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Study on a hydroxypropyl chitosan–gelatin based scaffold for corneal stroma tissue engineering
Applied Surface Science 255 (2009) 8701–8705
Contents lists available at ScienceDirect
Applied Surface Science journal homepage: www.elsevier.com/locate/apsusc
Short communication
Study on a hydroxypropyl chitosan–gelatin based scaffold for corneal stroma tissue engineering Shilu Wang a, Wanshun Liu a, Baoqin Han a,*, Lingling Yang b a b
College of Marine Life Sciences, Ocean University of China, Qingdao 266003, China Shandong Eye Institute, Qingdao 266071, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 26 November 2008 Received in revised form 18 April 2009 Accepted 19 April 2009 Available online 23 June 2009
Hydroxypropyl chitosan (HPCTS) was crosslinked with gelatin (GEL) and chondroitin sulfate (CS) by 1,4butanediol diglycidyl ether to synthesize a scaffold. In this study, this scaffold was tested in physical and biological characteristics as a bioactive corneal stroma surrogate. The results showed the scaffold exhibited 83–88% light transmission values at wavelengths of visible light. Besides that, the scaffold had 96% water content and allowed NaCl and glucose to permeate. Moreover, it was suitable for keratocytes growing on its surface. In the biological part, we compared the scaffold with CS-free ones to investigate the potential effect of CS and found out that CS notablely improved cell compatibility of the scaffold. ß 2009 Elsevier B.V. All rights reserved.
PACS: 68.37.Hk 78.40.Me 87.80.Rb 87.64.Tt Keywords: Corneal stroma Hydroxypropyl chitosan Gelatin Chondroitin sulfate Scaffold
1. Introduction In recent years, chitosan–gelatin scaffolds have been studied for applications in tissue engineering, for example, in cartilage and hepatic tissue engineering [1,2]. Chitosan has many excellent biological properties such as biocompatiblity, biodegradablity and antimicrobial activity for biomedical applications [3]. Corneal stroma counts for approximately 90% thickness of the whole cornea, and is mostly composed of regular spaced collagen fibers, between which the cells of corneal stroma (keratocytes) distribute orderly [4,5]. Gelatin is derived from partial degradation of collagen and has low antigenicity compared to its precursor [6]. Chondroitin sulfate is a mucopolysaccharide present in diverse forms of proteoglycans. There is a trace amount of chondroitin sulfate in human corneal stroma [7]. As a matter of fact, chondroitin sulfate is commonly added in cornea preservation medium and has an outstanding effect on corneal endothelial cell cultures [8,9].
* Corresponding author. Tel.: +86 532 82032105; fax: +86 532 82032105. E-mail address: [email protected] (B. Han). 0169-4332/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.apsusc.2009.04.206
In this study, a scaffold which was composed of crosslinked hydroxypropyl chitosan (HPCTS), gelatin (GEL) and chondroitin sulfate (CS) was inspected for its ability of mimicking corneal stroma. Chitosan becomes water-soluble only under diluted acid condition [10]. In order to combine its outstanding biological properties with improved water-solubility, a property required during this experiment, HPCTS as a water-soluble derivative of chitosan was chosen to be a component material of the scaffold [11]. As mentioned before, corneal stroma is abundant in collagen, so we used GEL as another bio-sourced material in the scaffold. We were interested in the potential effect of CS on keratocytes cultured in vitro. Therefore, in the biological properties part, we compared the HPCTS/GEL/CS with HPCTS/GEL scaffold on their abilities to support adhering and proliferating of cells. 2. Experimental 2.1. Materials preparation 4 wt.% HPCTS solution and 4 wt.% GEL solution were prepared by agitating for 4 h and 30 min, respectively, at 50 8C. 0.5 wt.% CS solution was prepared at room temperature. After that, 4 wt.% HPCTS, 4 wt.% GEL and 0.5 wt.% CS solutions in the ratio of 48:12:1
S. Wang et al. / Applied Surface Science 255 (2009) 8701–8705
8702
were mixed and adjusted to pH 9. And then, 0.5 wt.% 1,4butanediol diglycidyl ether was added into the mixed solution. This mixture was under 50 8C thermo-crosslinking for 12 h and 50 8C freeze–drying for another 12 h. Afterwards, the solid product was soaked in triply distilled water at room temperature for 2 h, and then dried at 50 8C for 24 h. 2.2. Physical properties 2.2.1. Optical property The optical transmittance spectra of scaffold (HPCTS/GEL/CS, immersed in D-Hanks solution, n = 3) was examined in the wavelength range from 250 nm to 800 nm by means of Ultraviolet Spectrometer (TU- 1 800S, PGeneral, China) at room temperature. 2.2.2. Moisture content Moisture content (MC) of the scaffold (HPCTS/GEL/CS, n = 5) was evaluated at 50 8C as all of the components were hightemperature intolerant. Weighing bottle mass (MW) was recorded after dried in ovens for 24 h, and then the wet scaffold was added into the weighing bottle. The mass of the weighing bottle and wet scaffold was recorded as ME. As they were dried to constant weight, we could then weighed the mass of the weighing bottle and dried scaffold (MD). The moisture content was calculated with the equation: MC = 1 (MD MW)/(ME MW). 2.2.3. Ion and glucose permeability Scaffold (HPCTS/GEL/CS), approximately 500 mm in thickness and 15 mm in diameter (Fig. 1), was used to measure the permeability. Sodium chloride solution (0.9 g NaCl, 100 ml, in triply distilled water) was at right side, and triply distilled water was at left side of the scaffold. The total test device was then assembled into 37 8C thermo-incubator. The conductivity of the left side liquid was recorded until it reached its equilibration. Glucose solution (0.1 g glucose, 100 ml, in triply distilled water) was prepared in the same way. Glucose contents of both sides were detected by DNS method at 540 nm. 2.3. Biological properties 2.3.1. L929 cells for testing in vitro cytotoxicity by MTT 200 ml (3 104 cells per ml) L929 cell suspension was added to each culture well of 96-well cell culture plates and then incubated in CO2 incubator (37 8C, 5% CO2) for 24 h. Scaffolds were transferred into DMEM with 10% FBS, equivalent of 3 cm2 total surface area (both sides combined) per 1 ml extracting medium,
Fig. 1. HPCTS/GEL/CS scaffold.
and then incubated for 24 h [12]. Afterwards, for experimental groups, extracting medium was added to the culture wells to replace primary medium. On the 2nd, 4th and 7th day respectively, MTT assay was performed to detect cytotoxicity of all groups by analyzing ultimate results of OD value. 2.3.2. SEM of rabbit keratocytes cultured on scaffolds Rabbit keratocytes were added into a 48-well cell culture plate, in which scaffolds 11 mm in diameter (HPCT S/GEL, HPCT S/GEL/ CS) had already been located. After cultured for 7 days, scaffolds were washed by D-Hanks solution for three times before ready for the SEM processing. Samples (HPCTS/GEL + Cells and HPCTS/GEL/ CS + Cells) were fixed by 2.5% glutaraldehyde in 0.1 M phosphate buffer for 1 h, dehydrated in ascending grades of alcohol, soaked in isoamyl acetate, CO2 critical point drying, sputtered coating with gold and then examined using a scanning electron microscope (KYKY-2800B, KYKY Technology Development Ltd., China) at an accelerating voltage of 25 kV. 2.3.3. Confocal microscope imaging of human keratocytes on scaffolds In this section, we compared HPCTS/GEL scaffold with HPCTS/ GEL/CS scaffold by means of laser confocal scanning microscope (C1 Plus, Nikon, Japan). Human keratocytes were labeled with 5,6carboxyfluorescein diacetate succinimidyl ester (CFSE) before seeded on scaffolds. Once CFSE enters cells, it will lose membrane permeability and obtain high fluorescence activity because CFSE will combine with cytoskeleton to form a fluorescent protein adduct. And this adduct will be allocated equally to two secondgeneration cells during proliferation. The labeled keratocytes were cultured as previously described (in SEM part) for 14 days. After that, we took out the scaffolds and performed PI staining. As is known, PI cannot permeate cell membrane, and so in this experiment, we used methanol to destruct membrane before staining. The scaffolds were examined by LCSM with excitation wavelengths at 408 nm, 488 nm and 544 nm, respectively. 2.3.4. LCSM image analysis We randomly selected six fixed-size parts of each image (Fig. 6B and E) and analyzed them using the software Image J 1.40 and SSPS 13.0. 3. Results and discussion 3.1. Physical properties results and comparisons Fig. 2 showed in the ultra-violet range of 250–350 nm that the scaffold possessed 55–76% light transmission values. In the wavelengths of visible light, the values were incrementally
Fig. 2. Optical property of the HPCT S/GEL/CS scaffolds.
S. Wang et al. / Applied Surface Science 255 (2009) 8701–8705
8703
Fig. 3. 0.9% sodium chloride solution was used for testing ion permeability of the HPCTS/GEL/CS scaffolds at 37 8C.
increased from 83% at 400 nm to 88% at 800 nm. Previous work of Rafat et al. [13] indicated in spectral regions (at 450, 500, 550, 600 and 650 nm), human cornea (from eye bank) light transmission values increased from 50% to 75%. The scaffold was obviously more transparent than human cornea according to the data. The moisture content was 0.95824 0.002351 (n = 5). As is known, human cornea has between 72% and 82% water content. This scaffold was remarkably higher in water content than human cornea. Fig. 3 showed that NaCl had permeated to the left side after 8 h, consequently the conductivity of it rose rapidly from 0.2 to 2090 ms/cm. After 96 h, the rate slowed down. The conductivity was 9470 ms/cm on the 9th day and 9500 ms/cm on the 10th day. The result indicated on the 10th day both sides reached their equilibrations. These results demonstrated the scaffold was NaCl permeable. As shown in Fig. 4, between 24 and 48 h, glucose went across the scaffold at a remarkable rate, and therefore the ratio declined from 19.6 to 4.3 accordingly. Afterwards, the rate slowed down. On the 10th day, the glucose contents ratio of the right side to the left side reached 1.05, close to 1. These results demonstrated the scaffold was glucose permeable. For comparison with published data, which reported human cornea glucose diffusivity was (2.6 0.33) 10 6 cm/s2[14], we analyzed raw data of the experiment as well (Table 1). Since there were 10 time intervals, we calculated the glucose diffusivity at each time respectively (Table 1). In accord with analysis of Fig. 4, results on 24 and 48 h exhibited significant difference (P < 0.05) with other results, so they were not suitable to be included in statistical result. Statistics analysis indicated that glucose diffusivity was (1.16 0.11) 10 6 cm/s2 (n = 8).
Fig. 4. 0.01% glucose solution was used for testing the main energy source—glucose permeability of the HPCTS/GEL/CS scaffolds at 37 8C.
3.2. Biological properties results and analysis Table 2 showed that both scaffolds (HPCTS/GEL, HPCTS/GEL/CS) extracts preformed no cytotoxicity. Furthermore, MTT test results of the 2nd day demonstrated there were significant differences between scaffold groups and control group (P < 0.05), which meant both scaffolds had positive impacts for the cells proliferation. On the 4th and 7th day, scaffold groups had no significant differences with control group. The reason is connected to the fact that the rapid growing cells fully covered the well, so there was no more room for cells proliferation. The total results approved the principle of adopting biocompatibility materials, and consequently the scaffolds were suitable for seeding cell on. Fig. 5 showed that the scaffolds (HPCTS/GEL, HPCTS/GEL/CS) were irregular superpositions (Fig. 5), which meant they had rough surfaces to support the cells growth. Simultaneously, it suggested the scaffolds had a connected structure and this point was important to evaluate the scaffolds’ potential to sustain cells growing even inside themselves. Rabbit keratocytes seemed to favor the edged area of the surfaces (Fig. 5a and c), consistent with the theory that roughness helps cells to adhere. As it is known, lamellipodia symbolizes cell attachment. We could observe keratocytes lamellipodia from both scaffolds (Fig. 5b and d), and therefore we could conclude that they both allowed adherence of keratocytes. Furthermore, considering secreting extracellular matrix (ECM) is a main function of activated keratocytes, Fig. 5b and d also indicated the scaffold with CS was much more suitable for activated cells growing on its surface,
Table 1 Glucose diffusivity results. Time (h) Glucose concentration (mg/ml) Glucose diffusivity (10 6 cm/s2)
24 0.745 2.24
48 0.676 1.57
72 0.614 1.37
96 0.578 1.20
120 0.533 1.15
144 0.505 1.07
168 0.433 1.23
192 0.426 1.10
216 0.401 1.09
240 0.388 1.03
Note: (1) glucose diffusivity = 0.566(1/C-1)/T. (2) C = glucose concentration. (3) T = time.
Table 2 OD values, relative growth rates (RGR) and cytotoxicity grades of scaffolds. Scaffold
HPCTS/GEL HPCTS/GEL/CS Control
2d
4d
7d
OD (means SD) (n = 6)
RPR (%)
Grade
OD (means SD) (n = 6)
RPR (%)
Grade
OD (means SD) (n = 6)
RPR (%)
Grade
0.2233 0.01751 0.2383 0.00983 0.2067 0.01211
108.0 115.3 –
0 0 –
0.2800 0.01673 0.2917 0.1472 0.2817 0.00408
99.4 103.5 –
1 0 –
0.2850 0.00548 0.2833 0.01506 0.2850 0.00548
100.0 99.4 –
0 1 –
8704
S. Wang et al. / Applied Surface Science 255 (2009) 8701–8705
Fig. 5. SEM images of (a and b) the HPCTS/GEL scaffold + rabbit keratocytes (200, 2000); (c and d) the HPCTS/GEL/CS scaffold + rabbit keratocytes (500, 2000).
Fig. 6. LCSM images of (A) keratocytes cytoplasm (CFSE, green), cytoblast (PI, red) and the HPCTS/GEL scaffold (blue); (B) cytoblast and the HPCTS/GEL; (C) cytoplasm and the HPCTS/GEL; (D) cytoplasm, cytoblast and the HPCTS/GEL/CS; (E) cytoblast and the HPCTS/GEL/CS; (F) cytoplasm and the HPCTS/GEL/CS. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
S. Wang et al. / Applied Surface Science 255 (2009) 8701–8705 Table 3 Random sampling results of scaffolds. Scaffold
HPCTS/GEL HPCTS/GEL/CS
Sample
Sample
Sample
Sample
Sample
Sample
1
2
3
4
5
6
16 41
17 26
21 38
22 44
28 34
17 30
as the cells apparently had secreted more ECM than cells on the CS-free one. Fig. 6 showed that the scaffolds launched a bright blue fluorescence, that because GEL contained aromatic amino acids. These amino acids can form fluorescence in near-ultraviolet region (408 nm in this experiment). The surface of the scaffolds consisted with that of the SEM images, rough and superposed. Keratocytes mostly located on the protruding part of scaffolds. The results of random samples of the images (Fig. 6B and E) were presented in Table 3. Independent samples T test analysis demonstrated there was a significant difference between the HPCTS/GEL group and HPCTS/GEL/CS group (t = 4.593, df = 10, P < 0.05). HPCTS/GEL/CS group had significantly more cells than HPCTS/GEL group. 4. Conclusion We can draw three conclusions. Firstly, the HPCTS/GEL/CS scaffold was transparent, with high water content, NaCl and glucose permeable. Secondly, the bio-sourced components and the
8705
scaffold’s rough surface help keratocytes to adhere and proliferate. Thirdly, CS, even just a small quantity was added, significantly improves cell compatibility of the scaffold. Acknowledgement This study was supported by grant from the National High Technology Research and Development Program of China (no. 2006AA02A1 32 and no. 2007AA09 1603). References [1] D.J. Griffon, M.R. Sedighi, D.V. Schaeffer, J.A. Eurell, A.L. Johnson, Acta Biomater. 2 (2006) 313. [2] C.R. Wittmer, J.A. Phelps, C.M. Lepus, W.M. Saltzman, M.J. Harding, P.R. Van Tassel, Biomaterials 29 (2008) 4082. [3] K. Kurita, Mar. Biotechnol. 8 (2006) 203. [4] L. Robert, J.M. Legeais, A.M. Robert, G. Renard, Pathol. Biol. 49 (2001) 353. [5] M.J. Doughty, W. Seabert, J.P.G. Bergmanson, Y. Blocker, Tissue Cell. 33 (4) (2001) 408. [6] Y.S. Choi, S.R. Hong, Y.M. Lee, K.W. Song, M.H. Park, Y.S. Nam, Biomaterials 20 (1999) 409. [7] R. Praus, I. Brettschneider, Ophthalmic Res. 7 (1975) 542. [8] H.E. Kaufman, E.D. Varnell, S. Kaufman, Am. J. Ophthalmol. 98 (1984) 112. [9] B.Y. Yue, J. Sugar, J.E. Gilboy, J.L. Elvart, Invest Ophthalmol. Vis. Sci. 30 (1989) 248. [10] E. Khor, L.Y. Lim, Biomaterials 24 (2003) 2339. [11] Y.F. Peng, B.Q. Han, W.S. Liu, X.J. Xu, Carbohydr. Res. 340 (2005) 1846. [12] The 28th U.S. Pharmacopeia. (87) Biological Reactivity Tests, In Vitro (http:// www.newdruginfo.com/). [13] M. Rafat, F. Li, P. Fagerholm, N.S. Lagali, M.A. Watsky, R. Munger, T. Matsuura, M. Griffith, Biomaterials 29 (2008) 3960. [14] B.E. McCarey, F.H. Schmidt, Curr. Eye Res. 11 (1990) 1025.
Contents lists available at ScienceDirect
Applied Surface Science journal homepage: www.elsevier.com/locate/apsusc
Short communication
Study on a hydroxypropyl chitosan–gelatin based scaffold for corneal stroma tissue engineering Shilu Wang a, Wanshun Liu a, Baoqin Han a,*, Lingling Yang b a b
College of Marine Life Sciences, Ocean University of China, Qingdao 266003, China Shandong Eye Institute, Qingdao 266071, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 26 November 2008 Received in revised form 18 April 2009 Accepted 19 April 2009 Available online 23 June 2009
Hydroxypropyl chitosan (HPCTS) was crosslinked with gelatin (GEL) and chondroitin sulfate (CS) by 1,4butanediol diglycidyl ether to synthesize a scaffold. In this study, this scaffold was tested in physical and biological characteristics as a bioactive corneal stroma surrogate. The results showed the scaffold exhibited 83–88% light transmission values at wavelengths of visible light. Besides that, the scaffold had 96% water content and allowed NaCl and glucose to permeate. Moreover, it was suitable for keratocytes growing on its surface. In the biological part, we compared the scaffold with CS-free ones to investigate the potential effect of CS and found out that CS notablely improved cell compatibility of the scaffold. ß 2009 Elsevier B.V. All rights reserved.
PACS: 68.37.Hk 78.40.Me 87.80.Rb 87.64.Tt Keywords: Corneal stroma Hydroxypropyl chitosan Gelatin Chondroitin sulfate Scaffold
1. Introduction In recent years, chitosan–gelatin scaffolds have been studied for applications in tissue engineering, for example, in cartilage and hepatic tissue engineering [1,2]. Chitosan has many excellent biological properties such as biocompatiblity, biodegradablity and antimicrobial activity for biomedical applications [3]. Corneal stroma counts for approximately 90% thickness of the whole cornea, and is mostly composed of regular spaced collagen fibers, between which the cells of corneal stroma (keratocytes) distribute orderly [4,5]. Gelatin is derived from partial degradation of collagen and has low antigenicity compared to its precursor [6]. Chondroitin sulfate is a mucopolysaccharide present in diverse forms of proteoglycans. There is a trace amount of chondroitin sulfate in human corneal stroma [7]. As a matter of fact, chondroitin sulfate is commonly added in cornea preservation medium and has an outstanding effect on corneal endothelial cell cultures [8,9].
* Corresponding author. Tel.: +86 532 82032105; fax: +86 532 82032105. E-mail address: [email protected] (B. Han). 0169-4332/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.apsusc.2009.04.206
In this study, a scaffold which was composed of crosslinked hydroxypropyl chitosan (HPCTS), gelatin (GEL) and chondroitin sulfate (CS) was inspected for its ability of mimicking corneal stroma. Chitosan becomes water-soluble only under diluted acid condition [10]. In order to combine its outstanding biological properties with improved water-solubility, a property required during this experiment, HPCTS as a water-soluble derivative of chitosan was chosen to be a component material of the scaffold [11]. As mentioned before, corneal stroma is abundant in collagen, so we used GEL as another bio-sourced material in the scaffold. We were interested in the potential effect of CS on keratocytes cultured in vitro. Therefore, in the biological properties part, we compared the HPCTS/GEL/CS with HPCTS/GEL scaffold on their abilities to support adhering and proliferating of cells. 2. Experimental 2.1. Materials preparation 4 wt.% HPCTS solution and 4 wt.% GEL solution were prepared by agitating for 4 h and 30 min, respectively, at 50 8C. 0.5 wt.% CS solution was prepared at room temperature. After that, 4 wt.% HPCTS, 4 wt.% GEL and 0.5 wt.% CS solutions in the ratio of 48:12:1
S. Wang et al. / Applied Surface Science 255 (2009) 8701–8705
8702
were mixed and adjusted to pH 9. And then, 0.5 wt.% 1,4butanediol diglycidyl ether was added into the mixed solution. This mixture was under 50 8C thermo-crosslinking for 12 h and 50 8C freeze–drying for another 12 h. Afterwards, the solid product was soaked in triply distilled water at room temperature for 2 h, and then dried at 50 8C for 24 h. 2.2. Physical properties 2.2.1. Optical property The optical transmittance spectra of scaffold (HPCTS/GEL/CS, immersed in D-Hanks solution, n = 3) was examined in the wavelength range from 250 nm to 800 nm by means of Ultraviolet Spectrometer (TU- 1 800S, PGeneral, China) at room temperature. 2.2.2. Moisture content Moisture content (MC) of the scaffold (HPCTS/GEL/CS, n = 5) was evaluated at 50 8C as all of the components were hightemperature intolerant. Weighing bottle mass (MW) was recorded after dried in ovens for 24 h, and then the wet scaffold was added into the weighing bottle. The mass of the weighing bottle and wet scaffold was recorded as ME. As they were dried to constant weight, we could then weighed the mass of the weighing bottle and dried scaffold (MD). The moisture content was calculated with the equation: MC = 1 (MD MW)/(ME MW). 2.2.3. Ion and glucose permeability Scaffold (HPCTS/GEL/CS), approximately 500 mm in thickness and 15 mm in diameter (Fig. 1), was used to measure the permeability. Sodium chloride solution (0.9 g NaCl, 100 ml, in triply distilled water) was at right side, and triply distilled water was at left side of the scaffold. The total test device was then assembled into 37 8C thermo-incubator. The conductivity of the left side liquid was recorded until it reached its equilibration. Glucose solution (0.1 g glucose, 100 ml, in triply distilled water) was prepared in the same way. Glucose contents of both sides were detected by DNS method at 540 nm. 2.3. Biological properties 2.3.1. L929 cells for testing in vitro cytotoxicity by MTT 200 ml (3 104 cells per ml) L929 cell suspension was added to each culture well of 96-well cell culture plates and then incubated in CO2 incubator (37 8C, 5% CO2) for 24 h. Scaffolds were transferred into DMEM with 10% FBS, equivalent of 3 cm2 total surface area (both sides combined) per 1 ml extracting medium,
Fig. 1. HPCTS/GEL/CS scaffold.
and then incubated for 24 h [12]. Afterwards, for experimental groups, extracting medium was added to the culture wells to replace primary medium. On the 2nd, 4th and 7th day respectively, MTT assay was performed to detect cytotoxicity of all groups by analyzing ultimate results of OD value. 2.3.2. SEM of rabbit keratocytes cultured on scaffolds Rabbit keratocytes were added into a 48-well cell culture plate, in which scaffolds 11 mm in diameter (HPCT S/GEL, HPCT S/GEL/ CS) had already been located. After cultured for 7 days, scaffolds were washed by D-Hanks solution for three times before ready for the SEM processing. Samples (HPCTS/GEL + Cells and HPCTS/GEL/ CS + Cells) were fixed by 2.5% glutaraldehyde in 0.1 M phosphate buffer for 1 h, dehydrated in ascending grades of alcohol, soaked in isoamyl acetate, CO2 critical point drying, sputtered coating with gold and then examined using a scanning electron microscope (KYKY-2800B, KYKY Technology Development Ltd., China) at an accelerating voltage of 25 kV. 2.3.3. Confocal microscope imaging of human keratocytes on scaffolds In this section, we compared HPCTS/GEL scaffold with HPCTS/ GEL/CS scaffold by means of laser confocal scanning microscope (C1 Plus, Nikon, Japan). Human keratocytes were labeled with 5,6carboxyfluorescein diacetate succinimidyl ester (CFSE) before seeded on scaffolds. Once CFSE enters cells, it will lose membrane permeability and obtain high fluorescence activity because CFSE will combine with cytoskeleton to form a fluorescent protein adduct. And this adduct will be allocated equally to two secondgeneration cells during proliferation. The labeled keratocytes were cultured as previously described (in SEM part) for 14 days. After that, we took out the scaffolds and performed PI staining. As is known, PI cannot permeate cell membrane, and so in this experiment, we used methanol to destruct membrane before staining. The scaffolds were examined by LCSM with excitation wavelengths at 408 nm, 488 nm and 544 nm, respectively. 2.3.4. LCSM image analysis We randomly selected six fixed-size parts of each image (Fig. 6B and E) and analyzed them using the software Image J 1.40 and SSPS 13.0. 3. Results and discussion 3.1. Physical properties results and comparisons Fig. 2 showed in the ultra-violet range of 250–350 nm that the scaffold possessed 55–76% light transmission values. In the wavelengths of visible light, the values were incrementally
Fig. 2. Optical property of the HPCT S/GEL/CS scaffolds.
S. Wang et al. / Applied Surface Science 255 (2009) 8701–8705
8703
Fig. 3. 0.9% sodium chloride solution was used for testing ion permeability of the HPCTS/GEL/CS scaffolds at 37 8C.
increased from 83% at 400 nm to 88% at 800 nm. Previous work of Rafat et al. [13] indicated in spectral regions (at 450, 500, 550, 600 and 650 nm), human cornea (from eye bank) light transmission values increased from 50% to 75%. The scaffold was obviously more transparent than human cornea according to the data. The moisture content was 0.95824 0.002351 (n = 5). As is known, human cornea has between 72% and 82% water content. This scaffold was remarkably higher in water content than human cornea. Fig. 3 showed that NaCl had permeated to the left side after 8 h, consequently the conductivity of it rose rapidly from 0.2 to 2090 ms/cm. After 96 h, the rate slowed down. The conductivity was 9470 ms/cm on the 9th day and 9500 ms/cm on the 10th day. The result indicated on the 10th day both sides reached their equilibrations. These results demonstrated the scaffold was NaCl permeable. As shown in Fig. 4, between 24 and 48 h, glucose went across the scaffold at a remarkable rate, and therefore the ratio declined from 19.6 to 4.3 accordingly. Afterwards, the rate slowed down. On the 10th day, the glucose contents ratio of the right side to the left side reached 1.05, close to 1. These results demonstrated the scaffold was glucose permeable. For comparison with published data, which reported human cornea glucose diffusivity was (2.6 0.33) 10 6 cm/s2[14], we analyzed raw data of the experiment as well (Table 1). Since there were 10 time intervals, we calculated the glucose diffusivity at each time respectively (Table 1). In accord with analysis of Fig. 4, results on 24 and 48 h exhibited significant difference (P < 0.05) with other results, so they were not suitable to be included in statistical result. Statistics analysis indicated that glucose diffusivity was (1.16 0.11) 10 6 cm/s2 (n = 8).
Fig. 4. 0.01% glucose solution was used for testing the main energy source—glucose permeability of the HPCTS/GEL/CS scaffolds at 37 8C.
3.2. Biological properties results and analysis Table 2 showed that both scaffolds (HPCTS/GEL, HPCTS/GEL/CS) extracts preformed no cytotoxicity. Furthermore, MTT test results of the 2nd day demonstrated there were significant differences between scaffold groups and control group (P < 0.05), which meant both scaffolds had positive impacts for the cells proliferation. On the 4th and 7th day, scaffold groups had no significant differences with control group. The reason is connected to the fact that the rapid growing cells fully covered the well, so there was no more room for cells proliferation. The total results approved the principle of adopting biocompatibility materials, and consequently the scaffolds were suitable for seeding cell on. Fig. 5 showed that the scaffolds (HPCTS/GEL, HPCTS/GEL/CS) were irregular superpositions (Fig. 5), which meant they had rough surfaces to support the cells growth. Simultaneously, it suggested the scaffolds had a connected structure and this point was important to evaluate the scaffolds’ potential to sustain cells growing even inside themselves. Rabbit keratocytes seemed to favor the edged area of the surfaces (Fig. 5a and c), consistent with the theory that roughness helps cells to adhere. As it is known, lamellipodia symbolizes cell attachment. We could observe keratocytes lamellipodia from both scaffolds (Fig. 5b and d), and therefore we could conclude that they both allowed adherence of keratocytes. Furthermore, considering secreting extracellular matrix (ECM) is a main function of activated keratocytes, Fig. 5b and d also indicated the scaffold with CS was much more suitable for activated cells growing on its surface,
Table 1 Glucose diffusivity results. Time (h) Glucose concentration (mg/ml) Glucose diffusivity (10 6 cm/s2)
24 0.745 2.24
48 0.676 1.57
72 0.614 1.37
96 0.578 1.20
120 0.533 1.15
144 0.505 1.07
168 0.433 1.23
192 0.426 1.10
216 0.401 1.09
240 0.388 1.03
Note: (1) glucose diffusivity = 0.566(1/C-1)/T. (2) C = glucose concentration. (3) T = time.
Table 2 OD values, relative growth rates (RGR) and cytotoxicity grades of scaffolds. Scaffold
HPCTS/GEL HPCTS/GEL/CS Control
2d
4d
7d
OD (means SD) (n = 6)
RPR (%)
Grade
OD (means SD) (n = 6)
RPR (%)
Grade
OD (means SD) (n = 6)
RPR (%)
Grade
0.2233 0.01751 0.2383 0.00983 0.2067 0.01211
108.0 115.3 –
0 0 –
0.2800 0.01673 0.2917 0.1472 0.2817 0.00408
99.4 103.5 –
1 0 –
0.2850 0.00548 0.2833 0.01506 0.2850 0.00548
100.0 99.4 –
0 1 –
8704
S. Wang et al. / Applied Surface Science 255 (2009) 8701–8705
Fig. 5. SEM images of (a and b) the HPCTS/GEL scaffold + rabbit keratocytes (200, 2000); (c and d) the HPCTS/GEL/CS scaffold + rabbit keratocytes (500, 2000).
Fig. 6. LCSM images of (A) keratocytes cytoplasm (CFSE, green), cytoblast (PI, red) and the HPCTS/GEL scaffold (blue); (B) cytoblast and the HPCTS/GEL; (C) cytoplasm and the HPCTS/GEL; (D) cytoplasm, cytoblast and the HPCTS/GEL/CS; (E) cytoblast and the HPCTS/GEL/CS; (F) cytoplasm and the HPCTS/GEL/CS. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
S. Wang et al. / Applied Surface Science 255 (2009) 8701–8705 Table 3 Random sampling results of scaffolds. Scaffold
HPCTS/GEL HPCTS/GEL/CS
Sample
Sample
Sample
Sample
Sample
Sample
1
2
3
4
5
6
16 41
17 26
21 38
22 44
28 34
17 30
as the cells apparently had secreted more ECM than cells on the CS-free one. Fig. 6 showed that the scaffolds launched a bright blue fluorescence, that because GEL contained aromatic amino acids. These amino acids can form fluorescence in near-ultraviolet region (408 nm in this experiment). The surface of the scaffolds consisted with that of the SEM images, rough and superposed. Keratocytes mostly located on the protruding part of scaffolds. The results of random samples of the images (Fig. 6B and E) were presented in Table 3. Independent samples T test analysis demonstrated there was a significant difference between the HPCTS/GEL group and HPCTS/GEL/CS group (t = 4.593, df = 10, P < 0.05). HPCTS/GEL/CS group had significantly more cells than HPCTS/GEL group. 4. Conclusion We can draw three conclusions. Firstly, the HPCTS/GEL/CS scaffold was transparent, with high water content, NaCl and glucose permeable. Secondly, the bio-sourced components and the
8705
scaffold’s rough surface help keratocytes to adhere and proliferate. Thirdly, CS, even just a small quantity was added, significantly improves cell compatibility of the scaffold. Acknowledgement This study was supported by grant from the National High Technology Research and Development Program of China (no. 2006AA02A1 32 and no. 2007AA09 1603). References [1] D.J. Griffon, M.R. Sedighi, D.V. Schaeffer, J.A. Eurell, A.L. Johnson, Acta Biomater. 2 (2006) 313. [2] C.R. Wittmer, J.A. Phelps, C.M. Lepus, W.M. Saltzman, M.J. Harding, P.R. Van Tassel, Biomaterials 29 (2008) 4082. [3] K. Kurita, Mar. Biotechnol. 8 (2006) 203. [4] L. Robert, J.M. Legeais, A.M. Robert, G. Renard, Pathol. Biol. 49 (2001) 353. [5] M.J. Doughty, W. Seabert, J.P.G. Bergmanson, Y. Blocker, Tissue Cell. 33 (4) (2001) 408. [6] Y.S. Choi, S.R. Hong, Y.M. Lee, K.W. Song, M.H. Park, Y.S. Nam, Biomaterials 20 (1999) 409. [7] R. Praus, I. Brettschneider, Ophthalmic Res. 7 (1975) 542. [8] H.E. Kaufman, E.D. Varnell, S. Kaufman, Am. J. Ophthalmol. 98 (1984) 112. [9] B.Y. Yue, J. Sugar, J.E. Gilboy, J.L. Elvart, Invest Ophthalmol. Vis. Sci. 30 (1989) 248. [10] E. Khor, L.Y. Lim, Biomaterials 24 (2003) 2339. [11] Y.F. Peng, B.Q. Han, W.S. Liu, X.J. Xu, Carbohydr. Res. 340 (2005) 1846. [12] The 28th U.S. Pharmacopeia. (87) Biological Reactivity Tests, In Vitro (http:// www.newdruginfo.com/). [13] M. Rafat, F. Li, P. Fagerholm, N.S. Lagali, M.A. Watsky, R. Munger, T. Matsuura, M. Griffith, Biomaterials 29 (2008) 3960. [14] B.E. McCarey, F.H. Schmidt, Curr. Eye Res. 11 (1990) 1025.