Preparation and characterization of a novel tobramycin-containing antibacterial collagen film for corneal tissue engineering

Acta Biomaterialia 10 (2014) 289–299

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Preparation and characterization of a novel tobramycin-containing antibacterial collagen film for corneal tissue engineering Yang Liu, Li Ren ⇑, Kai Long, Lin Wang, 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 25 April 2013 Received in revised form 13 August 2013 Accepted 26 August 2013 Available online 5 September 2013 Keywords: Cornea Antibacterial Collagen Cross-linking Drug release

a b s t r a c t Corneal disease is a major cause of blindness and keratoplasty is an effective treatment method. However, clinical treatment is limited due to a severe shortage of high-quality allogeneic corneal tissues and the bacterial infection after corneal transplantation. In this study, we develop a novel artificial and antibacterial collagen film (called Col-Tob) for corneal repair. In the Col-Tob film, the tobramycin, which is an aminoglycoside antibiotic to treat various types of bacterial infections, was cross-linked by 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide and N-hydroxysuccinimide onto the collagen. Physical properties, antibacterial property and biocompatibility of the films were characterized. The results indicate that the film is basically transparent and has appropriate mechanical properties. Cell experiments show that human corneal epithelial cells could adhere to and proliferate well on the film. Most importantly, the film exhibits excellent antibacterial effect in vitro. Lamellar keratoplasty shows that the Col-Tob film can be sutured in rabbit eyes and are epithelialized completely in15 ± 5 days, and their transparency is restored quickly in the first month. Corneal rejection reaction, neovascularization and keratoconus are not observed within 3 months. This film, which can be prepared in large quantities and at low cost, should have potential application in corneal repair. Ó 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction Corneal disease is a major cause of blindness, and keratoplasty is considered to be an effective method for visual rehabilitation of patients with corneal blindness. However, its clinical application is limited due to a severe shortage of high-quality allogeneic corneal tissues [1]. Therefore, various efforts have been made to develop corneal tissue substitutes by using natural biological materials [2–6]. A tissue-engineered cornea should be biocompatible and transparent, and have appropriate mechanical properties. Collagen, the main load-bearing component in connective tissues, has been extensively studied as a scaffold material for tissue engineering corneas [7–11]. As reported, after implantation, the collagen scaffolds could replace the pathological corneal tissue in animals [12] or humans [13]. Although the collagen scaffold has many advantages for replacing pathological corneal tissue, its application is still limited by bacterial infection after keratoplasty [14–16]. Dropping antibiotics ⇑ Corresponding authors. Address: School of Materials Science and Engineering, South China University of Technology, Guangzhou 510641, China. Tel./fax: + 86 20 22236088 (Y. Wang). Tel.: +86 20 39380255 (L. Ren). E-mail addresses: [email protected] (L. Ren), [email protected] (Y. Wang).

onto the wound in the first week is an efficient method to solve this problem, but it is difficult to fix the correct amount of the antibiotic, which could cause some of the drug to be wasted. In addition, dropping is also frequently troublesome. Drug carriers have been used for many years to encapsulate antibacterial agents for local administration. There are many such drug carriers, including calcium sulphate void filler and poly(lactic-co-glycolic acid) microspheres [17–21]. However, drug-loading in these physical delivery systems is always achieved by adsorption and encapsulation, which are difficult to control with regard to the drug-loading rate and drug release. As cornea is a thin, transparent and avascular tissue, these methods are not suitable for use with corneas because the presence of a physical carrier in the corneal repair materials may lead to a decrease in optical performance, mechanical properties or biocompatibility. In this paper, we use the chemical cross-linking method to add tobramycin, a new kind of aminoglycoside antibiotic from Micromonospora purpurea that is effective against most species of both Gram-positive and Gram-negative aerobic bacteria [20], to collagen to prepare a novel tobramycin-containing antibacterial collagen film (Col-Tob) for corneal repair. The tobramycin was grafted onto the surface of a collagen film using 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide

1742-7061/$ - see front matter Ó 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.actbio.2013.08.033

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(NHS) as cross-linking agents. The mechanical properties, light transmittance, antibacterial property and biological properties of the cross-linked film were characterized.

the mass of the residues in washing liquid. Cross-linked collagen film without tobramycin was also prepared as the control group. Scheme 1 displays the chemical reaction mechanism between Col and Tob.

2. Materials and methods 2.3. X-ray photoelectron spectroscopy (XPS) 2.1. Materials A Kratos Analytical (UK) model Axis Ultra X-ray spectrometer system was used with a single Al Ka X-ray source (hm = 1486.6 eV, 150 W). The binding energy was calibrated by C1s of C–C as 284.6 eV. The wide scanning was operated with a pass energy of 160 eV at a scan rate of 1 eV per step over a range of 1105 eV, while high-resolution region scans were gathered with a pass energy of 40 eV and a step size of 0.1 eV. Elemental analysis and quantification spectra from the individual peaks were obtained with 40 eV pass energy. A Gaussian function was assumed for the curve-fitting process.

Tobramycin (Tob) was purchased from Shandong Freda Biopharm Co., Ltd., China. Type I collagen (HM Biotech Ltd., Guangzhou, China) was extracted from bovine tendon. EDC and NHS were supplied by GL Biochem Ltd. (Shanghai, China). Phosphate-buffered saline (PBS) was prepared from tablet form (Calbiochem Corp, Germany). All cell-culture-related reagents were purchased from Sigma Chemical (St. Louis, MO, USA). Deionized water was obtained from a water purification system (Millipore S.A.S., France). New Zealand white rabbits of either gender (12 weeks old and 2.5–3 kg) were used as animal transplant recipients.

2.4. Fourier transform infrared (FTIR) spectroscopy 2.2. Preparation of films 1

The infrared structure of the films was analysed using Fourier transform infrared-attenuated total reflectance (FTIR-ATR; Vector 33, Bruker, Germany). Before acquiring the FTIR spectrum of a sample, a background spectrum was collected. All the spectra were obtained from 3800 to 500 cm1.

1

Collagen (6.5 mg ml ) was dissolved in 0.01 mol l HCl solution and tobramycin was dissolved in deionized water with the concentration of 15 mg ml1. Then the collagen solution was dispensed into a specific mold and dried to form a cornea-shaped film. The collagen film was immersed in 15 ml of tobramycin solution, and EDC (the cross-linking agent) and NHS (the catalyst) were added to the solution to form a mixture with a mass ratio of EDC:NHS:Col-Tob = 1:1:6. The cross-linking was carried out by stirring the solution for several hours. After that, the Col-Tob film was rinsed three times with deionized water and dried again. The loading mass of tobramycin was determined by subtracting

2.5. Swelling test Water absorption of Col and Col-Tob was measured by swelling them in PBS (pH 7.4) at 35 °C. After gently blotting the film surface with filter paper to remove the absorbed water, the wet weight of the samples was immediately weighed. Films with known

R1 O

O

+

OH

COL

R1

N

C

N

R2

NH CH

O

COL

NH

(Collagen)

(EDC) R1:

R1 O

O

O

R1

O

NH

COL

R2

CH 2CH3 ;

NH

O

+

CH

OH

N

COL

O

+

N

O

C

NH

NH O

O

R2

R2 HCl-

R2:

(NHS)

CH2 CH 2CH2

N+

CH3;

CH3 OH NH2

O

O

HO H2N

O

OH

+

O

COL

O

N

OH

O H2N

OH H2N

O H2N

NH2

O

H2N

OH

OH

O

OH

O

OH H2 N

NH2

(Tobramycin) Scheme 1. The chemical reaction mechanism between collagen and tobramycin.

O HO O HN

C CO L

O

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dimensions were swelled in PBS, then the thickness and surface area of the hydrated films were measured in a certain time interval. The water absorption and variations in thickness and surface area of the films are calculated by the following equations:

water absorption ¼ ðW t  W 0 Þ=W t  100%

ð1Þ

thickness increase ¼ Ht =H0

ð2Þ

surface area increase ¼ St =S0

ð3Þ

Ref. [19]. The percentage of tobramycin release was calculated by dividing the amount of drug in the release medium by the total tobramycin introduced to the Col-Tob film. Scheme 2 displays the mechanism of the sustained tobramycin release process from the Col-Tob film. The experiments for tobramycin release were carried out in triplicate. 2.9. Antibacterial test

Here, Wt represents the wet weight of the film at the target times, W0 is the initial dry weight of the samples. Ht and St are the thickness and surface area of the wet samples at target times, respectively, and 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. Light transmittance Before the transparency test, the films were immersed in PBS for more than 2 h to take up water until saturation, then the solution on the surface of the Col and Col-Tob films was absorbed by filter paper. After that, the films were fixed into the specimen chamber of a UV3802 ultraviolet–visible spectrophotometer (Shanghai UNICO, China). Light transmittance of Col and Col-Tob was quantitatively evaluated by visible-light transmission measurements. 2.7. Mechanical test Analyses of the mechanical properties of samples were using a uniaxial load testing equipment (Model #5567, Instron Corporation, Issaquah, WA, USA) at the normal physiological temperature of the cornea (35 °C). Before the test, the films were immersed in PBS for 2 h, then a segment of film was clamped at its cut ends for axial tensile test (n = 5). The crosshead speed was 1 mm min1 and the test was stopped when the load decreased by 15% after the onset of failure. 2.8. Assessment of drug release in vitro Tobramycin release trials were performed in vitro in an incubator at 37 °C. For the trials, 225 mg of Col-Tob film was soaked in 15 ml of PBS (pH 7.4), then 3 ml of the supernatant of the sample medium was collected at regular time intervals (1, 2, 4, 8, 12, 24, 48, 72, 96, 120, 144 and 168 h, respectively). The volume of the medium was kept constant by adding an amount of PBS equal to the sample withdrawn for analysis. The concentrations of tobramycin were measured using the quantified method described in

OH NH2 O

OH

H2N OH O H2N OH OH HO H2N O

NH2 O

H2N OH O H2N OH HO O OH HN 2

O

OH

A typically type of bacteria isolated from patients who acquire corneal inflammation from trauma or recent surgery is Staphylococcus aureus [21]. This was therefore used to evaluate the antibacterial activity of the studied films using previously published procedures [22]. Generally, the S. aureus (Microbial Culture Collection Center of Guangdong Province, China) was grown in LB broth (1% w/v tryptone, 0.5% w/v yeast extract, 0.5% w/v NaCl) at 37 °C with constant agitation under aerobic conditions before any further experiment. The S. aureus was collected by centrifugation for 10 min at 4 °C and washed three times with deionized water, then the bacterial pellet was resuspended in sterilized water. The concentrations of the prepared bacterial suspensions used for antimicrobial tests were 3–8  108 colony-forming units (CFU) ml1. The Col-Tob and Col films that had been immersed in PBS solution for 1, 3, 5 and 7 days were placed into 48-well plates (Corning, UK), covered with 0.5 ml of bacterial suspension in the logarithmic growth phase and incubated by gentle rotation for 24 h at 37 °C. The viable S. aureus in the buffer was quantified by plating serial dilutions on agar plates. The agar plates were incubated for 24 h at 4 °C, and the total CFU were counted visually (n = 5). 2.10. Cell experiment 2.10.1. In vitro corneal epithelial cell culture Human corneal epithelial cells (HCECs) were obtained from the State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, China. The cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco BRL) with high glucose, supplemented with 15% fetal bovine serum (Sijiqing, China), 5 lg ml1 insulin, 100 U ml1 penicillin, 5 lg ml1 human transferrin (Sigma), 2 mM L-glutamine, 100 lg ml1 streptomycin (HyClone) and 10 ng ml1 human epidermal growth factor (Gibco BRL). HCECs were incubated in a humidified atmosphere containing 5% carbon dioxide at 37 °C. The cell supernatant was replaced every other day to maintain an adequate supply of cell nutrients. 2.10.2. The response of HCECs to the film Before cell experiments, the Col-Tob film was washed three times in PBS under aseptic conditions, then sterilized by ultraviolet

NH2 O

H2N OH O H2N OH H O O OH H2N

O

OH NH2

Hydrolysis

OH

O H2N OH

O HO

O

HN

HN

HN

O C

O C

O C

Sustained release

H2N

O

OH

H2N

Collagen ൕൕ Collagen Scheme 2. The mechanism of tobramycin sustained release process from the Col-Tob film.

O

+ Col NH2

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radiation for 2 h, and washed three times in PBS again at last. After sterilization, the film was transferred to a 6-well tissue culture plate (Corning, UK). The seeded cell density was 5000 cells cm2. The cell-seeded film was then incubated in a humidified atmosphere (5% CO2, 37 °C). The culture medium was replaced every 2 days. The response of HCECs to the Col-Tob film and the morphology of HCECs were examined. Before observation with an inverted fluorescence microscope (Olympus IX-70, Japan), the film’s surface was washed with PBS.

2.10.3. The proliferation of HCECs to the film After the samples were transferred to 96-well tissue culture plates (Corning, UK), HCECs suspension was seeded onto the ColTob films (experimental group, n = 10) and Col films (control group, n = 10), respectively. The seeded cell density was 5000 cells cm2. After 1, 2, 3, 4 and 5 days, the proliferation activity of the HCECs on the films was determined by methylthiazol tetrazolium (MTT) assay.

2.11. Histology After the HCECs had been cultured on the films for a week, the films were fixed in 4% paraformaldehyde at room temperature for 24 h. The films were processed and dehydrated stepwise using ethanol, then immersed in xylene and embedded in paraffin. The paraffin sections were subsequently stained with hematoxylin and eosin (H&E) using standard histochemical techniques, and viewed under a light microscope (Axioplan 2 imaging, Carl Zeiss, Germany).

2.12. Lamellar keratoplasty in rabbits Adult New Zealand white rabbits of either gender, aged 12 weeks and weighed 2.5–3 kg, were used as animal transplant models (n = 6). All animals were treated in accordance to the ARVO statement on the use of animals in ophthalmic and vision research. Before the surgery, the Col-Tob film was immersed in PBS for more than 1 h to take up water until saturation. Col-Tob films with a diameter of 6.25 mm diameter were implanted into the right corneas of the rabbits by lamellar keratoplasty (LKP). Only one eye of each animal was operated on. Briefly, in LKP, a 6.25 mm diameter circular incision was made using a trephine under general anesthesia. The depth of the lamellar incision was about 200 ± 25 lm. A lamellar dissection was then performed using a microkeratome along a natural uniform stratum in the corneal stroma to remove the host epithelium and anterior stroma. The Col-Tob film graft was sutured into the recipient bed by using eight interrupted 10–0 nylon sutures. Dexamethasone eye ointment was used three times daily for 7 days after LKP. Clinical examinations were followed up, including sodium fluorescein staining to assess epithelial integrity, slit lamp to assess corneal optical clarity, neovascularization, corneal deformation and rejection reaction. All animal experiments were performed with permission from the Medicine Ethics Committee in Sun Yat-sen University, China.

2.13. Statistical analysis All data are shown as mean ± standard deviation. Experiments were analyzed using analysis of variance to determine the significant differences among the groups. Statistical significance was defined as p < 0.05.

3. Results 3.1. XPS analysis XPS survey scans of both Col and Col-Tob are shown in Fig. 1 and the elemental composition is tabulated in Table 1. After cross-linking, the oxygen content increased from 16.40 to 18.17%, while that of nitrogen increased from 13.26 to 15.05%. Correspondingly, the ratio of nitrogen to carbon increased from 0.19 to 0.23, and that of oxygen to carbon increased from 0.23 to 0.27. The increases in percentage content of oxygen and nitrogen indicate that tobramycin is successfully introduced by chemical conjugation. High-resolution XPS spectra of C1s and N1s are shown in Fig. 2. The C1s spectrum of the Col and Col-Tob film that displayed a peak at 285 eV could be resolved into four main component peaks of different carbon species (Fig. 2a and b). The peaks around 284.60, 285.16, 285.89 and 287.71 eV are assigned to C–C, C–N, C–O and C@O, respectively. The N1s spectrum of Col and Col-Tob film displayed a peak at 400 eV, which required two peaks for the curve fit – one at 399.60 eV and the other at 401.57 eV – corresponding to amine (–CNH2) and amide (–CONH), respectively (Fig. 2c and d). The increases in amine and amide content in the Col-Tob film compared with the Col film is because of the cross-linking of collagen with tobramycin through the condensation polymerization of amine and carboxyl. 3.2. FTIR spectra analysis Fig. 3 shows the ATR-FTIR spectrum of Col and Col-Tob film. The main characteristic bands associated with collagen are detected. These bands are attributed to the amide groups from the collagen structure, respectively amides A, B, I, II and III. Amide A, observed in the FTIR spectrum at 3317 cm1, corresponds to N–H stretching. The CH2 asymmetrical stretch from amide B shows a vibration mode at 2931 cm1. The amide I band is associated with stretching vibrations of C@O and appears at 1631 cm1; amide II, associated with C–N stretching and N–H vibration, has a band at 1550 cm1; and the band at 1222 cm1 of amide III is due to

O1s C1s N1s

(b) Col-Tob

O1s

C1s N1s

(a) Col

1200

1000

800

600

400

200

0

Binding energy (eV) Fig. 1. XPS survey spectra of the films: (a) Col film; (b) Col-Tob film.

Table 1 Surface elemental compositions of Col and Col-Tob films. Materials

C (%)

O (%)

N (%)

N/C

O/C

Col Col-Tob

70.34 66.77

16.40 18.17

13.26 15.05

0.19 0.23

0.23 0.27

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A

30000

30000

C-O 20000

C-N

C=O

B C-C

C-C

Intensity (CPS)

Intensity (CPS)

40000

C-O 20000

C=O C-N 10000

10000

0

0 294

292

290

288

286

284

282

294

280

292

288

286

284

282

280

30000

30000

C

D

20000

Intensity (CPS)

Intensity (CPS)

290

Binding energy (eV)

Binding energy (eV)

-C-NH2 -CONH10000

20000

-C-NH2

-CONH-

10000

0

0 410

408

406

404

402

400

398

396

394

392

390

410

408

406

404

402

400

398

396

394

392

390

Binding energy (eV)

Binding energy (eV)

Fig. 2. High-resolution XPS spectra of C1s and N1s spectra and peak fit analysis for Col (a and c) and Col-Tob (b and d). The C1s spectrum at 285 eV can be resolved into four main component peaks (a and b). The N1s spectrum at 400 eV can be resolved into two peaks at 399.60 eV and one at 401.57 eV (c and d).

3.3. Water absorption

0.6 (1) Col-Tob

Absorbance

0.5 0.4

3317, Amide A: (2) Col N-H stretching 2931 ,Amide B: CH2 asymmetrical stretching (1 )

1550, Amide II:C-N stretching and N-H vibration 1222, Amide III: N-H and C-N stretching

1709, Tobramycin epoxy absorption

0.3 0.2

1631, Amide I: C=O vibration

1078 , Tobramycin: C-O-C stretching

(2 )

0.1 0.0 4000

3500

3000

2500

2000

1500

1000

500

Wavenumber (cm-1) Fig. 3. Fourier transform infrared spectrum of the Col-Tob and Col film.

N–H and C–N stretching. Spectral changes for the two films are also evident. The spectrum in Fig. 1 shows that, by moving from Col toward the Col-Tob, the spectrum shifts upwards, towards higher absorption intensities. These increases in intensity are more pronounced at collagen amide characteristic peaks, e.g. at 3317 cm1 (amide A) and 1631 cm1 (amide I). Furthermore, some of the characteristic absorption bands of tobramycin are identified for Col-Tob film. The FTIR spectra of Col-Tob depict characteristic tobramycin absorption bands at 1709 and 1078 cm1, which are attributed to epoxy absorption and C–O–C stretching, respectively.

Fig. 4(A) shows the films’ water absorption. After the samples had been stored in PBS for 1 h, the water absorption of the films tended to be constant. The water absorption for the Col-Tob film is 80.8 ± 2.5%, which is quite similar to that of human cornea (78.0 ± 3.0%) [23] and significantly higher than that of Col film (65.1 ± 1.5%) (n = 10, p  0.05). The sizes of wounds are different for each patient during keratoplasty, so corneal repair materials should be easy to fabricate with various dimensions. Fig. 4(B) and (C) indicates the variations in thickness and surface area of the samples after the films had been immersed in PBS for 1 h; the dimensions of the samples tended to be constant (Fig. 4, n = 10, p  0.05). The thickness and surface area of the films are controlled and repeatable.

3.4. Light transmittance Fig. 5 shows the light transmittance curve of Col and Col-Tob film. The thickness of the wet samples is about 150 ± 10 lm. The transparency of the Col-Tob film was slightly different from that of the Col film, with the former being slightly higher than the latter. With increasing wavelength, the light transmittance of the films increased to its maximum (more than 90%). The transmittance of the two films tended to be constant in the range of visible light.

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Water absorption (%)

100

A

80

60

40

20

0

8 7

B

Col

Col Col-Tob

6 5 4 3 2 1 0 0

6

12

18

24

Surface area increase (multiple)

Thickness increase (multiple)

Native

Col-Tob

1.150 1.145

C

Col Col-Tob

1.140 1.135 1.130 1.125 1.120 0

5

10

15

20

25

Time (h)

Time (h)

Fig. 4. Water absorption percentages (A) of the films and native human cornea. Variation of the samples’ thickness (B) and surface area (C). Values are expressed as the mean ± standard deviation (n = 10).

Light transmittance (T%)

100

exhibits an exponential tendency. The burst release of tobramycin from Col-Tob in the first 4 h is about 40% of the total tobramycin loading amount, and that is followed by a slower release of the drug. From the first day after surgery, the sustained release of tobramycin from the Col-Tob film manifests as a slow process that takes less than a week: on day 7, the total tobramycin released from the Col-Tob film has exceeded 95%.

Col-Tob

90

80

70

Col

3.7. Anti-bacterial effect

60

50 400

500

600

700

800

Wavelength (nm) Fig. 5. Light transmittances of Col-Tob film and Col film over the wavelength range of 400–800 nm.

3.5. Mechanical property A comparison between the films shows that they have similar mechanical behaviour (Fig. 6, n = 5, p  0.05). The thickness of the wet films is about 150 ± 10 lm. The ultimate tensile strengths of Col-Tob and Col are is 9.73 ± 0.8 and 9.15 ± 0.9 MPa, respectively (Fig. 6A). However, the elongation at break showed no significant difference between Col-Tob (42.0 ± 1.7%) and Col (48.0 ± 2.5%) (Fig. 6B). Furthermore, the Young’s moduli of Col-Tob and Col are 23.2 ± 0.9 and 19.1 ± 1.0 MPa, respectively (Fig. 6C). The Col-Tob film exhibits suitable mechanical properties. 3.6. In vitro tobramycin release The investigation of tobramycin release (the cumulative release in vitro) from the Col-Tob film is shown in Fig. 7. The release profile

Fig. 8 shows the results of the antibacterial effect of the Col and Col-Tob film in 1 week. The S. aureus grows well on the pure collagen film. The bacterial colony is also clearly visible in the Col’s agar culture dish (Fig. 8A). Compared to pure collagen film, there are significantly fewer CFU of S. aureus on the Col-Tob’s agar culture dish (Fig. 8B) than on the Col sample. The results indicate that the Col-Tob film has a significantly better antibacterial effect than the Col film. Fig. 9 shows the quantitative results of the antibacterial experiment, with there being significantly fewer CFU on the Col-Tob’s agar culture dish than that of the Col film (Fig. 9, n = 5, p < 0.05). The results suggest that the Col-Tob film inhibits the bacterial successfully.

3.8. The morphology of HCECs on the film Fig. 10 shows the morphology of HCECs on Col-Tob film at different time points. After the cells had been incubated for 12 h (Fig. 10A), 24 h (Fig. 10B), 48 h (Fig. 10C) and 72 h (Fig. 10D), the HCECs attached to and grew well on the Col-Tob film. At 12 h, the seeded cells had adhered to the surface of the film quite well. The morphology of the HCECs also gradually changed from a round shape to a spindle shape, which is quite similar to the HCECs growing on normal corneal tissues [24].

295

10

A

Elongation at break (%)

Tensile strength (MPa)

Y. Liu et al. / Acta Biomaterialia 10 (2014) 289–299

8 6 4 2

50

B

40 30 20 10

0

0

Col Young's modulus (MPa)

Col-Tob 25

Col-Tob

Col

C

20 15 10 5 0

Col-Tob

Col

600 100

80

60

40

20

0 0

20

40

60

80

100

120

140

160

180

Time (h)

Total Colony Forming Units (CFU)

Cumulative release of tobramycin (%)

Fig. 6. Mechanical properties of Col-Tob and Col. The mechanical properties of the films were analyzed with a mechanical tester as follows: tensile strength (A), elongation at break (B) and Young’s modulus (C). Values are expressed as the mean ± standard deviation (n = 5).

Col Col-Tob

500

400

300

200

100

0

1 Fig. 7. Cumulative release of tobramycin from Col-Tob film, the tobramycin sustained release from the Col-Tob film can last for more than a week.

3

5

7

Time (d) Fig. 9. Total colony forming units of Col-Tob and Col film at 1, 3, 5 and 7 days.

Fig. 8. Photographs of agar media with colonies of S. aureus strains after incubation with Col and Col-Tob film. Bacterial colony is clearly visible in the Col’s agar culture dish (A), the colony forming units of S. aureus on Col-Tob’s agar culture dish (B) are significantly less than that of Col sample.

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Fig. 10. The morphology of HCECs on the Col-Tob film at different time points, 12 h (A), 24 h (B), 48 h (C) and 72 h (D), respectively. The morphology of HCECs was changed from round shape to spindle shape gradually.

2.5

A

OD Value

2.0

Col-Tob Col

1.5 1.0 0.5 0.0 1

2

3

4

5

Time (day)

Fig. 11. The proliferation of HCECs on the Col-Tob and Col film (A). Values are expressed as the mean ± standard deviation (n = 10). H&E-stained section of HCEC constructs grown on Col-Tob film consisting of 2–3 layers (B).

Fig. 12. Photographs of fabricated cornea and implantation method. Representative images of Col-Tob film (A) and just after LKP. The graft was held in place with 10–0 sutures (B).

3.9. The proliferation of HCECs and histological sections of the Col-Tob film The MTT test (Fig. 11A) shows the proliferation of HCECs on the Col-Tob film and Col film (control group) (Fig. 11A,

n = 10, p  0.05). After the HCECs had been seeded on the samples for 1 day, they proliferated rapidly. The film was almost completely covered by HCECs 72 h later. Fig. 11B shows an H&E-stained section of the Col-Tob film which had been used for HCECs culture for 1 week. The photograph indicates

Y. Liu et al. / Acta Biomaterialia 10 (2014) 289–299

that 2–3 layers HCECs had covered the whole surface of the films.

3.10. Integral function evaluation of Col-Tob film in the LKP model The Col-Tob film can be sutured tightly on the rabbit’s ocular surface (Fig. 12B). The surgical sutures were removed after 7 days. Following LKP, all of the rabbits survived without infectious or hemorrhagic complications. The wound-healing process was completed by about 15 days after implantation (Fig. 13A). The re-epithelialization time for the Col-Tob grafts is 15 ± 5 days

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(Fig. 13B). The transparency of the transplants is quickly restored in the first 30 postoperative days (Fig. 13A). The implants are well integrated into the recipient corneas. Corneal neovascularization and the corneal rejection reaction are not observed within 3 months of transplantation (Fig. 13A and B). After 15 days, the process of epithelial coverage on the surface of the samples has been basically completed. Compared with the epithelialization at 15 days, more surface epithelium has appeared by day 30, with 3–5 layers of rabbit corneal epithelial cells covering the entire surface of the films (Fig. 14A and B). In addition, these epithelial cells also secrete a lot of basement membrane collagen. At this time, keratoconus is not observed (Fig. 15A, B and C).

Fig. 13. Postoperative observation of LKP using Col-Tob film. The process of restoration of transparency (A). The process of re-epithelialization (B). The transparency of the transplants is restored quickly in the first postoperative 30 days.

Fig. 14. H&E-stained section of the Col-Tob graft which was transplanted to the rabbit’s ocular surface for 15 days (A) and 30 days (B). The process of epithelial coverage on the surface of the samples has been basically completed after 15 days; 3–5 layers rabbit corneal epithelial cells had covered the whole surface of the films after one month.

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Fig. 15. Slit lamp biomicroscope photographs at 3 months after implantation with biosynthetic corneal substitutes, left (A), centre (B) and right (C) of the eyeball. Implants were well integrated into recipient corneas. Keratoconus was not observed.

4. Discussion Currently, although corneal transplantation has a high success rate, the major problem is the shortage of high-quality donor tissues in most countries. Another problem is bacterial infection after keratoplasty. In this study, we hope that the antibiotic-containing collagen film is able to repair corneal tissue and also reducing the risk of corneal infection after keratoplasty. Films prepared from collagen I have demonstrated significant potential for corneal repair, and may therefore provide a suitable substitute for donor cornea [7–10]. In this study, the bovine collagen comprised primarily type I collagen, the main biochemical component of the cornea, making up 71% of the dry weight of the corneal extracellular matrix [25]. The antibiotic used in this study was tobramycin, which has been reported to have superior a antibacterial effect on most Gram-positive and Gram-negative aerobic bacteria, and some mycobacteria [20]. The unique molecular structure of tobramycin allows it to combine with collagen by both chemical and hydrogen bonding. The combination results of XPS and FTIR confirmed that the tobramycin was immobilized on the collagen film in this study. The inflammatory response is triggered by keratoplasty, and also bacterial infections occur regularly after this operation. Generally, antibiotics are dropped frequently onto the wound after surgery to treat this problem. However, this therapeutic method is difficult, and has a low bioavailability of drugs. Drug-loading by adsorption and encapsulation in some physical delivery systems is often used to solve these problems [19–21]. Cornea is a thin tissue, so the presence of some physical carrier in corneal repair materials may lead to a decrease in optical performance (the light transmittance of human cornea is about 87% [26]), mechanical properties or biocompatibility. Therefore, a tobramycin-containing collagen film that can avoid those shortcomings was developed and evaluated in this study. Notable differences in the antibacterial effects of Col and Col-Tob films are observed during the first week. The total number of CFU of S. aureus on Col-Tob’s agar culture dish is significantly smaller than that of the Col sample, suggesting that the Col-Tob film has a better antibacterial effect than the Col film. Further, the sustained release of tobramycin from the Col-Tob film can last for more than a week, which is the critical period to avoid bacterial infection after keratoplasty. About 40% of the total tobramycin is burst released in the first 4 h may because some tobramycin molecules are incorporated within the collagen film by hydrogen bonds, which are destroyed gradually under the soaking and scouring action of the buffer solution. The subsequent release of antibiotic during the first week manifests as a slow and steady process, which is caused by the slow hydrosis occurring between the amino groups of tobramycin and the carboxyl groups of collagen. Compared with the frequent application of eye drops, drug release in this chemical cross-linking system shows higher bioavailability. The antibacterial effect of the Col-Tob film on other bacterial strains will be further studied in the future. In developing corneal repair materials, we are concerned with function and biocompatibility [27]. In order to function, the

implant must have appropriate properties, such as mechanical strength, water absorption, suture performance and light transmittance [28]. The introduction of tobramycin has a tiny impact on the optical properties of the collagen film. The water absorption of ColTob is quite similar to that of human cornea (78.0 ± 3.0%) [23] and is significantly higher than that of Col film. This may be due to the introduction of tobramycin to collagen has brought a large number of hydrophilic groups, such as amino and hydroxyl. According to the desired wet film’s thickness and surface area, we can prepare the dry material with the corresponding size easily. To examine the mechanical properties of Col-Tob and Col, the ultimate tensile strength, elongation at break and Young’s modulus were measured. The ultimate tensile strength of the Col-Tob film is close to that of native corneal tissue (11 ± 0.5 MPa) [23]. Besides its antibacterial action, tobramycin takes on another active role: with the introduction of tobramycin molecules, the free volume of the functional groups of the cross-linked molecule is decreased and the steric hindrance is increased, as is the molecular deformation energy. Both the tensile strength and the elastic modulus of Col-Tob increased on the macro-level. The MTT test demonstrates that the Col-Tob film has no cytotoxic reactivity against HCECs. The morphology of HCECs on the Col-Tob film is quite similar to that of HCECs grown on normal corneal tissue. The H&E-stained section of Col-Tob film indicates that HCECs can bond well to the surface of Col-Tob film. The results of these studies show that the ColTob film has good biocompatibility. This drug loading method, by chemical cross-linking, was only studied with regard to corneal repair. Different results may be achieved with other drugs or tissues. Further studies will focus on investigating the optimum tobramycin concentrations needed to maximize the bactericidal property without harming normal tissues. Evaluation of the antibacterial effect of this method in vivo will be also be included in our future study. 5. Conclusions In this study, we have successfully developed a novel tobramycin-containing antibacterial collagen film for corneal repair. The results show that this film has similar water absorption, mechanical properties and light transmittance to native cornea, and shows an excellent antibacterial effect and biocompatibility. After implantation in the rabbit eye, the corneal rejection reaction, neovascularization and keratoconus are not observed within 3 months. This cross-linked film shows the potential to solve the bacterial infection after keratoplasty when used in corneal repair in the future. Acknowledgements This work is supported by the National Basic Research Program of China (No. 2012CB619104), National Science Foundation of China (No. 51273072, 50803018, 51232002), National Science & Technology Pillar Program in the Twelfth Five-year Plan of China

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