Poly(2-hydroxyethyl methacrylate

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A Morphology-Controllable Collagen/Poly(2-hydroxyethyl methacrylate) Porous Hydrogel with Paraffin Microsphere as Template Keke Chen, Xialian Fan, Keyong Tang, Guangming Wan, Xichan He, Xiumin Li, Qiyuan Chen, Min Shen, Yiwen Lv, and Fang Wang ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00264 • Publication Date (Web): 25 Oct 2018 Downloaded from http://pubs.acs.org on October 26, 2018

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ACS Applied Bio Materials

A Morphology-Controllable Collagen/Poly(2-hydroxyethyl methacrylate) Porous Hydrogel with Paraffin Microsphere as Template Keke Chen1, Xialian Fan1, Keyong Tang1,*, Guangming Wan2, Xichan He1, XiuminLi1, Qiyuan Chen1, Min Shen1, YiwenLv1, Fang Wang1 1School

of Materials Science and Engineering, Zhengzhou University, Zhengzhou 450001, China

2The

First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China *Corresponding

author: [email protected]

Abstract: In this study, porous poly(2-hydroxyethyl methacrylate) (PHEMA) matrix was fabricated by paraffin template method, which was used as substrate to adhere collagen fibers to form an inter-connective porous collagen/PHEMA (Col-PHEMA) composite hydrogel. Microscope and scanning electron microscope (SEM) were employed to characterize the morphology of paraffin microspheres and Col-PHEMA composite hydrogels.The paraffin microspheres with the diameter in the range of 100 µm to 200 µm were collected by a pre-set sieve. Whereafter the interface of uniform paraffin microspheres were thermal-bonded to form a contacted template, the derived Col-PHEMA composite hydrogels had an inter-connective porous microstructure. Fourier transform infrared spectroscopy (FTIR) indicated that new hydrogen bonds were formed between collagen fibers and PHEMA hydrogel. Besides, the Col-PHEMA composite hydrogels revealed high hydrophilicity, good mechanical properties, and good water uptake capacity. The porous Col-PHEMA composite hydrogels showed good biocompatibility and the collagen layer may promote the proliferation of fibroblast cells. The Col-PHEMA composite hydrogel is expected to find an application in corneal repairing. Key

word:

paraffin

template,

poly(2-hydroxyethyl

methacrylate),

collagen,

inter-connective pore, composite hydrogels

Introduction Cornea tissue is the outermost transparent layer of eyes, which serves as an optical

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element to transmit and focus light into eyes1-3. Nowadays, corneal diseases are the second primary cause of blindness, affecting more than 12.7 million people globally4. Corneal transplantation is an effective method to cure corneal diseases5. The major current problem in corneal transplantation is the shortage of cornea donation with the increasing numbers of patients every year6. Therefore, cheap and excellent artificial corneas are widely researched and prepared for cornea repairing. Among them, artificial corneas with "Center-skirt" structure have attracted wide attentions, which were consists of central optic membrane and annular skirt support7-10. Especially, the artificial cornea skirt scaffold with a three-dimensional (3D) porous microstructure is conducive to corneal epithelial cells adhesion and proliferation, allowing nascent tissue to grow in the porous composite. It was found that the adhesion, growth and migration of corneal cells on cornea skirt support materials were greatly influenced by the microstructure and porosity of scaffold11-14. So the fabrication of porous cornea scaffold by controlling preparation conditions and synthesis parameters should be a promising route to meet the needs of cornea treatment. Some polymer materials have been evaluated to be used in preparing artificial cornea. With non-woven polyurethane fabrics as skirt and polysiloxane or polyurethane as core, artificial corneaswere developed, whose retention time in eyes was not long enough15. Caldwell16 developed an artificial cornea with the transparent core from polyurethane and the skirt from PTFE. The artificial corneas couldact for one year or longer in animal corneas. Poly(methyl methacrylate) (PMMA) is usually used for fabricating artificial cornea, which is hard to be decomposed in-vivo.17 Subsequently, with the increasing need for artificial cornea, it becomes more and more imperative to develop

soft

and

hydrophilic

hydrated

matrices.

Compared

with

PMMA,

poly(2-hydroxyethyl methacrylate) (PHEMA) was obtained at the same preparation and external condition is low in swelling degree, making it easy to keep the shape and control degradation rate. Besides, the PHEMA can be hydrolyzed in-vivo to provide space for newly generated cells. Hence, PHEMA has been widely used in biomedical fields including drug delivery17,18, soft contact lenses19,20, and scaffolds for tissue engineering application21,22, due to its excellent processability, good hydrophilicity, and low cytotoxicity. Besides, PHEMA is good in biocompatibility and bio-chemical degradability23. So it is an ideal polymer to prepare scaffold for cell anchorage, and it


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could be degraded gradually and replaced by the renascent tissue24. Unfortunately, PHEMA is poor in cell adhension25. In this study, in order to overcome the shortcoming of cell adhension, collagen fibers were attached on the surface of PHEMA hydrogel to provide more adhesion pointson the surface and interior of composite hydrogels. Collagen is the most abundant protein in the human body with excellent biocompatibility, low toxicity, and low immunogenicity. Collagen could provide a good physiological environment to culture corneal cell26,27. There are plenty of carboxyl and amino groups in collagen, which could form new hydrogel bonds with the hydroxyl groups of PHEMA hydrogel to stabilize the collagen fibers and PHEMA hydrogel. The abundant collagen fibers on the surface of holes and hole-joints provide more space for cell adhesion and act as channels for cell migration. Due to its excellent performance and safety, collagen has been widely used for medical purpose, including drug delivery, burns/wounds sponges, surgical suture, hemostatic agents and tissue engineering. For instance, Fan and colleagues28 prepared porous TiO2/collagen-chitosan scaffold for wound repairing. This composite scaffold played a crucial role in the aggregation of red blood cells. Wang and colleagues29

used

1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide

(EDC),

N-hydroxysuccinimide (NHS) crosslink collagen with chitosan to prepare artificial livers with good hepatocyte compatibility and excellent blood compatibility. Nowadays, such new techniques as particulate leaching30,31, gas foaming23, and phase separation32,33 are being employed to prepare inter-connection porous scaffold. These techniques provide various advantages to porous hydrogels for tissue engineering. However, the pore size and structure of hydrogels is difficult to be controlled. Paraffin template method (PTM) was considered to be a high-efficiency route to fabricate porous scaffold, because of its simple operation, cost-efficiency and easy controllability34. PTM is usually conducted at low temperature, which limits the application inpreparationsome materials. The polymerization temperature of PHEMA hydrogel could be as low as 37oC by an ammonium persulphate-sodium metabisulphite redox initiation technique35. So it is feasible to prepare highly porous PHEMA hydrogel by PTM. In the present work, PHEMA hydrogel with inter-connective pore structure was prepared by a paraffin microspheres template method, and a novel porous Col-PHEMA composite hydrogel was obtained by immersing the PHEMA hydrogel precursor in collagen solution with different concentrations. The optimized Col-PHEMA composite



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hydrogel should have an inter-connective pore structure with collagen fibers attached to the surface of holes and hole-joints. The structure of Col-PHEMA composite hydrogel was shown in Fig. 1(A), in which collagen fibers were adhered on the surface of holes and hole-joints to form fiber network structure. Just like Fig. 1(B), cells fall on the surface of pure PHEMA hydrogel, which is easy to slip off. In this case, there are collagen fibers in the PHEMA hydrogel, resulting in stronger adhension for cells. PHEMA hydrogel and Col-PHEMA composite hydrogel were fabricated though a freeze-drying process, and scanning electron microscopic (SEM), X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy (FTIR) were used to characterize

the

structure.

The

mechanical

properties,

porosity,

degradation,

hydrophilicity and cytotoxicity of the composites were investigated systematically as well. It is hoped to be cheap route to prepare porous scaffold for cornea repairing.

Fig. 1. (A) Structural schematics of porous Col-PHEMA composite hydrogel; (B) schematic diagram of cell falling on the surface of pure PHEMA hydrogel and Col-PHEMA composite hydrogel.

2.

Experimental

2.1 Materials Type I collagen was extracted from pigskin according to our previous work36. Triethylene glycol dimethacrylate (TEGDMA) was purchased from Shanghai Macklin Biochemical Co. Ltd, China. Hydroxyethyl methacrylate (HEMA) was supplied from Xiya Chemical Reagent Technologies Co. Ltd. Sodium chloride, potassium chloride,


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hexane,

sodium

dihydrogen

phosphate,

dipotassium

phosphate,

ammonium

persulphate, and sodium metabisulphite were provided by Tianjin Fengchuan Chemical Reagent Co. Ltd. Paraffin with ceresin used for embedding ＆ sectioning was purchased from Shanghai Yiyan Biological Research Co. Ltd. All reagents were of analytical reagent. 2.2 Preparation of paraffin template The paraffin template was prepared according to previous literature37. In brief, 2.5 g gelatin and 20 g paraffin were added into 500 mL distilled water at 70oC. After being stirred continuously at 500 r/min for half an hour, the mixture was poured into 2000 mL cold water to solidify the paraffin spheres. The paraffin spheres were sieved by two sieves with the pore diameter of 100 μm and 200 μm to obtain the paraffin sphere particles sized 100-200 µm, as illustrated in Fig. 2. Finally, paraffin spheres were washed five times with distilled water and dried at room temperature. In order to get the paraffin template, the obtained paraffin spheres were poured into a hollow PVC cylinder mould with the diameter of 1.6 cm and the pressure of 10 N was applied until no obvious pores at room temperature. Solidification at 50oC was done for 20 minutes to get the paraffin template, which was then stored in a desiccator at 18oC for subsequent use. 2.3 Preparation of porous PHEMA hydrogel 0.07g ammonium persulphate and 0.14g sodium metabisulphite were dissolved in 3 mL 50% (v/v) aqueous acetone solution, and then 7 mL HEMA and 1.86 µL TEGDMA were added. The compound solution was poured into paraffin templateand put it in vacuum to ensure sufficient penetration. Then put the paraffin template at 37oC for 4h. The obtained hydrogel was washed by n-hexane in an orbital shaker for 1 week at a low speed. The obtained porous PHEMA hydrogel was washed five times with distilled water and freeze-dried at -55oC for 12 h to facilitate subsequent absorption of collagen. The obtained materials were stored in a desiccator at 18oC for subsequent use. 2.4 Preparation of inter-connective porous Col-PHEMA composite hydrogel



Collagen was dissolved into PBS (0.2 M, pH=7.4) solution containing 200 mM K+ and stored in refrigerator at 4oC for 24 h. The porous PHEMA scaffolds were immersed in various concentrations (0.5, 1.0, 2.0, 4.0 mg/mL) of collagen solution, and then put it in refrigerator at 4oC for 48 h to make sure that the collagen solution was absorbed into the PHEMA pores. The mixture was then transferred to a water bath at 37oC for 4 h for the self-assembly of collagen fibers.

Fig. 2. Scheme of preparation of Col-PHEMA porous composite hydrogel.

2.5 Characterization The hydrogels were freeze-dried at -55oC for 12 h before being characterized. The morphologies of various materials were recorded by a microscopy and scanning electron microscopy (Quanta250FEG, USA). The tensile strength of Col-PHEMA composite hydrogels were determined by a texture analyzer (TA. XT Plus, UK) at a stretching rate of 5 mm/min. X-ray photoelectron spectroscopy (XPS) of Col-PHEMA composite hydrogels were done by ESCALAB 250 spectrometer (Thermo Scientific Ltd, England) with Al Ka (1486.6 eV) radiation source. Fourier transform infrared spectroscopy (FTIR) (VERTEX70, Germany) was employed to characterize the composition of various


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samples in the range of 4000-400 cm-1. The contact angle was measured and analyzed by standard type contact angle meter (TC2000C1, China). 2.6 Porosity The porosity of Col-PHEMA composite hydrogels were accurately measured at least three times with the averages reported by automatic mercury porosimeter (PM60GF-18, USA). Briefly, pressure of Hg was increased continuously from 0.013 to 430 MPa and pore size distributions were determined using the Washburn equation (1) and (2) 38: 𝐷=

―4𝛾 𝑐𝑜𝑠𝜃

(1)

𝑃 𝑉1

(2)

𝜌 (%) = 𝑉2 × 100%

where D is the pore diameter, 𝜌 is the total porosity, 𝛾 is the surface tension, 𝜃 is the contact angle and P is the applied pressure, V1 and V2 were the volume of mercury was pressed into sample and the volume of sample, respectively. 2.7 Water content The water content of Col-PHEMA composite hydrogels were measured by immersing the freeze-dried scaffold sized 20 mm in diameter and 5 mm in height in 100 mL PBS (0.2 M, pH=7.4) at 37oC for 24 h, and the PBS buffer solution was changed every two hours. After gently wiping the PBS solution away from the surface of hydrogels with filter paper, the hydrogels were weighed. The water content of Col-PHEMA composite hydrogels were measured and calculated according to equation (3)28: 𝑊𝑎𝑡𝑒𝑟 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 =

𝑊𝑠 ― 𝑊𝑑 𝑊𝑠

× 100%

(3)

where Ws and Wd are the weights of PBS-immersed sample and freeze-dried sample, respectively. 2.8 Cytotoxicity analysis In this paper, MTT analysis was done to evaluate the cytotoxicity of PHEMA hydrogel and Col-PHEMA composite hydrogel. DMEM medium containing 10% calf



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serum was transferred to the scaffolds until it reached 0.2 g/mL, and the mixture were cultured at 37oC for 24 h. The mouse fibroblasts (Fbs) was used to prepare 105/mL cell suspension, and 200 µL suspension was put into each hole of the 96-well plate. After being cultured in 5% CO2 atmosphere at 37oC for 24 h, and the old medium was carefully replaced with fresh ones, additional culture was done for another 2 days, 4 days or 7 days. Each hole was added with 20 µL MTT and continuously cultured for 4 h, and 150 µL dimethyl sulfoxide (DMSO) was used to replace the medium and the plate was shook for 10 min. The absorbance (Abs) was analyzed using a Micro-plate Reader (Multiskan Go, Thermo, Finland) at 570 nm. Each sample was done five parallel experiments, and the average was reported. The relative growth rates (RGR) were calculated according to equation (4)28: 𝐴

(4)

𝑅𝐺𝑅 = 𝐴0 × 100%

where A and A0 are the Abs of experiment groups and control, respectively. The cytotoxicity of porous scaffolds was done according to ISO/TC194 file28. The RGR and cytotoxicity stage were shown in Table 1. Table 1. RGR and cytotoxicity stage

RGR (100%)

Cytotoxicity

stage

Cytotoxicity

≥100

0

non

75～99

I

non

50～74

Ⅱ

mild

23～49

Ⅲ

middle

1～24

Ⅳ

Middle

Ⅴ

Severe

﹤1 3. Results and discussion


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Fig. 3. Microscopic images of paraffin spheres sintered at 50oC for 0 min (A), 20 min (B), and 30 min (C).

The microscopic images of paraffin spheres sintered at 50oC for different times were shown in Fig. 3. The newly prepared paraffin microspheres unsintered were regular sphere and exposed to each other as shown (A). When the paraffin microspheres were heated at 50oC for 20 min, overlapped area appeared between them. However, when the heating time reaches more than 30 min, melting takes placein the paraffin microspheres and the volume becomes smaller. It was indicated that the overlapped area between microspheres grows bigger with increasing the sintering time. During the first 20 min, the paraffin microspheres were in good shape and a suitable overlapping area formed between the microspheres. If the paraffin microspheres were removed from the polymer materials, a stable inter-connected pore structure may be formed.



Fig.4. SEM images of the fracture surface of PHEMA hydrogel and Col-PHEMA composite hydrogels. The immersion concentration of collagen solution were 0.0 mg/mL (A1, A2); 0.5 mg/mL (B1, B2); 1.0 mg/mL (C1, C2); 2.0 mg/mL (D1, D2); 4.0 mg/mL (E1, E2), respectively.


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The morphology of PHEMA hydrogel and Col-PHEMA composite hydrogel were shown in Fig. 4. From Fig. 2(A1, A2), it could be found that the pure PHEMA hydrogels have a relatively intact inter-connected pore structure, indicating the paraffin template method an efficient route to controllably form rational porous structure. The diameter of pore was about 200 µm, and the overlapping area between microspheres was about 100 µm. The inter-connected pore structure may facilitate the transplantation of cells and nutrients when being used for bioengineering purpose. Besides, the attached collagen on the surface of holes and hole-joints of PHEMA hydrogel may improve its bioactivity. As shown in Fig. 4(B-E), the surface of holes and hole-joints of Col-PHEMA composite hydrogel were covered with collagen fibers formed at different concentrations of collagen solution. Little collagen fibers may be found attached on the surface of holes and hole-joints of Col-PHEMA composite hydrogel from 0.5 mg/mL collagen solution. When the concentration of collagen solution increases to 1.0 mg/mL, the attached collagen fibers formed a wrinkled thin collagen sponge, with the collagen fibers in the hole-joints like an arachnoid net. Collagen fibers not only could improve the biocompatibility of the porous PHEMA hydrogel, but also provide more sites for cell growth, adhesion and migration. However, if the concentration of collagen solution is higher than 2.0 mg/mL, the attached collagen fibers on the surface of holes became much denser, and the hole-joints were blocked with collagen fibers, which is not better for cell migration.



Fig.5. FTIR spectra of pure PHEMA, pure collagen, HEMA monomer and Col-PHEMA composite hydrogel made by 1.0 mg/mL collagen solution

HEMA monomer, pure PHEMA, pure collagen and Col-PHEMA (1.0 mg/mL) composite hydrogel were characterized by FT-IR. As shown in Fig. 5, the characteristic absorption peaks of collagen may be found around 1646 cm-1 (amide I, C=O stretching vibration), 1550 cm-1 (amide II, N–H bending vibrations and C–N stretching vibrations), 1240 cm-1 (amide III, C–N stretching and N–H in plane bending). From the spectra of PHEMA, the absorption peak at 1709 cm-1 and 1024 cm-1 was attribute the C=O stretching and C-O-C stretching of ester group, respectively. The peaks around 2970 ～ 2840 cm-1 and 1470～1380 cm-1 were stretching and bending vibrations of saturated CH2, which was consistent with reported results39,40. In addition, all peaks appeared in the spectra of Col-PHEMA composite hydrogel with 1.0 mg/mL collagen solution suggested the existence of collagen in the composite. In the FTIR spectrum of paraffin, the peaks around 3000 ～ 2800 cm-1 were the stretching vibrations of CH2 and CH3. Thus the characteristic peaks of paraffin and PHEMA overlap at 3000 ～ 2800 cm-1. And the existence of paraffin cannot be effectively excluded by FTIR. In addition, the red-shift occurred around 3400 ～ 3200 cm-1 should be due to the formation of hydrogen bonds between collagen and PHEMA.


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Fig.6. The C1s spectra of PHEMA hydrogel, Col-PHEMA composite hydrogel prepared from 1 mg/mL collagen solution (1-Col-PHEMA), and Col-PHEMA composite scaffolds prepared from 4 mg/mL collagen solution (4-Col-PHEMA).

Further evidence for the existence of collagen in the composite was provided by XPS. As shown in Fig. 6, for the samples of PHEMA, 1-Col-PHEMA and 4-Col-PHEMA composite hydrogel, typical C1s XPS signals appeared. The peaks at 284.5 eV, 285.8 eV, 286.2 eV, 287.3 eV and 288.5 eV, were attributed to the C-C, C-N, C-O, C=O and O-C=O bonds, respectively. Distinctly, the state of C element in PHEMA hydrogel revealed three C1s spectrum of C-C, C-O and -COO bonds. There are abundant amino groups in collagen molecules. By comparing with pure PHEMA hydrogel, a typical peak of C–N bond at 285.8 eV was found in both composite hydrogels of 1-Col-PHEMA and 4-Col-PHEMA. Furthermore, the intensity of C-N binding energy was improved for the Col-PHEMA composite hydrogel with increasing the collagen content. It was indicated that there are more collagen fibers in the composites prepared



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from collagen solutions with higher concentration, which was consistent with the results of SEM and FTIR.

Fig.7. Contact angle pictures (A) (a: PHEMA hydrogel, b: Col-PHEMA composite hydrogel made in 1 mg/mL collagen solution, c: Col-PHEMA composite scaffolds made in 4 mg/mL collagen solution); Water content (B) of Col-PHEMA composite hydrogels prepared in different concentrations of collagen solution. Table 2. Contact angle of Col-PHEMA composite hydrogel made from different concentrations of collagen solution

Sample

Concentration of collagen solution (mg/mL)

Contact angle (º)

a

0

87.5

b

1.0

43.5

c

4.0

39.5

The hydrophilicity and water content of freeze-dried porous PHEMA hydrogel and Col-PHEMA composite hydrogel were presented in Fig. 7 and Table 2. The contact angle of pure PHEMA hydrogel was 87.5o, and the contact angle of Col-PHEMA composite hydrogel decreases with increasing the concentration of collagen solution. Collagen is


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well-known a hydrophilic biopolymer with abundant such hydrophilic groups as amino and carboxyl. The amount of collagen in the Col-PHEMA composite hydrogel is conducive to the hydrophilicity of composite hydrogel. As shown in Fig. 7, the hydrophilicity of Col-PHEMA composite hydrogel were improved obviously with increasing the amount of collagen fibers on the surface, indicating that collagen plays a crucial role in improving the hydrophilicity of composite hydrogels. Water content is another crucial factor for artiﬁcial cornea skirt materials, which greatly inﬂuences the biocompatibility of hydrogel41. The water contents of PHEMA hydrogel and Col-PHEMA composite hydrogel were investigated in PBS (pH=7.4) solution at 37oC for 48 h. As shown in Fig. 7(B), pure PHEMA hydrogel showed high water content (70%) due to the existence of hydroxyl groups in HEMA monomer. The water content of Col-PHEMA composite hydrogelis little higher than that of pure PHEMA hydrogel, indicating that collagen is beneficial to improve the water content. However, the water content increase is not remarkable with increasing the collagen used. Therefore, collagen could improve the hydrophilicity and water content of the porous Col-PHEMA composite hydrogel. In this study, the water contents of various Col-PHEMA composite hydrogel were all higher than 70%, closed to the water content of natural cornea (78%)42.



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Fig.8. Tensile strength of Col-PHEMA hydrogels from different collagen concentrations (0 mg/mL, 0.5 mg/mL, 1 mg/mL, 2 mg/mL, and 4 mg/mL).

Cornea skirt acts as a supporter of the cornea tissue, which should have good mechanical strength. Good mechanical strength of Col-PHEMA composite hydrogel could ensure itself as cornea skirt material to support artificial cornea. In this study, PHEMA hydrogel and Col-PHEMA compositehydrogel were fully swelled in PBS solution (pH=7.4) at 37°C for 5 days, followed by tensile strength test. As shown in Fig. 8, the Col-PHEMA composite hydrogel exhibited much higher tensile strength than pure PHEMA hydrogel. The tensile strength of Col-PHEMA composite hydrogel increases significantly with the increase of collagen concentration. It is interesting to note that the tensile strength of Col-PHEMA composite hydrogel prepared from 4.0 mg/mL collagen solution was significantly increased by 107% (from 20 kPa to 41.4 kPa). The remarkable improvement of mechanical property should be attributed to the fiber network of collagen and the new hydrogen bonds between collagen and PHEMA. Meanwhile, the self-assembly of collagen can also make the fiber network structure more stable, which is beneficial to the improvement of the tensile strength. Table 3. Pore parameters of Col-PHEMA composite hydrogels

Collagen concentrations

Pore Size Range (μm)

Local porosity (%) Pore Size ≥ 20μm

Local porosity (%) Pore Size ≤ 20μm

Total Porosity (%)

0 mg/mL

2.77-247.61

88.53

1.59

90.12±2.11

0.5 mg/mL

0.64-237.96

85.21

3.86

89.07±1.56

1 mg/mL

0.35-202.30

74.23

7.12

81.35±0.93

2 mg/mL

0.01-189.45

53.19

17.54

70.73±3.14

4 mg/mL

0.01-49.44

36.67

25.92

62.89±1.22

The pore parameters of Col-PHEMA composite hydrogels were shown in Table 3. It


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was observed that with increasing the collagen solution concentration, the percentage of local porosity with the pore size greater than 20 μm and total porosity decreases, while the percentage of the local porosity with the pore size less than 20 μm increases. Collagen fibers were adhered to the surface of holes and hole-joints of the porous PHEMA hydrogel and a fiber network was formed, as shown in SEM images in Fig. 4. The porosity of pure PHEMA hydrogel was 90.12±2.11%, while the porosity of Col-PHEMA composite hydrogels prepared in 0.5, 1.0, 2.0, 4.0 mg/mL collagen solution was 89.07±1.56%, 81.35±0.93%, 70.73±3.14%, and 62.89±1.22%, respectively. So the porosity of the composite hydrogel could be regulated by adjusting the concentration of collagen solution. Hydrogel with rational porous structure may provide sufficient space for cell attachment and proliferation, and the high porosity is also conducive to facilitate the diffusion and transportation of nutrients. Many researchers have shown that the suitable minimum pore size for cell growth was about 20 μm7,11. In this study, the porosity of Col-PHEMA composite hydrogel prepared in 0.5 mg/mL collagen solution is higher than those of other three. At this collagen concentration, the collagen is not enough to fully distribute on the surface of the holes and hole-joints of Col-PHEMA composite hydrogel. The Col-PHEMA composite hydrogel from 1.0 mg/mL collagen solution exposed much more collagen deposition and the distribution of collagen was more homogeneous, and its porosity reached 81.35±0.93%.



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Fig.9. Viability of cells in the leachates of porous scaffold (porous PHEMA hydrogel and Col-PHEMA composite hydrogel (1.0 mg/mL)) Table 4. RGR and cytotoxicity level of porous hydrogel (2 day, 4 day and 7 day)

2 day

4 day

7 day

Control

PHEMA

Col-PHEMA

RGR (%)

100

115.5

152.9

Cytotoxicity Level

0

0

0

RGR (%)

100

99.8

124.4

Cytotoxicity Level

0

I

0

RGR (%)

100

94.4

122.8

Cytotoxicity Level

0

I

0

Cell toxicity is one of the most important ways to evaluate the biocompatibility of biomaterials. Therefore, cell toxicity of Col-PHEMA composite hydrogel was employed to evaluate its possible application in cornea transplant scaffold. Fig. 9 showed the viability of cells in the leachates of porous scaffold (porous PHEMA hydrogel and Col-PHEMA composite hydrogel (1.0 mg/mL)). The RGR and cytotoxicity level of porous hydrogels (2 day, 4 day and 7 day) were shown in Table 4. Cell viability of mouse fibroblasts in leachates of porous composites hydrogel for 2, 4 and 7 days was analyzed using MTT assay. Low cytotoxicity is conducive for the growth and differentiation of fibroblasts cells on the hydrogel. In Table 4, the RGR of pure PHEMA composite hydrogel was greater than 100% and the cytotoxicity stage located at level 0 in the first 2 days. The RGR of Col-PHEMA composite hydrogel was higher than pure PHEMA hydrogel, indicating that the collagen fibers are beneficial to improve the cell viability. However, when the culture duration was extended to 4 days and 7 days, the RGR of pure PHEMA hydrogel reduced to 99.8% and 94.4%, and the cytotoxicity stages kept in the range of level I. The RGR of Col-PHEMA composite hydrogels was all greater than


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100%, and the cytotoxicity stage keep in level 0. Collagen is the most abundant extracellular matrix (ECM) constituent of nature with weak antigenicity, good biodegradability and excellent biocompatibility, which may attach to the surface of holes and hole-joints to be conducive to cell adhesion and proliferation. 4. Conclusion Porous PHEMA matrix with inter-connected porous microstructure was fabricated using a paraffin template method, which was then used as substrate to adhere collagen fibers to prepare porous Col-PHEMA composite hydrogel. The optimized pore size of composite is about 100-200 µm, and the collagen fibers were attached to the surface of holes and hole-joints to form a network of collagen fiber, which is conducive to cell adhesion and proliferation. Benefited from the novel structure and hybrid advantages, the Col-PHEMA composite hydrogel prepared from 1 mg/mL collagen solution behaved good hydrophilicity, proper water content and high porosity. Besides, the biocompatibility of porous PHEMA hydrogel is improved significantly by the collagen fibers. It is suggested that the interconnected porous Col-PHEMA composite hydrogel could be a promising candidate for artificial cornea skirt. Acknowledgements The financial support by the National Natural Science Foundation of China (Grant No. 51673177, 51373158) and the National Key Research and Development Project of China (Grant No. 2017YFB0308500) is gratefully acknowledged. References (1) Kong, B.; Mi, S. Electrospun Scaffolds for Corneal Tissue Engineering: A Review. Materials, 2016, 9(8), 614. (2) Meek, K. M.; Knupp, C. Corneal Structure and Transparency. Prog. Retin. Eye. Res. 2015, 49, 1-16. (3) Zeng, L.; Li, G.; Zeng, W. Application of Bio-artificial Cornea and Its Research Progress. Ann. Eye. Sci. 2017, 2(8). (4) Simpson, F. C.; Griffith, M. Regenerative Medicine in The Cornea. Curr. Ophthalmol. Rep. 2017, 5(3), 187-192. (5) Liu, Y.; Ren, L.; Yao, H. Collagen Films with Suitable Physical Properties and Biocompatibility for Corneal Tissue engineering Prepared by Ion Leaching Technique. Mater. Lett. 2012, 87, 1-4.



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Poly(2-hydroxyethyl methacrylate

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