Materials Science and Engineering C 33 (2013) 196–201

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Crosslinked collagen–gelatin–hyaluronic acid biomimetic film for cornea tissue engineering applications Yang Liu, Li Ren ⁎, Yingjun Wang ⁎ School of Materials Science and Engineering, South China University of Technology, Guangzhou 510641, China National Engineering Research Center for Tissue Restoration and Reconstruction, Guangzhou 510006, China Guangdong Province Key Laboratory of Biomedical Engineering, South China University of Technology, Guangzhou 510006, China

a r t i c l e

i n f o

Article history: Received 24 April 2012 Received in revised form 23 July 2012 Accepted 13 August 2012 Available online 19 August 2012 Keywords: Cornea Collagen Gelatin Hyaluronic acid Tissue engineering Biocompatible materials

a b s t r a c t Cornea disease may lead to blindness and keratoplasty is considered as an effective treatment method. However, there is a severe shortage of donor corneas worldwide. This paper presents the crosslinked collagen (Col)–gelatin (Gel)–hyaluronic acid (HA) films developed by making use of 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) as the crosslinker. The test results on the physical and biological properties indicate that the CGH631 film (the mass ratio of Col:Gel:HA=6:3:1) has appropriate optical performance, hydrophilicity and mechanical properties. The diffusion properties of the CGH631 film to NaCl and tryptophan are also satisfactory and the measured data are 2.43×10−6 cm2/s and 7.97×10−7 cm2/s, respectively. In addition, cell viability studies demonstrate that the CGH631 film has good biocompatibility, on which human corneal epithelial cells attached and proliferated well. This biocompatible film may have potential use in cornea tissue engineering. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Cornea is the outermost layer of the eye and the main refractive element that is responsible for transmission of light to the retina [1]. Corneal disease is a major cause of blindness worldwide. About 10 million people worldwide suffer from vision loss probably caused by corneal disease [2]. Corneal transplantation has been one even the only one of the effective methods to treat some corneal diseases. However, its clinical utility is limited due to a severe shortage of high quality donor corneal tissues. The demand exceeds the supply in many countries, especially in the developing countries [3]. In addition, corneal grafts from donors can still stimulate host immune responses resulting in tissue rejection and neovascularization, or they can transfer diseases from unhealthy donor tissues. For these reasons, a tissue engineering approach involving natural biomaterials is promising in the process of cornea tissue repair and regeneration [4–6]. Recent reports on the cornea repair materials include a variety of sources, such as human amniotic membrane [7,8], collagen scaffolds [9], silk films [10,11], chitosan films [12], and so on. As the main load bearing component in connective tissues, collagen has been extensively studied in cornea tissue engineering [13–17]. Most of the present researches in this area focus on the gelatin because of

⁎ Corresponding authors at: School of Materials Science and Engineering, South China University of Technology, Guangzhou 510641, China. Tel.: +86 15521139818; fax: +86 20 22236088. E-mail addresses: [email protected] (Y. Wang), [email protected] (L. Ren). 0928-4931/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2012.08.030

its biocompatibility and low antigenicity [18]. Hyaluronic acid is an important component of the extracellular matrix, which has been found to facilitate the adhesion and proliferation of corneal epithelial cells [19]. Due to its important role in wound healing and cell growth, hyaluronic acid shows an ideal potential in the application of cornea tissue engineering and implant biomaterials. The objective of this study was to develop a corneal repair material for cornea tissue engineering applications. In this study, crosslinked collagen (Col)–gelatin (Gel)–hyaluronic acid (HA) films were fabricated. Physical properties (e.g. mechanical properties, light transmittance, water absorption, diffusion coefficient, contact angle, etc.) and biological properties (human corneal epithelial cell research) of the films were tested. 2. Materials and methods 2.1. Materials Hyaluronic acid with a molecular weight of 1 × 10 6 was purchased from Freda, (Shandong, China). The gelatin was obtained from Sigma-Aldrich (USA). The type I collagen was extracted from tendon. 1-Ethyl-3-(3-dimethyl aminopropyl) carbodiimide and Nhydroxysuccinimide were supplied by GL Biochem Ltd (Shanghai, China). Deionized water was obtained from a water purification system (Millipore S.A.S., France). All cell-culture related reagents were purchased from Sigma Chemical (St. Louis, MO, USA).

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2.2. Preparation of films

2.4. Surface contact angle measurements

First of all, collagen was dissolved in 0.1 mol/L HCl solution at 4 °C (6 mg/mL). Hyaluronic acid was dissolved in deionized water and gelatin was dissolved in 0.5 mol/L HCl solution. Then, the three solutions were mixed with a mass ratio of Col:Gel:HA = 6:3:1 (CGH631)/ 6:3:0.5 (CGH6305)/6:3:0 (CGH630). After that, EDC and NHS were added to the mixture and thoroughly mixed at 4 °C to form a solution with a mass ratio of EDC:NHS:mixture = 0.5:0.5:6. Crosslinking was carried out by stirring the solution for 4 h. After crosslinking, the homogenous mixture was dispensed into a specific mold, which has the same curvature of contact lens. The films were air dried and rinsed three times with deionized water. Scheme 1 shows the chemical reaction mechanism between the collagen, gelatin and hyaluronic acid. The crosslinking reaction occurred between the carboxyl groups of HA and the amino of collagen (gelatin). EDC was used as the crosslinking agent in this reaction, while NHS was used as catalyst.

Contact angles were measured using the sessile drop method. Test was carried out on Data Physics OCA 15 (Germany) with an injection volume of 1 μL distilled water as medium. CA calculations were performed using a circle segment function intersecting with a straight baseline representing the surface (n=5). 2.5. Swelling test Water absorption of the films was measured by swelling them in PBS (pH = 7.4) at the normal physiological temperature of the cornea. After the samples were immersed in PBS to saturation, the films were blotted quickly with a filter paper to remove the absorbed water. The water absorption of the films is calculated according to the following equation: Water absorption ¼ ðWt −W0 Þ=Wt  100%;

2.3. Mechanical test The mechanical properties of the samples were measured using a uniaxial load testing equipment (Model #5567, Instron Corporation, Issaquah, WA, USA) at 35 °C. The films were immersed in a phosphate buffer solution (PBS) for 2 h before the test, and then the samples (1.0 cm width ×2.0 mm length×and 0.108±0.01 mm thickness) were clamped for axial tensile testing (n=5). Test was continued until fracture of the specimen and the tensile speed was 1 mm/min.

ð1Þ

where Wt is the wet weight of the samples at target times and W0 is the initial dry weight of the samples. The values are expressed as the mean ± standard error (n = 10). It's worth noting that the sizes of wounds are different for each patient during keratoplasty, so cornea repair materials should be easy to fabricate with various dimensions. Samples with known dimensions were swelled in PBS, and then the thickness and surface area of the hydrated films were measured in each hour. The thickness and surface area of the films were measured by a micrometer caliper and a

Scheme 1. The chemical reaction mechanism between collagen (gelatin) and hyaluronic acid.

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ruler, respectively. Variations of thickness and surface area are calculated according to the following equations: Thickness increase ¼ Ht =H0 ;

ð2Þ

Surface area increase ¼ St =S0 :

ð3Þ

Here, Ht and St are the thickness and surface area of the wet samples at target times, respectively. H0 and S0 are the initial thickness and surface area of the dry films, respectively. The values are expressed as the mean±standard error (n= 10). 2.6. Diffusion coefficient assay Since the cornea is avascular, nutrients for cornea tissue are mainly depending on the aqueous humor and to a lesser extent on the limbal vasculature [20]. Ion diffusion and tryptophan permeability of the film were carried out at 35 °C using a device with two-compartment chambers (the CG631 film has better mechanical properties than the other two films and hence the sample is taken for further characterization studies). The CGH631 film was fixed between the permeate chamber (filled with NaCl solution or tryptophan solution) and receptor chamber (filled with deionized water), and then the solution in each chamber was homogeneously stirred by an electromagnetic stirrer. The concentrations of ions or tryptophan in the receptor chamber were checked in subsequent time by various methods. Ionic conductivity of the sodium chloride solution in the receptor chamber was determined by a DDS-11A conductivity meter (Jinmai, Shanghai, China). Colorimetric measurements of the tryptophan solution in the receptor chamber were made at 540 nm using a tryptophan assay kit (GAG020, Sigma‐Aldrich) with a UV3802 ultraviolet–visible spectrophotometer (Shanghai UNICO, China). The values were fit to the regression line of standard concentration. 2.7. Light transmittance assay Before the transparency test, the CGH631 film was immersed in PBS for more than 2 h to uptake water. Then, the sample was fixed into the specimen chamber of the UV3802 ultraviolet–visible spectrophotometer (Shanghai UNICO, China). Light transmittance of the sample was measured in the range from 400 nm to 800 nm. After that, the film was transferred into pure glycerol, and then repeated the above measuring procedures [21]. 2.8. Cell experiment 2.8.1. In vitro corneal epithelial cell culture Human corneal epithelial cells (HCECs) were obtained from the State Key Lab of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, China. HCECs were cultured in Dulbecco's Modified Eagle's Medium (DMEM; Gibco BRL) with high glucose, supplemented with

2.8.2. The response of HCECs to the film The CGH631 film was washed five times in PBS under aseptic conditions, sterilized by ultraviolet radiation for 2 h and washed three times in PBS again. After that, the film was transferred to a 6-well tissue culture plate (Corning, UK). A certain volume of HCEC suspension was seeded onto the film (5000 cells/cm 2). The culture medium was replaced every 2 days. The response of HCECs to the sample was examined after the film was washed with PBS. Microscopic photos were taken by an inverted fluorescence microscope (Olympus IX-70, Japan) for observing the cellular morphology. 2.8.3. The proliferation of HCECs to the film The CGH631 films were placed in 96-well tissue culture plates (Corning, UK). HCECs were seeded onto the surface of the samples (experimental group, n = 10) and polystyrene wells (control group, n = 10), respectively. The proliferation of the HCECs on the films was quantitatively determined by methylthiazol tetrazolium (MTT) assay at the optical density (OD) value of 490 nm with a microplate reader. 3. Results and discussion A comparison between the samples shows that they had different mechanical behaviors (Fig. 1). The ultimate tensile strength of the films was 18.15 ± 1.02 MPa for CGH630, 21.02 ± 1.23 MPa for CGH6305 and 26.20 ± 1.41 MPa for CGH631 (Fig. 1A). The tensile strength improved with the addition of hyaluronic acid content in the composite films. However, the elongation at break has no significant difference between the samples (about 31.31% for CGH630, 32.97% for CGH6305 and 33.54% for CGH631) (Fig. 1B). Based on the above work, the Young's modulus of the films was 57.97 ± 1.21 MPa for CGH630, 63.75 ± 1.33 MPa for CGH6305 and 78.12 ± 1.47 MPa for CGH631 (Fig. 1C). The introduction of HA leads the cross-linked Col–Gel–HA molecule to have a greater molecular weight. Besides, with the introduction of HA, the free volume of the functional groups of the Col–Gel–HA molecule has decreased and the steric hindrance has increased. Also the molecular deformation energy has increased. The performance of the tensile strength and elastic modulus of the CGH film increased in the macro. These results indicate that the CGH631 film has the best mechanical properties among the three samples. The variation of contact angle versus time of the films is given in Fig. 2. Water spreading on the CGH630 film occurred within 30 s and the contact angle decreased from 81.7° to 64.6°. Likewise, the contact angle of CGH6305 and CGH631 decreased from 80.4° to 62.3° and 79.2°

C

25 20 15 10 5 0 6:3

6:3:0.5

6:3:1.0

Young's modulus (MPa)

B

30

Elongation at break (%)

Tensile strength (MPa)

A

15% fetal bovine serum (Sijiqing, China), 2 mM L-glutamine, 5 μg mL−1 insulin, 5 μg mL−1 human transferrin (Sigma), 100 U mL−1 penicillin, 10 ng mL−1 human epidermal growth factor (EGF; Gibco BRL) and 100 μg mL−1 streptomycin (HyClone). The cells were incubated in a humidified atmosphere containing 5% carbon dioxide at 37 °C.

40 30 20 10 0 6:3

6:3:0.5

6:3:1.0

80 70 60 50 40 30 20 10 0 6:3

6:3:0.5

6:3:1.0

Fig. 1. Mechanical properties of the films, tensile strength (A), elongation at break (B) and Young's modulus (C). Values are expressed as the mean ± standard deviation (n = 5).

84 82 80 78 76 74 72 70 68 66 64 62 60 58 56

A CGH630 CGH6305 CGH631

0

5

10

15

20

25

30

Time (s)

Thickness increase (multiple)

Contact angle (deg)

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6 CGH631 CGH6305 CGH630

5 4 3 2 1 0 0

6

12

Fig. 2. Variation of contact angle versus time for the composite films. Values are expressed as the mean ± standard deviation (n = 5).

18

24

Time (h)

1.040 CGH631 CGH6305 CGH630

1.035 1.030 1.025 1.020 1.015 1.010 1.005 1.000 0

5

10

15

20

25

Time (h) Fig. 4. Variation of the samples' thickness (A) and surface area (B). Values are expressed as the mean ± standard deviation (n = 10).

On the basis of the above results, the CGH631 composite film displays the best mechanical properties and hydrophilicity among the three samples. Hence, we chose CGH631 to carry out the further assays.

A -ln[( [(C0-2C)/C0]

to 58.0°, respectively. The contact angle of the sample decreased with the addition of hyaluronic acid content. Therefore, we concluded that CGH631's hydrophilicity is better than that of CGH630 and CGH6305. As reported previously [22], hyaluronic acid has a complex chemical structure with amine, hydroxyl and carboxyl groups, which results in a higher molecular polarity of the CGH film than that of the CG film, so the CGH film exhibited a minor contact angle. The hydrophilicity and water absorption of the films increased with these hydrophilic groups' addition. Because of liquid sorption and retention, the wettability and hydrophilicity of the solid surface would increase with the increase of solid–liquid contact time. Our results are consistent with the former conclusion. Water absorption for CGH630, CGH6305 and CGH631 was about 66.7%, 73.2% and 79.8%, respectively (Fig. 3). The water absorption of the samples in the PBS solution improved with the addition of hyaluronic acid content. The water uptake capability of the CGH631 film is quite similar to that of the human cornea (78.0 ± 3.0%) [15]. Fig. 4A shows the variation of the samples' thickness versus time. After the samples were stored in PBS at 4 °C for an hour, water absorption of the films tended to be constant. The CGH631 film thickened approximately fourfold. The thickness change of CGH6305 is lower than that of CGH631 but a little higher than that of CGH630. Fig. 4B shows the variation of the samples' surface area versus time. The CGH631 film's surface area changed tinily. The surface area change of CGH6305 is lower than that of CGH631, but, a little higher than that of the CGH630 film. This may also be caused by the different contents of hyaluronic acid. The results demonstrate that these films can be fabricated with various dimensions easily.

Surface area increase (multiple)

B

0.18 0.16 0.14 0.12 0.10 0.08 0.06 0.04 0.02 0.00 -0.02 0

50

100

150

200

250

300

350

Time (min)

B

80

0.0012 0.0010

-ln[( [(C0-2C)/C0]

Water absorption (%)

100

60

40

0.0008 0.0006 0.0004 0.0002

20

0.0000 0 Native

6:3

6:3:0.5

6:3:1.0

0

100

200

300

400

500

600

Time (min) Fig. 3. Water absorption percentages of the samples and native human cornea. Values are expressed as the mean ± standard deviation (n = 10).

Fig. 5. Ion diffusion (A) and tryptophan permeability (B) of the CGH631 film.

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Transmittance (%)

100

A

B

80 60 40

In PBS In Glycero l

20 300

400

500

600

700

800

Wavelength (nm) Fig. 6. The light transmittance of the CGH631 film immersed in PBS solution and pure glycerol, respectively (A). Representative image of CGH631 immersed in PBS solution (left) and subsequently transferred into pure glycerol (right) (B).

According to Fick's law, the relationship of the ion (tryptophan) concentration of the receptor chamber and the diffusion time can be described according to the following formulas:   2C 2PS ¼ t; − ln 1− C0 Vd P¼

 − ln 1− 2C C0 Vd ; 2St

ð4Þ

ð5Þ

where P denotes the diffusion coefficient; V and S are the volume of the solution in the chambers and the round through-hole area between the chambers, respectively; d is the thickness of the wet film; t is the

diffusion time; C0 is the initial ion concentration of the permeate chamber; C is the ion concentration of the receptor chamber at target time. Fig. 5A and B shows the ion and tryptophan diffusion of the CGH631 film, respectively. The gradient (K) of the fitted curve was about 5.55 × 10−5 (Fig. 5) and 1.82 × 10−6 (Fig. 6), respectively. The diffusion coefficient of the film can be calculated according to the following formula: P¼

KVd : 2S

ð6Þ

The through-hole area between the chambers was 3.14 cm2 (S). The volumes of the solution in the two-compartment chambers were both

A

B

C

D

Fig. 7. The morphology of HCECs on the CGH631 film at different time points, 12 h (A), 24 h (B), 48 h (C) and 72 h (D) (A, B: ×100; C, D: ×50).

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hydrophilicity, optical performance and mechanical properties. The diffusion property of the CGH631 film is similar to the human cornea. HCEC viability study results demonstrate that the film has good cellular compatibility. This biocompatible material may have potential use in cornea tissue engineering. Acknowledgments This work is supported by the National Basic Research Program of China (no. 2012CB619100), National Science Foundation of China (no. 50803018), National Science & Technology Pillar Program in the Twelfth Five-Year Plan of China (no. 2012BAI17B02), and Guangdong Important Scientific and Technological Special Project (no. 2010A080407008).

Fig. 8. The proliferation of human corneal epithelial cells on the CGH631 film and polystyrene (PS).

250 mL (V) and the thickness of the wet film was 110 ± 5 μm (d). On the basis of the above formulas, the diffusion coefficient of the samples to the NaCl solution was about 2.43 × 10−6 cm2/s, which is comparable to that of the human cornea (2.5 × 10 −6 cm2/s) [23]. The diffusion coefficient of the films to the tryptophan solution was 7.97× 10−7 cm2/s, which is also a stable diffusion. Fig. 6A shows the light transmittance curve of the CGH631 film that was immersed in PBS and glycerol, respectively. Light transmittance of the CGH631 film, which was estimated at an average of 450–600 nm in the visible region, was about 53%. After the sample was immersed in glycerol for dehydrating, light transmittance of the film increased to more than 90%. With the increase of wavelength, transmittance of the sample reached to its maximum and tended to be constant, which is similar to that of a native cornea [15]. Fig. 6B shows the images of CGH631 that had been immersed in PBS solution (left) and subsequently transferred into pure glycerol (right). Fig. 7 shows the morphology of the HCECs on the CGH631 film at different time points. After the cells were incubated for 12 h (Fig. 7A), 24 h (Fig. 7B), 48 h (Fig. 7C) and 72 h (Fig. 7D), the HCECs attached, grew and proliferated well on the CGH631 film. The seeded cells adhered to the surface of the film within 12 h. Besides, the morphology of the HCECs changed from a round shape to a spindle shape gradually, which is similar to the appearance of the normal HCECs. After HCECs were seeded on the films, they proliferated rapidly. The film was almost completely covered by HCECs 72 h later. The MTT test (Fig. 8) shows the proliferation of human corneal epithelial cells on the CGH631 film and polystyrene (PS) plates. Compared with the collagen scaffold [24] or gelatin scaffold [25], the CGH631 film has better biocompatibility. After the HCECs were seeded, they have a similar growth rate on the first day. However, the cell's growth rate on the PS plates is lower than that of the CGH631 film in the subsequent few days. The cell number on the PS plates reached the peak on the fourth day, and then began to decline, which can be that the number of HCECs had reached its saturation on the surface of the PS plates. 4. Conclusions In this study, crosslinked Col–Gel–HA films with different compositions were developed and tested. The CGH631 film has appropriate

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Crosslinked collagen-gelatin-hyaluronic acid biomimetic film for cornea tissue engineering applications.

Cornea disease may lead to blindness and keratoplasty is considered as an effective treatment method. However, there is a severe shortage of donor cor...
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