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

Super Mechanical Stimuli Responsive Hydrogel: Dynamic Cues for Cell Applications Ravichandran H. Kollarigowda,†,‡ Anu Stella Mathews,† and Sinoj Abraham*,†,§ †

Department of Chemical & Materials Engineering, Donadeo Innovation Centre for Engineering, and §Mechanical Engineering Department, Donadeo Innovation Centre for Engineering, University of Alberta, 9211 116th Street Northwest Edmonton, Alberta T6G 1H9, Canada

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S Supporting Information *

ABSTRACT: In this work, we fabricated a thermosensitive hydrogel based on NIPAM that incorporated an oxygen-permeable hyaluronic acid (HA) polysaccharide unit. The interpenetrating network (IPN) of NIPAM-HA-MA hydrogel was fabricated by using an ultraviolet (UV) cross-linking process. Importantly, we explored the real behavior kinetics of the hydrogel via the mechanical properties of Young’s modulus and the shear strain. The mechanical properties were reduced when the temperature was raised from 25 to 35 °C, and lower temperatures increased the rigidity of the IPN gel. This dynamic change occurred because of NIPAM’s thermosensitive character. Further, IPN hydrogel exhibits good transparency with the incorporation of MA-HA and this stimuli-responsive hydrogel was biocompatible with a human dermal fibroblasts cell line (HDF). The cells adherence was densest when the temperature reached 37 °C and was reduced when the temperature was lowered to 25 °C. We envision using such hydrogels as mimics of cell-instructive biomaterials in tissue engineering applications. KEYWORDS: stimuli-responsive hydrogels, PNIPAM hydrogel, hyaluronic acid, IPN gel, cell interaction materials

1. INTRODUCTION Hydrogels closely mimic the physical properties of the extracellular matrix (ECM), and as a result, they have been used for a wide range of biomedical applications.1−3 Because of their good biocompatibility, flexibility in manufacturing, variable composition, and desirable physical characteristics (similar to the physiological conditions), hydrogels alone or combined with cells have many biomedical applications. They can serve as scaffolds that provide a three-dimensional (3D) structure for tissue engineering (TE), as bearers of cell encapsulation (EC)2 or gene delivery/drugs,3 as adhesives or barriers between the fabric and material surfaces,4,5 or act as cell sheets for the reversible control of cell adhesion.6,7 The structure of the network of hydrogels in the wet state is swollen because of the presence of reticulation chemistry or physics.8,9 Chemically cross-linked hydrogels are also known as thermosetting hydrogels or permanent gels.10−14 Hydrogels that suffer relatively large and abrupt changes in their behavior, inflammation network structure, permeability, and/or mechanical resistance in response to small environmental changes are called stimuli-sensitive hydrogels.13−18 Stimuli-sensitive hydrogels are also called smart or environmentally sensitive hydrogels;19,20 some examples are pressure-, pH-, temperature-, ionic-, and light-sensitive hydrogels/materials.15−18 One of the interesting and promising areas in the biomedical field of hydrogels is sensitivity to temperature, as well as other © XXXX American Chemical Society

important properties of hydrogels that are improved, such as structural stability and mechanical resistance behaviors. Among them, thermosensitive hydrogels have received considerable attention and have been widely investigated during the past decade because of the importance of temperature in biomedicine and other systems. In particular, poly(N-isopropylacrylamide) (PNIPAM) is a thermosensitive hydrogel material that undergoes a phase transition around the lower critical solution temperature (LCST) of 32 °C in aqueous media.21 The thermosensitive PNIPAM hydrogel absorbs water to swell at temperatures lower than the LCST and collapses with a sharp decrease in volume when the temperature reaches the LCST with rapid alteration from hydrophilicity to hydrophobicity.22 Thus, PNIPAM hydrogels have been utilized in a variety of applications, such as bioseparation, TE,23 and the systematic distribution of controlled drugs.24,25 Given that a rapid response to changes in temperature is essential for the majority of applications, the low response rate of PNIPAM due to the formation of a dense layer of skin has to be improved. In our case, the PNIPAM was embedded with hyaluronic acid (HA), which produces a slight Received: October 8, 2018 Accepted: December 5, 2018 Published: December 5, 2018 A

DOI: 10.1021/acsabm.8b00595 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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ACS Applied Bio Materials

chloride was added dropwise into the reaction flask, whereas 0.1 mol sodium hydroxide was simultaneously added dropwise at 0−4 °C to maintain the pH 8, see Scheme S1. A basic pH of 8 was maintained throughout the reaction. The reaction was stopped and kept for dialyzing in water for 3 days, during which the water in the system was changed every 4 h. The dialyzed solution was precipitated in acetone; next, the precipitate was filtered and finally lyophilized. The MA−HA was analyzed using 1H NMR to confirm the methacrylation group on the HA. Synthesis of NIPAM−MA−HA Hydrogel. Thirty milligrams (w/w) of NIPAM was dissolved in 1 mL of PBS solution, and then 60 mg (w/w) of 100% MA−HA was added to the solution and sonicated for 15 min while maintaining a low-temperature bath. Nine milligrams of bis(acrylamide) cross-linker was added to the above solution and further sonicated for 5 min. One milligram of the Irga 2959 was dissolved in 25 μL of methanol (Irga was not soluble in water). The initiator solution was added to the solution flask containing the NIPAM, the MA−HA, and the cross-linker. 65 μL of the mixed solution was added to a 24-vial plate and kept under UV light for 2 h. The cured hydrogel was washed with PBS solution 3−4 times, part of the hydrogel was lyophilized for morphology and other studies and the rest of the sample was used for cell culture studies. Cell Culture. For 2D culture, NIPAM, MA-HA and NIPAM-HAMA hydrogel were loaded into a sterile 24-well plate with 24 h incubation, and then 500 μL of culture medium was carefully added onto the gel and then incubated overnight at 37 °C. The supernatant medium was carefully removed and the gel was washed three times with 500 μL of culture medium within an interval of 2 h to equilibrate the gel to the physiological pH prior to cell seeding. The final density of subconfluent human dermal fibroblasts (HDF) used for the 2D assay was 15 000 cells/well. Briefly, 15, 00 cells in 500 μL of medium were carefully added on to the settled gel and then incubated for 2 days to observe cell elongation.

alteration in the LCST of PNIPAM and tunes the mechanical properties of the thermosensitive hydrogel. In contrast, HA, also known as hyaluronan, is a polysaccharide of high molecular weight found in the ECM, especially in the soft connective tissue, and is a glycosaminoglycan.26−29 HA consists of units of repeated disaccharide taking the [D-glucuronic acid (1-B-3) of N-acetyl-D-glucosamine (1-B-4) structure and can be obtained from different natural sources, such as the vitreous humor of the eyes, the joint fluid, the comb del Gallo, and the umbilical cord.30 Although today there is a great amount of work on the use of NIPAM as well as HA on hydrogel synthesis and application in TE,31−33 we found few research works related to the synthesis of this polymer system in the presence of natural HA polymer in the reviewed literature. Ohya et al.34 studied PNIPAM, grafted PNIPAM−hyaluronan (PNIPAM−HA), and PNIPAM-grafted gelatin (PNIPAM−gelatin), and these materials show a sol−gel transformation at physiologic temperature and were applied as controls to adhesions of postsurgical tissues. These researchers also published the interrelationship between the microscopic structure and mechanical properties of the regions of the cell surface and adhesiveness. To the best of our knowledge, a cross-linker of methacrylate has been modified on HA with a maximum of 5−20%,35 there is no information on the 100% methacrylation of HA. Here, we report the first protocol and the condition for the synthesis of 100% methacrylate cross-linker with HA. Further, 100% of MA−HA was reacted with thermosensitive NIPAM to form the network of an interpenetrating hydrogel. A scanning electron microscope (SEM) revealed the surface dynamics of structural changes, from smooth surface to rough surface when the temperature goes from 25 to 37 °C. Further, mechanical properties of Young’s modulus and the shear strain showed a decrease in the rigidity of the hydrogel when the temperature goes from 25 to 35 °C. This dynamic change is because of NIPAM’s thermosensitive nature. Another key information, IPN gel exhibits a good transparency with the help of MA-HA incorporation in the main chain of IPN. These were tested with hydrogel fibroblast cell line for biocompatibility and we found that the fibroblast cell line is biocompatible. In fact, when the temperature goes from 25 to 37 °C, we realized that the cells were fully adhered to the surface more densely than at a lower temperature. This is because when the temperature changes, the NIPAM−MA−HA hydrogel structure dynamically changes from a smooth porous surface to a rough surface structure, this situation favors cell line adherence on the hydrogel. Basically, the cell interaction is more favorable on a roughly patterned scaffold than on a flat patterned scaffold.36 In addition, the adhesion and growth of fibroblast cells for the 37 °C scaffolds were significantly better than that of the smooth scaffold. This demonstrates that morphology changes in the scaffold could improve the adhesion and a growth of fibroblast cells particle porous scaffolds, which could be useful for applications in TE. We believe strongly that these materials could be the good biomaterials for cell instructive materials (CIMs).

3. RESULTS AND DISCUSSION 3.1. Functionalization and Hydrogel Fabrication Process. The HA was amended with the cross-linker of methacrylate in the presence of basic conditions; HA has two possible functional groups to modify in the spine of the polysaccharide, acid and another hydroxyl group. In general, glycidyl methacrylate was used to modify the acid via esterification,37,38 but glycidyl methacrylate can form a transesterification reaction during the methacrylation with the acid group, which restricts the degree of substitution, See Scheme S1. In view of this, we found a new way to achieve the 100% methacrylation of the HA backbone via the hydroxyl group. The mole ratio of methacryloyl chloride varied between 10−100% with respect to the hydroxyl group of HA at 0−4 °C under basic conditions. The 10% methacrylation was reached with 10 molar excess of methacryloyl chloride and reached 100% when use exceeds the 100% methacryloyl chloride (see Scheme S1), more details on the synthetic procedure can be found in the Experimental Section. The methacrylation was confirmed using 1H NMR; the double bonds of CH2CH2 appeared at 5.3 and 5.7 ppm and the methacrylate group of − CH3 was assigned to 1.9 ppm, see Figure 1 and Figure S1. The injectable interpenetrated hydrogel of the thermosensitive network was synthesized using NIPAM and 100% MA− HA. First, the hydrogel of NIPAM and MA−HA was made using Irga 2959 as a photoinitiator with UV irradiation with a wavelength of 365 nm for 2 h; see the Experimental Section for the synthetic procedure. The Schematic view of interpenetrating network (IPN) of hydrogel formation is presented in Figure 2. 3.2. Morphology Performance and chemical composition. The NIPAM hydrogel appeared as a white, thickened

2. EXPERIMENTAL SECTION Materials and Methods. All reagents and solvents used were purchased from Sigma-Aldrich and used as received. Synthesis of MA−HA (General Procedure). In a 500 mL roundbottom flask, 1 mol of HA was dissolved in 250 mL of water: acetone mixture (70:30). Ten to one-hundred percent excess methacryloyl B

DOI: 10.1021/acsabm.8b00595 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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and with a 9:1 ratio of cross-linker with respect to photoinitiator at room temperature and cured for 2 h under UV light (365 nm wavelength). When 10% MA−HA was reacted with NIPAM, the scaffold looked white, like unreacted NIPAM hydrogel (data not shown). In fact, the MA-HA composition at this concentration was not enough to change the scaffold’s structure. Next, when we increased the concentration to 30% the hydrogel was transparent and the texture was different from pure NIPAM hydrogel. When we increased the concentration to 35% and 40%, we had difficulty in solubilizing the MA−HA in the PBS medium (because HA molecular weight was high 1.8MDa). Therefore, we decided that 30% is the best concentration of MA−HA to combine with the NIPAM material. NIPAM− MA−HA was characterized by FTIR to confirm the interaction of MA−HA with NIPAM and we found that MA−HA was completely cross-linked with the NIPAM gel, see Figure S3. The wavenumber of the hydroxyl group was 3500 cm−1 and was completely reduced after the NIPAM reacted with the MA−HA. Further, the morphology of the hydrogel was characterized by SEM micrographs. First, NIPAM−MA−HA hydrogel was lyophilized and then liquid nitrogen was poured on the hydrogel just before placing it into the SEM chamber. The morphology of the NIPAM−MA−HA hydrogel was a porous structure with pores almost 2 μm in size. After that, the material was taken out and dried at 37 °C and again placed into the SEM chamber to determine the morphology of the hydrogel. Interestingly, we noticed that after drying the sample, the morphology of the hydrogel was changed into a rough, shallow microparticle with a porous crust patterned scaffold, see Figure 3c, d. 3.3. Mechanical Properties. To understand the phenomenon of thermosensitivity of the hydrogel, we have determined the mechanical properties, storage modulus (SM) and loss modulus (LM), of NIPAM−MA−HA with different temperature cycles. SM was 128 Pa at 25 °C (LM: 85 Pa); at 30 °C, SM was reduced to 98 Pa (LM: 68 Pa); and when it reached 35 °C, the SM was 48 Pa (LM: 48 Pa) with eight frequencies, see Figure 4a. Interestingly, when the temperature returns to 30 °C, the SM relaxed to 88 Pa (LM: 65 Pa) and when it reaches 25 °C the gel completely relaxed to the original situation, see Figure 3a. We also analyzed the oscillation torque (OT) and complex viscosity (CV), see Figure 4b. The OT at 25 °C was 7.6 Pa/s, but at 35 °C increases to 18 Pa/s, and when the temperature returns to 25 °C, the OT relaxed to the original value. The cycle was repeated four times and the results were consistent. The real thermosensitive character was observed using the mechanical properties of the hydrogel. This is because of the thermoresponsive character of the NIPAM hydrogel; the mechanism cycle of this hydrogel is shown in Figure 7 and Figure S4b. 3.4. Transmittance Performance. For thermosensitive NIPAM based hydrogels, structures in homogeneities usually prevent their applications in many fields. Particularly the transparencies of the cross-linked IPN hydrogels were always poor with high NIPAM network. Hence, enhancing the mechanical strength of NIPAM-MA-HA hydrogels is significant and also improving their optical transparencies of the IPN is also another key factor for the current research. In this work, our result shows that synthesized IPN of hydrogel not only have good mechanical properties but also display protuberant optical transparencies, as shown in Figure 5.

Figure 1. 1HNMR spectra of hyaluronic acid (black), 100% methacrylated hyaluronic acid (red) spectra and chemical structure of MA-HA with attributed chemical shifts.

Figure 2. Schematic illustration of thermosensitve hydrogel fabrication using UV cross-linking process. MA-HA, NIPAM, and bis(acrylamide) crosslnker solutions were added into the well plate. Next, photoinitiator (irgacure) was added and plate was kept under UV light for 2 h. Cured hydrogels were washed with buffer (pH 7.4 PBS) solution.

gel, but in the case of MA−HA it was transparent in this state, see Figure S2. SEM micrographs revealed that the NIPAM gel had a porous microparticle structure, whereas MA−HA was 2μm porous material, see Figure 3a, b. Therefore, MA−HA was

Figure 3. SEM images of hydrogels. Morpopholgies of (a-c) NIPAM, MA-HA and NIPAM-MA-HA at 37 °C. Morphological structure of (d-f) NIPAM, MA-HA and NIPAM-MA-HA was taken by cooling to ∼25 °C. (scale bar 2 μm).

of high molecular weight and was quite difficult to solubilize with higher concentrations, we tested the incorporation of MA−HA with NIPAM between 10 and 30% (w/w). When we increased the MA−HA to 40% (w/w), we noticed that during the mixture with NIPAM, it solidified and was coming out from the system before curing with the NIPAM. Therefore, we decided 30% (w/w) MA−HA was the maximum ratio to form an interpenetrating network with NIPAM. The NIPAM−MA−HA cross-linked hydrogel was synthesized by using 30% (w/w) MA−HA, 60% (w/w) of NIPAM C

DOI: 10.1021/acsabm.8b00595 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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The change of optical transparency was explored by using UV spectrophotometer, by increasing the HA content in the IPN we control the transparency of IPN gel. Although the temperature was below LCST the sole NIPAM gel shows zero transparency because of the opaqueness, but in the case of sole MA-HA and NIPAM-MAHA, IPN shows 98 and 85% transparency, respectively. When temperature was increased step-by-step from 25 to 40 °C, the transparency started going down with IPN hydrogel, but MA-HA gel did not show much difference. When the temperature reached 34 °C, the transparency was around 65% with NIPAM-MA-HA hydrogel, See Figure 5. This is considered as the LCST of the IPN hydrogel in this work. The presence of NIPAM in this hydrogel under goes transition with temperature which is comparable with previously reported values of LCST for NIPAM hydrogels.39,40 However when the temperature reached 35 °C, the transmittance was still 58% (even though we increased the temperature to 40 °C, the transmittance was not reduced). This result showed that although NIPAM is a strongly temperature-dependent material, the incorporation of MAHA provides stability and good transparency for this IPN gel. To confirm the above mechanism, we executed the same form of radical polymerization of NIPAM for 24 h with a target of 60 000 g/mol molecular weight, see Figure S5. The synthesized polymer was dissolved in water at ambient temperature and then heated to 32 °C and within 5 min the poly(NIPAM) clear solution became milky white. When cooled to room temperature, the NIPAM solution returned to clear. Although this thermosensitive behavior of NIPAM is well-known in our case, this sensitive character gave a new route to NIPAM-MA-HA scaffolds in biological applications. The nature of the water in the hydrogel can be determined through the permeation of liquids, nutrients, or cell products through this gel. When a dry hydrogel starts to absorb water, the first water molecules penetrating the gel matrix will hydrate the more hydrophilic and polar complexes of the system, leading to the creation of the so-called primary bound water.40 After the polar groups are hydrated (primary bound water), the network starts to swell and, thanks to this swelling character of hydrogel, the polymer chain disentangles and the molecules have more space to move, thus exposing the hydrophobic groups which also interact with the water molecules leading to the bound water−hydrophobic group interactions called secondary bound water. Into scientific literature it is common to encompass and simplify the terms primary bound water and secondary bound water under the terms total bound water.40 3.5. Oxygen Permeable Performance. Oxygen is one of the critical limiting factors for maintaining cell viability and function, a great deal of effort is being focused on improving the oxygen supply to cellular constructs. In order to increase the oxygen permeability of IPN hydrogel, HA substitution is the key factor. In our work, hyaluronic acid was efficiently incorporated in the main IPN gel. One hundred percentage of methacrylation helps to cross-link with the NIPAM main network. Importantly, the higher the oxygen permeability increases the stiffness with the decrease of water content of the hydrogel. The oxygen permeability of fabricated IPN was 130 ± 4 cm mL O2, see Figure S5. Oxygen permeability (Dk/t) was calculated by using eq 1 and (2).

Figure 4. Mechanical properties of thermosensitive hydrogel. )a) Storage modulus and loss modulus cycle of NIPAM-MA-HA, 25−35 °C. And (b) oscillation torque and complex viscosity cycle of NIPAM-MA-HA, 25−35 °C.

Figure 5. Transparency of NIPAM, MA-HA and NIPAM-MA-HA IPN gels with different temperature. (a) UV transmittance of all the three hydrogels (glass substrate used standard for calibration) and (b) Inserted images are photographs of fabricated hydrogels on printed paper. Reversible cycle inserted images of IPN optical image at 25−35 °C. D

DOI: 10.1021/acsabm.8b00595 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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ACS Applied Bio Materials (I − Id) Dk (preliminary) = 5.804 × 10−2 t pA A

−1 ij (2.35t ) zyz Dk DK zz (corrected) = (preliminary)jjj1 + j t t D cathode z{ k

morphology of the hydrogel because of NIPAM’s thermoresponsive character. Lowering the temperature below the biological condition for a short time is a compatible process, which helps us understand the gel chemistry of the smooth and rough surfaces of the morphologic changes of the NIPAM-MAHA surface. The thermosensitive character of NIPAM changes the surface roughness, which increases the surface area and favors the cell adhesion density and proliferation. The shape of the cells, the orientation, and the proliferation seem to depend on the morphology/texture of the hydrogel network and probably also on the thermoresponsive character of the NIPAM and the adjacent hydrogel material.45−48 Interestingly, most of the cells inside the porous area were arranged on the rough surface (adhered), as can be seen in Figure 6a. This behavior has not been noticed in pure HA gel (25 °C) (Figure S8). NIPAM-MA-HA scaffold better enhanced cell adhesion and growth on the gels at 37 °C compared with 25 °C (with 5 min of sonication,).18,49,50 The results of enhanced cell adhesion and growth at 37 °C of NIPAM-MA-HA hydrogel were compared with that at 25 °C, and these results were consistent thanks to the marker, which helped us mark the area to recheck the confirmation. Because our primary results of this material were promising, this could lead to CIMs using this thermoresponsive biomaterial for TE applications. The mechanism of this hydroge l is presented in Figure 7. Important task is here to know about the biocompatibility of

(1)

(2)

Where D is the diffusion coefficient (cm2/s), k is oxygen solubility (mL of O2/mL of material mmHg), t is sample thickness (mm), I is current (A), Id is dark current (A), pA is atmosphere pressure less the vapor pressure (hPa), A is area of oxygen sensor (cm2), and Dcathode is cathode diameter (mm). Incorporation of MA-HA in the IPN gel gives good oxygen permeability to the network to construct the cells on the surface. 3.6. Biological Performance. NIPAM-MA-HA hydrogel was tested with a fibroblast cell line and it was found that these novel biomaterials were biocompatible with this cell line. The cells were seeded on the scaffold and monitored for 1, 2, and 5 days and we found that the cells were alive and adhered to the surface even after 5 days (see Figure 6). To see the viability of

Figure 6. Phase contrast images of fibroblast cells on NIPAM-MAHA hydrogel at (a) 37 °C adhered on the surface of hydrogel, (b) less dense and after cooling to 25 °C the fibroblast cells as compared to the higher temperature, (c) when heated back to 37 °C cells after 24 and 48 h, and (d) adhered again (scale bar 5 μm).

Figure 7. Summary with graphical illustration of the NIPAM-MA-HA interpenetrating hydrogel at 37 and 25 °C. (a) Phase contrast image of fibroblast cells at 37 and 25 °C and (b) graphical representation of IPN hydrogel with well-illustrated hydrogel fibroblast cells along with porous hydrogel, left highlighted images stands for graphical representation of IPN network morphology.

the HDF cell line at 25 °C, we seeded cells on the hydrogel surface, monitored them for 6,12, and 24 h, and found that cells were alive even after 24 h, see Figure S7. Hence, we conclude that these biomaterials were biocompatible with HDF fibroblast cell line. The adhesion of fibroblast cells on the NIPAM-MA-HA at 37 °C were more pronounced than at 25 °C. Micrographs of fibroblast cells growth on NIPAM-HA-MA at 25 and 37 °C were obtained after the cells were stained, see Figure 6a, c. Overall, the adhered cells on the NIPAM-MA-HA scaffold at 37 °C were denser and had greater spread of cells than at 25 °C with 5 min of sonication. Our hydrogels were found to be biocompatible by seeding fibroblast cells, which are known to be quite sensitive to surface morphology and whose alignment is of particular interest in TE for achieving functional vascularization.41−44 The sonication could help spread the condition for faster and can create a mechanical force, which changes the

ultrsonication with the cell experiment. A recently reported article18 has performed a similar kind of experiments with HUVEC cell line different time zones and proved the ultrasonication biocompatibility. Many good works have been reported regarding stimuliresponsive biomaterials (such as elastin-like polypeptides and PNIPAM) that can instead exhibit an LCST in physiologically relevant conditions and have been used to capture and release proteins at surfaces to control cell adhesion.50−52 But in our case, the MA-HA incorporation deviates from the reported information and this hydrogel provides good oxygen permeability along with the excellent mechanical properties of IPN thermosensitive character. The gross morphology, as well as the microtopography and chemistry of the surface, determine which molecules can adsorb and how cells will attach and align themselves.45 Hence, we strongly believe that this type of hydrogel can be considered cell guiding biomaterials in tissue engineering applications. E

DOI: 10.1021/acsabm.8b00595 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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4. CONCLUSIONS In summary, 100% methacrylation was achieved on the backbone of the HA. In addition, the NIPAM-MA-HA crosslinking network hydrogel was fabricated. Thanks to the thermoresponsive character of NIPAM, we noticed that at 25 °C, the morphology of the hydrogel is a porous material with a smooth scaffold, but when heated to 37 °C, the structure changes into a porous microparticle state. Further, study of the mechanical properties was carried out with NIPAM-MA-HA, and the storage and loss modulus confirm the dynamic changes of rigidity of IPN gel by varying the temperature from 25 to 35 °C. when temperature goes from 25 to 35 °C, the rigidity decreases and when it comes back to 25 °C, the rigidity increases. But the oscillation torque increases when the temperature rises to 35 °C, and torque is reducedwhen the temperature falls back to 25 °C. This phenomenon was observed because of the thermosensitve character of NIPAM. Importantly, IPN gel shows good transparency even if it reaches LCST, thanks to MA-HA incorporation that helps it give good oxygen permeability. These materials were found to be biocompatible with the fibroblast cell line. Further, cell adherence was denser on the microparticle pattern scaffold when the temperature was 37 °C, and cell density was reduced the temperature was 25 °C. These results promise that these materials can be considered as new biomaterials in TE. However, the mechanisms for orientation of the cells with porous materials need to be further investigated in detail.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsabm.8b00595.

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Article

Synthetic scheme of MAHA, FTIR spectra of hydrogels, photograph of the hydrogels, and experimental part of mechanical properties (PDF)

AUTHOR INFORMATION

Corresponding Author

*Email: [email protected]. ORCID

Ravichandran H. Kollarigowda: 0000-0001-8376-218X Sinoj Abraham: 0000-0001-7501-6144 Present Address

‡ R.H.K. is currently at Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana−Champaign, 405 N. Mathews Ave., Urbana, IL 61801, USA

Funding

This work was supported by the Province of Alberta, Alberta Innovates (AI) and National Research Council. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge the National Research CouncilEdmonton and Nanofab of University of Alberta for the analytical tools and research instruments.

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DEDICATION Dedicated to the memory of Prof. Carlo David Montemagno F

DOI: 10.1021/acsabm.8b00595 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acsabm.8b00595 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX