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Hyper-Elastic Nanocellulose-Reinforced Hydrogel of High Water Content for Ophthalmic Applications Gopi Krishna Tummala, Thomas Joffre, Viviana R. Lopes, Aneta Liszka, Oleksiy Buznyk, Natalia Ferraz, Cecilia Persson, May Griffith, and Albert Mihranyan ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.6b00484 • Publication Date (Web): 04 Oct 2016 Downloaded from http://pubs.acs.org on October 6, 2016

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ACS Biomaterials Science & Engineering

Hyper-Elastic Nanocellulose-Reinforced Hydrogel of High Water Content for Ophthalmic Applications Gopi Krishna Tummala1, Thomas Joffre2, Viviana R. Lopes1, Aneta Liszka3, Oleksiy Buznyk3, 4, Natalia Ferraz1, Cecilia Persson2, May Griffith3, Albert Mihranyan1* *Correspondence to Prof. Albert Mihranyan. E-mail: [email protected]. 1

Nanotechnology and Functional Materials, Department of Engineering Sciences, Box 534

Uppsala University, 75121 Uppsala, Sweden. 2

Materials in Medicine, Division of Applied Materials Science, Department of Engineering

Sciences, Box 534 Uppsala University, 75121 Uppsala, Sweden. 3

Department of Clinical and Experimental Medicine, Linköping University, 581 85 Linköping,

Sweden. 4

The Filatov Institute of Eye Diseases and Tissue Therapy of the NAMS of Ukraine, 65061,

Frantsuzskyi Boulevard 49/51, Odessa, Ukraine. KEYWORDS: ophthalmic prosthesis, contact lens, cellulose nanocrystals, biocompatibility, polyvinyl alcohol.

ABSTRACT:

ACS Paragon Plus Environment

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A nanocellulose-reinforced polyvinyl alcohol hydrogel material of exceptionally high water content for ophthalmic applications is presented (>90% wt), which also features hitherto unprecedented combination of optical, mechanical, viscoelastic, oxygen permeability, and biocompatibility properties. The hydrogel combines the desired softness with remarkable straindependent mechanical strength and thereby demonstrates hyper-elastic, rubber-like mechanical properties. The observed unusual mechanical behavior is due to both high water content and combination of relatively stiff cellulose nanowhiskers entangled in a soft polymer matrix of polyvinyl alcohol (PVA), thus mimicking the structural characteristics of cornea’s main constituents, i.e. water and collagen.

Hydrogels are ubiquitously used in eye-care as lenses to correct refractive errors, cosmetic and decorative ocular devices, therapeutic drug delivery vehicles, bandage lenses, scleral buckling materials, cornea regeneration scaffolds,1 and even integrated ocular wireless electronic sensors for diagnostic purposes.2, 3 For use as a contact lens, an ideal ophthalmic hydrogel device should not only feature desirable optical properties but also be comfortable to wear, conform to the eye’s shape, be oxygen permeable, biocompatible and withstand substantial mechanical shear. If used as a cornea regeneration implant, it should further be sufficiently strong to be sutured. Thus, hydrogels for ophthalmic use should mimic the natural tissue and thereby combine inherently conflicting properties such as high water content, softness and sufficient mechanical strength. If the ophthalmic lens is too stiff it may form a gap at the edge resulting in foreign body sensation in the eye, lens awareness, and mechanically induced inflammation.4


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Apart from optical and mechanical properties, in order to be used in vivo, the ophthalmic devices should be highly biocompatible and not elicit any immunogenic or toxic response. Because the eye is a sensitive and highly protected organ, it is rich in various proteins, which may deposit on the surface of the device and change their conformation, thereby inducing foreign body immune response and inflammation.5 Therefore, hydrogels for ophthalmic use as contact lenses should further be resistant to deposit formation, such as proteins, lipids, or minerals. In all, designing ophthalmic devices is highly complex. The development of ophthalmic contact lens or keratoprosthesis biomaterials has gone a long way from rather rigid to relatively soft ones, including those with both high and low water content. In general, high water content hydrogels are preferable as they are more comfortable to wear, have innate lubricity, better resistance against deposition of proteins and lipids, and feature good oxygen permeability.6 However, one common problem with hydrogel materials of high water content is their insufficient mechanical strength. Typically, the water content of siliconbased contact lenses varies between 20-40%, while that for soft hydrogel lenses is around 5075%.7 The most common contact lens materials include various synthetic polymers such as siliconbased materials and hydrogel forming polymers, e.g. polyhydroxyethylmethacrylate (PHEMA), polymethylmethacrylate (PMMA), polyvinylpyrrolidone, and cellulose acetate butyrate (CAB). One synthetic material that has been used for ophthalmic applications for decades is polyvinyl alcohol (PVA). It is commonly used as a viscosity-enhancing agent in eye-drops and as an internal lubricant in contact lenses.8, 9 It can also be used on its own as a matrix-forming material for contact lenses10, 11 or as a scleral buckling material.12 The choice of PVA for ophthalmic applications is stipulated by its high transparency, good wettability, excellent biocompatibility,


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and non-adherent properties, related to the abundance of hydroxyl groups along its chains. A modified UV-cross linked PVA contact lens called Nelfilcon A (69% water content) is marketed under the name of Contact Dailies.7 Here we report a nanocellulose-reinforced PVA hydrogel material of high water content, i.e. > 90% wt. It has been argued that equilibrium water content is the single most important property for a hydrogel for ophthalmic use because it influences transparency, permeability of dissolved species, biocompatibility, lubricity, and elasticity.6 It was previously reported that inclusion of cellulose fibrils in PVA matrix enhances the mechanical properties.13, 14 The inclusion of nanocellulose imparted the necessary reinforcement to achieve a highly desirable combination of various properties. Although composites of bacterial cellulose (BC) with PVA have been described as corneal implants, their reported transparency could be as low as 75% at 610 nm.15 Therefore, highly transparent native cellulose based ophthalmic hydrogels including contact lenses are still rather uncommon. In order to bypass the opacity problem of some nanocellulose materials, we used carboxylated nanocellulose whiskers (CNC) produced from pharmaceutical grade microcrystalline cellulose by TEMPO-mediated oxidation.16 The choice of carboxylated nanocellulose was further motivated by its enhanced biocompatibility as shown in a number of recent publications.17-19 Fig. 1a is an AFM image of the carboxylated cellulose nanowhiskers. The nanocellulose whiskers were added to a PVA 10% solution in hot DMSO:water (80:20 ratio) solution and stirred. The abundance of hydroxyl groups on PVA chains suggests a multitude of possibilities for hydrogen bonding with cellulose whiskers.


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Figure 1. CNC-PVA hydrogel lens: (a) AFM image of carboxylated cellulose nanowhiskers, (b) Self-standing lens, (c) Lens showing conformability, (d) Transparent sheet of hydrogel and (e) SEM image of freeze-dried lens, (f) Picture of CNC-PVA hydrogel implant sutured to ex vivo porcine cornea.


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Fig. 1b and 1c are images of the molded CNC-PVA hydrogel lens. Upon visual examination, as illustrated in Fig. 1b and 1c, the lens was transparent, self-standing and, yet, soft, elastic, and capable of retaining its convex shape. Fig 1d shows transparency of a 1mm thick hydrogel sheet. Fig. 1e shows the SEM image of a freeze-dried lens. It is seen in this image that the composite matrix consists of an open and macroporous honeycomb structure. Despite its high water content, the lens had rubber-like properties. It could be manually pulled without rupture and readily returned to its original form without deformation upon release. Fig 1f shows suturability of the hydrogel and is discussed in later part of the text. Table 1 gives a summary of the physical and mechanical properties of CNC-PVA hydrogels, specifically, their optical, mechanical, oxygen permeability properties. In particular, it was observed that the lens featured a transparency above 95% in the visible range, while also moderate UV-absorption properties were evident, see Table 1. Normally, UV-absorption properties are desirable to reduce the risk of developing cataract or pterygium. While the UV-blocking properties of the lens were generally inferior to Type I (i.e. blocking 90% UV-A and 99% UV-B) and Type II (i.e. blocking 70% of UV-A and 95% of UV-B) UV-blocking lenses, the lens was still superior to ordinary contact lenses, which normally exhibit residual UV-blocking properties not exceeding 10% for UV-A and 30% for UV-B.20 The high transparency is a combined result of several factors such as (i) the width and thickness of CNC being less than visible light wavelength,21 (ii) refractive index similarities between PVA and CNC, and (iii) very good interface between PVA and CNC ensuring no air scattering. It should further be noted that the measured water contact angle of CNC-PVA hydrogel material was about 12º. Clinically, low contact angle ensures high wettability of the ophthalmic contact lens or keratoprosthesis and good tear film spreading.5


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The refractive index of the lens was nearly identical to that of distilled water, i.e. 1.33, and differed only in the third-fourth decimal, see Table 1. The latter implies that the CNC-PVA hydrogel lens is essentially invisible, when immersed in water. For comparison, the typical refractive index values for conventional hydrogel contact lenses are between 1.38-1.42, including Nelfilcon A.7 Thanks to its extra-high water content the CNC-PVA hydrogel lens features high oxygen permeability, i.e. 66x10-11 Dk, exceeding that of commercially available Nelfilcon A contact lenses. Table 1. Comparison of the physical properties of lenses based on PVA with typical soft contact lenses.

CNC–PVA hydrogel

PVA hydrogel, Kita et al22

Nelfilcon A, cross-linked modified PVA23

Typical soft contact lens

Polymer content, %

7

25

31

26 - 67

Water content, %

93

78

69

33 - 74

Optical properties VIS (610 nm), T%

> 95 %

> 99 %

> 92 %

> 95 %

UV-A (320 – 400 nm), T%

80

N.A.

N.A.

90

UV -B (290 – 320 nm), T%

60

N.A.

N.A.

70

1.33

N.A.

1.38

1.38 – 1.42

26 x 10-11

22-130 x 10-11

Refractive index

Permeability O2 permeability, Dk [(cm2/sec) (ml O2 /ml x mm Hg)]

66 x 10-11

44 x 10-11

Mechanical properties Stress to failure, MPa Strain to failure, %

0.15±0.02

1.2

N.A.

0.4-3.0

383±15

500

N.A.

178-245


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Toe point Modulus, MPa

0.016± 0.003

N.A.

N.A.

N.A.

Young’s modulus, MPa

0.071± 0.007

4.61

N.A.

0.3-1.6

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It was further observed that PVA-CNC hydrogel lens features a rubber-like mechanical profile, which is unusual for an ophthalmic contact lens or keratoprosthesis material.24, 25 Normally, contact lens material exhibits a stress-strain curve typical for ductile materials as depicted in Fig. 2a. Such a profile is characterized by two regions, i.e. initially steep linear elastic deformation region followed by moderately ascending region, which corresponds to irreversible plastic deformation until breakage. The stress value at the inflection point is defined as the yield strength and the slope of the initial linear region is defined as the Young’s modulus. The Young’s modulus is the measure of stiffness and is an important parameter related to contact lens wear comfort and handling. Notably, soft lenses possess the benefit of providing easier fit and higher comfort for the wearer.26 The mechanical properties of contact lenses have been reported in the literature before,5, 6, 25, 27, 28 and are summarized in Table 1 The stress-strain curve of the CNC-PVA hydrogel shows an unusual shape, which is more typical for hyper-elastic materials than normal contact lenses as shown in Fig. 2a and 2b. Hyper-elastic materials such as soft tissues and elastomers are characterized by non-linear stress-strain curves where low stress is sufficient to achieve substantial stretching without permanent deformation (i.e. toe region) until a certain point is reached, known as the heel. After this point the stress needed to further stretch


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Figure 2. Stress-strain curves for (a) typical ductile, elastic and brittle materials (illustration), (b) CNC-PVA hydrogel sample (c) viscoelastic properties of CNC-PVA hydrogel. the material grows steeply, until finally the material breaks under the load. While water present in the hydrogel undoubtedly softens the composite, its presence alone cannot explain the observed mechanical profile. Such behavior is normally detected in collagenous tissues.29 In particular, in the relaxed state collagen consists of macroscopically disordered array of wrinkled


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chains intermitted by crystalline clusters. Under strain the chains realign themselves in the direction of the applied load, and the heel point indicates a transition from the disordered, random-orientation state to one that is highly aligned. This aligned structure is significantly stiffer than the material in the relaxed state. In analogy with collagen, the observed stiffening of CNC-PVA hydrogel above the heel point during the tensile test is attributed to the cellulose nanowhisker reorientation in the loading direction and could be due to substantial entanglement between relatively stiff cellulose nanowhiskers and softer PVA chains. By considering an in-plane reorientation of the cellulose nanowhiskers from continuum mechanics and a mean field approach,30, 31 analytical modelling was used to simulate the change in the mechanical response due to the reorientation of the cellulose nanowhiskers in the soft PVA matrix. To predict the stiffness of the composite undergoing small deformations, a rule of mixtures for random in plane fiber orientation could also be used.32, 33 However, such an approach will not allow to predict the stiffening of the composite, which occurs in large deformations. Here the results are in good agreement (r2=0.98) with the concept of straininduced reorientation, as shown in Figure 2b. Because the cellulose nanowhiskers are at least 100 times stiffer than PVA,34, 35 we assume that the strain-induced stiffening of the hydrogel is dominated by cellulose nanocrystals. Additionally, in order to further experimentally verify the reorientation of cellulose nanowhiskers, the strain-induced birefringence of the hydrogel was visualized when illuminated with a polarized light source, see Fig. 3. It is known that when anisotropic crystalline objects orient in a particular direction, e.g. by the imposition of a strain field, or a flow in one direction, their refractive index depends on the light polarization. The latter phenomenon is known as birefringence and is useful to study local reorientation in anisotropic elastomeric materials.36 As


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it is seen in Fig. 3, when the CNC-PVA sample is pulled along its vertical axis, the hydrogel gradually brightens under the strain, when illuminated with polarized light, until it eventually breaks and turns dark again. The time-lapse video demonstrating the strain-induced birefringence is provided in Supporting Information. It should be mentioned that the local differences in brightness observed in Fig. 3 do not necessarily reflect the anisotropic distribution of CNC whiskers inside the hydrogel but are due to the light source consisting of an array of diodes. Nonetheless, the increase of brightness between Fig 3a, 3b, 3c, 3d and 3e is the result of a change in birefringence of the material. The observed strain-induced birefringence is in full accordance with the results reported elsewhere for soft elastomeric materials reinforced with clay nanotubes.37, 38 Since the high water content hydrogel exhibits rubber-like behavior, its viscoelastic properties are of interest, see Figure 2c. The viscoelastic behavior of the hydrogel can be described by an elastic (storage) modulus G’ (also known as the dynamic rigidity), which reflects the reversibly stored energy, and a viscous (loss) modulus G’’, which reflects the irreversible energy loss. It is seen in Figure 2c that the system is characterized by an exceptionally high elastic modulus G’, i.e. ca. 16 kPa, and a viscous modulus G’’, i.e. ca. 450 Pa. The moduli are well separated and parallel to each at the tested frequencies between 1 and 20 Hz, suggesting true viscoelastic solid behavior. In this region, the overall system is characterized by a damping factor δ as low as ca. 1.5°, which is remarkable given its water content above 90%. As the frequency is increased it is further seen that the elastic modulus G’ slightly increases, probably due to strain-induced stiffening, and then abruptly falls, suggesting that the viscoelastic properties of the hydrogel break at frequencies above 60 Hz. In all, the results of the dynamic rheology analysis support the hyper-elastic behavior of the hydrogel.


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Figure 3. Strain induced birefringence curves for CNC-PVA hydrogel. Time-lapse images of PVA-CNC hydrogel (a-f) illuminated by a polarized light source. The pulling was set at 50 mm/min, and the increase in CNC alignment along the direction of loading is indicated by increased brightness in polarized light.


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As it is further seen in Fig. 2b, CNC-PVA hydrogel could be stretched to more than 300% of its original size before it breaks. The strength at failure for CNC-PVA hydrogel is 0.15 MPa, suggesting soft, hyper-elastic properties characteristic for natural tissue with high water content. For comparison, the previously reported PVA-BC contact lenses behaved either as brittle or as stiff ductile materials with Young’s modulus values between 24 and 63 MPa, strain values between 50 and 150%, and tensile strength values between 3.2 and 7.2 MPa15. Thus, the produced PVA-CNC hydrogel lenses of exceptionally high water content are not only soft but also sufficiently strong to be handled. The remarkable combination of mechanical properties of the PVA-CNC hydrogel is illustrated further in the suturing experiments, which showed that PVA-CNC hydrogels were able to tolerate placements of 12 interrupted sutures without any tearing. Fig. 1f illustrates the CNC-PVA hydrogel implant sutured ex vivo to porcine cornea. The suturability of the hydrogel was assessed as follows. Each suture was evaluated on a qualitative score between 0 and 3, where 0 is no visible material break; 1 - minimal material break, 2 - well visible break, and 3 - full break, suture comes out of material. The total score from all 12 sutures is then summed. If the numerical value of the total score is larger than 10, the prosthetic device is deemed too fragile. To clarify the procedure we present the data from all 3 sets tested, each involving 12 sutures in Table 2. Despite its exceptionally high water content, i.e. > 90%, the hydrogel scored an overall value of 8±1 (n=3, where n is number of samples), which is below the threshold value of 10, above which the prosthetic device is deemed too fragile. Minor shearing was noted but overall, the hydrogel implants exhibited good suturability without need for further reinforcement or particular care during handling. Further, CNC-PVA hydrogel material showed excellent biocompatibility, and no toxic effects due to leaching were detected. Fig. 4a summarizes the results of indirect cytocompatibility test


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using human corneal epithelial cells (HCE-2). It is seen that the viability of cells cultured in extract medium of CNC-PVA hydrogel was well above the cytocompatibility limit of 70% defined by ISO standard 10993:539 and significantly higher than the positive control (5% DMSO in culture medium) (p < 0.01). Table 2. Suturability evaluation results Sample ID

Suture # 1

2

3

4

5

6

7

8

9

10

11

12

Total

1

0

0

2

1

1

1

0

0

2

1

0

0

8

2

0

0

2

1

1

0

0

1

1

0

1

0

7

3

0

0

2

0

1

2

0

1

1

0

1

1

9

Furthermore, when performing a direct cytocompatibility test with HCE-2 cells, it was observed that CNC-PVA hydrogel promoted cell growth as shown in Fig. 4b, 4c and 4d. The HCE-2 cells grew rapidly on the surface of CNC-PVA hydrogel within 3 days, suggesting that this material may also show potential as a corneal implant. Fig 4e, 4f and 4g show HCE-2 cell growth over a period of 3 days on a TCP which is used as control. In conclusion, the CNC-PVA hydrogel has higher water content than commercial contact lenses. Yet its key properties are either similar to or outperform those of its analogues. Furthermore, the hydrogel possesses a mechanical profile that resembles that of natural tissue, combining the softness and elasticity with remarkable mechanical strength. These properties of CNC-PVA hydrogels make them highly promising biomaterials for a range of ophthalmic applications, including disposable contact lenses and cornea regeneration implants.


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Figure 4. Biocompatibility study (a) Cell viability of human corneal epithelial cells (HCE-2) cultured in extract medium of CNC-PVA hydrogel and in the presence of 5% DMSO (positive control). The data are expressed as percentage of the negative control (tissue culture plate extract medium) and represent the mean ± SEM (** p < 0.01)). Cell viability values greater than 70% of the negative control indicate a non-cytotoxic response.39 (b, c, d) Confocal microscopy images of GFP labelled human corneal epithelial cells (HCE-2) cultured on CNC-PVA hydrogel i.e., (b) Growth after 24h, (c) Growth after 48h, (d) Growth after 72h. (e, f, g) Confocal microscopy images of GFP labelled human corneal epithelial cells (HCE-2) cultured on TCP, (e) Growth after 24h, (f) Growth after 48h, and (g) Growth after 72h.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 1. Time-lapse video demonstrating the strain-induced birefringence. 2. Materials and Methods

AUTHOR INFORMATION Corresponding Author *Correspondence to Prof. Albert Mihranyan E-mail: [email protected].


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Author Contributions G.T., A.M. designed the material and conceived project. G.T., T.J., A.L., V.L., and O.B. carried out the experiments. G.T., A.M., T.J., V.L., O.B., N.F., C.P., and M.G. contributed to data analysis. G.T. and A.M. prepared the manuscript. ACKNOWLEDGMENTS This work has been funded by the Wallenberg Wood Science Center. One of the authors (A.M.) is Wallenberg Academy Fellow and thanks the Knut and Alice Wallenberg Foundation for their long-term financial support. The indirect cytocompatibility studies were performed at the BioMat platform, Science for Life Laboratory, Uppsala University. Work performed at the Griffith’s lab is funded by a Vinnova grant (dnr 2013-04645).

ABBREVIATIONS PMMA, polymethyl methacrylate; PHEMA, polyhydroxy ethylmethacrylate; CAB, cellulose acetate butyrate; PVA, polyvinyl alcohol; DMSO, dimethyl sulfoxide; TEMPO, 2,2,6,6Tetramethylpiperidine-1oxyl radical; BC, bacterial cellulose; CNC, cellulose nanocrystals; MCC, microcrystalline cellulose; AFM, atomic force microscopy; SEM, scanning electron microscopy; UV, ultra violet light; HCE-2, human corneal epithelial cells; ISO, international organization for standardization; TCP, tissue culture plate; AB, Alamar blue, KSFM, keratinocyte-serum free medium, PBS, phosphate buffered saline; GFP, green fluorescence protein.


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TOC Graphic Hyper-Elastic Nanocellulose-Reinforced Hydrogel of High Water Content for Ophthalmic Applications Gopi Krishna Tummala, Thomas Joffre, Viviana R. Lopes, Aneta Liszka, Oleksiy Buznyk, Natalia Ferraz, Cecilia Persson, May Griffith, Albert Mihranyan*


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Hyperelastic Nanocellulose-Reinforced Hydrogel ... - ACS Publications

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