Smart pH-responsive antimicrobial hydrogel scaffolds prepared by

Subscriber access provided by University of South Dakota

Article

Smart pH-responsive antimicrobial hydrogel scaffolds prepared by Additive Manufacturing Carolina García, Alberto Gallardo, Daniel López, Carlos Elvira, Asma Azzahti, Elena López-Martínez, Aitziber L. Cortajarena, Carmen M. González-Henríquez, Mauricio A Sarabia-Vallejos, and Juan Rodriguez-Hernandez ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00297 • Publication Date (Web): 13 Sep 2018 Downloaded from http://pubs.acs.org on September 13, 2018

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 29 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 Applied Bio Materials

216x121mm (96 x 96 DPI)

ACS Paragon Plus Environment

ACS Applied Bio Materials 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

Page 2 of 29

Smart pH-responsive antimicrobial hydrogel scaffolds prepared by Additive Manufacturing

Carolina Garcia,1 Alberto Gallardo,1* Daniel López,2 Carlos Elvira,1 Asma Azzahti,1 Elena Lopez-Martinez,3 Aitziber L. Cortajarena,3,4 Carmen M. González-Henríquez5 Mauricio A. Sarabia-Vallejos6 and Juan Rodríguez-Hernández1*

1. Polymer Functionalization Group (FUPOL). Instituto de Ciencia y Tecnología de Polímeros (ICTP), Consejo Superior de Investigaciones Científicas (CSIC), C/Juan de la Cierva 3, 28006 Madrid, Spain. Email: [email protected], [email protected] . 2. Macromolecular Engineering Group (MacroEng). Instituto de Ciencia y Tecnología de Polímeros (ICTP), Consejo Superior de Investigaciones Científicas (CSIC), C/Juan de la Cierva 3, 28006 Madrid, Spain. 3. CIC biomaGUNE, Parque Tecnológico de San Sebastián, Paseo Miramón 182, 20014 Donostia-San Sebastián, Spain 4. Ikerbasque, Basque Foundation for Science, Mª Díaz de Haro 3, 48013 Bilbao, Spain 5. Universidad Tecnológica Metropolitana, Facultad de Ciencias Naturales, Matemáticas y del Medio Ambiente, Departamento de Química, P.O. Box 9845, Correo 21, 7800003, Santiago, Chile. 6. Pontificia Universidad Católica de Chile, Escuela de Ingeniería, Departamento de Ingeniería Estructural y Geotecnia, 3. Departamento de Química. P.O. Box 306, Correo 22, 7820436, Santiago, Chile.

1 ACS Paragon Plus Environment

Page 3 of 29 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


Abstract We report on the fabrication of 3D printed pH-responsive and antimicrobial hydrogels with micrometer-scale resolution achieved by stereolithography (SLA) 3D printing. The preparation of the hydrogels was optimized by selecting the most appropriate difunctional polyethylene glycol di(meth)acrylates (testing crosslinking agents with chain lengths ranging from 2 up to 14 units ethylene glycol) and introducing acrylic acid (AA) as a monofunctional monomer. As a result of the incorporation of AA, the hydrogels described are able to reversibly swell and shrink upon environmental changes on the pH and the swelling extent is directly related to the amount of AA and can be thus finely tuned. More interestingly, upon optimization of the UV penetration depth employing a photoabsorber (Sudan I), a reliable procedure for the fabrication of 3D objects with high model accuracy is showed. Finally, the antimicrobial properties of all the hydrogels were demonstrated using Staphylococcus aureus as a bacterial model. We found that even those hydrogels with a low amount of AA monomeric units presented excellent antimicrobial properties against S. aureus.

Keywords Rapid prototyping, microstereolithography, 3D microstructure, scaffolds, 3D hydrogels, antibacterial materials, stimuli-responsive.



Page 4 of 29

Introduction The fabrication of hydrogels with intricate structures has been largely pursued during the last decade. In biological applications and, in particular, for tissue engineering purposes, the geometrical parameters of the scaffolds are essential to offer the most suitable structure for cell permeation, adhesion and proliferation inside the material1 but also for nutrient transport and the appropriate cell-matrix interactions2-3. As a result, in the fabrication of hydrogel scaffolds the structural design at different length scales (including macro, micro, and also at the nano level) is one of the priorities in current ongoing hydrogel materials research. The macroarchitecture refers to the complete form of the final device, which can be intricate due to the patient and organ specificity or the anatomical features. The microarchitecture includes among others pore size, shape, porosity, spatial distribution or pore interconnection. Some studies have reported the ideal porosity composition of scaffolds for tissue engineering to be between 60 and 80%, with pore sizes ranging from 100 to 500 µm.1 In addition to the porosity, the type of application will determine the stiffness required for the material.4 In this context, hydrogels are typically more adapted to fabricated scaffolds for soft tissues.5 Different methods have been reported and widely employed in the past to fabricate 3D scaffolds. These include solvent casting, woven and nonwoven fiber-based fabrics, phase separation, particulate leaching, melt molding, high-pressure processing, forging, injection molding, or hot and cold pressing.1, 6-12 However, most of the above mentioned approaches are rather manual and some of them require multiple steps for the preparation of the desired geometry.4 As an example, as reported by Guan et al.13 scaffolds prepared by thermally induced phase separation requires a polymer mixed with a solvent to be injected into a glass mold. Then, upon several tedious steps that include: 3 h in liquid nitrogen, 7 days incubation in alcohol to remove the solvent, and dry, the scaffold is completed. Finally, an additional issue of traditional techniques is the lack of control of the scaffold porosity.14 Usually, these methods produce materials with a heterogeneous porosity and with a wide range of pores sizes.


Page 5 of 29 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 comparison to the above mentioned methodologies, additive manufacturing (AM), usually referred to as 3D printing, offers important advantages. As depicted by Ventola

15

one of the

major advantages is the possibility to fabricate custom-made medical products or even equipment. Moreover, in comparison with the traditional manufacturing methods, AM is cost effective for small production runs and due to the recent advances in the different AM technologies high precision 3D parts with intricate geometries can be straightforwardly prepared only based on the appropriate CAD design.2, 16 Among the different AM technologies available, stereolithography (SLA) (also known as vat photopolymerization (VPP)) has been the most extended for the preparation of 3D engineered hydrogel scaffolds. SLA uses photosensitive mixtures that upon precise irradiation can be selectively crosslinked in the desired areas with micrometer-scale resolution thus forming a rigid material. The successive coating of the part with additional photosensitive mixture and further photopolymerization permits the formation of a 3D printed object. Several groups have already employed this strategy for the fabrication of hydrogels for different applications including the fabrication of tissue engineering constructs,17 heterogeneous aortic valve conduits18-19 oral modified-release dosage forms20 or for the elaboration of 3D printed microfluidic chips with patterned cell-laden hydrogel constructs.21 In addition to the geometrical requirements, a major issue in the use of hydrogel materials as supports is the eventual contamination by microorganisms and, mainly, by bacteria.22-23 Microorganism contamination has been usually prevented by working in aseptic conditions.24-25 However, these strategies still require further improvement since they reduce but are not able to completely avoid the adhesion of microorganisms. The material selection is, as a consequence, the focus of intensive research in order to obtain novel hydrogels that perfectly combine the mechanical and the chemical properties to produce the desired biological response while preventing its contamination with microbes.26 In this context, herein we report the fabrication of 3D printed hydrogels with accurate geometries that present both modulated pH-response and excellent antimicrobial properties. As will be thoroughly discussed the design of the 4 ACS Paragon Plus Environment


Page 6 of 29

photosensitive mixture that leads to the final material is crucial and is directly related to the material response.

Materials and methods The monomers polyethyleneglycol methacrylate (PEGMA300) and acrylic acid (AA) were used as received. The crosslinking agents employed in this study were, diethyleneglycol dimethacrylate (DEGDMA, units of ethylene oxide: NEO = 2), tetraethylene glycol dimethacrylate (TEGDMA, units of ethylene oxide: NEO = 4), polyethyleneglycol dimethacrylate

(PEGDMA550,

average

units

of

ethylene

oxide:

NEO

=

10)

and

polyethyleneglycol diacrylate (PEGDA700, average units of ethylene oxide: NEO = 14). Phenylbis(2,4,6-trimethylbenzoyl)phosphine oxide (IRG 819) was used as photoinitiator and 1(Phenyldiazenyl)naphthalen-2-ol (Sudan I) as UV absorber. All other solvents including ethanol and Millipore water were purchased from Sigma Aldrich and used as received. Characterization ATR-FTIR spectra were recorded in a FTIR spectrometer Spectrum One of Perkin-Elmer. Using ATR with and internal reflection elements diamond/ZnSe. The region analyzed corresponds to a 2 µm depth. Micro-computed tomography (Micro-CT). A CT-SCAN Nikon XT H-160 was employed to precisely investigate the structure of the 3D printed scaffolds in liquid media using a molybdenum target. For that purpose, the fabricated scaffolds were immersed in ethanol and fixed with cotton to prevent any rotation. The experimental conditions employed for these measurements were: Laser 71 kV and 138 µA. The exposure time for each projection was 708 ms and 1000 images were recorded at a separation of 0.36º (step size). Each projection, used for the 3D reconstruction, is the average of 4 images. The voxel resolution of the 3D models was, isotropically, ~22,7 µm, this value was obtained through VoxelSize.


Page 7 of 29 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


Hydrogel swelling and mesh size The swollen cylinders were accurately weighted and then allowed to dry until a constant weight was reached. The swelling (S) was defined as the amount of solvent (water or ethanol) per gram of dry network in the equilibrium state (equation (1)).

(1)

Where Wh is the swollen weight and Wd is the dried weight. Molecular weights between crosslinks, Mc, have been determined from the swelling data by using the methodology described by Brannon-Peppas and Peppas 27 for pH-sensitive hydrogels. (2) where equation (2) is the Flory-Rhener equation, Mc is the molecular weight between

crosslinks; ν2,s is the polymer volume fraction in the swollen state, which is calculated from the swelling; ͞Mn is the number average molecular weight of linear polymer chains, which is usually considered large enough to neglect the term 2/Mn, as it has been done here; ͞ν is the specific volume of the polymer, which has been obtained by averaging the values of the different components; V1 is the molar volume of ethanol (58,39 cm3/mol); χ is the Flory interaction parameter, which has been also obtained by averaging the values corresponding to the different components in ethanol 0,7 for PEO and 0,492 for PMMA.

Methods Fabrication of the 3D printed hydrogels by stereolithography (SLA) The 3D scaffolds were either designed using Autodesk Inventor 2015 or using the design provided by CaptainPicar3d (https://www.thingiverse.com/thing:918775) and manufactured using a stereolithographic (SLA) printing technology. For that purpose, a Projet 1200 3D printer from 3D Systems was employed equipped with a LED projector at 405 nm wavelength. The 3D printing is an additive process where the final object is constructed by coating down successive 6 ACS Paragon Plus Environment


Page 8 of 29

layers of material. The resolution achieved was 30 µm in Z-axis and around 56 µm (effective 585 dpi) in XY plane. The 3D printer software employed was Geomagic. The Project 1200 3D printer has an approximately 15 ml reservoir in which the desired photosensitive mixture was placed after oxygen removal (achieved bubbling with N2). For this purpose, the commercial green resin cartridges were empty and filled with the appropriate photosensitive mixture. In our particular case, no additional precautions were taken into account. The first series of experiments were focussed on the fabrication of hydrogels with variable crosslinking density and the analysis of the incorporation of acrylic acid (AA) on the final hydrogel properties. For this objective, a photosensitive mixture composed of the crosslinking agent (DEGDMA, TEGDMA, PEGDMA550 or PEGDA700) and a variable amount of monofunctional monomer, either PEGMA300 was employed first to construct square shaped structures (1cm x 1cm x 2mm). For 15 ml of the mixture crosslinking agent, 100 mg of photoinitiator (IRG 819) were employed. Then, provided the best crosslinking agent (herein PEGDMA550) different hydrogels with variable amount of AA ranging from 5 up to 30wt% were prepared using the same proportion of photoinitiator as depicted above. The second series of experiments involved the structure optimization. In order to fabricate parts with micrometer-scale resolution a photoabsorber that limits the UV-light penetration was employed. Sudan I was used as photoabsorber and the amount of to be employed was optimized: 3D printed parts were fabricated using 15 ml of a mixture di(meht)acrylate/AA, 100 mg of IRG 819 and an amount of Sudan I ranging from 0,5 mg to 10 mg (Figure S2 Supporting information). According to our results, an amount of 2 mg appeared to be optimal. On the one hand, less Sudan I did not permit to precisely reproduce the CAD design. On the other hand, higher amounts of Sudan I produced thinner crosslinked layers and, in some cases, the UV penetration is too low and no connection between two consecutive layers was observed thus producing holes and spaces in the structure of the 3D printed part. Mechanical properties of the hydrogels


Page 9 of 29 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


Mechanical properties were measured by dynamic compression test. Measurements were performed on 3D printed cylindrical scaffolds (Figure 5) at equilibrium water-swollen gels at 25ºC and using a universal test system MTS ® QTest1/L Elite uni-axial testing machine equipped with a 100 N load cell in compression mode. Samples were placed between compression platens. Each sample was subsequently deformed at 0.1 mm/min. All measurements were performed by triplicate. Bacterial adhesion and viability assays For this study, S. aureus was used as model bacteria since it is a common pathogen responsible for many hospital-acquired infections, through a strain that has acquired resistance to antibiotics (Methicillin-resistant Staphylococcus aureus, MRSA) and also of many common skin infections. For these experiments, the hydrogel samples are cylinders with a 1cm in diameter and 2mm height. This shape will allow us to evaluate the bacterial activity within the appropriate 24-well cells. Staphylococcus aureus strain RN4220 carrying the plasmid pCN57 for green fluorescent protein (GFP) expression was grown overnight at 37°C in Luria–Bertani (LB) media with erythromycin (10 µg mL−1). The cells were centrifuged and washed three times in PBS saline buffer (150 mM NaCl phosphate 50 mM, pH 7.4 0.05% Tween). The solution was adjusted to a cell concentration that corresponds to an optical density (OD) at 600 nm of 1.0 corresponding to approximately 1.5 109 colony forming units (CFU)/ml. The different scaffolds were incubated for 1 hour with bacterial suspensions in PBS at OD = 1.0. After incubation, the scaffolds were washed with PBS buffer three times for 15 minutes. After washing, bacterial adhesion was monitored by fluorescence microscopy using a Leica DMI-6000 fluorescence microscope. Images were acquired using a x 41.6 magnification objective and the corresponding set of filter for imaging green fluorescence corresponding to the GFP expressed in the bacteria. 8 ACS Paragon Plus Environment


Page 10 of 29

The bactericidal properties of the hydrogels were measured by staining the adhered bacteria with a red fluorescent dye that only penetrates permeabilized, i.e. died cells. Fluorescent images were acquired in order to calculate the percentage of live and dead bacteria under each experimental condition. In order to perform bacteria viability experiments, propidium iodide staining was chosen and used as indicated in the LIVE/DEAD BacLight Bacterial Viability Kit. Propidium iodide is a red-fluorescent nucleic acid stain that penetrates only cells with disrupted membranes and intercalates DNA. The different hydrogels with attached bacteria were incubated with propidium iodide (5 mg mL−1) for 15 minutes, followed by rinsing with PBS solution (3 times). Phase contrast, green and red fluorescence microscopy images were taken at x41.6 magnification. The number of total bacteria (alive and dead) is quantified from the green bacteria, and the dead bacteria are stained also in red. The bacterial cell density and the viability were quantified from the microscopy images using ImageJ software. Preliminary biocompatibility tests All hydrogels were sterilized by rinsing four times with a 70% ethanol solution during 10 min., washed with PBS four times and placed under UV radiation for 20 min. Then, two washes with incomplete DMEM culture medium were made, and finally two washes with complete culture medium (FBS and antibiotics) were performed. The cell studies were carried out using C2C12-GFP, a mouse pre-myoblast cell line (CRL 1772™, obtained from ATCC®, USA). Green Fluorescent Protein (GFP) was expressed due to previous lentivirus infection of this cell line. Routine passaging of the cell line was performed with DMEM high in glucose (GIBCO, UK), supplemented with 10% fetal bovine serum (FBS, 10500-064, Gibco, UK) plus antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin sulfate) (Gibco, UK). The medium was refreshed every two or three days. These cells were chosen as in vitro model because they are able to differentiate toward osteoblastic or myoblastic phenotype, depending on the surrounding microenvironment. Another interesting characteristic


Page 11 of 29 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


of these cells concerns their self-fluorescence. As a result of this property, they can be analyzed through some non-opaque/translucent surfaces, such as the polymer films employed here. For culturing cells over the porous films, the three cell types were seeded singly on the samples at different densities in supplemented DMEM, and the polymers were plated in a 24-well plate in maintenance medium, incubated at 37 °C with 5% CO2 in a humidified incubator. For experiments on PS-copolymers, cells were seeded at a density of 5x103/polymers.

Metabolic activity study: Alamar Blue assay. Metabolic activity of cells was measured by Alamar Blue assay, this was performed following the manufacturer’s instructions (Biosource, CA, USA). Assays were performed in triplicate on each sample type. This method is non-toxic, scalable and uses the natural reducing power of living cells, generating a quantitative measure of cell viability and cytotoxicity. Briefly, Alamar Blue dye (10 % of the culture volume) was added to each well, containing living cells seeded over films, and incubated for 90 minutes. The fluorescence (λex/λem 535/590 nm) of each well was measured using a plate-reader (Synergy HT, Brotek).

Results and discussion Preparation of the 3D printed hydrogels The preparation of the 3D hydrogels was carried out by stereolithography (SLA) that permits to precisely construct the object with micrometer resolution. The parts, designed using a CAD software, were printed using a Projet 1200 SLA printer (3D Systems). For that purpose, the monomer mixture was introduced into the reservoir and exposed to UV light in the required areas. The scaffold was constructed by coating down the successive layers of material and sequential UV exposure. The 3D printed parts were washed with ethanol to remove the residual



Page 12 of 29

unreacted monomer/crosslinking mixture and then exposed to a post-curing process that, finally, increased the photopolymerization conversion. Initial trials were conducted to determine the best crosslinking agent and the effect of the addition of AA on the hydrogel structure. Therefore, for these tests the photosensitive mixture employed comprises a monofunctional monomer, either PEGMA or AA, crosslinking agent (DEGDMA, TEGDMA, PEGDMA550 or PEGDA700) and a photoinitiator (IRG 819 in this case). Whereas the amount of photoinitiator was maintained constant (6.7 wt%), the relative quantity of monofunctional monomer to crosslinking agent was varied in order to fabricate hydrogels with different crosslinking density. The first series of experiments were focused on the analysis of different crosslinking agents (CLA) in the formation of stable 3D hydrogel networks using PEGMA300 as monofunctional monomer. These initial tests were carried out on 3D printed parts with the following dimensions: 10 x10 x 2 mm. Four different di(meth/-)acrylates were employed, i.e. DEGDMA, TEGDMA, PEGDMA550, and PEGDA700 with a variable amount of EO units between the acrylate terminal groups. Depicted in Figure 1 are the relative swelling values obtained for the hydrogels in water and ethanol prepared using the different crosslinking agents and a variable amount of mono/di-functional monomer employed. As can be observed, for a particular crosslinking agent, the same tendency can be detected, i.e. an increase of the CLA amount leads to hydrogel networks with less capacity to swell indicating the formation of hydrogels with a larger density of crosslinking. Nevertheless, important differences were obtained in the use of the different CLA. Only TEGDMA330 and PEGDMA550 series could be studied completely. The 3D printed parts using DEGMA leads to rigid parts that finally upon swelling easily break, and therefore they could not be included in the graph. The use of PEGDA700 produced hydrogels only easy to handle when very large amounts of PEGDA700 were employed and were extremely breakable when the PEGDA700 content is below 40 wt%. Comparing TEGDMA330 and PEGDMA550 series, the latter presented larger swelling in comparison with TEGDMA330 while maintaining the appropriate mechanical properties. Using PEGDMA550, hydrogels swellings varying from 0.35 and 1 were readily obtained.


Crosslinker employed PEGMA300/PEGDA700

1,2

PEGMA300/PEGDMA550 1,0

PEGMA300/TEGDMA330

0,8 0,6 0,4 0,2 0,0 20

30

40

50

60

70

80

90

100

Swelling [(wet weight-dry weight)/dry weight)]

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


Swelling [(wet weight-dry weight)/dry weight)]

Page 13 of 29

1,2

Crosslinker employed PEGMA300/PEGDA700 PEGMA300/PEGDMA550

1,0

PEGMA300/TEGDMA330 0,8 0,6 0,4 0,2 0,0 20

30

Amount of crosslinking agent (wt%)

40

50

60

70

80

90

100

110

Amount of crosslinking agent (wt%)

Figure 1. Swelling behavior of SLA 3D printed parts in water (left) and ethanol (right) varying the type and amount (wt %) of crosslinking agents. In order to obtain further information about the molecular structure of the hydrogel, the average molecular weight between crosslinks for the different systems was estimated. Figure 2 shows the evolution of the average molecular weight between crosslinks as a function of the molar content in crosslinker, for the series of hydrogels PEGMA330 / TEDEGMA330 (Figure 2a) PEGMA330 / PEGMA550 (Figure 2b) and PEGMA330 / PEGMA700 (Figure 2c). The experimental values, determined by applying the Flory-Rhener equation, are represented together with the theoretical ones calculated considering an ideal network and using equation (3).

Mc=Mr/2Y *(1-x)+Mca * x

(3)

Where Mc is the average molecular weight between crosslinks; Mr is the molecular weight of the monomer; Y is the ratio moles of crosslinker to moles of monomer; x is the percentage of crosslinker and Mca is the molecular weight of the crosslinker. This equation considers that there is a double population of values of the average molecular weight between crosslinks. One derived from the crosslinking reaction itself (first term of equation 3) and a second population of molecular weights, which is equal to the molecular



Page 14 of 29

weight of the crosslinking reagent, since it cannot be considered as punctual in space (second term of equation 3). The behavior is quite different for the three systems studied, as can be seen in Figures 2a-c. In the case of the PEGMA330 / TEDEGMA330 system (Figure 2a), the average molecular weight between crosslinks obtained experimentally is always below the theoretically predicted value. Both values approach as the percentage of crosslinker decreases. In the case of the PEGMA330 / PEGMA550 system (Figure 2b), the behavior is similar but the difference between both values is lower and for crosslinker compositions below 40 wt % there is a crossover, being the values obtained experimentally above the theoretical ones. Finally, in the case of PEGMA330 / PEGMA700 (Figure 2c), at high crosslinking compositions the experimental and theoretical values are quite close. Below a crosslinker composition of 70 wt%, the theoretical values are under the experimental ones, which highly increase at low crosslinking concentrations. Negative deviations from the average molecular weight between crosslinks values are indicative of cross-linking densities higher than those predicted through the stoichiometry, which could be an indication of the occurrence of additional cross-linking reactions, probably in the post-cure process. On the other hand, positive deviations indicate a lower cross-linking density that may be related to imperfections in the network, such as structural inhomogeneities (zones with high density of crosslinks alternating with areas of low density), formation of chains with free ends, cycles, etc. 28-30.


Page 15 of 29

(a)

1400

PEGMA300/TEDGDMA330

Mc calc Mc theo

1200

Mc calc

1000 800 600 400 200 0 20 30 40 50 60 70 80 90 100 110

%

(b)

1400

PEGMA330/PEGMA550

Mc calc Mc theo

1200

Mc calc

1000 800 600 400 200 0 20 30 40 50 60 70 80 90 100 110

%

(c)

1400

PEGMA330/PEGMA700

Mc calc Mc theo

1200 1000

Mc calc

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


800 600 400 200 0 20 30 40 50 60 70 80 90 100 110

%

Figure 2. Calculated molecular weight between crosslinks as a function of the amount (wt%) of crosslinking agent for the series of hydrogels: (a) PEGMA330 / TEDEGMA330, (b) PEGMA330 / PEGMA550 and (c) PEGMA330 / PEGMA700 PEGDMA550 gave the best performance in terms of swelling/mechanical properties, and was the crosslinker selected for the following studies centered on the incorporation of acrylic acid (AA) both to deliver pH responsiveness and also antimicrobial properties. For this purpose, AA was incorporated to the formulation varying the ratio between AA and PEGDMA550. Once the objects were printed, washed and post-cured (following the same protocol as depicted above) a compositional analysis of the resulting hydrogel was carried out by FT-IR. Additionally, as it is shown in Figure 3(a) polyethyleneglycol dimethacrylate550 (PEGDMA550) based network and 14 ACS Paragon Plus Environment


poly(acrylic acid) (PAA) hompolymer have been added to the graph for comparative purposes. By analyzing the region comprised between 1500 cm-1 and 1800 cm-1 the signal corresponding to the carbonyl groups can be identified. Whereas the carbonyl group of PPEGMA appears at 1724 cm-1 a different wavelength was observed for the carbonyl group associated to PAA, i.e. 1693 cm-1. As a result, the compositional variation can be estimated by comparing the intensity of the signals at these two wavelengths. In Figure 3(b) are depicted the relative intensity of these two signals that, as expected, evidencing a gradual enrichment of the final hydrogel in AA. As a result, the chemical composition of the hydrogels obtained by SLA can be easily controlled by finely tuning the feed composition.

(b) C=O (PEGDMA) -1 1724cm

-1

C=O (AA) -1 1693 cm

-1

(a)

IBand 1693cm /IBand 1724 cm

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

Page 16 of 29

PEGDMA 10% AA 25% AA 50% AA 75% AA PAA

750

1000 1250 1500 1750 2000 -1 Wavelength (cm )

2,0

1,5

1,0

0,5

0,0 0

20

40

60

80

100

wt % of acrylic acid in the feed

Figure 3. (a) FT-IR spectra of 3D printed parts with variable amount of AA. For comparative purposes, a PEGDMA network and PAA have been included. (b) Variation of the relative intensity of the carbonyl signals of PEGDMA and AA and calculated composition for the different hydrogels.

Similar to the hydrogels prepared in the first series of experiments comprising exclusively PEDGMA and PEGMA, the AA based hydrogels were able to swell both in ethanol and water. As depicted in Figure 4, the swelling has been estimated using ethanol and water at two different pH values, either acidic (pH below 3.5) and therefore below the pKa of the acrylic acid functional group (between 4.2-4.5 according to reported values) and at basic pH where the acid 15 ACS Paragon Plus Environment

Page 17 of 29 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


groups are deprotonated and thus negatively charged. As expected, the tendency in both ethanol and acidic pH is rather similar with a slight increase of the swelling when the amount of PEGDMA decreases. Interestingly, the swellings observed when the hydrogel is immersed in a basic pH aqueous solution are significantly higher for the same compositions. This clearly indicates that the swelling depends on the ionization of the AA functional groups. In the range of compositions explored from 5 up to 30 wt% of AA in the initial mixture the swelling in the ionized states varies between 30 up to 70 wt%. It is worth mentioning that hydrogels with larger amounts have been prepared but upon immersion in water the hydrogels were not stable and easily break. As a result, these swelling values are rather fair in comparison with other hydrogels reported due to the large amount of crosslinking agent required to fabricate the 3D objects using SLA. Nevertheless, some of the hydrogels obtained are able to almost duplicate their initial weight by incorporation of water within the structure and, most importantly, as has been already mentioned, can be straightforwardly fabricated with unlimited 3D geometries with micrometer-scale resolution. Since the swelling is directly related to the ionization degree of the AA groups and the latter is related to the environmental pH, another interesting feature of these hydrogels is the response to changes in the acidity/basicity of the aqueous solution. In order to analyze this feature, the swelling was measured upon several cycles by alternatively immersing the samples in aqueous acidic pH solutions (pHa) and basic aqueous solutions (pHb). In Figure 5 is depicted the swelling by changing the water pH between 3.5 and 10 for hydrogels prepared using between 0 and 30 wt% of AA. Interestingly, the hydrogels were able to respond to pH by changing their swelling back and forth depending on the environmental pH. At higher pH values the hydrogels with the AA in the charged state are able to swell to a larger extent in comparison to the same hydrogels at acidic pH. For instance, the hydrogels prepared using 30 wt% of AA are able to swell between 70-75 wt% at basic pH and the swelling is significantly reduced down to 20-25 wt% at acidic pH. Another interesting feature of these materials is that the pH response is directly related to the amount of AA within the hydrogel structure. The graph depicted in



Figure 5(b) represents the average swelling increase defined as the difference in swelling between basic and acid pH for hydrogels prepared using a variable amount of AA. As can be observed, the difference in the swelling at both pH values increases linearly with the amount of AA incorporated in the feed. As a result, both the swelling extent and also the intensity of the

Swelling ((Wwet - Wdry)/Wdry)

pH response can be easily modulated by controlling the chemical composition of the hydrogel.

Ethanol Water pH 3.5 Water pH 10

80

60

40

20

0

5

10

15

20

25

30

Amount of AA in the feed (wt %)

Figure 4. Swelling of the hydrogels prepared using a variable ratio AA to PEGDMA: from 100 and 70 wt% and AA from 0 to 30 wt%.

(a) 1,0

0,8

0,6

0,4

0,2

pHb(6)

pHa(6)

pHb(5)

pHa(5)

pHa(4)

pHb(4)

pHb(3)

pHa(3)

pHb(2)

pHa(2)

pHa(1)

0,0

Average swelling increase pHb - pHa

(b) 0 wt% AA 10 wt% AA 20 wt% AA 30 wt% AA

pHb(1)

Swelling [(wet weight - dry weight) / dry weight]

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

Page 18 of 29

0,8

0,6

0,4

0,2

0,0

0

10 20 30 Amount of AA in the feed (wt%)

Figure 5. (a) Hydrogel swelling measured after six consecutive cycles changing between acid pH (pHa) and basic pH (pHb) for four different monomer/crosslinking agent mixtures: 100%


Page 19 of 29 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


PEGDMA, 90%PEGDMA/10%PAA, 80%PEGDMA/20%PAA and 70%PEGDMA/30%PAA. The values correspond to: pH acid ~3,5 and pH basic ~ 10. The number after each pH indicates the cycle of the measurement. (b) Average swelling difference between basic and acid pH as a function of the hydrogel composition. The above-depicted experiments evidenced the possibility to vary the chemical composition of the hydrogels and therefore obtain variable pH response. These were carried out in 3D printed parts with simple geometries. However, the complexity of the scaffold can be significantly increased by using the CAD design. However, the first attempts to print complex 3D scaffolds without using photoabsorber lead to 3D printed parts that could not perfectly reproduce the design. For this reason, Sudan I was added to the photosensitive mixture. Sudan I is a widely employed photoabsorber that limits the penetration depth of the UV light improving the resolution of the 3D printed parts

31-34

and can be easily removed by extensive washing in

ethanol. In particular, the amount of Sudan I added to the mixture was optimized and fixed to 2 mg in 15 ml of monomer/crosslinking agent mixture (see supporting information). In Figure 6 are depicted the micro-CT images of the hydrogels fabricated using four different designs measured in ethanol using photosensitive mixtures containing 80 wt% of PEGDMA550, 20 wt% of AA and 0.014 wt% of Sudan I. As observed in Figure 6, by using a small amount of Sudan I, high-resolution 3D printed hydrogels were readily obtained. An attempt to quantify the accuracy of the 3D printed part has been carried out by using the micro CT images as well as the images of the models. By using the Image J software several dimensions (see Supporting information - Figure S5) to detect the deviation of the 3D printed part. According to our analysis in all cases the deviations remain below the 4 %. Since our 3D printed parts are around 1 cm height the eventual deviations are below 0.4 mm.



Page 20 of 29

(i)

(ii)

(iii)

(iv)

Figure 6. Left: Models (i) to (iv) selected for the fabrication of the antimicrobial objects. Right: respective lateral and top 3D views of the micro-CT images. These hydrogels were fabricated using a photosensitive mixture containing 80 wt% of PEGDMA550, 20 wt% of AA, 6.7 wt% of photoinitiator and 0.014 wt% of Sudan I.

The mechanical properties of the printed scaffolds were evaluated by compression tests. First of all, the contribution of the addition of AA on the final mechanical properties was evaluated by using one of the scaffold geometries. For this series of experiments, the geometry (iii) was selected (note that for this experiments Sudan I was not employed). As presented in Figure 7(a), a gradual decrease of the modulus is observed upon incorporation of AA remaining however in values above 20 MPa in all the compositions up to 25 wt% of AA and only 19 ACS Paragon Plus Environment

Page 21 of 29

decreasing to 15 MPa for the hydrogels with 30 wt% of AA. Therefore, an increase in the amount of AA has associated a diminution on the mechanical properties. As has been mentioned, these tests were carried out on printed parts that due to the reduced resolution appears to be cylindrical tubes. In order to evaluate the mechanical properties of the different designed geometries, a second series of experiments was carried out for a particular hydrogel fabricated using 20 wt% of AA and introducing Sudan I to exactly reproduce the design. The moduli of the different geometries are represented in Figure 7(b). The modulus of the 3D printed scaffolds using Sudan I decreased between 50-60% and all presented similar values (within the error of the measurements) between 6 and 8 MPa. Without any doubt, Sudan I significantly improved the resolution of the printed parts and thus hydrogels with more accurate shape could be fabricated. However, these parts are less resistant to elastic deformations. Nevertheless, according to the literature, these values are rather high in comparison to others obtained in view of the preparation of hydrogel scaffolds. For instance, Bryant et al. reported the preparation of PEG based hydrogels with modulus in the kPa range measured in solid cylindrical probes.35 More recently, Chatterjee evidenced that PEO hydrogels with moduli ranging between 12 and 306 kPa, but still largely below the values reported here.36

(a)

(b)

30

10

Modulus (MPa)

25

Modulus (MPa)

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


20 15 10

8 6 4 2

5 0

0

0

30 20 10 25 Amount of AA in the feed (wt %) 0

(i)

(iii) (ii) Scaffold geometry

(iv)

Figure 7. Mechanical properties of the hydrogels. (a) Geometry (iii) printed without the use of Sudan I. The final part is a cylinder. (b) Different geometries ((i) to (iv)) printed with a 2wt% of Sudan I.



Page 22 of 29

Provided a well-defined chemical composition and a controlled response to environmental changes, the bacterial adhesion and antimicrobial efficiency was investigated. The adhesion and antibacterial efficiency were evaluated by incubating the 3D printed hydrogels with bacteria during 1h. Upon rinsing, the adhered bacteria were imaged using a fluorescence microscope. As depicted in the illustrative images in Figure 8, the hydrogels prepared without AA presented moderate to low bacterial adhesion that significantly decreased upon addition of AA. The addition of 20 wt% of AA leads to a 10-fold reduction of bacterial adhesion from the beginning of the experiment (Figure 8e). As a result, based on previous findings two different explanations can be found for this result. On one hand, the PEG groups together with the negatively charged groups of the AA monomer units can repel the bacteria taking into account that the bacterial cell wall is also negatively charged. On the other hand, AA, with the charged carboxylic acid groups has been proved to be an excellent antimicrobial. Thus, in addition to the low bacterial adhesion, probably those bacteria adhering to the hydrogel surface are effectively killed and released from the surface, although some bacteria remain attached to the surface. More interestingly, after 24 h of hydrogel treatment, there were less bacteria on the surfaces, this effect was more significant on the samples with lower AA %, since the samples with higher AA % presented less bacteria attached already at t=0 (Figure 8f). Moreover, almost all bacteria that remained attached were dead in all the hydrogel bearing AA (Figure 9) while they partially remain alive in the reference hydrogel without AA (see Supporting information – Figure S6). Therefore, the role of AA is key to the bacterial adhesion to the hydrogels and their viability. It is worth mentioning that AA is covalently attached since it copolymerizes the PEGDMA and the residual unreacted monomer is removed by extensive washing in EtOH. As a result, the antimicrobial activity observed is most probably due to the interaction of the carboxylic acid groups with the bacteria. The mechanism of this interaction is still under investigation. However, previous works carried out by us

37

and others 38 found similar antimicrobial activity

in PAA based systems. 21 ACS Paragon Plus Environment

Page 23 of 29 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


Figure 8. Fluorescence microscopy images at 41.6X magnification of the hydrogel surfaces: (a) 25 wt% of AA, (b) 20 wt% (c) 15 wt% (d) 10 and (e) 0 wt% of AA in the feed. Scale bars correspond to 20 µm (f) Bacterial density on the surface (number/ cm2) at time t=0 hours after 1 hour incubation with bacteria. Error bars correspond to the standard deviation of the number of bacteria counted in 5 different random areas of each sample.



Page 24 of 29

Figure 9. Bacterial viability on the different hydrogels with a variable amount of AA. Fluorescence micrographs at 41.6X magnification, after 24 hours after bacteria adhesion. (a), (c) and (d) correspond with the emission of all bacteria, while (b), (d) and (f) correspond to the emission of propidium iodide, correlated to dead bacteria. (a) and (b) 25% wt AA, (c) and (d) 20% wt AA; (e) and (f) 10% wt AA. Scale bars correspond to 20 µm. In order to use these hydrogels as scaffolds or other tissue engineering applications, a crucial aspect is the biocompatibility towards mammalian cells. In this context, several preliminary experiments have been carried out to assess this issue. First of all, the preparation protocol was optimized (note that the bacterial experiments reported above were carried out using this optimized protocol). On the one hand, the use of post-curing treatments (UV-light) allow us 23 ACS Paragon Plus Environment

Page 25 of 29 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


achieve a high conversion degree so that the amount of unreacted monomer is minimal. On the other hand, extensive washing with ethanol permits to completely remove potential residual unreacted monomer. This is essential since acrylates and methacrylates are known to be cytotoxic. Biocompatibility assays were prepared using 90%PEGDMA/10%PAA at three different stages: (a) samples obtained directly after the 3D printing step, (b) samples obtained after 3D printing and the post-curing step and (c) samples obtained after 3D printing, post-curing and extensively washed with ethanol (Supporting information S7). These initial tests evidence a clear improvement after introducing the post-curing step remaining, the metabolic activity, significantly lower than a standard tissue culture plate (TCP). However, after extensive washing with ethanol the metabolic activity increases and remains close to the values found in TCP. In summary, a combination of both post-curing and ethanol washing appears to be an excellent combination to improve the biocompatibility. These results are in good agreement with those reported by Macdonald et al.39 that adapted the Fish Embryo Test (FET) to evaluate the biocompatibility of commercial SLA resins. In particular, they found that the biocompatibility of the resins significantly increased by using 3D printed parts that were treated during 72h in EtOH.

Conclusions This manuscript describes the precise fabrication of stimuli responsive antibacterial hydrogels by additive manufacturing. By optimizing the photosensitive mixture, hydrogels with variable crosslinking density and chemical composition could be synthesized. More precisely, stable hydrogels with moduli up to 20 MPa and bearing up to 30 wt % of AA were readily prepared. Moreover, by using a small amount of photoabsorber the 3D printed structures perfectly correspond to the designed CAD scaffolds.



Page 26 of 29

Finally, the pH response, as well as the antibacterial properties, were investigated. Hydrogels with AA amounts below 30% were able to swell and shrink by varying the environmental pH between acid and basic. These swelling and shrinking processes were repeated during several cycles without affecting the hydrogel integrity. Equally, these hydrogels have shown excellent antimicrobial properties. Within the compositional range of the explored (5-30 wt% of AA) all the 3D printed hydrogels were able to readily kill S. aureus on contact. 3D hydrogel scaffolds with smart behavior and antibacterial properties may find potential applications among others for tissue engineering purposes, for the elaboration of smart biosensors with precise geometry or as actuators in biological media. Other potential applications of these hydrogels involve the incorporation of both biological and electronic modalities for the development of an anatomically correct 3D printed parts capable of detecting electromagnetic frequencies. Supporting information Swelling of the hydrogels prepared by using different diacrylates. Effect of the addition of Sudan I on the resolution of the 3D printed parts. Removal of Sudan I using ethanol. Micro-CT images of the different models. Calculations made to analyse the accuracy of the 3D printed parts. Bacterial counting on the hydrogels with different amount of acrylic acid (AA). Preliminary biocompatibility evaluation of the hydrogels. Acknowledgments The authors gratefully acknowledge support from the Consejo Superior de Investigaciones Científicas (CSIC). Equally, this work was financially supported by the Ministerio de Economía y Competitividad (MINECO) through MAT2013-47902-C2-1-R, BIO2016-77367-C2-1-R MAT2013-42957-R and MAT2016-78437-R (FEDER – EU). The authors acknowledge financial support given by FONDECYT Grant N° 1170209. M.A. Sarabia acknowledges the financial support given by CONICYT through the doctoral program Scholarship Grant. Finally,


Page 27 of 29 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


this study was funded by VRAC Grant Number L216-04 of Universidad Tecnológica Metropolitana. References 1. Annabi, N.; Nichol, J. W.; Zhong, X.; Ji, C.; Koshy, S.; Khademhosseini, A.; Dehghani, F., Controlling the Porosity and Microarchitecture of Hydrogels for Tissue Engineering. Tissue Eng., Part B 2010, 16 (4), 371-383. 2. Hockaday, L.; Kang, K.; Colangelo, N.; Cheung, P.; Duan, B.; Malone, E.; Wu, J.; Girardi, L.; Bonassar, L.; Lipson, H., Rapid 3D Printing of Anatomically Accurate and Mechanically Heterogeneous Aortic Valve Hydrogel Scaffolds. Biofabrication 2012, 4 (3), 035005. 3. Mondschein, R. J.; Kanitkar, A.; Williams, C. B.; Verbridge, S. S.; Long, T. E., Polymer Structure-Property Requirements for Stereolithographic 3D Printing of Soft Tissue Engineering Scaffolds. Biomaterials 2017, 140, 170-188. 4. Gross, B. C.; Erkal, J. L.; Lockwood, S. Y.; Chen, C.; Spence, D. M., Evaluation of 3D Printing and Its Potential Impact on Biotechnology and the Chemical Sciences. Anal. Chem. 2014, 86 (7), 3240-3253. 5. Hollister, S. J., Porous Scaffold Design for Tissue Engineering. Nat. Mater. 2005, 4 (7), 518. 6. Hutmacher, D. W.; Sittinger, M.; Risbud, M. V., Scaffold-Based Tissue Engineering: Rationale for Computer-Aided Design and Solid Free-Form Fabrication Systems. Trends Biotechnol. 2004, 22 (7), 354-362. 7. Yang, S.; Leong, K.-F.; Du, Z.; Chua, C.-K., The Design of Scaffolds for Use in Tissue Engineering. Part I. Traditional Factors. Tissue Eng 2001, 7 (6), 679-689. 8. Kemppainen, J. M.; Hollister, S. J., Tailoring the Mechanical Properties of 3D‐Designed Poly(Glycerol Sebacate) Scaffolds for Cartilage Applications. J. Biomed. Mater. Res., Part A 2010, 94 (1), 9-18. 9. Zhang, P.; Hong, Z.; Yu, T.; Chen, X.; Jing, X., In Vivo Mineralization and Osteogenesis of Nanocomposite Scaffold of Poly(lactide-co-glycolide) and Hydroxyapatite Surface-Grafted with Poly (L-lactide). Biomaterials 2009, 30 (1), 58-70. 10. Pham, Q. P.; Sharma, U.; Mikos, A. G., Electrospinning of Polymeric Nanofibers for Tissue Engineering Applications: a Review. Tissue Eng. 2006, 12 (5), 1197-1211. 11. Bajaj, P.; Schweller, R. M.; Khademhosseini, A.; West, J. L.; Bashir, R., 3D Biofabrication Strategies for Tissue Engineering and Regenerative Medicine. Annu. Rev. Biomed. Eng. 2014, 16, 247-276. 12. Bártolo, P. J.; Domingos, M.; Patrício, T.; Cometa, S.; Mironov, V., Biofabrication Strategies for Tissue Engineering. In Advances on Modeling in Tissue Engineering, Springer: 2011; pp 137-176. 13. Guan, J.; Fujimoto, K. L.; Sacks, M. S.; Wagner, W. R., Preparation and Characterization of Highly Porous, Biodegradable Polyurethane Scaffolds for Soft Tissue Applications. Biomaterials 2005, 26 (18), 3961-3971. 14. Yeong, W.-Y.; Chua, C.-K.; Leong, K.-F.; Chandrasekaran, M., Rapid Prototyping in Tissue Engineering: Challenges and Potential. Trends Biotechnol. 2004, 22 (12), 643-652. 15. Ventola, C. L., Medical Applications for 3D Printing: Current and Projected Uses. Pharm. Ther. 2014, 39 (10), 704-711. 16. Yang, S.; Leong, K.-F.; Du, Z.; Chua, C.-K., The Design of Scaffolds for Use in Tissue Engineering. Part II. Rapid Prototyping Techniques. Tissue Eng. 2002, 8 (1), 1-11. 17. Dhariwala, B.; Hunt, E.; Boland, T., Rapid Prototyping of Tissue-Engineering Constructs, Using Photopolymerizable Hydrogels and Stereolithography. Tissue Eng. 2004, 10 (9-10), 1316-1322.



Page 28 of 29

18. Duan, B.; Hockaday, L. A.; Kang, K. H.; Butcher, J. T., 3D Bioprinting of Heterogeneous Aortic Valve Conduits with Alginate/Gelatin Hydrogels. J. Biomed. Mater. Res., Part A 2013, 101 (5), 1255-1264. 19. Duan, B.; Kapetanovic, E.; Hockaday, L. A.; Butcher, J. T., Three-Dimensional Printed Trileaflet Valve Conduits Using Biological Hydrogels and Human Valve Interstitial Cells. Acta Biomater. 2014, 10 (5), 1836-1846. 20. Wang, J.; Goyanes, A.; Gaisford, S.; Basit, A. W., Stereolithographic (SLA) 3D Printing of Oral Modified-Release Dosage Forms. Int. J. Pharm. 2016, 503 (1), 207-212. 21. Stephanie, K.; Chu Hsiang, Y.; Fulya, E.; Sharareh, E.; Ali, K.; Savas, T., 3D-Printed Microfluidic Chips with Patterned, Cell-Laden Hydrogel Constructs. Biofabrication 2016, 8 (2), 025019. 22. Gopinathan, U.; Stapleton, F.; Sharma, S.; Willcox, M.; Sweeney, D.; Rao, G.; Holden, B., Microbial Contamination of Hydrogel Contact Lenses. J. Appl. Microbiol. 1997, 82 (5), 653-658. 23. Wu, Y. T.-Y.; Willcox, M.; Zhu, H.; Stapleton, F., Contact Lens Hygiene Compliance and Lens Case Contamination: A Review. Contact Lens and Anterior Eye 2015, 38 (5), 307316. 24. Maltseva, I.; Morris, C. A.; Khong, K.; Luk, A., Protection of Contact Lenses from Microbial Contamination Caused by Handling. Google Patents: 2015. 25. Galante, R.; Pinto, T. J.; Colaço, R.; Serro, A. P., Sterilization of Hydrogels for Biomedical Applications: A Review. J. Biomed. Mater. Res., Part B 2017, 106 (6), 2472-2492. 26. Kang, K. H.; Hockaday, L. A.; Butcher, J. T., Quantitative Optimization of Solid Freeform Deposition of Aqueous Hydrogels. Biofabrication 2013, 5 (3), 035001. 27. Brannon-Peppas, L.; Peppas, N. A., Equilibrium Swelling Behavior of pH-Sensitive Hydrogels. Chem. Eng. Sci. 1991, 46 (3), 715-722. 28. Naghash, H. J.; Okay, O., Formation and Structure of Polyacrylamide Gels. J. Appl. Polym. Sci.e 1996, 60 (7), 971-979. 29. Tobita, H.; Hamielec, A., Crosslinking Kinetics in Polyacrylamide Networks. Polymer 1990, 31 (8), 1546-1552. 30. Fernández, E.; López, D.; López-Cabarcos, E.; Mijangos, C., Viscoelastic and Swelling Properties of Glucose Oxidase Loaded Polyacrylamide Hydrogels and the Rvaluation of Their Properties as Glucose Sensors. Polymer 2005, 46 (7), 2211-2217. 31. Zheng, X.; Deotte, J.; Alonso, M. P.; Farquar, G. R.; Weisgraber, T. H.; Gemberling, S.; Lee, H.; Fang, N.; Spadaccini, C. M., Design and Optimization of a Light-Emitting Diode Projection Micro-Stereolithography Three-Dimensional Manufacturing System. Rev. Sci. Instrum. 2012, 83 (12), 125001. 32. Kamarudin, K. Parameter Optimization for Photo Polymerization of Mask Projection Micro-Stereolithography. Universiti Tun Hussein Onn Malaysia, 2013. 33. Khairu, K.; Raman, I.; Mohamed, M. A. S.; Ibrahim, M.; Saidin, W. In Parameter Optimization for Photo Polymerization of Microstereolithography, Advanced Materials Research, Trans Tech Publ: 2013; pp 420-424. 34. Ibrahim, R.; Raman, I.; Ramlee, M. H. H.; Mohamed, M. A. S.; Ibrahim, M.; Saidin, W. In Evaluation on the Photoabsorber Composition Effect in Projection Microstereolithography, Applied Mechanics and Materials, Trans Tech Publ: 2012; pp 109-114. 35. Bryant, S. J.; Anseth, K. S., Hydrogel Properties Influence ECM Production by Chondrocytes Photoencapsulated in Poly(ethylene glycol) Hydrogels. J. Biomed. Mater. Res. 2002, 59 (1), 63-72. 36. Chatterjee, K.; Lin-Gibson, S.; Wallace, W. E.; Parekh, S. H.; Lee, Y. J.; Cicerone, M. T.; Young, M. F.; Simon, C. G., The Effect of 3D Hydrogel Scaffold Modulus on Osteoblast Differentiation and Mineralization Revealed by Combinatorial Screening. Biomaterials 2010, 31 (19), 5051-5062. 37. Vargas-Alfredo, N.; Martínez-Campos, E.; Santos-Coquillat, A.; Dorronsoro, A.; Cortajarena, A. L.; del Campo, A.; Rodríguez-Hernández, J., Fabrication of Biocompatible and Efficient Antimicrobial Porous Polymer Surfaces by the Breath Figures Approach. J. Colloid Interface Sci. 2018, 513, 820-830. 27 ACS Paragon Plus Environment

Page 29 of 29 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


38. Gratzl, G.; Paulik, C.; Hild, S.; Guggenbichler, J. P.; Lackner, M., Antimicrobial Activity of Poly (acrylic acid) Block Copolymers. Mater. Sci. Eng. 2014, 38, 94-100. 39. Macdonald, N. P.; Zhu, F.; Hall, C.; Reboud, J.; Crosier, P.; Patton, E.; Wlodkowic, D.; Cooper, J., Assessment of biocompatibility of 3D printed photopolymers using zebrafish embryo toxicity assays. Lab Chip 2016, 16 (2), 291-297.


Smart pH-responsive antimicrobial hydrogel scaffolds prepared by

Recommend Documents