On the Correlation between the Microscopic Structure and Properties

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Cite This: ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

On the Correlation between the Microscopic Structure and Properties of Phosphate-Cross-Linked Chitosan Gels Pasquale Sacco,*,† Francesco Brun,‡ Ivan Donati,† Davide Porrelli,§ Sergio Paoletti,† and Gianluca Turco§ †

Department of Life Sciences, University of Trieste, Via Licio Giorgieri 5, I-34127 Trieste, Italy Department of Engineering and Architecture, University of Trieste, Via A. Valerio 6/1, I-34127 Trieste, Italy § Department of Medicine, Surgery and Health Sciences, University of Trieste, Piazza dell’Ospitale 1, I-34125 Trieste, Italy ‡

S Supporting Information *

ABSTRACT: Ionic chitosan gels fabricated using multivalent anions, tripolyphosphate (TPP) or pyrophosphate (PPi), respectively, have been investigated as potential biomaterials to be used in tissue engineering. Starting from the hypothesis that the polymer mesh texture at the microscale affects the final performance of the resulting materials, an innovative image analysis approach is presented in the first part of the article, which is aimed at deriving quantitative information from transmission electron microscopy images. The image analysis of the (more extended) central area of the gel networks revealed differences between both the cross-linking densities and pore size distributions of the two systems, the TPP gels showing a higher connectivity. Chitosan−TPP gels showed a limited degradation in simulated physiological media up to 6 weeks, reasonably ascribed to the texture of the (more extended) central area of the gels, whereas PPi counterparts degraded almost immediately. The release profiles and the calculation of diffusion coefficients for bovine serum albumin and cytochrome c, herein used as model payloads, indicated a different release behavior depending on the polymer network homogeneity/inhomogeneity and molecular weight of loaded molecules. This finding was ascribed to the marked inhomogeneity of the PPi gels (at variance with the TPP ones), which had been demonstrated in our previous work. Finally, thorough in vitro studies demonstrated good biocompatibility of both chitosan gels, and because of this feature, they can be used as suitable scaffolds for cellular colonization and metabolic activity. KEYWORDS: chitosan, gel, image analysis, molecule release, degradation, tissue engineering

1. INTRODUCTION Polymer-based scaffolds, either in dried or wet state, play a pivotal role as biomaterials in tissue engineering and regenerative medicine because of their ability to retain a large amount of water and to resemble the natural three-dimensional (3D) tissue architecture of different tissues, the extracellular matrix, thus aiding cellular growth and proliferation.1−5 Besides providing mechanical support, scaffolds host cells and/or molecules−macromolecules such as drugs or growth factors within their network, enabling cells to properly grow (or differentiate) into desired tissues. Such features frame these matrices as smart depots for the in situ delivery of molecular therapeutics and/or cells. Additionally, entangled polymer networks might conceal their payload, preventing the recognition by immune system cells such as macrophages, thereby warranting a controlled and sustained release of such molecules.6 The fine regulation of the cross-linking density at the microscale is therefore a critical issue for obtaining positive clinical outcomes when designing biomaterials.7 This is mainly due to three aspects: (i) the ability of cells to infiltrate and subsequently colonize scaffolds; (ii) the possibility to tuning the © XXXX American Chemical Society

leakage of molecules according to desired purposes, thus (iii) favoring tissue remodeling and, at the same time, limiting the failure of implants because of uncontrolled immune response upon implantation of biomaterials. Chitosan is a biopolymer of natural origin, which has been extensively studied as smart depot of molecule therapeutics8 because of its low toxicity, biodegradability, and FDA-approval as a material for the development of medical devices.2 From a structural point of view, chitosans are considered as a family of water-soluble polysaccharides composed of two building units: β-1 → 4 linked, i.e., N-acetyl-glucosamine (A unit, the minor component) and its deacetylated form glucosamine (D unit, the major component), randomly distributed along the polymer chain.9 The fraction of D-units (FD) greatly affects the physical−chemical properties of chitosans: the primary amine groups of D-units are protonated in acidic conditions, thus ensuring chitosans to behave as polycations; furthermore, they Received: January 31, 2018 Accepted: March 13, 2018

A

DOI: 10.1021/acsami.8b01834 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

2. MATERIALS AND METHODS

may be chemically modified, e.g., by covalent addition of chemical groups. As a result of these considerations, chitosans can ionically/covalently interact with specific molecules, simply defined as gelling agents, thereby increasing point-by-point connectivity among chains and consequently assembling into matrices endowed with peculiar texture, mesh and pore sizes.10 To date, thermally responsive chitosan gels have been reported as the results of an intriguing effort for the preparation of parenteral in situ forming depots.11,12 They consist of chitosan solutions (at low temperature), which turn into semisolid gels when administered in the human body. However, critical aspects for these systems concern their lack of storage stability at/below room temperature, inadequate mechanical performance, and inability to finely tune the release of loaded molecules. Some of the aforementioned limitations were overcome by changing the gelling agent13 or by combining chitosan mixtures with solid-state biomatrices.14,15 On the basis of our previous contributions,16−18 the present article focuses on chitosan−tripolyphosphate (TPP) and chitosan−pyrophosphate (PPi) gels as potential biomaterials to be used in tissue engineering. Contrary to most available systems (nanoparticles and nano/microgels) based on chitosan and multivalent anions like TPP or PPi,19−21 these gels are solid cylindrical networks fabricated by a dialysis-based technique, characterized by mechanical properties (shear modulus, G) in the range of 103−105 Pa.18 A qualitative analysis of transmission electron microscopy (TEM) images has demonstrated that the type of cross-linker strongly affected the homogeneity/ inhomogeneity of their mesh at the microscale.18 A quantitative approach is certainly needed to get further insight into such chitosan networks. In the first part of this article, a new image analysis approach aimed at deriving quantitative information about the polymer density and the mesh size is presented. By considering image segmentation with subsequent automated analysis, the distribution of the pore thickness (i.e., the void space surrounded by polymer chains) is assessed directly from TEM images by adopting the concept of “maximal inscribed circle”. The computed values are supposed to correlate with the average mesh size and therefore they can provide insight into the texture and space orientation of the considered structures. The inspection of TEM images is in fact an established tool for deriving information about the texture of gels. Several reports have been published in the literature aiming at this, and some of them also include image analysis approaches to quantitatively support the visual inspection of the acquired images. Quantitative texture analysis by the computation of the fractal dimension and the gray-level cooccurrence matrix was proposed for instance by Brun et al.,22 whereas the application of the watershed segmentation to assess the average mesh size was used in ref 23. In ref 24, classical stereology25 was considered for the assessment of the mean volume of the pores from binarized TEM images. In the second part of this work, we report on different properties as correlated with the results of the texture analysis, thus for the first time providing a rational base for the use of our systems for tissue engineering purposes. Specifically, we describe how the different reticulation at the microscale affects the degradation of chitosan gels in simulated physiological media, whereas the release of selected payloads depends on both polymer/cross-linker network and molecular weight of the loaded molecules. Finally, we report a thorough investigation about the in vitro cytocompatibility of gels and show how the latter represent adequate substrates for cellular anchoring.

2.1. Materials. Medium-molecular-weight chitosan (fraction of acetylated units, FA = 0.23 determined by 1H NMR) was purchased from Sigma-Aldrich and purified by precipitation with isopropanol, followed by dialysis against deionized water. The relative molar mass of chitosan was determined by intrinsic viscosity measurements (conditions: AcOH/AcNa buffer, 20 mM, pH 4.5; NaCl, 100 mM, T = 25 °C) with [η] = 836 mL/g. The viscosity-average molecular weight ( M v ) was calculated using the Mark−Houwink−Sakurada equation reported in ref 26 for the used conditions, with K = 0.00843 and α = 0.92 as parameters, and it was found to be 270 000.18 By considering the contribution of N-acetyl-glucosamine (molecular weight of the monomer, 203 g/mol) and that of glucosamine units (molecular weight of the monomer in its base form, 161 g/mol), the molecular mass of the chitosan repeating unit resulted 171 g/mol, corresponding to a viscosity-average degree of polymerization, DPv , of about 1580. Sodium tripolyphosphate pentabasic-Na5P3O10-(TPP ≥ 98.0%), sodium pyrophosphate tetrabasic-Na4P2O7-(PPi ≥ 95%), NaCl, CaCl2·2H2O, Na2HPO4, D-glucose, glycerol (ReagentPlus ≥ 99.0%), bovine serum albumin (BSA, molecular mass 66 430), cytochrome c (molecular mass 12 384) from equine heart, Triton X-100, Hoechst 33258, and phosphate buffer saline (PBS) were all purchased from Sigma-Aldrich Chemical Co. Other chemicals for the preparation of Hank’s Balanced Salt Solution (HBSS) were purchased from Carlo Erba, Italy. The Lactate Dehydrogenase Activity Assay Kit (“LDH mixture”) and AlamarBlue reagent were purchased from Sigma-Aldrich Chemical Co. 2.2. Chitosan Gel Preparation. Wall-to-wall cylindrical gels were obtained by the slow ion diffusion technique.16 Briefly, a solution composed of chitosan (3% w/v) and glycerol (5% v/v) in 0.2 M acetic acid was casted into a mold (diameter = 22 mm, thickness = 2.5 mm) closed by two dialysis membranes (average flat width 33 mm, cutoff 14 000; Sigma Aldrich, Chemical Co.) and fixed by double circular stainless iron rings. Dialysis membranes with cutoff 2000 were used only for the preparation of gels containing cytochrome c as payload (see below). The system was hermetically sealed and immersed into a gelling solution (V = 50 mL) of 0.2 M acetic acid containing multivalent anions (TPP or PPi), NaCl (150 mM), and glycerol (5% v/v). The concentration of TPP and PPi in the gelling solution was 29.4 mM in both cases; thus, the molar ratio (r) of the cross-linker to the repeating unit of chitosan (r = [cross-linker]/[chitosan]r.u.) was set to 3.8 at the beginning of the dialysis.18 Ion diffusion proceeded for 24 h under moderate stirring at room temperature, allowing gel formation. It was already shown that, at the end of dialysis, only a limited amount of either cross-linker diffused within chitosan solutions.16 2.3. Image Acquisition. TEM imaging at 1000× and 5000× was carried out on TPP and PPi gel cross sections. Gel slices of approximately 1 mm thickness were dried in ethanol, 70% v/v, and overnight contrasted using uranyl acetate, 0.2% w/v. Subsequently, slices were totally dried in ethanol, 100% v/v, and finally embedded in epoxy resin after complete removal of the alcohol. Ultrathin sections were cut using a microtome (equipped with a diamond blade) and gently deposited onto Nickel grids. The images were acquired using a Philips EM transmission electron microscope. 2.4. Image Analysis. TEM images were segmented to classify polymer pixels and void pixels by applying in the following order: background estimation and subtraction,27 edge-preserving smoothing,28 and global thresholding. Starting from the segmented (“black and white”) images, a first measure of density, i.e., the number of polymer pixels with respect to the total number of pixels, can be derived. Then, the Euclidean distance transform was computed and all of the values below a visually assessed threshold were forced to be 0 (black) to avoid spurious (small) void areas and to capture only the essential meshes. This operation is sometimes called h-minima transform.29 Each connected component of the modified distancetransformed image is then assumed as a single mesh for characterization. For each mesh, a skeletonization algorithm was then applied. The maximum value of the (unmodified) distance transform along the B


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Figure 1. Image analysis steps to assess the polymer density from the pore size distribution for the PPi case. (A) Original image as acquired by the TEM system. (B) Pre-processing composed of background subtraction and edge-preserving smoothing. (C) Threshold analysis of the image. (D) Distance transform of white pixels in image (C). (E) Skeletonization of the filtered distance-transformed image. (F) Superimposition of the maximal inscribed circles within the void areas used to characterize the pore size. The scale bar is 5 μm. [Only one image at 1000× magnification is presented, but images at 5000× were also considered.]

Figure 2. Image analysis steps to assess the polymer density from the pore size distribution for the TPP case. (A) Original image as acquired by the TEM system. (B) Pre-processing composed of background subtraction and edge-preserving smoothing. (C) Threshold analysis of the image. (D) Distance transform of white pixels in image (C). (E) Skeletonization of the filtered distance-transformed image. (F) Superimposition of the maximal inscribed circles within the void areas used to characterize the pore size. The scale bar is 5 μm. [Only one image at 1000× magnification is presented, but images at 5000× were also considered.] are summarized in Figure 1 for the PPi case and in Figure 2 for the TPP case. 2.5. Release Studies. BSA and cytochrome c were selected as model payloads to be encapsulated within gels. The solution

skeleton is assumed as the radius of the maximum inscribed circle. The distribution of the diameter of these circles is considered here as pore size distribution, and it is supposed to correlate with the mesh size of the considered polymer chains. The considered image processing steps C


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[(Wt/W0) − 1] × 100, where W0 is the weight of gels at time 0, whereas Wt is the weight of the same at a selected time. 2.7. Cell Culture and Preparation of Gels for in Vitro Tests. Mouse fibroblast-like (NIH-3T3) cell line (ATCC CRL1658) was used for the in vitro experiments. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), High glucose (4.5 g/L), with NaPyruvate (PromoCell GmbH, Germany), supplemented with 10% heat-inactivated fetal bovine serum (Sigma Aldrich, Chemical Co.), 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM Lglutamine in a humidified atmosphere of 5% CO2 at 37 °C. In all experiments (except for cell adhesion/colonization tests), 25 000 cells/ well were plated on 24-well plates using 300 μL of medium for each well. After complete adhesion, the culture medium was discarded and the cells were rinsed with PBS once. Finally, 600 μL of fresh medium was added to the cells before being treated with gels. For in vitro studies, the solution composed of chitosan (3% w/v) and glycerol (5% v/v) in 0.2 M acetic acid was supplemented with 22 μL of 100 U/mL penicillin and 100 μg/mL streptomycin as final concentration and casted into the mold. At the end of dialysis, TPP− chitosan or PPi−chitosan gels were washed for 4 h with 20 mL of PBS (VPBS/Vgel = 21) to eliminate all residual traces of unbound crosslinker and UV-sterilized for 1 h (30 min of irradiation for each side of sample). Macroscopic gels were subsequently cut under sterilized conditions into small cylinders (diameter = 4 mm, thickness = 2.5 mm) using a disposable biopsy punch (Kai Medical, Japan) and stored at 4 °C until their use. Tested materials were conditioned in PBS buffer at 37 °C for 1 h and thereafter deposited on the cell layer (Vmedium/Vgel = 19). 2.8. Evaluation of Membrane Damage: Lactate Dehydrogenase (LDH) Assay. In vitro cytotoxicity of TPP−chitosan and PPi−chitosan gels toward NIH-3T3 cells was evaluated by assessing the cell membrane damage and consequent release of the LDH enzyme from the cytoplasmic compartment. After 24 and 72 h of treatment, culture media (65 μL) of treated/untreated cells were collected and centrifuged at 100 × g for 5 min. Resulting supernatants (45 μL) were transferred into a 96-well plate and mixed with 90 μL of LDH mixture. Samples were incubated at room temperature for 25 min under dark conditions. The reaction was stopped by adding 13.5 μL of HCl (1 M). The absorbance of samples was measured using a TECAN Microplate Reader at wavelengths of 490 and 690 nm. Untreated cells were used as a negative control, whereas lysed cells were used as a positive control. Each material test was conducted at least in triplicate. Data are expressed as percentage by normalizing the absorbance of treated/untreated cells at time investigated (24 and 72 h) to the absorbance of the lysed cells. Data are reported as the mean of three independent experiments (±SD). 2.9. Evaluation of Cellular Metabolism: AlamarBlue Assay. After collecting samples for LDH assay, cell culture media were discarded whereas tested gels were carefully removed from each well. Cells were rinsed with PBS once and incubated with 300 μL/well of AlamarBlue reagent (10% v/v in complete DMEM medium) for 4 h at 37 °C. At the end of this time frame, 150 μL of incubation medium were transferred in a black 96-well plate and the fluorescence was measured using a FLUOStar Omega-BMG Labtech spectrofluorometer (λex = 544 nm; λem = 590 nm). Each material test was conducted at least in triplicate. The metabolic activity of cells is expressed as percentage by normalizing the fluorescence of treated cells at time investigated (24 and 72 h) to the fluorescence of untreated cells. Data are reported as the mean of three independent experiments (±SD). 2.10. Evaluation of Cell Morphology: Optical Microscopy. In vitro cytotoxicity of TPP−chitosan and PPi−chitosan gels toward NIH-3T3 cells was also evaluated qualitatively in terms of cell morphology and spreading. Photographs of treated/untreated cells were taken at time investigated (24 and 72 h) with an Exacta Optech microscope equipped with a Pentax digital camera using a 40× objective. The cytotoxic scale described in30 was used as a qualitative parameter to grade the cytotoxicity level of TPP- and PPi gels. 2.11. Cell Adhesion/Colonization Test. UV-sterilized small chitosan gel cylinders (diameter = 6 mm, thickness = 2.5 mm) were conditioned in PBS buffer at 37 °C for 1 h and placed in a 96-well

composed of chitosan (3% w/v) and glycerol (5% v/v) in 0.2 M acetic acid was supplemented with either BSA or cytochrome c (22 mg as final mass) and casted into the mold. At the end of the dialysis, the excess of gelling solution was gently removed using filter papers and gels were immediately immersed in PBS (VPBS/Vgel = 16, final volume = 15 mL). Samples were subsequently placed at 37 °C under mild shaking. At selected times, 2 mL of incubation medium were withdrawn and replaced with the same amount of fresh PBS. The concentration of released payloads was quantified spectrophotometrically at a wavelength of 279 nm in the case of BSA and 550 nm in the case of cytochrome c using an Ultrospec 2100 Pro UV/visible spectrophotometer. PBS buffer was used as blank. Quartz cuvettes with 1 cm optical path were used to record the absorbance. A calibration curve (R2 > 0.99) using PBS as the medium was performed prior to release experiments to identify the linear correlation between absorbance (range 0−1) and the concentration of payloads. The drug diffusion coefficients were calculated fitting the release data by Fick’s second law (eq 1) in the first 4 h of incubation. This allows neglecting contributions from gel degradation and swelling.

D ∂ ⎛⎜ ∂C ⎞⎟ ∂C ∂ 2C R = +D 2 R ∂R ⎝ ∂R ⎠ ∂t ∂Z

(1)

where D is the drug diffusion coefficient in the gel, t is the time, C is the drug concentration (mass/volume) in the gel, and R and Z are the radial and axial axes, respectively. The initial condition is (eq 2)

C(Z , R , t = 0) = C tot,0 0 ≤ Z ≤ Zc 0 ≤ R ≤ R c Cr(t = 0) = 0 (2) where Zc and Rc are the gel height and radius, respectively, whereas Ctot,0 is the initial drug concentration in the gel and Cr is the drug concentration in the release medium. The gel domain was subdivided into 50 control volumes in the radial direction and 25 control volumes in the axial direction. Interfacial diffusion coefficient Di was assigned to the external surface of the control volumes facing the release environment, whereas diffusion coefficient D was assigned to the inner part of the gel (see Supporting Information for further details). The external surface is defined as ZδZ = Zc/25 and RδR = Rc/50 for the axial and radial axes, respectively. The boundary conditions are defined as (eq 3)

C tot(Z , R , t ) = C in(Z in − 2Z δZ , R in − R δR , t ) + Cout(Z δZ , R δR , t )

(3)

Zc = Z in + 2Z δZ ; R c = R in + R δR where Cin and Cout are the protein concentrations in the inner part and in the external surface of the gel, respectively. The protein present in the reservoir at each time point as a function of protein released is calculated as (eq 4) VrCr(t ) = πR c2ZcC in −

Zc

∫0 ∫0

Rc

C tot(Z , R , t )2πR dR dZ

(4)

where Vr is the volume of the release medium. 2.6. Degradation Studies. The structural stability of gels was verified in homemade Hank’s Balanced Salt Solution (HBSS). The composition of HBSS is as follows: NaCl 8 g/L, NaHCO3 0.35 g/L, KCl 0.4 g/L, KH2PO4 60 mg/L, MgSO4·7H2O 0.2 g/L, CaCl2·2H2O 0.185 g/L, Na2HPO4 48 mg/L, D-glucose 1 g/L, and final pH 7.4. Gels were fabricated as indicated in Section 2.2. At the end of dialysis, TPP−chitosan or PPi−chitosan gels were laid down on filter papers to blot the excess of gelling solution and weighed using an analytical balance (time 0). Subsequently, gels were immersed in HBSS (VHBSS/ Vgel = 11, final volume = 10 mL). Samples were then placed at 37 °C under mild shaking. At selected times, samples were removed from incubation medium, properly blotted, and weighed. Finally, gels were placed again in fresh HBSS (which is replaced at each time point). Data are reported as % of mass gained/lost with respect to the initial weight of three samples (±standard deviation (SD)), calculated as D


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ACS Applied Materials & Interfaces plate with 200 μL of a cell suspension (200 000 cells/mL) in complete DMEM medium. After 24 h of incubation, samples were transferred in clean wells, washed with PBS, and incubated with 200 μL of AlamarBlue reagent as described above. After 4 h of incubation at 37 °C, the fluorescence of the collected medium was measured. Fluorescence values were recorded together with those from the blank signal, measured from gels without cells. Confocal laser scanning microscopy (CLSM) analyses were performed to evaluate the ability of NIH-3T3 cells to adhere atop gels and, consequently, colonize matrices. After 24 h of incubation, samples were washed with PBS and fixed with paraformaldehyde (4% v/v in PBS) for 30 min at room temperature. Cells were permeabilized using 0.1% v/v Triton X-100 in PBS for 15 min at room temperature and blocked with 1% w/v BSA in PBS for 15 min at room temperature. Samples were washed with PBS and incubated for 5 min at room temperature with Hoechst (5 μg/mL) in PBS to stain cell nuclei. Finally, samples were washed in PBS and quickly rinsed with deionized water to remove the residual PBS salts and mounted on glass microscope slides using Mowiol mounting medium (Sigma Aldrich). CLSM analyses were performed using a Nikon Eclipse C1si confocal laser scanning microscope with a Nikon Plan Apochromat 40× objective. Resulting stacks of images were analyzed using ImageJ software. 2.12. Statistical Analyses. Experimental data derived from biological tests were analyzed using the one-way analysis of variance followed by Tukey’s multiple comparison test to compare all groups. A confidence interval of 95% (significant level, α = 0.05) was selected to identify significant differences among groups. No statistical differences are indicated in the article as NS. Statistically significant differences between the pore size distributions of PPi and TPP gels obtained by TEM image analysis were tested using SPSS Statistics 21 (IBM SPSS Statistics; SPSS Inc., Chicago, IL). Because the normality (Kolmogorov−Smirnov test) and equality of variance (Levene test) assumptions were not valid, a nonparametric Mann−Whitney U-test was used. Statistical significance was pre-set at α = 0.05.

what already demonstrated in our previous contribution using Flory’s theory.18 By combining images at different scales (1000× and 5000×), it is possible to refine the pore size distribution by including the effect of the small pores. In this work, the proposed image analysis approach was limited to the 1000× and 5000× images only. These magnifications were considered as adequate to assess the differences in the considered materials, as confirmed by the box plot presented in Figure 3. The Mann−Whitney test

Figure 3. Box plot for the pore size distributions of chitosan gels reticulated by either PPi or TPP considering TEM images. A significant difference has been revealed by comparing the two systems (Mann−Whitney test, *p < 0.05).

applied to the distributions of chitosan−PPi and chitosan−TPP gel pore sizes revealed a statistically significant difference between the two groups (*p < 0.05). The calculated pore size distribution was in the range of 0.1−1.2 μm for both the systems, with a prevalence of small pores noticed for TPP gels. It is worth noting that the proposed image analysis approach presents a few advantages with respect to the methods briefly reviewed in the Introduction. Rather than producing dimensional textural indices as done in ref 22, the proposed approach computes values that directly correlate to real distances in micrometer (knowing the pixel size of the TEM images). Moreover, the method is able to derive a complete distribution of pore sizes instead of computing only a global average value as produced by classical stereology,25 where a standard deviation is usually computed by considering more than one image. Therefore, the presented method can be used also for a local (i.e., in a specific user-defined region-of-interest) inspection of a single TEM image. Also, it avoids the oversegmentation produced by image analysis approaches based on the watershed segmentation, where large pores are separated by spurious watershed lines.23 3.2. Stability of Gels. The physical properties of ionically cross-linked gels are susceptible to pH changes, to which the degree of ionization of both polymer and multivalent ions used as cross-linkers are correlated. When chitosan−TPP/chitosan− PPi gels were placed in HBSS medium and incubated at 37 °C, a similar behavior was noticed for the two gels, albeit largely modulated on the time axis (Figure 4). In general, an initial increase of mass, because of water uptake, is followed by a more or less gradual loss, which can even reach complete gel degradation in the case of PPi. In more detail, chitosan−TPP gels (after a very slight [−8 ± 3%] deswelling in the first 24 h of incubation, in line with our previous findings16 and with those of Franzén et al.31) showed a notable increase of mass (up to about 80%) up to 6 days of incubation. It was abruptly followed by a loss of mass observed up to 2 weeks of incubation, when

3. RESULTS AND DISCUSSION 3.1. Image Analysis. The results shown in Figure 6C of our previous work18 clearly demonstrate that the radial distribution of the polymer chains for the case of the PPi gels is quite different from that of TPP. Whereas in the latter case the distribution is rather homogeneous throughout the sample (but for a minor decrease in the central part), the PPi gel shows a marked radial inhomogeneity, having a definitely larger amount of polymer accumulated at the boundary as compared to that in the core. In a preliminary TEM analysis, such finding was confirmed: the ratio of the average pore size in the boundary zone over that at the central part was found to be 0.76 for the TPP gel and only 0.09 for the PPi case (data not shown). Given the difficulty of clearly identifying a reliable position on the boundary and the need of providing a larger image sampling area for reasons of statistical significance, it was decided to focus the investigation on the central area of both PPi and TPP gels. Figures 1A and 2A show images of the internal structure of chitosan−PPi and chitosan−TPP gels, respectively. A qualitative comparison of the TEM images at 1000× already points to a clearly reduced number of polymer chains per surface unit in the case of chitosan−PPi gels. Confocal imaging of asprepared gels, i.e., without any dehydration step, corroborated this finding (Figure S1 in Supporting Information). The computed average polymer density values (mean ± SD of a set of three images per group) are 0.28 ± 0.02 mm2/mm2 (or 28 ± 2%) for the PPi and 0.43 ± 0.01 mm2/mm2 (or 43 ± 1%) for the TPP case. The different cross-linking densities confirm E


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the gels. The equilibrium interplay of the above factors can be quite complex, not to say of the possibility that kinetic effects manifest on different time scales for the different factors. Clearly, the two systems seem to follow a similar path as a response to a pH increase (namely, swelling followed by deswelling); however, the PPi gel is much faster in covering the time trajectory and, on the observed time scale, it even reaches complete dissolution. Overall, it is evident that the chitosan gel cross-linked with TPP modifies its structure much more slowly and is able to withstand the pH stress much better than that in the PPi case, limiting the degradation in simulated physiological media because of stronger TPP−chitosan binding and ensuing higher cross-linking density (see Section 3.1). 3.3. Release Experiments. The release of BSA and cytochrome c from gels was monitored up to 24 h at pH 7.4 and 37 °C. By comparing the release profiles of payloads (Figure 5A,B), it is clear that both chitosan−TPP and chitosan−PPi gels are more prone to release cytochrome c with respect to BSA in the time frame investigated. Specifically, higher amounts of cytochrome c were quantified in the release medium after 24 h of incubation. These findings can be safely traced back to the different molecular weight of payloads (12 384 for cytochrome c vs 66 430 for BSA), which make the former more rapidly released from the gel networks. The calculation of diffusion coefficients, D, in the first 4 h of incubation (see Table I) validated our assumption, where the best fit of the experimental data for chitosan−TPP gels gave diffusion coefficients of 2.21 × 10−7 and 5.19 × 10−7 cm2/s in the case of BSA and cytochrome c, respectively, meaning that the latter is released more quickly. In parallel, the diffusion coefficients for chitosan−PPi gels were 0.75 × 10−7 and 2.91 × 10−7 cm2/s for BSA and cytochrome c, respectively. The ratio of the D value for chitosan−PPi gels over that of the chitosan−TPP ones clearly indicates that the former are substantially less pervious to solute leakage than the latter ones. A structural consideration stems from the different amount of payloads released by chitosan−TPP and chitosan−PPi gels, where the former system seems to favor the leakage of encapsulated molecules. This is particularly evident in the case of BSA (Figure 5A), where a different release profile was observed up to 24 h. Similar trends were observed in the case of cytochrome c for chitosan−TPP and chitosan−PPi gels (Figure 5B), where both curves tend to overlap after 6 h of incubation. A slight difference in the molecule leakage was observed during the first 4 h of incubation, again with larger quantities of payload released from chitosan−TPP gels. By comparing the

Figure 4. Variation of chitosan−TPP (black circles) and chitosan−PPi (orange circles) gel weights during incubation in HBSS medium at 37 °C and pH 7.4. The weight of gels calculated at each time point was normalized to that at time 0, and data were reported as % of mass gained or lost (±SD, n = 3). Dashed lines are drawn to guide the eye.

about 50% of the original gel mass was eroded. An almost constant slight degradation was then evidenced in the next 4 weeks, as shown in Figure 4, with approx. 65% of total gel mass eliminated after 6 weeks. Chitosan−PPi gels behaved very differently when placed in the same medium, showing a fast, but slighter as compared to that of TPP, swelling in the first 24 hours, followed by a rapid degradation. Herein, the increase of pH tremendously weakened the internal gel structure, which completely degraded after 4 days of incubation. Overall, it is evident that chitosan gels cross-linked with TPP are more able than their PPi counterparts to limit the degradation in simulated physiological media, very reasonably due to their higher cross-linking density (see Section 3.1) and to a stronger TPP binding strength toward chitosan with respect to PPi. Different (and mutually correlated) factors contribute to modulating the interaction of cationic gels with the solvent and with the ionic cross-linkers upon changing pH. Clearly, they all stem from the decrease of charge of the polyelectrolyte network, which, in turn, brings about a series of consequences: (i) a decrease in the number of interaction sites with the anionic cross-linkers, with an ensuing effective decrease in the cross-linking density and in gel stability and strength (below a certain critical value, this may even lead to the breakdown of the 3D integrity of the gel structure); (ii) in turn, the weakening of the entangled polymer network reduces the elastic retraction force and makes the gels more prone to absorb water (swelling phase), as noticed by the initial mass increment; and (iii) reduction in the electrostatically driven osmotic swelling, which leads to a partial release of water from

Figure 5. Cumulative release of BSA (A) and cytochrome c (B) from chitosan−TPP/chitosan−PPi gels in PBS buffer at pH 7.4 and 37 °C. Dashed lines are drawn to guide the eye. F


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Table I. Values of the Diffusion Coefficient (D) and the Interfacial Diffusion Coefficient (Di) for BSA and Cytochrome c in Chitosan−TPP and Chitosan−PPi Gels D (cm2/s)

Di (cm2/s)

system

chitosan−TPP A

chitosan−PPi B

ratio B/A

chitosan−TPP C

chitosan−PPi D

ratio D/C

BSA cytochrome c

2.21 × 10−7 5.19 × 10−7

0.75 × 10−7 2.91 × 10−7

0.34 0.56

7.71 × 10−7 8.53 × 10−7

2.50 × 10−7 3.63 × 10−7

0.32 0.43

Figure 6. Effect of chitosan−TPP and chitosan−PPi gels on cell morphology, membrane damage, cellular metabolism, and ability to host cells. (Panel A) Optical microscopy of mouse fibroblast-like (NIH-3T3) cells treated with gels or untreated. Images were acquired with a digital camera using a 40× objective. (Panel B) LDH assay: cells were treated with gels or untreated, and the release of LDH enzymes from cytoplasm was considered as a parameter of cell membrane integrity. Data were normalized for lysed cells, herein, defined as 100% of LDH released. AlamarBlue assay: cells were treated with gels, and the percentage of viability was normalized to that of untreated cells, herein, defined as the 100% of viability. Data are expressed as mean (±SD) of three independent determinations with at least three replicates in each experiment. NS: no statistical differences. (Panel C) Distribution of NIH-3T3 cell nuclei embedded within chitosan−TPP and chitosan−PPi gel slices. Images were acquired by confocal laser scanning microscopy, and the 3D rendering was performed by ImageJ software.

different polymer network distribution throughout the gels.18 In this context, chitosan−PPi gels are inhomogeneous, albeit continuous, networks, with the prevalence of polymer at the boundary (polymer-rich section) with respect to that in the core (polymer-poor section). Conversely chitosan−TPP gels present a more homogeneous polymer profile. In other words, one should assume that the slower diffusion observed for chitosan−PPi gels is controlled by the denser external boundary (which acts as a rate-determining step), thus hampering the diffusion of loaded molecules with respect to chitosan−TPP gels.

data in Figure 5A,B, it can be argued that differences in terms of molecule release in the two systems are clearly evident for higher-molecular-weight payloads. Obviously, the lower the molecular weight of encapsulated molecule, the lower the influence of gel internal microstructure (cross-linking density and pore size). The higher ability observed for chitosan−TPP gels to promote the release of selected payloads might, at first, be considered as unexpected on the basis of the different average polymer density values, namely, 0.28 ± 0.02 mm2/mm2 for the PPi and 0.43 ± 0.01 mm2/mm2 for the TPP case. However, they could be reasonably accounted for considering the G


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to control. A similar percentage of released LDH was noticed among groups even for a longer time of exposure (72 h), thus suggesting only a slight cytotoxicity by the gels in the early phase of incubation. This was reasonably ascribed to the partial cross-linker leakage from solid networks because both TPP and PPi could give rise to mild/severe cytotoxicity depending on their concentration in the cell culture medium.32,33 The AlamarBlue assay pointed to a limited reduction of cellular metabolism, in line with a slight decrement in cell number, of −11 and −7% for chitosan−TPP- and chitosan− PPi-treated cells, respectively, after 24 h of incubation, which once again can be considered as not statistically significant as compared with control (Figure 6B). These results confirmed the negligible cytotoxicity of the gels in the first 24 h of incubation. For a longer incubation time (72 h), cell proliferation reduction of 10% was still detectable in the case of fibroblasts treated with chitosan−TPP gels, whereas negligible differences were observed for cells incubated with chitosan−PPi gels with respect to the control group. Overall, the present data indicate a moderate reduction in cell number for the former gels, thus validating visual observations discussed above. In spite of this, statistical analyses enable us considering the present gels as safe, at most endowed with a mild cytotoxicity.30 The ability of NIH-3T3 to adhere (and consequently colonize) over chitosan−TPP/chitosan−PPi gels was verified by incubating cells with scaffolds for 24 h. Confocal laser scanning microscopy images on different gel sections highlighted the presence of intact cell nuclei distributed throughout the matrices (Figure S3). Strikingly, nuclei appeared to be spread out along the whole range of stacks analyzed (Figures 6C and S3), suggesting that cells migrated from the external cell culture medium toward inner gel networks. These findings wellcorroborated the AlamarBlue assay performed under the same experimental conditions (Figure S4), where the higher fluorescence values recorded for gels incubated with cells proved the presence of viable NIH-3T3 within networks. Herein, it has to be stressed that gels were extensively washed before performing the assay, thereby avoiding any artifact due to nonadherent/embedded cells. The similar amount of nuclei noticed for both the gel types (Figure 6C), together with no statistical differences between fluorescence intensities evidenced for chitosan−TPP and chitosan−PPi systems (Figure S4), would indicate that both scaffolds were prone to host cells regardless of the cross-linker used for the synthesis of gels.

To calculate the diffusion rate at the gel/solution interface, we resorted to calculate an interfacial diffusion coefficient, Di (see Supporting Information for further details). The rationale for analyzing Di is to assess the persistence of the observed difference in the (rate-determining) boundary structure neighboring the interface of the two types of gels, as opposed to the leveling effect of the destructive force exerted by the solvent over the gel integrity. In all cases, the values of Di are larger than the corresponding values of D, demonstrating a reduced ability of the interface of both gels to withstand the action of the solution. Still, in both payload cases, the values of the interfacial diffusion coefficients of the PPi-based systems are lower than those of the corresponding TPP-based ones (Table I), confirming the higher diffusive resistance of the gel boundary for chitosan−PPi with respect to that for chitosan− TPP gels. Again, the diffusive resistance at the gel surface increases with the molecular mass of loaded molecules for both PPi and TPP systems. 3.4. Evaluation of Chitosan−TPP/Chitosan−PPi Gel Cytotoxicity and Ability to Host Cells. Generally, cytotoxicity of polymer-based materials toward mammalian cells is mostly ascribed to the leakage of the cross-linker and/or polymer and consequent interactions with cells. To study the biocompatibility of chitosan−TPP and chitosan−PPi gels, mouse fibroblast-like (NIH-3T3) cells were selected as the model. In more detail, gels were laid down upon twodimensional cellular monolayers, and their impact on cell morphology, metabolism, and membrane damage was investigated. Preliminarily, we observed that when gels were placed immediately after sterilization in contact with cells, the cell culture medium turned yellowish, thus suggesting a pH variation. This is likely ascribed to acetic acid release from networks. Such an aspect negatively affected the viability of cells: after 24 h of incubation, fibroblasts were almost detached from wells, whereas remaining cells showed chiefly a roundshaped morphology with the presence of dense chromatin aggregates and apoptotic bodies. According to ref 30, we graded both chitosan−TPP and chitosan−PPi gels as severely cytotoxic in such experimental conditions. To limit this flaw, gels were preincubated in PBS buffer at 37 °C for 1 h to favor acetic acid release and consequent substitution with PBS buffer, thereby increasing the pH within networks. First, we did not observe any color change of the medium after 24 h by adopting this experimental setup, thus suggesting that the pH of the culture medium was amenable to cell growth and proliferation. Second, fibroblasts showed a normal cell morphology after 24 and 72 h of treatment, as pointed out in Figure 6A, albeit few cellular aggregates were still evident in the case of cells treated with chitosan−TPP gels. Moreover, a limited reduction in the total number of cells was also detectable by visual analyses. Conversely, cells treated with chitosan−PPi gels seemed unaffected by the presence of networks, showing a higher similarity in number and spreading with respect to those in the control. To quantitatively verify the cytotoxicity of gels toward fibroblasts, LDH and AlamarBlue assays were carried out. The former was designed to assess cellular membrane damage. LDH enzymes released from the cytoplasm of treated cells were mostly similar to those from untreated fibroblasts (Figure 6B). In more detail, +13 and +6% released enzymes were noticed for chitosan−TPP- and chitosan−PPi-treated cells, respectively, after 24 h of incubation; these differences were not statistically significant (one-way analysis of variance, α = 0.05) with respect

4. CONCLUSIONS In this work, we synthesized chitosan gels ionically reticulated by multivalent anions TPP and PPi and TEM images were acquired to study the internal mesh distribution of the resulting networks. Quantitative parameters such as cross-linking density and pore size distribution were computed by an innovative image analysis approach aiming at adopting the concept of maximal inscribed circle among polymer meshes as an analytical tool. The results pointed at a different average cross-linking density (0.28 ± 0.02 for PPi vs 0.43 ± 0.01 mm2/mm2 for TPP gels) and pore size distribution in the range 0.1−1.2 μm. The different cross-linking density, together with the different crosslinker binding strength toward chitosan, was found to heavily influence the stability of gels in simulated physiological media, where the higher reticulation for chitosan−TPP gels ensured them to be moderately eroded up to 6 weeks, whereas PPi counterparts degraded within a few days. At the same time, the H


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(7) Drury, J. L.; Mooney, D. J. Hydrogels for Tissue Engineering: Scaffold Design Variables and Applications. Biomaterials 2003, 24, 4337−4351. (8) Bhattarai, N.; Gunn, J.; Zhang, M. Chitosan-Based Hydrogels for Controlled, Localized Drug Delivery. Adv. Drug Delivery Rev. 2010, 62, 83−99. (9) Vårum, K. M.; Smidsrød, O. Structure-Property Relationship in Chitosans. In Polysaccharides: Structural Diversity and Functional Versatility; CRC Press, 2004; Vol. 30, pp 625−642. (10) Berger, J.; Reist, M.; Mayer, J. M.; Felt, O.; Peppas, N. A.; Gurny, R. Structure and Interactions in Covalently and Ionically Crosslinked Chitosan Hydrogels for Biomedical Applications. Eur. J. Pharm. Biopharm. 2004, 57, 19−34. (11) Klouda, L. Thermoresponsive Hydrogels in Biomedical Applications: A Seven-Year Update. Eur. J. Pharm. Biopharm. 2015, 97, 338−349. (12) Supper, S.; Anton, N.; Seidel, N.; Riemenschnitter, M.; Curdy, C.; Vandamme, T. Thermosensitive Chitosan/glycerophosphate-Based Hydrogel and Its Derivatives in Pharmaceutical and Biomedical Applications. Expert Opin. Drug Delivery 2014, 11, 249−267. (13) Supper, S.; Anton, N.; Boisclair, J.; Seidel, N.; Riemenschnitter, M.; Curdy, C.; Vandamme, T. Chitosan/glucose 1-Phosphate as New Stable in Situ Forming Depot System for Controlled Drug Delivery. Eur. J. Pharm. Biopharm. 2014, 88, 361−373. (14) Meng, Q.; Man, Z.; Dai, L.; Huang, H.; Zhang, X.; Hu, X.; Shao, Z.; Zhu, J.; Zhang, J.; Fu, X.; Duan, X.; Ao, Y. A Composite Scaffold of MSC Affinity Peptide-Modified Demineralized Bone Matrix Particles and Chitosan Hydrogel for Cartilage Regeneration. Sci. Rep. 2015, 5, No. 17802. (15) Huang, H.; Zhang, X.; Hu, X.; Dai, L.; Zhu, J.; Man, Z.; Chen, H.; Zhou, C.; Ao, Y. Directing Chondrogenic Differentiation of Mesenchymal Stem Cells with a Solid-Supported Chitosan Thermogel for Cartilage Tissue Engineering. Biomed. Mater. 2014, 9, No. 035008. (16) Sacco, P.; Borgogna, M.; Travan, A.; Marsich, E.; Paoletti, S.; Asaro, F.; Grassi, M.; Donati, I. Polysaccharide-Based Networks from Homogeneous Chitosan−Tripolyphosphate Hydrogels: Synthesis and Characterization. Biomacromolecules 2014, 15, 3396−3405. (17) Sacco, P.; Travan, A.; Borgogna, M.; Paoletti, S.; Marsich, E. Silver-Containing Antimicrobial Membrane Based on Chitosan−TPP Hydrogel for the Treatment of Wounds. J. Mater. Sci.: Mater. Med. 2015, 26, No. 128. (18) Sacco, P.; Paoletti, S.; Cok, M.; Asaro, F.; Abrami, M.; Grassi, M.; Donati, I. Insight into the Ionotropic Gelation of Chitosan Using Tripolyphosphate and Pyrophosphate as Cross-Linkers. Int. J. Biol. Macromol. 2016, 92, 476−483. (19) Rampino, A.; Borgogna, M.; Blasi, P.; Bellich, B.; Cesàro, A. Chitosan Nanoparticles: Preparation, Size Evolution and Stability. Int. J. Pharm. 2013, 455, 219−228. (20) Calvo, P.; Remunan-Lopez, C.; Vila-Jato, J. L.; Alonso, M. J. Novel Hydrophilic Chitosan−Polyethylene Oxide Nanoparticles as Protein Carriers. J. Appl. Polym. Sci. 1997, 63, 125−132. (21) Huang, Y.; Lapitsky, Y. Determining the Colloidal Behavior of Ionically Cross-Linked Polyelectrolytes with Isothermal Titration Calorimetry. J. Phys. Chem. B 2013, 117, 9548−9557. (22) Brun, F.; Accardo, A.; Marchini, M.; Ortolani, F.; Turco, G.; Paoletti, S. Texture Analysis of TEM Micrographs of Alginate Gels for Cell Microencapsulation. Microsc. Res. Tech. 2011, 74, 58−66. (23) Turco, G.; Donati, I.; Grassi, M.; Marchioli, G.; Lapasin, R.; Paoletti, S. Mechanical Spectroscopy and Relaxometry on Alginate Hydrogels: A Comparative Analysis for Structural Characterization and Network Mesh Size Determination. Biomacromolecules 2011, 12, 1272−1282. (24) Schuster, E.; Eckardt, J.; Hermansson, A.-M.; Larsson, A.; Lorén, N.; Altskär, A.; Ström, A. Microstructural, Mechanical and Mass Transport Properties of Isotropic and Capillary Alginate Gels. Soft Matter 2014, 10, 357−366. (25) Gundersen, H. J.; Bendtsen, T. F.; Korbo, L.; Marcussen, N.; Møller, A.; Nielsen, K.; Nyengaard, J. R.; Pakkenberg, B.; Sørensen, F. B.; Vesterby, A. Some New, Simple and Efficient Stereological

homogeneity/inhomogeneity of the polymer network, together with the molecular weight of loaded molecules, has shown to guarantee a different release behavior for BSA and cytochrome c, herein, used as model payloads. Given the very good cytocompatibility of both the systems after acetic acid removal and substitution with physiological media, together with the feature of being suitable milieus for cellular colonization and metabolic activity, chitosan−TPP and chitosan−PPi gels can be considered as promising biomaterials for tissue engineering purposes, especially for the co-delivery of cells and growth factors in muscle/cartilage regeneration.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.8b01834. (i) Protocol for the synthesis of fluorescein isothiocyanate-labeled chitosan, (ii) confocal imaging (3Drendering) of chitosan−PPi and chitosan−TPP gel internal structure, (iii) fitting of the release data, (iv) 3D-rendering of cell nuclei embedded within gel matrices and evaluation of cellular metabolism, and (v) some details of the calculation of diffusion coefficients (PDF) (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +39-040-5588733. ORCID

Pasquale Sacco: 0000-0002-4483-5099 Ivan Donati: 0000-0003-3752-8346 Davide Porrelli: 0000-0002-6437-7646 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by the University of Trieste-FRA 2016 to Ivan Donati (FRADONATI2016-17). The authors acknowledge the support in part by the ERA-MarineBiotech project Mar3Bio. Dr. Michela Cok is acknowledged for helping in the acquisition of TEM images. Confocal images reported in this article were generated in the Microscopy Center of the University of Trieste at the Department of Life Sciences, funded as detailed at www.units.it/confocal.

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REFERENCES

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On the Correlation between the Microscopic Structure and Properties

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