (determined by SEC-MALS). Intrinsic viscosity of HA was supplied by the manufacturer as 2.8 m3/kg.

The viscosity-average

molecular weight of HA was extrapolated from intrinsic viscosity vs. molecular weight correlation,64 and determined as 1,700 kDa. The apparent viscosity (η) of CHI-G was provided as 130 mPa.s according to the manufacturer. This apparent viscosity (η) was first converted to intrinsic viscosity [η] by using the relevant relationship for a similar DD of chitosan.65 The viscosity-average molecular weight of CHI-G was then calculated as 345.5 kDa by extrapolating the [η] vs. MW correlation.65 CHI, which was pure from endotoxins, included 0.12% (wt.%) protein as impurity. According to the certificate of analysis, chitosan-glutamate and chloride included 43% (wt.%) glutamate and 13% (wt.%) chloride, respectively. The polymers were used without further purification. Milli-Q water (Millipore, Milford, MA, USA) was used throughout the experiments. All solutions were filtered using 0.45 µm cellulose acetate membrane (Sartorius, Germany). Sodium chloride (NaCl) and calcium chloride (CaCl2) were purchased from Sigma-Aldrich. Hydrochloric acid (HCl) and sodium hydroxide (NaOH) were purchased from Merck. Coacervate Preparation. CHI (0.09 % w/v) and HA (0.07 % w/v) were dissolved separately in NaCl or CaCl2 solutions to have an ionic strength (I) of 300 mM (Scheme 1). The complete dissolution of both polymers was achieved by constant stirring overnight. The pH of each polymer solution was adjusted to 6.25 before being sterilized by filtering through a 0.45 µm pore size in a laminar flow hood. The HA solution was added dropwise into the CHI solution at different mixing ratios while keeping the mixture stirred. These HA/CHI mixtures were used for the examination of droplet formation, turbidity, dynamic light scattering (DLS), and zeta potential measurements at different charge ratios of negative ACS Paragon Plus Environment

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(deprotonated carboxyls) to positive (protonated amines) groups in the polymers ([-]/[+]). Degrees of deacetylation of chitosan (given above) and degrees of ionization of HA and CHI at pH = 6.25 were taken into consideration for calculation of the number of charges. Relevant potentiometric titration data was taken from our previous work.33 For further in vitro experiments, a charge ratio ([-]/[+]) of 0.31 was applied (please refer to the “Results and Discussion” section for a detailed discussion on the selection of this ratio). The prepared coacervate suspension was centrifuged at 4,000 rpm for 23 minutes to facilitate its separation into the coacervate and dilute (supernatant) phases. After centrifugation, the supernatant was carefully decanted without disturbing the coacervate phase.

HA/Chitosan Glutamate

NaCl HA/Chitosan Chloride

CaCl2

HA/Chitosan Chloride

Scheme 1. Sample types used in this study Turbidimetric Titrations. Turbidity was measured using a multiwell plate reader equipped with a UV-Vis spectrophotometer (BioTek Instruments, USA) at a wavelength of 630 nm at room temperature (RT). None of the polymers absorb light at this wavelength. After stirring the solutions for ten minutes, turbidity was recorded in absorption units (a.u.) while samples were applied as 100 µL into a 96-well plate. Turbidity of the HA/CHI mixture was measured after adding 0.07% (w/v) HA solution into 0.09% CHI (w/v) solution in varying volumes. This led to turbidity values at different charge ratios of HA:CHI for I = 300 mM and pH = 6.25. All samples were referenced against the respective salt solution. All experiments were run in triplicate.



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Dynamic Light Scattering. Dynamic light scattering (DLS) experiment was performed on a Zetasizer Nano ZS (Malvern Instruments, UK), which records data at a scattering angle of 173° with a 633 nm He/Ne laser. All experiments were done at 25°C. Cumulants analysis (“general purpose mode”) was employed for fitting of correlation curves, which were used to determine mean apparent translational diffusion coefficients. The Stokes-Einstein equation was then utilized to convert diffusion coefficients to Z-average hydrodynamic diameter, i.e. intensity weighted mean diameter (dH). The mean of three measurements of dH with a standard deviation was reported. Intensity was recorded as derived count rate, which is defined as 0.3% of the measured mean count rate. Zeta Potential. Zeta potential measurements were performed on a Zetasizer Nano ZS (Malvern Instruments, UK). Coacervate suspensions were loaded into folded capillary cells (DTS1060). The electrophoretic mobility of HA/CHI mixtures was converted to zeta potential (ζ) by the Malvern software, which applies the Smoluchowski equation. Values of zeta potential were presented as the mean with standard deviation of three measurements. Optical Microscopy. The presence of coacervate droplets was confirmed using optical microscopy (Leica DMI 6000B, Germany). HA/CHI mixtures placed on glass slides were observed as spherical droplets or irregular shaped aggregates for coacervates or precipitates, respectively. Environmental Scanning Electron Microscopy (ESEM) and Scanning Electron Microscopy (SEM). Coacervate suspensions were centrifuged for 23 min at 4,000 rpm to collect the HA/CHI coacervates, which were formed at the bottom of the falcon tubes. These coacervate samples were washed with sodium cacodylate buffer before being incubated with a 2.5% gluteraldehyde solution at room temperature (RT) for 90 minutes. For ESEM. Coacervate samples were directly observed with Environmental Scanning Electron Microscopy (Carl Zeiss EVO LS 15, Germany). ESEM was applied at an accelerating voltage of 5 kV and at different magnifications. The mean pore size was estimated using ImageJ software (NIH Image, Bethesda, MD, USA).


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For SEM. Fixed coacervate samples were frozen at – 80°C. After freezing, samples were lyophilized overnight and kept at RT until observation with SEM. Samples were coated (Bal-Tec SCD 005, Germany) with gold (10 nm) and observed with SEM (Carl Zeiss EVO Scanning Electron Microscope, Germany). Dry Weight Analysis of Coacervates. After the centrifugation of the coacervate suspensions, the collected HA/CHI coacervates were weighed to determine the wet weight. Then the samples were completely dried overnight at 60°C in the oven before recording the dry weight of the coacervates. All experiments were conducted in triplicate. The degree of water content (%WC) of coacervates was calculated according to the equation: % =

× 100

where Ww is the wet weight of the coacervate while Wd is the dry weight of the coacervate. Degradation Behavior of the Coacervates. The rate of coacervate degradation was evaluated by measuring mass loss for up to 16 weeks. Triplicate samples of HA/CHI and HA/CHI-G coacervates prepared in NaCl were weighed and then immersed individually in 1X PBS solution with 0.09% (w/v) of sodium azide in sealed falcon tubes. The coacervates were incubated at 37°C under agitation (60 rpm) for 1, 7, 14, 30, 60, and 120 days without refreshing the buffer. At the end of predetermined incubation intervals, scaffolds were removed from the buffer, rinsed in deionized water to remove salts, and then weighed. The percentage of weight loss (%WL) – i.e. the degradation rate of coacervates – was calculated according to the equation: % = 100 −

× 100

where Wi is the initial weight of scaffold and Wf is the weight of the scaffold after incubation in the PBS-sodium azide solution for various periods of degradation.



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Isolation, Culture, and Characterization of Mesenchymal Stem Cells. Six-week-old male Sprague-Dawley rats were sacrificed. Their femurs and tibias were carefully cleaned from their skin and cut at the ankle bone. Bone marrow was flushed with standard growth medium (SGM) (α-MEM – Glutamax (Gibco, Invitrogen, USA) supplemented with 10% (v/v) Fetal Bovine Serum (FBS) (Gibco, Invitrogen, USA) and 100 unit/mL of penicillin/streptomycin (Pan Biotech, Germany) and incubated in an incubator at 37°C, in a humid atmosphere containing 5% CO2. The media were changed three times a week. The cells were trypsinized when they reached confluence. The cells were characterized using cell surface markers by fluorescence-activated cell sorting analysis (FACSCalibur – BD Pharmingen, USA). For FACS, the cells were stained with various fluorescently labeled monoclonal antibodies (BD Pharmingen, USA). In brief, the cells were detached from the culture flasks by a 0.25% trypsin-EDTA solution (Gibco, Invitrogen, USA) and then washed with PBS (Gibco, Invitrogen, USA). In each tube, 3×105 cells (in 100 µL PBS) were mixed with the fluorescentlylabeled antibody (FITC anti-rat CD11a, FITC anti-rat CD90, PE anti-rat CD31, PE-Cy anti-rat CD45, FITC anti-rat CD29) and incubated in the dark at RT for 45 minutes. Following rewashing with PBS, excess antibodies were removed. Finally, labeled cells were analyzed by a FACSCalibur flow cytometer. Cell Encapsulation, Viability and Morphology. Rat bone marrow stem cells (rBMSCs), which were obtained from passage 2, were detached from the culture flasks by a 0.25% trypsin-EDTA solution. The detached cells were counted by an automated cell counter (Countess® II FL, USA). Polymer solutions were prepared as described earlier. After optimal cell density was reached (500,000 cells/coacervate-sample ~ 1,000,000 cells/mL), cells were resuspended in the HA solution. HA/cell suspension was then added dropwise into the CHI solution by maintaining the 0.4:1 (v/v) HA:CHI mixing ratio (corresponding to [-]/[+] = 0.31). HA/CHI/cell suspension was centrifugated at 4,000 rpm for 23 minutes. Obtained coacervates were


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carefully transferred to a 24-well plate containing 500 µL standart growth medium and incubated at 37°C, 5% CO2 throughout the experiment. The Live/Dead assay (Thermo-Fisher Scientific, USA) was applied on encapsulated cells within HA/CHI coacervates at RT for 30 minutes using calcein-AM (2 µM) and ethidium homodimer-1 (4 µM). Samples were visualized by fluorescent microscope (ZEISS Vert.A1, Germany) after 1, 7, 14, and 21 days of incubation. To assess cell viability, three different zones were processed for calculating the number of green and red spots using the ImageJ software. The ratio (cell-viability percentage) of the number of live cells to the number of total cells was calculated. One-within (time), one-between (salt type) ANOVA was applied to the data using IBM SPSS Statistics, version 23 (Armonk, NY). For morphology stainings, encapsulated cells within HA/CHI coacervates were first fixed with 3.7% formaldehyde (Merck Millipore, USA) for 30 minutes followed by permeabilization with 0.1% Triton X-100 (Thermo-Fisher Scientific, USA) for 10 minutes and PBS washing. These samples were incubated in dark at RT with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, USA) and Alexa Fluor 546 Phalloidin (Thermo-Fisher Scientific, USA) for the staining of nucleus and of cytoskeleton, respectively. Confocal microscope (ZEISS LSM 700, Germany) was used to visualize the samples after 72 hours of incubation. RESULTS AND DISCUSSION Cell viability is crucial for tissue engineering purposes. For the long-term survival of cells, coacervate/cell constructs are preferably prepared in physiological conditions, i.e. pH 7.4 and I = 150 mM. However, chitosan is not soluble at pH ≥ 6.7 due to the deprotonation of more than 75% of its amine groups at this pH.33 Meanwhile, ionic strength (I) of 50 mM and 150 mM at pH = 6.25 did not result in coacervates. Coacervation between HA and CHI, on the other hand, could be observed at pH = 6.25, I = 300 mM, and [-]/[+] = 0.31 (Figure 1A,D,E). Thus, we have proceeded with these experimental conditions for the cell viability and morphology experiments. Also, in this study, sodium hyaluronate of



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1,700 kDa was used instead of 234 kDa (molecular weight of HA used in our previous study)33 to prepare a cell-encapsulating scaffold with high viscosity that would stay in the injection location in vivo. The use of a high MW polymer in our system enhances phase separation. This result is attributed to the relatively lower entropy loss of longer chains when they are entrapped in the dense environment of coacervates.33 Effect of Chloride as a Chitosan Salt in the NaCl or CaCl2 Solutions. The charge ratio of two oppositely charged biopolymers is one of the parameters that determines phase separation behavior.66,67 The nature of this phase separation can be determined by combining light microscopy results with visual inspection of the samples (Figure 1). Spherical droplets are accepted as distinctive features of a coacervate suspension.68-70 For the HA/CHI system at pH = 6.25 and I = 300 mM, spherical droplets with submicrometer size (diameter) were observed at charge ratio ([-]/[+]) of 0.31 (Figure 1A,B) whereas precipitates appeared as solid flocs with irregular shapes at [-]/[+] of 0.77 (Figure 1C). At higher charge ratios of 0.46 and 0.61 in the NaCl solution, coacervate droplets coexisted with precipitates (Figure S1), for which a similar observation was made for a synthetic polycation/anionic mixed micelle system.71 On the other hand, precipitates were the most dominant phase-separated species of the HA/CHI mixture at charge ratio of 0.69 and above (Figure S2). This result was attributed to the mismatch in charge spacings of HA and CHI chains, which are 1.3 nm and 0.6 nm, respectively. At equivalent charge spacing of polymers, contact-ion pairs may form, leading to precipitation as a result of concurrent release of counterions and water molecules. On the other hand, mismatch in charge spacing would lead to a weak level of charge complementarity that might have resulted in loops rather than contact ion-pairs. Thus, loops would help to sustain a certain degree of hydration, favoring coacervation. At higher charge ratios ([-]/[+] > 0.46), a higher number of carboxyl groups would reduce the charge mismatch, favoring precipitation. By visual inspection of the HA/CHI mixtures, a one-phase turbid solution was detected up to a charge ratio of 0.61 (Figure 1E). However, this method did not help to distinguish the range of charge ratios


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where coacervates and precipitates coexisted. Nevertheless, precipitates could be observed distinctly at charge ratios of 0.69 and 0.77, agreeing with the light microscopy images described above (e.g. Figure 1C). After the centrifugation of the HA/CHI mixture at [-]/[+] = 0.31, a polymer-rich phase (semiopaque in appearance) and a dilute solution phase could be distinctly seen (Figure 1D). The semiopaque appearance of the coacervate phase might be attributed to the level of purity of the biopolymers.

Figure 1. Optical microscopy images for the HA/CHI mixtures at pH = 6.25 and I = 300 mM (A) [-]/[+] = 0.31 (coacervate suspension) prepared with NaCl solution, (B) [-]/[+] = 0.31 (coacervate suspension) prepared with CaCl2 solution. (C) [-]/[+] = 0.77 (precipitate) prepared with NaCl solution. The scale bars represent 10 µm. (D) Picture of the coalesced HA/CHI coacervate phase ([-]/[+] = 0.31) after



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centrifugation. Visual appearance of HA/CHI mixtures in (E) NaCl and (F) CaCl2 solution. The charge ratios ([-]/[+]) are given above each cuvette. Turbidity, which is proportional to scattering at all angles, shows a non-monotonic behaviour with charge ratio for the HA/CHI system (Figure 2A). This non-monotonic behaviour was also observed in measurements of total scattering intensity (count rate) by DLS (Figure 2B). However, the maxima from these two techniques for HA/CHI mixtures dissolved in NaCl appears at different [-]/[+] values; i.e. at []/[+]max = 0.61 and at [-]/[+]max = 0.31 by turbidity and DLS measurements, respectively. As demonstrated above with light microscopy, the former [-]/[+]max value corresponds to the simultaneous observation of coacervate liquid droplets and solid precipitates. On the other hand, for the latter []/[+]max value, coacervate droplets were the only kind of phase-separated species present in the HA/CHI mixture. Thus, it can be concluded that turbidity was not sensitive to the conditions where coacervate droplets and precipitate particles coexisted in solution; i.e. turbidity only dropped where macroscopic separation was visible to the naked eye (Figure 1E,F). Meanwhile, scattering intensity is relatively more susceptible to the appearance of precipitate particles; i.e. count rate drops at [-]/[+] values where both coacervate droplets and precipitate particles are present (Figure 2B). The Z-average hydrodynamic diameter (henceforth dH) was constant at 609 ± 6 nm between [-]/[+] of 0.08 and 0.31 for HA/CHI mixtures (Figure 2C). The “objects” with a dH of 609 nm were attributed to coacervate droplets which would coalesce to form the coacervate phase upon centrifugation. Here, it should be added that this hydrodynamic size did not originate from that of pure biopolyelectrolytes since the dH of the biopolymer solutions was much lower; i.e. 130 ± 24 nm for CHI and 73 ± 13 nm for HA. These “objects”, which correspond to coacervate droplets in HA/CHI mixtures, had positive zeta potential values at low [-]/[+] values (Figure 2D). Charge reversal only occurred at [-]/[+] > 0.69, where precipitate particles dominated the HA/CHI mixture as confirmed by light microscopy. Observation of coacervation at non-stoichiometric charge ratios agreed well with the literature.33,72 In our previous ACS Paragon Plus Environment

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work,33 we have considered the contribution of several factors for the formation of non-stoichiometric coacervates. One of the factors, charge mismatch between polymers, is explained above. The second important factor is the semi-flexibility of HA and CHI chains. Compared to the degrees of freedom of polymers in the nonphase-separated state, the chains lose their conformational entropy since chains exist in a more constrained conformation in the coacervate phase. Since the polymers chosen were semiflexible (persistence length of 6.5 nm and 4 nm for CHI and HA, respectively),73,74 the loss in conformational entropy would be less compared to flexible polymers. This would favor nonstoichiometric coacervation. The last factor contributing to positive zeta potentials for coacervate suspensions is related to both intracomplex and intercomplex disproportionation.75 According to intracomplex disproportionation, coacervate droplets might include species with a charge-neutral core, which fulfills the requirement of charge neutrality for coacervation, and a tail where excess cationic charges are located. On the other hand, if intercomplex disproportionation takes place, the migration of polycations will lead to the coexistence of a neutral coacervate drop with a more highly charged aggregate. Either kind of disproportionation will explain the origin of positive zeta potential values for coacervate suspensions, and subsequently non-stoichiometric coacervation. The fact that size stays constant but turbidity and intensity increase up to [-]/[+] = 0.31 might be due to the rise in the number of scattering objects. The excess addition of HA after this point caused formation of precipitate particles, which later dominated the HA/CHI mixture and settled down, eventually reducing the turbidity (Figure 2A). In the literature, ionic strength, salt valence and salt type have been found to affect the formation or dissociation of polyelectrolyte/polyelectrolyte coacervation, complexation and multilayers.59-62 However, among these three parameters, the most important one is ionic strength due to its charge screening effect. In fact, it is shown that complex coacervation is disfavored at high ionic strength regardless of the type of macromolecules in the system.33,76,77 Thus, it would be highly unlikely to get HA/CHI coacervates at I = 900 mM, which is the condition where Ca+2 and Na+ would have the same



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concentrations. Also, at I = 900 mM, (i) the number of cells and their cellular integrity would be negatively affected, which might inhibit their proliferation as well as their differentiation capacity,78-80 and (ii) increase of extracellular osmolality would cause cells to shrink. For all these reasons, ionic strength was also set to 300 mM for the experiments done in CaCl2. Maxima in the plot of derived count rate (total scattering intensity) versus charge ratio for HA/CHI mixtures prepared in CaCl2 occurred at the same [-]/[+] as that of samples prepared in NaCl, i.e. [-]/[+] = 0.31 (Figure 2B). This charge ratio of maximum intensity was chosen to carry out ESEM and cell encapsulation studies (see below) since (i) spherical droplet morphology observed at [-]/[+] = 0.31 indicated coacervation and (ii) the coacervate yield was optimum at this maximum intensity. Zeta potential values of the HA/CHI mixtures prepared in CaCl2 were also similar with the ones prepared in NaCl at different charge ratios. For coacervate suspension in CaCl2, DLS gave a Z-average hydrodynamic diameter (dH) of 668 ± 118 nm, which was also similar, within error limits, to the value for mixtures prepared in NaCl solution (Figure 2C). Dry weight analysis of coacervates was conducted to get a better understanding of this similarity in DLS and zeta potential results despite the use of different types and valencies of salt ions. Water content in the coacervate (after centrifugation) prepared in CaCl2 and NaCl solutions was determined to be 94 ± 0.1% and 92 ± 0.4%, respectively. The relatively higher water content for coacervates in CaCl2 solution was expected because of the larger hydration number (number of water molecules per ion) for Ca+2 than that for Na+.62 In a recent study,60 the effect of various salts (including NaCl and CaCl2) on phase separation of poly(acrylic acid sodium salt) (pAA)/poly(allylamine hydrochloride) (pAH) system was investigated by keeping ionic strength constant. Coacervation of pAA/pAH was disfavored with CaCl2, which was attributed to the increased solubility of polyelectrolytes in chaotropic salts. Counterions would rather stay with the polyelectrolytes than be released after the two oppositely charged polyelectrolytes interacted and formed coacervates. Thus, higher water content was observed in coacervates prepared in CaCl2 solutions compared to NaCl, where Ca+2 is relatively more chaotropic.


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In addition to the dry weight data, DLS provided further proof to this subtle difference in phase separation. First, the deviation of the plot of dH vs. charge ratio from its baseline was assigned as the point of transition from coacervate droplets to precipitates. Then, this charge ratio at the transition point was read as 0.39 ± 0.01 and 0.35 ± 0.01 for the HA/CHI system in NaCl and in CaCl2, respectively (Inset of Figure 2C). In other words, the coacervation region extends to a relatively higher charge ratio for the HA/CHI system in NaCl than in CaCl2, i.e. favoring coacervation over precipitation for the former.

Figure 2. (A) Turbidity, (B) Total scattering intensity (count rate), (C) Z-average (hydrodynamic) diameter and (D) Zeta potential as a function of HA to CHI charge ratio, [-]/[+]. Ionic strength was adjusted to 300 mM with CaCl2 (filled square) or NaCl (empty circle). pH of the biopolymers was adjusted to 6.25 before mixing them. Inset: Zoomed-in graph of Z-average diameter versus charge ratio (Data points are fitted to straight lines. Transition point is determined as the point where the two lines intersected. Dashed lines are for NaCl while the solid lines are for CaCl2).



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Effect of Glutamate as a Chitosan Salt in the NaCl Solution. Although small ions are usually used as counterions of polyelectrolytes for complex coacervation, it is imperative to study the potential of chitosan-glutamate for cell encapsulation studies. Here, it should be emphasized that glutamate is not covalently attached to the chitosan chain in any way but rather is a counterion. For the same degree of deacetylation, chitosan-chloride (CHI) and chitosan-glutamate (CHI-G) have different monomer molecular weights, which are taken as 198.6 g/mole and 308.3 g/mole, respectively.81 Meanwhile, their pKa’s and charge behaviour at different pH’s are assumed to be the same.82 The analysis of the plot of dH vs. charge ratio (Figure 3C) demonstrated [-]/[+] of 0.56 ± 0.02 as the point of transition from coacervates to precipitates. For HA/CHI-G system, light microscopy (Figure S3A-C) and visual inspection images (Figure S3E) indicate that the maximum in the plot of turbidity vs. charge ratio (Figure 3A) corresponded to the point where coacervate droplets were no longer detected, i.e. precipitate particles were the only species present at [-]/[+] > 0.71. It was surprising to see that the coacervation region was extended to [-]/[+] = 0.71 for the HA/CHI-G mixture, which was 0.61 for HA/CHI. The intensity vs. charge ratio (Figure 3B) plot also shows a more extended window for the coacervation of the HA/CHI-G system than of the HA/CHI. This result can be explained considering the Hofmeister series. Chaotropic ions increase the solubility of polymers while kosmotropic ions have the opposite effect. Here, Cl- ion, being in the middle of the Hofmeister series, is assumed to be unresponsive to polymer solubility.60 Meanwhile, glutamate is considered as a chaotropic ion as hydrogen bonds are broken following the protonation of water molecules by glutamate.83 The increased solubility of CHI-G compared to CHI then causes the coacervation window to extend for the former polymer. Also, Perry et al.60 have attributed the inhibition of coacervation to the hardness of the ions. Cl- is a more compact/hard ion with a higher charge density than glutamate, for which charge is dispersed due to both the larger size of the ion and its zwitterionic character. Thus, it becomes inevitable for the HA/CHI-G system to give a larger coacervation region in comparison to HA/CHI. As for the HA/CHI pair, stoichiometric charge neutralization was not fully accomplished for HA/CHI-G. In other words, zeta potential (ζ) of coacervate suspension was not near or equal to zero ACS Paragon Plus Environment

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even for samples with highest turbidity, i.e. ζ = 6.19 ± 0.67 mV at [-]/[+] = 0.71 (Figure 3A,D). This result can also be explained with the same factors (charge mismatch, chain flexibility and disproportionation) as above. Although these results allowed us to work at higher charge ratios with the HA/CHI-G system than for HA/CHI, we decided to exclude HA/CHI-G coacervation for cell encapsulation studies that will be described below. The main reason behind the exclusion of HA/CHI-G was that this system had not reached scattering intensity maxima at [-]/[+] of 0.31, which was the point of optimum coacervation for the HA/CHI system. In fact, preliminary studies done at charge ratio of maximum scattering intensity, []/[+] = 0.48 for HA/CHI-G system did not indicate it as a substantially more preferential medium for cells (data not shown). This postulation is supported by a study on alginate/chitosan coacervate capsules, where cell viability was higher when chitosan-chloride was used instead of chitosan-glutamate at the same polymer molecular weight and degrees of deacetylation.84

Figure 3. (A) Turbidity, (B) Total scattering intensity (count rate), (C) Z-average (hydrodynamic) diameter and (D) Zeta potential as a function of HA to CHI charge ratio, [-]/[+]. Filled triangle: ACS Paragon Plus Environment


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chitosan-glutamate (CHI-G); empty circle: chitosan-chloride (CHI). Ionic strength was adjusted to 300 mM with NaCl. pH of the biopolymers was adjusted to 6.25 before mixing them. Inset: Zoomed-in graph of Z-average diameter versus charge ratio (Data points are fitted to straight lines. Transition point is determined as the point where the two lines intersected. Dashed lines are for HA/CHI while the solid lines are for HA/CHI-G.) Microstructure of HA/CHI Coacervates. The microstructure of HA/CHI coacervates was first examined by ESEM. With this technique, it is usually possible to examine the pore structure of the coacervates in a saturated water vapor environment which enables imaging with minimal drying. The application of fixative on coacervates before ESEM had no significant impact on the pore sizes when compared to native coacervates (Figure S4). In HA/CHI coacervates (three days after centrifugation), there were numerous small pores inside the bigger pores, resulting in a highly porous structure (Figure 4A,C). Those pores established an interconnected network even though their sizes were not uniform. Coacervates prepared in either NaCl or CaCl2 had pore diameters ranging from 5 to 500 µm. Mean pore sizes varied with salt type use; i.e. average mean diameters of 26 µm and 19 µm were detected for pores within coacervates in CaCl2 and NaCl solutions, respectively (Figure 4A,C). Since water content was relatively less in coacervates where ionic strength was adjusted with NaCl, pores were somewhat shrunk in this relatively less hydrated network. After lyophilization, HA/CHI coacervates were also examined by SEM. SEM micrographs showed that the pores of the lyophilized coacervates were much smaller than the native (wet) ones (Figure 4B,D). This result can be attributed to the collapse of the pores and shrinkage of the microstructure after the removal of water by freeze-drying.


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Figure 4. ESEM and SEM images of HA/CHI coacervates where ionic strength was adjusted to 300 mM with (A,B) CaCl2 and (C,D) NaCl after 72-hours of incubation (Samples could be kept only partially hydrated during the ESEM imaging). Scale bar represents 200 µm in (A), 50 µm in (C), and 20 µm in (B,D). Since the aim of this study was to encapsulate cells within HA/CHI coacervates, their internal pore architecture was expected to influence cell attachment and differentiation by creating interfaces for cells to live. Pore sizes larger than 10 µm would allow adequate exchange of gases, transport of nutrients and the removal of cellular wastes in addition to preservation of viability and cellular function of the encapsulated cells.85 HA/CHI coacervates were found to have a microstructure that supports attachment and/or spreading of cells across the pores of the coacervate. Moreover, choice of HA as a component of our coacervates has made it advantageous for cell attachment and proliferation since HA is known to interact with CD44 or RHAMM cell surface receptors.86-88 Degradation Behaviour of the Coacervates. Biodegradation of a scaffold is a very important factor for successful regeneration of the tissues. Degradation should only create non-toxic by-products while providing an adequate environment for the newly grown/formed tissue.89 Degradation rate of HA which ACS Paragon Plus Environment


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was found to be dependent on the ratio of HA to CHI in a hydrogel90 could be slowed down by addition of catalase, if needed.91,92 In our study, in vitro degradation of HA/CHI and HA/CHI-G coacervates at various time points were also investigated (Figure 5). Both types of coacervates degraded substantially during the first week, i.e. 63.2 ± 0.9 % and 62.4 ± 1.5 % of original weight were lost for HA/CHI and HA/CHI-G coacervates, respectively. The degradation rate slowed down in the following two weeks after which the weight remained constant at 34.6 ± 2.4% for HA/CHI and 29.9 ± 2.6% for HA/CHI-G coacervates. The shape of the coacervates remained intact until the end of 120 days of degradation. The high degree of weight loss in the first few days would be a result of water loss from the coacervate due to the increment of the pH in coacervate (data not shown), while integrity and size of the coacervate-scaffold did not change as much. Otherwise, it would not be possible to observe appearance of ECM after 21 days (as shown later by results of morphology staining). In order to show whether or not the instability of the hydrogel led to cell loss over time, Quant-iT™ PicoGreen® dsDNA Assay (Thermo-Fisher Scientific, USA) was also carried out for the dsDNA quantification (data not shown). The pattern of increase in DNA amount, indicating cell proliferation for each coacervate sample, was observed throughout 14 days of incubation. Thus, we concluded that coacervate degradation did not lead to a considerable amount of cell loss over time.


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Figure 5. In vitro degradation behavior of HA/CHI and HA/CHI-G coacervates in PBS containing sodium azide (pH = 7.5) at different incubation time intervals (up to 120 days). Values are reported as mean ± SD (n = 3). Characterization of rBMSCs and Cell Encapsulation. Before the encapsulation of cells within coacervates, cultures of rBMSCs were analyzed for the expression of MSC-specific cell-surface markers. In accordance with the standards of International Society for Cellular Therapy (ISCT) and literature on the most common surface marker molecules to describe MSCs,93-99 two criteria were followed: (i) expression of CD29, CD44, CD73, CD90, CD105, CD146, CD166 and STRO-1 are the positive markers and (ii) CD11a, CD2, CD31, CD34, CD45, CD117 and HLA-DR are the negative markers for the MSCs. According to flow cytometry experiments performed with cells before encapsulation, rBMSCs were negative for the hematopoietic lineage markers of CD11a (1.27%), CD31 (2.10%) and CD45 (1.40%) and positive for CD29 (96.59%) and CD90 (98.26%) (Figure 6).



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Figure 6. Flow cytometry histogram obtained by FACSCalibur, (A) CD90, (B) CD45, (C) CD29, (D) CD31 and (E) CD11a labeled rBMSCs. Positive gated cell fractions were indicated on histograms. To assess the potential of HA/CHI coacervates as a cell encapsulation system, it was examined whether the biopolymers or the encapsulation process affected the viability and the morphology of the rBMSCs. For cartilage tissue engineering, the use of a 3D matrix and high cell-density cultures were found necessary for chondrogenesis.100-103 To study the viability and spreading of the cells, rBMSCs were encapsulated at a concentration of 500,000 cells/coacervate-sample (1,000,000 cells/mL of coacervate) and evaluated via Live/Dead assay staining after three weeks in culture. By using this assay, live cells within the coacervates were stained as green while dead cells within coacervates were stained in red. Figure 7A,E show stained rBMSCs in coacervates one day post-encapsulation (ionic strength was adjusted to 300 mM with either NaCl or CaCl2). The vast majority of the rBMSCs survived after the coacervation process; i.e. cell viabilities were around 90 ± 2.8% and 93 ± 0.8% (Figure 7I) for coacervates prepared using CaCl2 and NaCl, respectively. According to ANOVA analysis, cell viability did not significantly change with time (F(3) = 1.038 and p = 0.411). Based on stainings at different time points, rBMSCs, which initially had a rounded morphology, started to extend their ECM after 7 days of incubation as shown in Figure 7B,F and Figure S5. At days 14 (Figure 7C,G) and 21 (Figure 7D,H; Figure 8A-D) cells perceptibly exhibited a more spread morphology (fibroblast-like) when compared to earlier time points, while still showing good cell viability (≥84%) with little cell death.


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Figure 7. Images of encapsulated rBMSCs after Live/Dead assay analysis representing the cell viability inside HA/CHI coacervates prepared using (A-D) CaCl2 and (E-H) NaCl solutions after (A,E) 1 day, (B,F) 7 days, (C,G) 14 days and (D,H) 21 days of incubation. Living cells were stained by calcein-AM (green), and dead cells were stained by ethidium homodimer-1 (red). The scale bars represent 200 µm, (I) the graph shows the mean values ± standard deviations for live cells from 3 different zones for each experimental group.



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Figure 8. Images of encapsulated rBMSCs after Live/Dead assay analysis representing the cell viability and morphology inside HA/CHI coacervates prepared using (A,C) CaCl2 and (B,D) NaCl solutions after 21 days of incubation. Living cells were stained by calcein-AM (green), and dead cells were stained by ethidium homodimer-1 (red). The scale bars represent (A,B) 50 µm and (C,D) 20 µm. Further, morphology staining was applied after 3 and 21 days of incubation, i.e. DAPI stained the nucleus in blue while the Alexa Fluor 568 phalloidin stained the actin filaments in red color. Similar cytoskeleton organization was observed in both coacervates using different salt solutions (Figure 9A,B). Cells exhibited a spherical cellular morphology with little spreading after 72 hours of incubation. The spherical shape of cells up to three days of incubation was also confirmed with optical light microscopy without cell-staining (Figure S6). Here, it should be noted that coacervates were transparent enough for direct visualization of cells. After 21 days of incubation following the encapsulation process, cells gained a well-spread morphology with distinguishable F-actin filaments unless stimulated by special inducers such as growth factors (Figure 9C,D). However, there was still no big difference in terms of morphology among the spread cells with the usage of different salt solutions after 21 days of incubation.


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Figure 9. Images of encapsulated rBMSCs stained with phalloidin/DAPI after (A,B) 3 days, (C,D) 21 days of incubation, representing the cell morphology inside HA/CHI coacervates prepared with (A,C) CaCl2, (B,D) NaCl solutions. The scale bars represent 10 µm. The results of cell morphology and Live/Dead studies confirmed that the HA/CHI coacervates offered a favorable scaffold for cell encapsulation and spreading, meaning that both coacervates established a notably biocompatible platform. Many studies suggested that using HA and CHI polymers together could play a significant role for cellular organization.16,17,104-106 Tan et al.16 and Remya et al.17 prepared hydrogels using N-succinylchitosan (S-CS) with aldehyde hyaluronic acid (A-HA) and chitosan with hyaluronic acid dialdehyde (CHDA), respectively. It was shown that these hydrogels allowed survival of chondrocytes after encapsulation and preserved their morphology in vitro. However, the formation of both hydrogels required chitosan or hyaluronic acid to be chemically modified which is both tedious and costly especially considering large-scale production. In addition, further in vivo studies of CHDA hydrogel were only partially successful in regeneration of hyaline and fibrous cartilage.107



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CONCLUSION Coacervate-based scaffolds have become increasingly advantageous for tissue regeneration. In this study, it was demonstrated that HA/CHI coacervates prepared using either NaCl or CaCl2 salt solutions could provide favourable microenvironments to support the cultivation and proliferation of rBMSCs in in vitro cultures. For this purpose, prepared coacervate suspensions were first optimized to find the regions and transition points of phase separation as coacervation or precipitation. At charge ratio of [-]/[+] = 0.31, coacervate suspensions had the highest yield (after centrifugation) with better droplet morphology and good size distributions. Moreover, the centrifuged suspensions confirmed the formation of an immiscible coacervate layer which was qualified as scaffolding material. According to our rheology measurements (data not shown), HA/CHI coacervates have two-three orders of magnitude higher zero-shear rate viscosities over physical gels prepared either with HA or CHI above their entanglement concentrations. The high viscosity and gel-like response of HA/CHI coacervates, which will be explored systematically in our future studies, make them especially suitable as scaffolds. This study also presents a successful protocol for the preparation of a novel scaffold for tissue engineering and biomaterial purposes. As a scaffold, coacervates are non-cytotoxic and have an efficient cell entrapment capacity. This coacervate-based encapsulation system and used polymers (being a member of glycosaminoglycans (GAGs), or having a structural similarity to GAGs as in HA and CHI, respectively) may have remarkable potential for the treatment of cartilage defects for future trials. These scaffolds will be further used for in vitro chondrogenic differentiation and in vivo experiments to achieve cartilage-like tissues.


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ASSOCIATED CONTENT Supporting information. Detailed optical microscopy images of HA/CHI and HA/CHI-G mixtures (Figure S1-S3), ESEM images of HA/CHI coacervates with and without glutaraldehyde fixation (Figure S4), higher magnification images of stained rBMSC encapsulated coacervates using Live/Dead assay (Figure S5) and semi-opaque appearance of rBMSC encapsulated coacervate (Figure S6). Supporting Information is available free of charge on the ACS Publications website. AUTHOR INFORMATION Corresponding Author * Gamze Torun Kose, e-mail: [email protected]; * A. Basak Kayitmazer, e-mail: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Conflicts of Interest The authors declare no competing financial interest. ACKNOWLEDGEMENT We thank Dr. Bengu Borkan for her help with ANOVA analysis. This research was supported by Yeditepe University Research Funds.



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