Nanopillared Chitosan-Gelatin Films: A Biomimetic Approach for

Subscriber access provided by Nottingham Trent University

Bio-interactions and Biocompatibility

Nanopillared Chitosan-Gelatin Films: A Biomimetic Approach for Improved Osteogenesis Sevde Altuntas, Harkiranpreet Kaur Dhaliwal, Nicole Joy Bassous, Ahmed Eid Radwan, Pinar Alpaslan, Thomas J Webster, Fatih Buyukserin, and Mansoor Amiji ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.9b00426 • Publication Date (Web): 11 Aug 2019 Downloaded from pubs.acs.org on August 16, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 57 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Biomaterials Science & Engineering

Nanopillared Chitosan-Gelatin Films: A Biomimetic Approach for Improved Osteogenesis Sevde Altuntas, †, ‡, § Harkiranpreet K. Dhaliwal, ‡ Nicole J. Bassous,∥ Ahmed E. Radwan,⊥,# Pinar Alpaslan † Thomas Webster,∥ Fatih Buyukserin*† and Mansoor Amiji*‡

†Department

of Biomedical Engineering, TOBB University of Economics and

Technology, 43 Sogutozu Street, 06560, Ankara, TR ‡School

of Pharmacy, Bouvé College of Health Sciences, Northeastern University, 360

Huntington Avenue, 02115, Boston, MA, USA §Brigham

and Women`s Hospital, Renal Division, 4 Blackfan Circle Street, 02115,

Boston, MA, USA ∥Department

of Chemical Engineering, Northeastern University, 360 Huntington Avenue,

02115, Boston, MA, USA ⊥Brigham

and Women`s Hospital, Department of Radiology, Harvard Medical School, 72

Francis Street, 02115, Boston, MA, USA 1 ACS Paragon Plus Environment

ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

#Chemistry

Page 2 of 57

and Physics Department, Simmons University, 300 The Fenway,Boston,

MA, USA *Corresponding Authors e-mail: [email protected], [email protected] KEYWORDS: nanopillared chitosan:gelatin film, osteogenic differentiation, mineralization

ABSTRACT Biomimicry strategies, inspired from natural organization of living organisms, are being widely used in the design of nanobiomaterials. Particularly, nonlithographic techniques have shown immense potential in the facile fabrication of nanostructured surfaces at large scale production. Orthopedic biomaterials or coatings possessing extracellular matrix (ECM)-like nanoscale features induce desirable interactions between bone tissue and implant surface, also known as osseointegration. In this study, nanopillared chitosan-gelatin (C:G) films were fabricated using nanoporous anodic alumina molds, and their antibacterial properties as well as osteogenesis potential was analyzed by comparing to the flat C:G films and tissue culture polystyrene (TCP) as 2 ACS Paragon Plus Environment

Page 3 of 57 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


controls. In vitro analysis of the expression of RUNX2, OPN and OCN genes for mesenchymal stem cells (MSCs) as well as osteoblast-like Saos-2 cells was found to be increased for the cells grown on nano C:G films indicating early stage osteogenic differentiation. Moreover, the mineralization tests (quantitative calcium analysis and alizarin red staining) showed that nanotopography significantly enhanced the mineralization capacity of both cell lines. This work may provide a new perspective of biomimetic surface topography fabrication for orthopedic implant coatings with superior osteogenic differentiation capacity and fast bone regeneration potential.

INTRODUCTION

Titanium and its alloys (such as Ti-6Al-4V) are commonly used as implants due to their biocompatible nature, desirable mechanical properties and high corrosion resistivity. Biocompatibility of these materials is related to the chemical composition and integrity of the native oxide layer present on the implant surface, however this coating may not be able to prevent leaching of toxic metal ions (such as Al, V or Cr) from the underlying material.1 It has been reported in the literature that these metal ions may cause several 3 ACS Paragon Plus Environment


Page 4 of 57

health problems including Alzheimer`s disease, retinal degeneration, and long term fertility issues for both genders.1-3 Additionally, the limited osteoconductive character of the oxide layer may cause insufficient osseointegration between the bone and implant surface, eventually leading to implant failure.4 To overcome these drawbacks and induce effective osteogenesis, the chemical and topographic properties of the bone extracellular matrix (ECM) milieu have been mimicked in various recent studies by using coating materials of inorganic or organic origin.5

Hydroxyapatite (HA) (Ca5(PO4)3(OH)) is a major component of the bone ECM and is frequently used as a biomimetic inorganic implant coating material. HA coatings on the metal implant surfaces have previously been shown to exhibit promising results because of their biocompatibility, bioactivity and osteoconductive properties compared to the native oxide layer on the implants.5 Despite these positive prospects, due to the costly coating procedures (e.g., plasma spraying and ion beam deposition), and infection related implant failures caused by the inherent binding affinity of bacteria towards its Ca2+ doped positive surface, the wide spread use of HA has been limited. Alternatively,

4 ACS Paragon Plus Environment

Page 5 of 57 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


coating strategies utilizing organic matrixes (such as natural polymers or proteins) have also been investigated by various groups as reported in the literature.6-8 For instance, Liu and coworkers have successfully used bone morphogenic protein-2 (BMP-2) coated implants to induce stem cell differentiation and osteoblast regeneration.6 In addition to biological molecules, increasing the interaction between bone and implant surface through the use of polymer-based coating approaches have also been investigated. To create an ECM-mimetic environment for optimal osteogenesis, the selection of polymers or polymer combinations is of prime importance. Here, biodegradable polymer systems such as poly(D,L-lactic-co-glycolic acid) (PLGA) copolymer9, polylactic acid (PLA)6 or nano-HA/chitosan7 are generally preferred.

Chitosan is considered as one of the most promising natural polymers for tissue engineered scaffolds owing to its abundance, biocompatibility, biodegradability, and antibacterial properties and hence is widely used for bone tissue engineering applications. These advantages generally are caused by the hyaluronic acid-like structure of the chitosan molecule.10 However, its mechanical properties are not



Page 6 of 57

compatible for hard tissues11 because of its fragility and water-soluble nature, which makes it difficult to produce a sustainable implant coating model. To improve its mechanical properties, the use of gelatin-doped chitosan solution has been recommended by previous studies.12 The cell adhesive property of gelatin (due to the presence of Arg-Gly-Asp (RGD) sequences) and antibacterial property of chitosan creates a good mixture of a polymer which has immense potential to serve as a scaffold for tissue engineering applications with enhanced mechanical properties.12,13

Improving osteointegration through mimicking the topographical aspects of the bone ECM, which dominantly is consisted of nanoscale structures, has also gained increasing recent attention. Improved cellular response to substrates featuring nanomorphology in terms of cellular attachment, differentiation and osteointegration/osteogenesis has been reported.14,15 Fabrication of cell interfacing biomaterials with nanoscale features can be conducted via various techniques including electrospinning, lithography, anodization and template/molding approaches.16-19


Page 7 of 57 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


For bone tissue engineering, electrospun chitosan scaffolds with gelatin doping are one of the promising materials in terms of mechanical properties, thermal stability and biocompatibility.20 Improving the cell-biomaterial interactions with such scaffolds is primarily influenced by the focal adhesion kinase regions, the size of which is mainly related to the features of surface topography such as roughness, hierarchy or structure density per area21 Interestingly, it has been reported that electrospun patches do not provide large focal adhesion kinase regions as much as nanopillared surfaces.22 As electrospinning technology inherently cannot provide vertically alligned nanoarrays on a surface, nanopillared structures fabricated by alternative approaches can be good candidates for implant coating studies in terms of cellular adhesion and differentiation.

Anodic alumina molds (AAMs) are unique materials that have arrays of hexagonally packed nanopores with controllable topographical parameters involving pore depth, pore size and porosity. The potential of this inert and durable membrane as a biomaterial has been demonstrated both in the free membrane form23, its functionalized versions24 as well as a coating on metal implants.25 AAMs are also frequently used as molds to create



Page 8 of 57

functional nanotopographical patterns within different setups such as atomic layer deposition26, photolithography or hot embossing27. However, there are no reports about the use of these molds for the nanoscale structuring of natural polymers. The novelty of this work lies in the utilization of AAMs to form such nanostructured biopolymers for inducing osteogenesis for the first time. In the present study, we have fabricated nano Chitosan: Gelatin (C:G) films possessing arrays of ordered nanopillars by using AAMs as molds via drop casting approach. We have shown that the nanopillared films possess excellent bactericidal properties and then investigated the potential of these films for osteogenesis/ differentiation of mesenchymal stem cell lines (MSCs) and osteoblast like cells (Saos-2). The effect of nanotopography on the differentiation and mineralization of both cell lines were studied at the genomic level by PCR analysis and through conducting various mineralization assays. Our results suggest that the nanoscale morphology is critical in the timescale of osteogenic differentiation and nanostructured films are promising antibacterial implant coating materials that can improve mineralization, and thus facilitate the osteointegration of a metal implant.


Page 9 of 57 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


MATERIAL AND METHODS

Fabrication and Modification of Anodic Alumina Molds (AAMs)

The well-known two-step anodization protocol 16, 28 (see details at supporting information) was utilized to fabricate the nanoporous AAMs from high purity Al foils. After the fabrication process, the AAMs were coated with a hydrophobic octadecyltrimethoxysilane (ODS, Alfa Aesar) solution to decrease the surface energy. For this process, a 0.1% (v/v) ODS solution was prepared in hexane and the AAMs were incubated in this solution for 16 h. The molds were then dried at 100°C overnight prior to producing the nanostructured C:G polymer films. A similar coating process was also applied to a Si wafer , which was employed for the fabrication of flat polymer film controls.

Preparation of PEGDE Crosslinked C:G Films

A C:G solution (1.25: 0.25 w/w, 1.25: 0.25 w/w, medium molecular weight chitosan,190-310 kDa, 75-85% deacetylation degree, Sigma Aldrich; skin derived bovine



Page 10 of 57

gelatin, Helavet Ind.) was prepared in a 0.3% (v/v) acetic acid solution. The solution was mixed at 1000 rpm for 2 days at 45°C until it was completely dissolved. In order to increase the stability of the film, poly (ethylene glycol) diglycidylether (PEGDE, Sigma Aldrich) was added to the C:G solution (0.3% (v/v)) at pH 7.5.29 After 15 mins, the solution was carefully spread on the AAMs or Si wafers, and the substrates with cast polymer solution was left in moisture-free incubator at 25°C. After drying at 25°C, the films were then peeled off from the nanoporous AAMs or flat Si wafers.

Morphological Characterization of AAMs and C:G Films

The morphology of the molds and the films were determined using an environmental scanning electron microscope (SEM, Quanta 200, FEI, Hillsboro, OR, USA, 10 mm working distance, 5 kV acceleration voltage). The samples were coated with goldpalladium using a precision etching-coating system (GATAN) before imaging and Image J Software were used to determine size of structures. Atomic force microscopy (ez-AFM, Nanomagnetics) was used to examine the 3D surface structure of the C:G films and to measure the roughness values of the nano and flat films. The tapping mode AFM scans 10 ACS Paragon Plus Environment

Page 11 of 57 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(3 µm × 3 µm ) were obtained at 2.5 µm/s scanning rate for both directions with SSS cantilever (NANOSENSORS™ SSS-NCL).

Surface Energy and Contact Angle Analysis

Surface wettability of the ODS-treated and non-treated AAMs were measured by using a contact angle measurement device (Biolin Scientific, Attention optical tensiometer, UK) using 5 water droplets (10 µl), for three different batches of AAMs. The C:G surface energy and wettability of C:G films were investigated with a drop shape analysis system. The contact angle of 16 µl sessile drops was measured at randomly selected locations on both C:G nano and flat films. The Owens/Wendt equation (Equation 1) was used to calculate surface energy, hence distilled water, glycerol and ethylene glycol were used to obtain contact angle values for the films.

𝝈𝑳(𝒄𝒐𝒔 ϴ + 𝟏) 𝟏𝟐 𝟐(𝝈𝑫 𝑳)

𝑷 𝟏 𝟐𝝈 𝑳

= (𝝈𝑷𝑺)

𝟏𝟐

𝟏𝟐 𝝈𝑷𝑳

+ 𝝈𝑫 𝑺

Equation 1

𝟏𝟐

Here, and are representative dispersive and polar component of the surface tension of the wetting liquid, respectively. and are representative dispersive and polar



Page 12 of 57

component of the surface tension of the solid surface, respectively. is the average contact angle values of the drops.

Confirmation of Crosslinking

Fourier Transform Infrared (FTIR, Mattson 1000) Spectrometry, 1H-Nuclear Magnetic Resonance (H-NMR, Varian MR 400 MhZ) and Differential Scanning Calorimetry (DSC, Q2000 V24.11 Build 124) were used for the chemical and/or thermal analysis of the crosslinked and non-crosslinked C:G films and to confirm film crosslinking. Experimental details of these methods are given in the supporting information section.

Swelling and Weight Loss Measurements

To measure swelling performance, the films (D = 6 mm) were incubated in 1 ml phosphate buffered saline (PBS, pH 7.2, Sigma Aldrich) at 37°C for 20 days. After removal of excess water from films, the weight of each film was measured immediately. The swelling ratio (Q) was calculated from the Equation 2.

Q= (W2-W0) ×100/W0

Equation 2


Page 13 of 57 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


where, W2 is the weight of film after 1, 2, 3, 4, 5, 10, and 20 days of swelling, respectively. W0 is the initial weight of the film.

To measure the weight loss of the samples, circular films (D=6 mm) were soaked in a solution containing 0.02% (w/v) sodium azide and 0.5% (w/v) NaCl, pH 7.4. The samples were collected at the mentioned time points and were lyophilized for 24 h in a freeze-drier (Labconco, FreeZone 6Plus). The Equation 3 was then used to calculate the remaining weight ratio (Wr).

Wr= (W2) ×100/W0 Equation 3

Here, W2 and W0 represent the remaining and initial weights of the films, respectively.

Antimicrobial Activity of C:G Films

To evaluate the antimicrobial activity of the nano and flat films, colony counting method and live-dead assay were performed. Two types of pathogenic bacteria, namely Pseudomonas aeruginosa (ATCC 27853; gram-positive) and Staphylococcus aureus



Page 14 of 57

(ATCC 12600; gram-negative) were used for testing the antimicrobial activity of the films. The bacteria were grown in Tryptic Soy Broth (TSB, Sigma Aldrich) medium for overnight at 37°C. On the following day, bacterial solutions were diluted to 109 CFU/ml utilizing optical density (OD) value of bacterial solutions measured at 562 nm. The final bacterial suspensions were diluted to 106 CFU/ml for the assays. For colony counting assays, films were placed into 96 well plates and the bacteria cultures were added on top of the films. Antibiotic solutions and polystyrene well plates were used as controls. After 4 and 24 h of incubation, solutions were collected from each well and were further diluted to one million and ten million folds. 10 µl of the diluted solutions were dropped on the agar TSB plates and the plates were incubated at 37°C and 5% CO2 for overnight. The number of colonies was counted for each sample on the following day. The experiment was performed in triplicates. To visualize the antimicrobial activity of the films, live-dead assays were performed using prodium iodide and SYTO-9 (see supporting information for details).

Mammalian Cell Culture


Page 15 of 57 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Human osteosarcoma cells (Saos-2, ATCC® HTB-85™) were kindly provided by Dr. Thomas Gardella at Massachusetts General Hospital and Harvard Medical School. Bone marrow-derived human mesenchymal stem cell lines (MSCs, ATCC® PCS-500-012™) were purchased from the American Type Culture Collection (ATCC). The Saos-2 cell line was grown in McCoy`s 5A medium supplemented with 10% heat-inactivated fetal bovine serum (FBS, Invitrogen) and a 1% antibiotic-antimitotic solution (ATCC PCS-99902TM). Routinely, the cell lines were maintained in humidified incubator at 37°C/5% CO2/ 95% humidity and cell media was changed every 3 days. To induce the osteogenic activity of cells, the initial media was changed with osteogenic media, which was supplemented with 5 mM β-glycerophosphate (Sigma Aldrich), 50 mM ascorbic acid (Sigma Aldrich) and 10 nM dexamethasone (Sigma Aldrich). The passage number of the cell lines used for the experiments was between 1-5. MSCs were maintained in a basal medium supplemented with 7% FBS, 2.4 mM recombinant human insulin like growth factor-1, 5 ng/ml fibroblastic growth factor-β, 2.4 mM L-Alanyl-L-Glutamine, and a 1% Penicillin-Streptomycin-Amphotericin B solution. To induce MSCs differentiation towards mineralizing cell phenotypes, cells were incubated in 10 mM β-glycerophosphate, 50 15 ACS Paragon Plus Environment


Page 16 of 57

mM ascorbic acid and 10 nM dexamethasone for a period of two weeks. The differentiation media was changed every 3−4 days. The passage number of MSCs used was between 5-10. Cells grown on tissue culture polystyrene (TCP) were used as the control group for all tests conducted with films.

The immunological response of the films was measured using J774A.1 (ATCC® TIB67™) murine macrophage cell line. The cell line was maintained in 10% FBS supplemented DMEM and was grown in a humidified incubator at 37°C/5% CO2/ 95% humidity. The cells were grown on films for 24 h and were then collected from the surface via trypsinization and qPCR analysis was performed for TNF-alpha gene expression, which is a pro-inflammatory cytokine marker.

Cell Viability

Briefly, the Saos-2 or MSC cells (104 cells/ well) were seeded on the films in a 96-well tissue culture plate at 37°C and 5 % CO2 / 95 % humidity environment for 3 days. The


Page 17 of 57 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


films were cut using 6 mm size biopsy punches and 9.3 mm2 surface area of the films was used for experiments. To test the cell viability, the cells were washed with serum free media, and media was replaced with 100 µl of fresh phenol red free DMEM media supplemented with 10 µl of a 2.4 M 3-[4, 5-dimethylthiazole-2-yl]-2, 5diphenyltetrazolium bromide salt (MTT) solution and the cells were incubated for 2 h at 37°C . To dissolve tetrazolium crystals, the solution was then replaced with 100 µl of dimethyl sulfoxide (DMSO) and the samples were further incubated for 10 min at 37°C. The absorbance value of the solution was recorded at 540 nm.

Alkaline Phosphatase Activity Test

Alkaline phosphatase (ALP) activity levels of Saos-2 and MSCs were quantified by using an assay involving conversion of p-nitro phenyl phosphate into p-nitro phenol. Accordingly, cells seeded on the films were cultured in osteogenic media and the media was changed every 3 days. After culturing for 1, 3, 7 and 10 days, the cells were trypsinized and were then lysed using a ProtinExTM protein extraction solution. Total protein amount was determined and normalized using a bicinchoninic acid assay (BCA, 17 ACS Paragon Plus Environment


Page 18 of 57

Thermo Scientific Inc.). For the ALP assay, 2 µl of 0.67 M p-nitro phenyl phosphate was added into 96 µl of a 10 M diethanolamine and 0.50 mM MgCl2 mixture. The solution was incubated at 37°C for 1 h and afterwards, 20 µl of the control and test samples were added to the wells. Optical absorbance was recorded at 405 nm wavelength. ALP activity of MSCs was measured at 14- and 21-day time points. All the experiments were performed in triplicates.

Alizarin Red Staining, Quantification of Mineral Deposition and Protein Adsorption

To demonstrate the mineral accumulation on the cell membranes of Saos-2 and MSCs, cells (104 cells/ well) were seeded on nano and flat films and were initially grown in regular cell media overnight. Next day, the cell media was replaced by osteogenic media and at the end of the day 3, 10, 14 and/or 21 cells were trypsinized from film surfaces and transferred to TCP surface. The cells were fixed using formalin and then stained with 2% alizarin red dye for 30 min following one day incubation. The cell images were taken using optical microscope (Keyence, Japan). Additionally, the extent of


Page 19 of 57 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


mineralization and protein adsorption were also quantified on different substrates, the details of which were provided in the supporting information section.

qPCR Analyses

Saos-2 and MSCs were seeded on nano C:G nano and flat films, at a density of 2×105 cells per well of a 12 well plate. After day 1 of incubation, the media was replaced with osteogenic media. At the end of day 3, 10, and 21 cells were collected using trypsinEDTA and the cell pellets were obtained. After the mentioned incubation time, the cells were collected and were washed with PBS and were used for qPCR analysis. The following isolation, quantification and primer design details were given in the supporting information section.

Statistical Analysis

For statistical analysis, data was collected for at least three independent experiments and the results were represented as mean ± standard deviation. One-way ANOVA



Page 20 of 57

analysis was used to show the significance of the difference between the test and control samples. p-values less than 0.05 were considered as significant.

RESULTS AND DISCUSSION

Fabrication and Physicochemical Characterization of Nano C:G Films

In this study, nano C:G films were produced via a non-lithographic solvent casting approach using nanoporous AAM molds, and they were subsequently tested for their osteogenic potential. Schematic representation of the nano C:G film fabrication is shown in Figure 1a. The molds were fabricated using a double-step anodization process as previously reported.16, 24, 30-32 First, the AAMs were modified with a hydrophobic silane (ODS) to decrease the surface energy and ease film removal process. Wettability measurements showed that the contact angle of the molds increased from 46±9° to 119±18° after ODS coating (data not shown). Morphological characterization of the molds and the resultant films were carried out with SEM. Representative micrographs display arrays of uniform cylindrical pores on the AAM molds with 104 ± 4 nm diameter (Figure 1b), that translate into nanopillar arrays of the natural polymer solution with ~ 90 20 ACS Paragon Plus Environment

Page 21 of 57 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


nm pillar diameter, ~ 300 nm height and ~ 50 nm pitch between pillars (Figure 1c).The slight difference between the mold and pillar diameter is thought to arise from the mechanical stress during the film peeling process as well as the evaporation of solvent which normally fills the pores together with the polymer chains.16, 32 It is worth mentioning that similar to the dimension of these fabricated C:G nanopillars, the bone ECM predominantly is consisted of nanoscale components spanning from few nm apatite minerals 33 to collagen fibers (50-500 nm diameter) as well as to aggrecan structures that carry hierarchal glycosaminoglycan branches of contour lengths around 400 nm.34 Interestingly surfaces displaying aggrecan-mimetic components with 100 nm diameter and 300 nm height (similar to our C:G nanopillars) caused large size and number of focal adhesion regions between an implant and tissue.21



Page 22 of 57

Figure 1. Fabrication and morphological characterization of nano C:G films (a) Schematic representation of the nano C:G film fabrication. SEM micrographs of (b) AAM molds, and (c) nano C:G films (t=0). The inset scale bar represents 500 nm. (d) 3D AFM scans, and (e) the Owens/Wendth plots of the nano C:G films.


Page 23 of 57 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


It is also reported that columnar structures mainly control stem cells differentiation and osteogenic activity while 3D fiber patches are mostly effective for proliferation and migration activities of the cells.35 Additionally, nanopillared surfaces have some advantages compared to fiber-networks in terms of antibacterial activity36 since they have more potential to increase surface tension of bacteria membrane compared to fiber-networks. Thus, it can be easily hypostasized that our nanopillared films with dimensions similar to ECM components can potentially trigger osteogenic and antibacterial activities.

In order to investigate the influence of surface topography and energy on bacterial/cellular response, flat C:G films were casted using ODS-coated Si wafers and used as control substrates. Investigation of their roughness values with AFM revealed that the nanotopographic film (roughness (R=190±26 nm) is exceedingly rougher than the flat counterpart (R=7±9 nm) as expected, (Figure 1d, Figure S1a). The surface energy of the C:G films was calculated using the Owens/Wendt equation.37 The extracted values are 118 mN/m and 42 mN/m for flat and nano C:G films, respectively,



Page 24 of 57

(Figure 1e, Figure S1b). The effect of these parameters on bacterial/cellular behavior as well as protein adsorption is discussed in the following sections.

PEGDE, a well-known biocompatible crosslinker, was used for the stabilization of C:G films in aqueous media by coupling the hydroxyl or deprotonated amine groups of the polymers.38 To examine the extent of crosslinking, 1H NMR was performed, and spectra were analyzed for presence of different functional groups, (Figure 2). The peaks at 3.54.0 ppm and 3.1 ppm are due to H-2 protons of glucosamine residues, which represent primary amine groups of chitosan, (Figure 2a). In the same figure, the peaks around 1.52.0 ppm correspond to proline rich environment of gelatin.39 With the addition of the PEGDE crosslinker (Figure 2b), the peak at 3.7 ppm appears that belongs to –OCH2 protons PEGDE and the signal represents ring opening of epoxide groups.40 Comparing the two spectra also reveals that the area under the amine peaks (i.e. 3.1 ppm) are reduced (ca. 23.7 %) indicating that the functional epoxide groups of PEGDE reacts with free amine in the polymer mixture.38, 41


Page 25 of 57 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Similar information can be extracted from FTIR data (Figure S2a) through comparing the signature peaks of non-crosslinked and crosslinked C:G films. For the noncrosslinked films, a peak at 1148 cm-1 was observed, which arises due to typical –NH2 vibration of chitosan. This peak disappeared after the crosslinking between amine and epoxy groups. An additional peak at 1733 cm-1 was also observed in the crosslinked films, which corresponds to bonding between PEGDE and chitosan.41 Gelatin-PEGDE crosslinking was further confirmed by the presence of a shoulder observed around 1117 cm-1 that belongs to C–O–H functional group.39 Additionally, the peak observed around 2870 cm-1 represents the –CH2 vibration of PEGDE, and the peak around 1524 cm-1 indicates the electrostatic interaction between amino groups of chitosan and carboxyl groups of gelatin.42

Finally, thermal characterization of the films was conducted via DSC measurements.43, 44

The results of the non-crosslinked C:G films showed that the glass transition

temperature (Tg) and melting point (Tm) of C:G films was 58 °C and 64 °C, respectively (Figure S2b).44, 45 Additionally, film decomposition started to appear around 205 °C



Page 26 of 57

(Figure 2c, black arrow). These peaks disappeared in the crosslinked samples (Figure 2d, S2c) indicating the covalent bonding between the natural polymers and the crosslinker 46, and confirming the NMR and FTIR data.

Figure 2. Chemical and thermal characterization of the nano C:G films. 1H NMR spectrum of (a) non-crosslinked (PEGDE-free), and (b) crosslinked C:G films. DSC thermographs of (c) non-crosslinked, and (d) crosslinked C:G films. Measurements were performed in duplicate.


Page 27 of 57 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


In addition to chemical and thermal properties, water uptake as well as degradation behavior of the films were also examined.47 The nano and flat C:G films were first tested for swelling capacity and the results demonstrated that these materials could absorb water up to ~ 30% of their original weight (Figure 3a), which can be explained by the hydrophilic character of the constituent natural polymers. Both films were stable up to 5 days and started to degrade afterwards, losing about ~ 40 % of their initial dry weight at day 20, (Figure 3b). To test the stability of the nanopillars, the films were incubated in DMEM cell culture media for 1, 3 and 24 h. SEM micrographs taken after 1 h showed arrays of slightly swollen but intact nanopillars on the film surface, (Figure 3c). The swollen nanopillars were found to be joint together at 3 h. Here, initial signs of degradation were also observed, and it is plausibly responsible from the formation of the void portions on the films, (Figure 3d). These voids can also be a result of the swollen nanocraters seen in Figure 3c that occur due to the local imperfections of the honeycomb structure of AAM molds.



Page 28 of 57

Figure 3. Swelling characteristics of the nano and flat C:G films. (a) Percent swelling of crosslinked nano and flat C:G ﬁlms. (b) Remaining weight ratios of lypholized films. The films were incubated in PBS at 37 °C, up to 20 days. SEM scans of nano C:G films postincubation in cell culture media for (c) 1 h, (d) 3h, and (e) 24 h. The insets scale bars represent 500 nm. The data presented is average ± standard deviation (n=3), * represents p

Nanopillared Chitosan-Gelatin Films: A Biomimetic Approach for

Recommend Documents