Engineered 3D Microenvironments with Starch Nanocrystals

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Engineered 3D Microenvironments with Starch Nanocrystals as Cell-instructive Materials Susanna Piluso, Marianne Labet, Chen Zhou, Jin Won Seo, Wim Thielemans, and Jennifer Patterson Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.9b00907 • Publication Date (Web): 06 Sep 2019 Downloaded from pubs.acs.org on September 7, 2019

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Biomacromolecules

Engineered 3D Microenvironments with Starch Nanocrystals as Cell-instructive Materials Susanna Piluso,¥‡† Marianne Labet,§ Chen Zhou,¥ Jin Won Seo,¥ Wim Thielemans,§ and Jennifer Patterson¥‡‖†* ¥Department ‡Prometheus,

of Materials Engineering, KU Leuven, 3001 Leuven, Belgium

Division of Skeletal Tissue Engineering, KU Leuven, 3000 Leuven, Belgium

§Renewable

Materials and Nanotechnology Research Group, Department of Chemical

Engineering, KU Leuven, Campus Kulak Kortrijk, 8500 Kortrijk, Belgium ‖Department

of Imaging & Pathology, KU Leuven, 3000 Leuven, Belgium

Hydrogels, topography, starch, gelatin, photo-crosslinking, morphology

ACS Paragon Plus Environment

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Naturally, cells reside in 3D microenvironments composed of biopolymers that guide cellular behavior via topographical features as well as through mechanical and biochemical cues. However, most studies describing the influence of topography on cells’ behavior are performed on rigid and synthetic 2D substrates. To design systems that more closely resemble native microenvironments, herein we develop 3D nanocomposite hydrogels consisting of starch nanocrystals (SNC) embedded in a gelatin matrix. The incorporation of different concentrations of SNC (0.05, 0.2, 0.5 wt.%) results an increase of compressive modulus when compared to hydrogels without SNC, without affecting the swelling ratio, thus providing a tunable system. Confirming the cytocompatibility of the novel composites, the viability of encapsulated L929 fibroblasts is >90% in all hydrogels. The cellular metabolic activity and DNA content are similar for all formulations and increase over time, indicating that the fibroblasts are proliferating within the hydrogels. After 4 d of culture, Live/Dead staining and F-actin/nuclei staining show that the encapsulated fibroblasts develop an elongated morphology in the hydrogels. On the other hand, encapsulated chondrogenic progenitor ATDC5 cells also maintain a viability around 90% but display a rounded morphology, especially in the hydrogels with SNC, indicating a potential application of the materials for cartilage tissue engineering. We believe that topographical and mechanical cues within 3D microenvironments can be a powerful tool to instruct cells’ behavior and that the developed gelatin/SNC nanocomposite warrants further study.


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1. INTRODUCTION In natural tissues, cells reside in a highly sophisticated three-dimensional (3D) microenvironment consisting of nano- and microscale topographical structures that play a key role in cellular processes such as differentiation, migration, and proliferation.1-2 As cells contain nanoscale features such as focal adhesion complexes and fine processes (e.g., cilia and filopodia), they can sense and respond to physical cues as small as approximately 10 nm.3-4 A number of studies have investigated the influence of nanotopographical cues on the behavior of cells and have shown that cell morphology, adhesion, alignment, and proliferation depend strongly on the cell type and the dimensions of the physical cues.5-11 For example, engineered topographical gradients of nanoparticles with three different average diameters (16, 38, and 68 nm) have been used to study the response of fibroblasts and osteoblasts. Interestingly, features of 16 nm promoted the adhesion of both cell types, whereas only fibroblasts adhered on the nanotopography of 38 nm. Furthermore, it was shown that there was a critical density between 50 and 140 nanoparticles per µm2 where both cell types adhered in the greatest numbers.9 However, most of these investigations on the effects of topography were performed on stiff, two-dimensional (2D) surfaces that do not fully resemble the natural microenvironment of the cell. An emerging approach to design 3D systems that can recreate the topography of the native microenvironment is to prepare nanocomposite hydrogels, which are highly hydrated polymer networks that incorporate nanoparticles or nanofibers within the matrix. Nanocomposite hydrogels have been prepared using various types of nanomaterials such as ceramic, polymeric, and metallic nanoparticles as well as carbon-based nanomaterials.12 Furthermore, the addition of the nanomaterial can strongly influence the behavior of cells encapsulated within the hydrogels. For example, the incorporation of disk-shaped nanosilicates within gelatin hydrogels enhanced the


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osteogenic differentiation of encapsulated human mesenchymal stem cells (hMSCs) as evident from an increase in alkaline phosphatase activity compared to hMSCs in the pristine hydrogels.13 In another recent study, the incorporation of gold nanoparticles within gelatin hydrogels promoted the proliferation and osteogenic differentiation of human adipose-derived stem cells (ADSCs) in a dose-dependent manner and significantly increased bone formation when implanted in a rabbit calvarial defect.14 While a main rationale behind the incorporation of nanomaterials within hydrogels is to create extracellular matrix (ECM)-like structures, the nanocomposites can also compensate for other scaffold limitations such as the absence of cell adhesive moieties, lack of conductive properties, or weak mechanical properties.15 For example, nanodiamonds have been used to increase the compressive properties of gelatin hydrogels for bone regeneration applications. The compressive modulus of the gelatin hydrogels increased to nearly double that of gelatin alone by increasing the concentration of nanodiamonds from 0.05 to 0.2% (w/v). Moreover, human ADSCs exhibited higher traction forces when cultured on the surface of the nanocomposite hydrogels compared to gelatin alone, due to the increase in substrate stiffness.16 Likewise, the addition of carbon nanotubes (CNTs) successfully increased the compressive modulus of gelatin hydrogels from 15 kPa (gelatin only) to approximately 60 kPa when 0.5 mg mL-1 of CNTs were incorporated without affecting the porosity of the matrix or the growth of encapsulated NIH-3T3 cells and hMSCs. Interestingly, both cell types displayed spreading patterns in the CNT-containing hydrogels similar to those observed in softer gelatin hydrogels.17 In recent work by our group, addition of CNTs to polyethylene glycol (PEG) hydrogels significantly increased the compressive modulus of the hydrogels without changing their swelling ratio, and the initial viability of encapsulated fibroblasts was higher in the hydrogels with CNTs.18


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As previously mentioned, the majority of nanocomposite hydrogels have been fabricated using ceramic, metallic, or carbon-based nanoparticles. However, in the last few years, polysaccharidebased nanomaterials, such as nanocrystals derived from starch and cellulose, have been gaining attention due to their interesting properties, such as high crystallinity, high stiffness, high aspect ratio (ribbons for cellulose nanocrystals and platelets for starch nanocrystals), and the presence of reactive hydroxyl groups on their surface.19-20 The main advantages of polysaccharide nanocrystals over other currently used nanomaterials include high availability, renewability, low cost, biodegradability, and non-toxicity.19, 21 They have also been shown to be biodegraded in aqueous environments.22 Starch nanocrystals (SNC) can be prepared using different methods, including acid or enzymatic hydrolysis, regeneration via self-assembly after dissolution, and physical disintegration, leading to a platelet-like morphology.20 A crucial step in the preparation of SNC is to maintain the integrity of the amylopectin crystalline regions while completely removing the amorphous/dislocated amylose and amylopectin regions. Therefore, acid hydrolysis methods are preferred over physical disintegration as they can be more carefully controlled and diffusion limitations restrict their action to the non-crystalline regions. Among the acid hydrolysis methods, the most common one uses sulfuric acid due to the higher yield and shorter preparation time compared to hydrolysis with hydrochloric acid.23 SNC have mainly been used in the biomedical field and waste water treatment and to increase the strength and stiffness of polymer networks.20, 24-25

For example, the tensile modulus of waterborne polyurethanes and natural rubbers increased

remarkably with increasing content of nanocrystals.24 They are excellent stabilizers for Pickering emulsions making it possible to carry out emulsion polymerization followed by composite film formation without the need for additional processing steps.26-27 SNC have also been used to reinforce chitosan/poly (vinyl pyrrolidone) blends for in vitro wound healing applications. In this


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case, the presence of the SNC did not affect the cytocompatibility or in vitro blood compatibility of the nanocomposites; however, the cytotoxicity was only evaluated by culturing NIH3T3 cells with extracts (growth medium in which the nanocomposites had been immersed for 24 h).28 To the best of our knowledge, our study is the first to investigate the effect of SNC on cellular behavior in 3D hydrogels. While SNC have not been significantly studied as biomaterials, previous studies have described the use of starch polymers for tissue engineering applications, including cartilage regeneration, due to their biodegradability, cytocompatibility, and processing versatility.29-31 For example, when adipose stromal cells were encapsulated in chitosan hydrogels containing starch, they exhibited higher viability, proliferation, and chondrogenic differentiation compared with cells in chitosan hydrogels without starch.30 Additionally, starch-based materials have been tested for bone regeneration in vivo, showing a minimal inflammatory response when implanted either intramuscularly or in cortical bone defects in goats.32 Hybrid scaffolds composed of oxidized starch and collagen also led to skin wound healing in a rat model with no chronic inflammation.33 Therefore, we believe that SNC should likewise be promising as biomaterials, including for cartilage tissue engineering.34-35 Herein, we describe the fabrication of 3D engineered microenvironments consisting of SNC embedded within gelatin hydrogels. We hypothesize that this combination may result in advantageous effects that the individual materials cannot achieve and that the nanocomposites can positively influence the behavior of encapsulated cells. Gelatin was selected because, as it is derived from the native ECM molecule collagen, it contains cell adhesion moieties, is biodegradable, and has shown excellent biocompatibility in previous studies.36-38 For example, minimal cytotoxicity towards various cell types (fibroblasts, keratinocytes, endothelial cells) in


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vitro and a minimal foreign body reaction after subcutaneous implantation in vivo were observed with a gelatin-based hydrogel.37 We first evaluated the effect of the addition of different amounts of SNC on the physical and mechanical properties of the nanocomposite hydrogels. In a next step, the effect of SNC on proliferation and morphology of two cell types that were encapsulated in the 3D hydrogels was investigated. L929 fibroblasts are standardly used in cytotoxicity assays and were utilized to demonstrate the cytocompatibility of this novel combination of materials. ATDC5 cells, isolated from mouse teratocarcinoma fibroblastic cells, exhibit rapid proliferation and chondrogenic differentiation in vitro, and they have been extensively used to evaluate the effect of materials on cell viability, proliferation, and chondrogenesis.39-41 Therefore, ATDC5 cells were embedded in the nanocomposite hydrogels to evaluate their potential for cartilage regeneration applications. 2. EXPERIMENTAL SECTION 2.1 Materials Type A gelatin (Bloom 330) was kindly provided by Rousselot (the Netherlands). Lithium Phenyl(2,4,6-trimethylbenzoyl)phosphinate (LAP) was purchased from BDL (Czech Republic). Cell culture reagents including the Live/Dead staining kit for mammalian cells, Presto Blue reagent, Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), and penicillin/streptomycin were obtained from Thermo Fisher (Belgium). Waxy maize starch (Waxylis 200, > 99% amylopectin) was gratefully supplied by Roquette SA (France). All the other chemicals were obtained from Sigma Aldrich (Belgium) unless otherwise indicated. 2.2 Preparation of starch nanocrystals (SNC) Sulfuric acid-hydrolyzed SNC were prepared from waxy maize starch, as previously described.20-21, 42 Briefly, waxy maize starch was hydrolyzed under constant stirring for 5 d at 40


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°C in a 3.16 M aqueous H2SO4 solution. After hydrolysis, the nanocrystals were separated from the acid by centrifugation at 10,000 rpm at 10 °C. Subsequent washing with distilled water and centrifugation was performed until neutrality of the eluent (about 8 cycles). A homogeneous aqueous dispersion of SNC at 5.3 wt.% was obtained using an Ultra Turrax T25 homogenizer for 5 min at 13,500 rpm. 2.3 Characterization of SNC To characterize the SNC, transmission electron microscopy (TEM), elemental analysis, and dynamic light scattering (DLS) were performed. TEM was carried out with a probe-corrected microscope (ARM200F cold-FEG, JEOL) operated at an acceleration voltage of 80 kV. The samples were prepared for TEM by placing a few drops of the SNC dispersion onto 200 mesh copper TEM grids that were coated with a carbon film (Agar Scientific). Elemental analysis (C, H, N, S) data were collected on a calibrated Thermo Flash 2000 elemental analyzer. The content of carbon, hydrogel, nitrogen, and sulfur was reported as a mass percentage of the sample. DLS was measured on a Brookhaven NanoBrook Omni instrument with the detection range limited from 5 nm to 800 nm to avoid measurement of large (> 800 nm) dust particles. 2.4 Preparation of photocrosslinked Gel/SNC hydrogels The synthesis of methacrylated gelatin (GelMA) was performed according to a published protocol.43 Briefly, type A gelatin was mixed at 10% (w/v) in phosphate buffered saline (PBS, pH 7.4) at 50 °C and stirred until fully dissolved. Methacrylic anhydride (10-fold excess, 5 mL) was added dropwise to the gelatin solution while stirring and allowed to react for 2 h. During the methacrylation reaction, the mixture was stirred vigorously, and the pH of the solution was kept between 7.0 and 7.4 using NaOH. After 2 h, the reaction mixture was dialyzed for 4 d against distilled water at room temperature. Then, the product was lyophilized and stored at – 20 °C. The


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degree of substitution was determined using a 2,4,6-trinitrobenzene sulfonic acid (TNBS) assay following a published protocol.44 The nanocomposite hydrogels were prepared using different concentrations of SNC as indicated in Table 1. The SNC were first dispersed at the desired concentration in a solution of the photoinitiator [LAP at 0.3% (w/v) in PBS] by stirring to obtain a uniform distribution of nanocrystals. Then, GelMA was added to the dispersion of SNC, to reach a final concentration of 5% (w/v) GelMA, and the mixture was vigorously stirred at 50 °C in a water bath for 30 min, protected from light, until the GelMA was dissolved and a homogeneous mixture was obtained. To prepare the hydrogels, 50 or 200 µL of the precursor solution was injected into molds (5 mm in diameter for swelling and cell culture studies; 9 mm in diameter initially and cut with a biopsy punch into disks of 8 mm in diameter for mechanical testing) and exposed to UV light (1200 µW cm-2, 365 nm, 7 min exposure time). After photocrosslinking, the hydrogels were washed with PBS to remove the photo-initiator. Table 1. Composition of Gel/SNC hydrogels. Sample Name GelMA (% (w/v))a Starch nanocrystals (wt.%) Gel/SNC-0 5 0 Gel/SNC-0.05 5 0.05 Gel/SNC-0.2 5 0.2 Gel/SNC-0.5 5 0.5 aFor all the hydrogels, the concentration of the LAP photo-initiator was 0.3 % (w/v). 2.5 Equilibrium swelling of the hydrogels To assess the swelling properties, after crosslinking, the nanocomposite hydrogels were transferred to 1.5 mL vials and immersed in 1 mL of ultrapure water at 37 °C for 24 h to reach equilibrium. The weight of the swollen samples was recorded after blotting off the excess water using filter paper. The hydrogels were then lyophilized to obtain the dry weight. The swelling ratio was defined according to equation (1):


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𝑺𝒘𝒆𝒍𝒍𝒊𝒏𝒈 𝒓𝒂𝒕𝒊𝒐 =

𝑴𝒔 ― 𝑴𝒅

𝑴𝒅

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(1)

where Ms in the weight of the swollen hydrogel and Md is the weight of the lyophilized hydrogel. The swelling ratio was determined using 3-5 replicates for each hydrogel composition. 2.6 Mechanical properties Compression testing was performed on disc-shaped swollen hydrogels with a diameter of 8 mm and thickness of about 1.2 mm using a dynamic mechanical analyzer (DMA Q800, TA Instruments, UK). All the measurements were performed at 37 °C with a liquid-filled sample holder to prevent water evaporation from the hydrogels. Experiments were conducted in controlled force mode with a force ramp rate of 1 N min-1 up to 18 N. The elastic modulus was determined from the slope of the stress-strain curve at 5-10% strain. A minimum of 4 replicates for each hydrogel formulation was used for the analysis. 2.7 Cell culture and encapsulation L929 fibroblasts (used between passage 7 and 9) were cultured in DMEM with glutaMAX-1 supplemented with 10% FBS and 1% penicillin/streptomycin in a humidified incubator at 37 °C with 5% CO2. ATDC5 cells were cultured in two different culture media: maintenance medium consisting of DMEM/Ham’s F12 (1:1) supplemented with 5% FBS, human transferrin (10 mg mL1),

and sodium selenite (3×10−8 M) or proliferation media consisting of DMEM/Ham’s F12 (1:1),

2 mM glutamine, and 5% FBS. Both compositions contained penicillin/streptomycin at 1%. The cell culture medium was changed every 3 d, and the cells were subcultured once they reached 8090% confluence. Prior to cell encapsulation, the hydrogel precursors (GelMA and LAP) were sterile filtered. Sterile dispersions of SNC were prepared by adding 70% ethanol followed by centrifugation and washing steps with sterile PBS. L929 fibroblasts or ATDC5 cells were harvested using trypsin and


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resuspended at a density of 1 x 106 cells mL-1 in the GelMA-SNC solutions containing LAP. This suspension (40 µL) was then pipetted into a sterile 1 mL syringe with a cut off tip and exposed to UV light for 7 min. Afterwards, the nanocomposite hydrogels were rinsed with their respective growth medium and cultured for 1 w in 96 well plates with 200 µL of growth medium. The media were refreshed every 2 d. 2.8 Cell viability, metabolic activity, and proliferation Cell viability was evaluated at 1, 4, and 7 d of culture with a Live/Dead assay. Briefly, the nanocomposite hydrogels were rinsed with PBS and then incubated with calcein AM (2 µM) and ethidium homodimer-1 (4 µM) in PBS for 30 min. The gels were then rinsed with PBS and imaged using confocal microscopy (LSM 510 inverted microscope, Zeiss) at 10× magnification (green channel for calcein: λex = 488 nm and λem = 525 nm; red channel for ethidium homodimer: λex = 543 nm and λem = 590 nm). For each hydrogel, three z-stacks (5 µm slices over a total depth of 100 µm; 0.88 µm x 0.88 µm pixel size) were taken, and the maximum intensity projection images were used for analysis. The live and dead cell numbers were determined with ImageJ (NIH, USA). The viability was determined as the number of live cells divided by the total number of cells. The circularity was determined using ImageJ and ranges from 0-1, with 1 indicating a completely round shape. Images were taken for at least n=4 (two samples each of two independent experiments). The metabolic activity of the encapsulated cells was evaluated using a Presto Blue assay. At 1, 4, and 7 d of culture, Presto Blue reagent was added directly to the growth media for a final concentration of 10%, and the hydrogels were incubated for 3 h at 37 °C. After incubation, aliquots of the media were transferred to a 96-well plate, and the fluorescence was measured using a plate reader (λex = 540 nm, λem = 590 nm). Cell proliferation was evaluated via DNA quantification at 1, 4, and 7 d. The hydrogels were first digested overnight in 0.5 mg mL-1 proteinase K in phosphate


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buffered EDTA (5 mM EDTA in PBS, 10 mM Tris, and 0.1% Triton X-100) at 60 °C. The DNA concentration was determined using a Quant-iT Picogreen dsDNA assay (Invitrogen). Briefly, the digested hydrogels and standards were mixed with the Picogreen reagent and incubated at room temperature for 5 min. Then, the DNA content was measured with a fluorimeter. The metabolic activity and DNA quantification assays were conducted on n=6 (three samples each from two independent experiments). 2.9 F-actin staining To evaluate cell morphology, cells within the hydrogels were stained for F-actin and nuclei using phalloidin and DAPI staining, respectively. At 1, 4, and 7 d, the hydrogels were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100 for 50 min. After fixation, the samples were rinsed with PBS and incubated with glycine (0.1 M in PBS) to block the free aldehyde groups. Then, the cells were stained by incubation with Alexa Fluor 488-conjugated phalloidin (0.8 U mL-1 in 1% BSA solution in PBS) for 2 h followed by DAPI (2.5 µg mL-1 in PBS) staining for 30 min. The samples were imaged with confocal microscopy (LSM 880; Zeiss), and images were taken of 3 random fields of view at 40× magnification (Alexa Fluor 488: λex = 488 nm and λem = 530 nm; DAPI: λex = 405 nm and λem = 447 nm). Images were taken for at least n=4 (two samples each of two independent experiments). 2.10 Statistics All data are represented as mean ± standard deviation with the number of replicates indicated elsewhere in the experimental section or in the figure captions. Statistical comparisons were performed using the one-way (or two-way when necessary) analysis of variance (ANOVA) with GraphPad Prism 8 software. A value of p

Engineered 3D Microenvironments with Starch Nanocrystals

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