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chitosan porous scaffold for skin tissue engineering
Materials Science and Engineering C 32 (2012) 2361–2366
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Facile fabrication of the glutaraldehyde cross-linked collagen/chitosan porous scaffold for skin tissue engineering Yunyun Liu, Lie Ma ⁎, Changyou Gao MOE Key Laboratory of Macromolecular Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China
a r t i c l e
i n f o
Article history: Received 11 January 2012 Received in revised form 20 June 2012 Accepted 4 July 2012 Available online 10 July 2012 Keywords: Collagen Chitosan Scaffold Cross-linking Tissue engineering
a b s t r a c t Porous scaffold is one of the key factors in skin tissue engineering. In this study, a facile method was developed to prepare the glutaraldehyde (GA) cross-linked collagen/chitosan porous scaffold (S2). The properties of S2 were compared with the scaffolds prepared by the traditional method (S1). Compared to the rough surface and collapsed inner structure of S1, S2 showed a smooth surface and controlled size. After treated by GA with same concentration, S1 and S2 showed the similar swelling ratios, which are big enough to ensure the nutrient supply in the early stage of wound healing. The effects of the fabrication methods as well as the GA concentration on the cross-linking degree and in vitro degradation degree of the scaffolds were studied. It was found that the cross-linking degree of S2-0.25% was much higher than that of S1. Investigation of the tensile and compression properties of the scaffolds found that the mechanical property of S2-0.04% is closest to that of S1. High performance liquid chromatography (HPLC) was applied to determine the residual GA. The results proved that, compared to water rinse, oven drying is a feasible and effective method to remove the residual GA. Finally, the cytocompatibility of S2 was evaluated by in vitro culture of fibroblasts. The results of cell morphology and cell viability proved that S2-0.04% could retain the original good cytocompatibility of S1 to accelerate cell infiltration and proliferation effectively. All these results indicate that it is a feasible method to prepare the GA cross-linked collagen/chitosan scaffold. © 2012 Elsevier B.V. All rights reserved.
1. Introduction As the largest and highly complex organ of human body, skin, which is mainly composed of epidermis and dermis, plays a crucial role in thermoregulation, microbial defense and sensing [1]. However, because of trauma, burn and chronic disease, skin lose has become one of the most serious problems in clinic and millions of people are suffering from skin lose annually. Many treatments including skin transplantation, reconstructive surgery and wound dressing are applied for skin repair [2]. However, given the problems such as donor limitation and immune response [3–7], none of these approaches is ideal to fully accomplish the purpose of skin repair. Recently, the advances of tissue engineering and regenerative medicine make it possible and bring a bright future for clinicians to restore skin defects [8]. In the past decades, many kinds of skin substitutes such as Integra, Apligraf and Dermagraf, have been developed and applied for the treatment of full thickness skin defects in hospital [9–11]. However, many problems such as the limited effect, lack of appendixes and scarring are still the main hurdles to realize the full repair of the damaged skin. As one of the crucial factors determining the properties of the skin substitutes, porous scaffold offers an extracellular matrix analog which functions as the artificial dermis template for host cell ⁎ Corresponding author. Tel./fax: +86 571 87951108. E-mail address: [email protected] (L. Ma). 0928-4931/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2012.07.008
infiltration, proliferation and differentiation. Nowadays, many kinds of materials obtained from natural feedstocks such as collagen, chitosan and hyaluronic acid, or from synthetic feedstocks such as poly (lactic-co-glycolic acid) (PLGA), poly (glycolic acid) (PGA), are used in the scaffold fabrication [12–15]. Among them, collagen and chitosan are widely used as the main materials for tissue engineering scaffold. Collagen is one of the major constituents of extracellular matrix and is well known for the low antigenicity, excellent biocompatibility and biodegradability. In the development or healing of tissues and organs, collagen is involved in controlling the cell behaviors such as adhesion, proliferation and differentiation and plays an important role in the formation of extracellular matrix. Chitosan, an amino polysaccharide (poly-1,4-D-glucoamine) derived from chitin by deacetylation, has been widely used in a variety of biomedical fields such as wound dressings and drug delivery systems because of its good biocompatibility and biodegradability [16–19]. Furthermore, it was reported that chitosan can function as a bridge to increase the cross-linking efficiency of glutaraldehyde in the collagen/chitosan scaffolds because of its large number of amino groups in molecular chain [20]. Therefore, the scaffold fabricated by collagen and chitosan has been widely used in the repair of many kinds of tissue and organ such as vessel, skin, tendon and cartilage. In our previous work, a bilayer dermal equivalent consisting of a collagen/chitosan porous scaffold and silicone membrane was reported [21]. In vivo animal tests proved that the collagen/chitosan scaffold
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has no obvious cytotoxicity and has the property of reconstructing the damaged dermis within 4 weeks [22,23]. However, the previous fabricating method is quite complicated, which consists of many steps such as freeze-drying, cross-linking and refreeze-drying. Mainly due to the complicated process, problems such as surface collapse, crack formation and size deformation frequently occurred. To overcome the problems of previous method and enhance the quality of collagen/chitosan scaffolds, a facile method to fabricate the GA cross-linked collagen/chitosan scaffold was reported in this study. The effect of the different fabricating methods as well as the GA concentration on the macro and microstructure, cross-linking degree and degradation behavior, and the mechanical properties are investigated, and then the influence on the residual GA and the corresponding cytotoxicity are evaluated.
The mean pore size of the scaffolds was determined by analysis of the corresponding SEM images. The porosity of each kind of scaffold was measured by immersing the scaffolds (initial weight, W0) into ethanol at room temperature for 24 h (wet weight, W) and calculated by the formulas below.
2. Materials and methods
The swelling ratios of scaffolds were measured as the previous report [21]. Briefly, the scaffold was incubated in Milli-Q water at 37 °C and then the wet weight (W) was measured at different time points. The swelling ratio is defined as the ratio of weight increase (W − W0) to the initial weight (W0). Each value was averaged from three parallel measurements and expressed as mean ± standard deviation (SD).
2.1. Materials Collagen type Ι was isolated from fresh bovine tendon with the method of trypsin digestion and acetic acid dissolution as described previously [20]. Chitosan (Mη: 5×105, 85% deacetylation degree) was purchased from Jinan Haidebei Marine Bioengineering CO., LTD. Glutaraldehyde (GA, 25% water solution) was purchased from Sinopharm Chemical Reagent Co., Ltd. All other reagents and solvents were of analytical grade and used as received. 2.2. Preparation of the GA cross-linked collagen/chitosan porous scaffolds The GA cross-linked collagen/chitosan porous scaffolds were fabricated by two routes. The first route has been described in previous studies [20]. Briefly, collagen and chitosan were dissolved in 0.5 M acetic acid solution respectively, and then were homogenized to form a 0.5% (w/v) collagen/chitosan blend in a mass ratio of 9:1. The collagen/ chitosan blend was injected into a mold and freeze-dried to form the uncross-linked scaffold. Then the scaffold was rehydrated and further treated with 0.25% (w/v) GA at 37 °C for 4 h. After rinsed with Milli-Q water for six times to remove the excess GA, the scaffold was freeze-dried again to obtain the GA cross-linked collagen/chitosan scaffold (S1). In this study, a facile route was adopted to fabricate the GA cross-linked collagen/chitosan scaffold. The collagen/chitosan blend was prepared as the same way of S1. Then, the blend was injected into a container, and 2.5% GA solution were added to ensure the final concentration of GA in a setting value. The volume of the added 2.5% GA can be calculated from the below formula:
Porosity ð%Þ ¼
ðW−W0 Þρ1 100% ρ1 W þ ðρ2 −ρ1 ÞW0
where ρ1 and ρ2 represent density of collagen (1.21 g/ml) and ethanol (0.79 g/ml) respectively. 2.4. Swelling ratio
2.5. Cross-linking degree and in vitro degradation Since amino groups are consumed with the GA cross-linking treatment, the cross-linking degree of S1 and S2 can be defined as the amino group ratio of the GA treated scaffold to the uncross-linked one. Briefly, a weighed amount of scaffold (11 mg) was incubated in 1.0 ml 4% (w/v) NaHCO3 solution and 1.0 ml 0.5% (w/v) 2, 4, 6-trinitrobenzene sulfonic acid (TNBS) at 40 °C. After 4 h, 3.0 ml 6 M HCl was added and the reaction was carried out at 120 °C for 1 h. The resulting solution was diluted with 5.0 ml Milli-Q water and then extracted by 20 ml ethyl ether. Decanted 5.0 ml solution from aqueous phase and heated for 15 min at 37 °C. After cooled to room temperature, the solution was diluted again with 15 ml water and the absorbance was measured at 346 nm by UV–vis (Shimadzu, UV-2550) [24]. In vitro biodegradation test of S1 and S2 was performed by collagenase digestion as the previous studies [20]. The specimen was immersed in phosphate buffered saline (PBS, pH 7.4) containing 0.1 mg/ml (265 u/mg) collagenase (type I, Sigma) at 37 °C for 24 h. Following centrifugation at 1500 rpm for 10 min, the clear supernatant was hydrolyzed with 6 M HCl at 120 °C for 12 h. The content of hydroxyproline released from scaffold was measured with UV–vis. The biodegradation degree is defined as a percentage of the released hydroxyproline of the specimen to the uncross-linked one (%). 2.6. Mechanical performance
2:5% VGA ¼ C ðVGA þ Vblend Þ where VGA is the volume of the added 2.5% GA, Vblend is the volume of collagen/chitosan blend, and C is the final GA concentration in the blend. After stirred at 37 °C for 4 h, the collagen/chitosan blend was injected into a mold and freeze-dried. To remove the excess GA, the freeze-dried scaffold was treated under vacuum condition for 5 h at 37 °C to form the GA cross-linked collagen/chitosan porous scaffold (S2).
The mechanical performances of the scaffolds in both dry and wet conditions were tested with Instron Materials Test System (Instron 5543A) at room temperature. For the tensile test, the rectangular specimens (60 mm× 20 mm, with the thickness of 2 mm) were used. The distance of two air clamps was 20 mm, and the speed of crosshead was set as 6 mm/min. The round specimens with diameter of 15 mm and the thickness of 10 mm were applied in the compressive test. The compressive velocity was set as 2 mm/min. The wet samples were prepared by immersing in PBS at 37 °C for 24 h. 2.7. Analysis of residual GA
2.3. Macroscopic shape and microstructure observation Macroscopic images of S1 and S2 were taken by a digital camera (Canon, A480). The corresponding surface and cross section microstructure were observed by scanning electron microscopy (SEM, FEI SIRION-100) with an accelerating voltage of 25 kV after the samples were sputter-coated with a thin gold layer.
To detect the residual GA in scaffolds, S1 and S2 were immersed in 100 ml Milli-Q water at 37 °C for 96 h, and then the supernatants were analyzed by high performance liquid chromatography (HPLC, Agilent 1100 Series) with 0.25% (w/v) GA standard sample as control. Column temperature of HPLC was maintained at 30 °C. The volume of injected sample was 20 μl with acetonitrile/water (60:40) as the mobile
Y. Liu et al. / Materials Science and Engineering C 32 (2012) 2361–2366
phase and the flow rate was set as 0.14 ml/min. UV detection wavelength was 235 nm. 2.8. Cytotoxicity evaluation The cytotoxicity of scaffolds was evaluated by CLSM observation and cell viability test. Before cell culture, scaffolds were immersed in 75% ethanol for 12 h for sterilization, followed by solvent exchange with PBS for 6 times. Then the sterilized scaffold was placed on 48-well polystyrene plate and seeded with 100 μl human dermal fibroblast suspension at a density of 3 × 106 cells/ml. The fibroblast-seeded scaffold was cultured in a 5% CO2 incubator at 37 °C and the culture medium was changed every two days. At day 1 and day 7, cells seeded in the scaffold were observed by confocal laser scanning microscopy (CLSM, Leica TCS SP5). The specimen was washed with PBS for 2 times and then fixed with 4% formalin for 48 h. Following with the removal of the residual formalin in PBS, the specimen was stained with 5 mg/ml 4′,6-diamidino-2-phenylindole (DAPI) solution at 37 °C for 30 min. Therefore, the fibroblasts existed in the scaffold were distinct from the FITC labeled scaffold under CLSM. Cell viability as a function of culture time was evaluated by MTT assay according to the methods of Mosmann with minor modification [25]. Briefly, at each culture interval, the media in the scaffolds were removed and 200 μl MTT (5 mg/mL, Sigma) solution was injected into the scaffolds. After being incubated for 4 h, the residual MTT in the scaffolds was removed and 200 μl dimethyl sulfoxide (DMSO) was added for each scaffold and incubated at 37 °C for 15 min. Finally, the absorbance of violate substance was measured at 570 nm by using a microplate reader (Biorad 680). 3. Results and discussion 3.1. Macroscopic shape and microstructure To investigate the effects of the different fabrication methods on the structure of the collagen/chitosan scaffolds, which plays a crucial role in determining the infiltration and proliferation of cells, the macroscopic shape and microstructure of S1 and S2 are compared in Fig. 1. As shown in Fig. 1A, S1 shows a rough surface with several cracks. Comparably, the S2 scaffold shows a round and smooth surface (Fig. 1D). The corresponding microstructures from both surface and cross section view were observed by scanning electron microscopy (SEM) (Fig. 1B, C, E and F) and the structure parameters of the scaffolds were summarized in Table 1. From the surface view, the microstructures of S1 and S2 are quite different (Fig. 1B, E). A sheet-like
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structure with a pore size of 82 ± 19 μm is observed in the case of S1. However, for S2, a structure with more fibers is found and the slightly larger pores with an average size of 87 ± 16 μm were obtained. From the cross-section view, porous structures were mostly preserved in both kinds of scaffolds (Fig. 1B, F). However, an obvious collapse could be found in the case of S1, the pore size of which decreased to 66 ± 11 μm. For S2, the size of the pores in the cross-section image, i.e. 83 ± 8 μm on average, was similar to those on the surface, which is more suitable for the infiltration of fibroblasts [26]. As shown in Table 1, the porosity of S1was about 98.9%, whereas a slightly bigger porosity (with a value of 99.4%) was obtained for S2, indicating that the effect of the fabrication methods on the scaffold porosity is limited. Indeed, the collapse of S1 has been observed in our previous study. The explanation of this phenomenon is thought mainly due to the fabrication route, especially the refreeze-drying process. As described in the preparation of S1, the collagen/chitosan porous scaffolds were freeze-dried again after the GA treatment. During the refreeze-drying process, the newly-formed ice crystals will break the original structure of the scaffold. However, because of the GA cross-linking treatment, the biomacromolecules are hard to reorganize during the refreeze-drying treatment and therefore a collapse structure is obtained by the S1 method. 3.2. Swelling ratio In the early stage of wound healing, the nutrients that cells need will entirely depend on tissue fluids surrounding the wound. Therefore the ability of the scaffold to absorb water is one of the important factors in determining the biological activity of a dermal equivalent. The swelling ratios of S1 and S2 with the immersing time were compared in Fig. 2. For all the samples, the swelling ratios increase with the immersing time and reach to a platform after immersed for about 12 h. It is shown that S1 has the similar swelling ratio to that of S2-0.25%. However, in the S2 group, the swelling ratios changed with the concentration of GA. For S2 cross-linked by 0.1% GA (S2-0.1%), the highest swelling ratio was obtained. However, by increasing the GA concentration to 0.25% or decreasing to 0.04%, both resulted in the decrease of the swelling ratios. The swelling ratio of the scaffold was largely dependent on the hydrophilicity of the materials and the property to maintain the three-dimensional structure. In general, with the increase of GA concentration, the hydrophilicity of the scaffold became worse because of the consumption of the amino group. However, the cross-linking treatment can increase the ability of scaffold to maintain the 3D structure. Therefore, at the same GA
A
B
C
D
E
F
Fig. 1. Macroscopic shape of S1 (A) and S2 (D), and the corresponding microstructure from the surface (B, E) and cross-section view (C, F).
Y. Liu et al. / Materials Science and Engineering C 32 (2012) 2361–2366
A
Table 1 Comparison of the structure parameters of S1 and S2. S2
Freeze-drying times Macroscopic shapes Pore size (surface, μm) Pore size (cross-section, μm) Porosity (%)
2 Rough and shrinkage 82 ± 19 66 ± 11 98.9 ± 0.2
1 Smooth and controllable size 87 ± 16 83 ± 8 99.4 ± 0.1
concentration, S1 showed lower swelling ratio than S2 because of the structure collapse. Compared to S2-0.1%, the decrease of GA concentration in the case of S2-0.04% will result in an increase of hydrophilicity, but a decrease of the ability to maintain the 3D structure, the balance of these two effects finally determines a lower swelling ratio. Nevertheless, the swelling ratios of all the scaffolds are big enough to absorb a large amount of nutrients to support the cell growth in the early stage of wound healing. 3.3. Cross-linking degree and in vitro biodegradation It is known that the ideal scaffold should have a proper degradation behavior to match the regenerating process of the damaged skin [27]. Therefore, the cross-linking method, as well as the cross-linking degree is regarded as one of the most important parameters for the scaffold fabrication. In this study, the scaffolds were cross-linked by GA with two different methods. In Fig. 3A, the effects of the different treating methods, as well as the GA concentration, on the cross-linking degree of the scaffold were investigated. When treated with 0.25% GA, the scaffolds fabricated with different ways, i.e. S1 and S2-0.25%, possessed different cross-linking degree. The cross-linking degree of S2 is significantly higher than S1. It may be attributed to the instability of the formed Schiff base structure, which may be decomposed when S1 was washed with water for many times. With the increase of GA concentration, the cross-linking degree of S2 increased. However, no significant difference was observed between the cross-linking degrees of S2-0.1% and S2-0.25%. It is probably because of the saturation of the cross-linking degree when the GA concentration reached to 0.1%. The biological stability of the cross-linked scaffolds was evaluated by in vitro collagenase degradation test (Fig. 3B). After incubated in collagenase solution for 24 h, the uncross-linked collagen/chitosan scaffold had been thoroughly biodegraded, which indicates 100% degradation. For all of S2 scaffolds, with the increase of GA concentration, the ability to resist collagenase digestion gradually strengthens, which has a good consistence with the results of cross-linking degree in Fig. 3A. However, although the cross-linking degree of S1 is obviously lower than that of S2, no significant differences of biodegradation degree between S1
Swelling ratio (100%)
S2-0.04% S2-0.1% S2-0.25% S1
120
80
40
Cross-linking degree (%)
S1
160
45
*
40
Comparison
*
35 30 25 20 15 10 5 0 S1
B Degradation degree (%)
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S2-0.04%
S2-0.1%
S2-0.25%
90
*
75 60 45 30 15 0 S1
S2-0.04%
S2-0.1%
S2-0.25%
Fig. 3. The cross-linking degree (A) and in vitro degradation degree (B) of S1 and S2 treated by GA with different concentration.* denotes statistically significant difference (pb 0.05).
and S2 are found. It is probably because of the collapse structure of S1, which affects the infiltration of the collagenase solution into the S1 scaffold. Moreover, the concentration of collagenase may be another reason, which is probably too high to distinguish the difference of cross-linking degree between these two kinds of scaffolds. 3.4. Mechanical properties To evaluate the mechanical properties of the scaffolds, which are quite important for the clinical operation, the tensile and compressive tests of the different scaffolds are compared in Fig. 4. For the tensile test under dry state (Fig. 4A), the modulus of S1 is much higher than S2-0.25% although they are treated by GA with the same concentration. In the S2 groups, the modulus decreased when the GA concentration increased from 0.04% to 0.1%, which can be contributed to the increased cross-linking degree of the scaffold (Fig. 3A). However, there is no significant difference between S2-0.1% and S2-0.25%. One can notice that at dry state, the moduli of the scaffolds are higher than that at wet state. Here, water is thought as a kind of plasticizer of the scaffold. In the compressive test (Fig. 4B), the compression modulus of S1 at dry state is similar to that of S2 treated by 0.04% and 0.1% GA. When the GA concentration increased to 0.25%, a significant higher modulus was detected. In contrast to the results of tensile test, the compression modulus of the scaffold under wet condition was higher than that under dry state. It may be attributed to the absorbed water in the scaffolds. At this state, a higher compressive stress is needed to reach the same compressive ratio. 3.5. The amount of residual GA in scaffold
0 0
5
10
15
20
25
Time (d) Fig. 2. The swelling ratios of S1 and S2 with the immersing time. S2-0.04% indicates S2 scaffolds cross-linked by 0.04% GA. Values are mean ± SD (n = 3).
The previous results showed that S2-0.04% has the similar swelling ratio, biodegradation degree and mechanical property to S1, the healing property of which has been proved in a porcine model. Therefore, the GA concentration was set as 0.04% in the following tests.
Y. Liu et al. / Materials Science and Engineering C 32 (2012) 2361–2366
A
2.0
* *
1.6
Residual GA (mg/g scaffold)
1.8
Young's modulus (MPa)
500
* *
A
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*
1.4
dry
1.2
wet
1.0 0.8 0.6 0.4
400
300
200
100
0.2 0
0.0 S1
S2-0.1%
2
S2-0.25%
B
4.5 4.0
* dry
3.5
wet
3.0 2.5 2.0 1.5 1.0 0.5
3
4
5
6
Rinsing times
Residual GA (mg/g scaffold)
2
Compression modulus(×10- MPa)
B
S2-0.04%
350 300 250 200 150 100 50 0
0.0 S1
S2-0.04%
S2-0.1%
0
S2-0.25%
Fig. 4. The tensile (A) and compression (B) moduli of S1 and S2 in dry and wet states. * denotes statistically significant difference (p b 0.05). Values are mean± SD (n = 3).
It is known that the residual GA is the main reason for the cytotoxicity of the GA treated scaffold. Therefore, the analysis of the residual GA of the scaffold should be indispensable when comparing these two fabrication routes. Herein, HPLC was applied to detect the residual GA of the scaffolds under different treatments. For S1, the residual GA was removed by rinsing with a large amount of water. Therefore, the amount of the residual GA with the rinsing times was plotted in Fig. 5A. It is shown that with the increase of the rinsing times, the amount of residual GA in S1 decreased gradually. After rinsed for 6 times, the residual GA is low than 9.4 mg/g scaffold. It means that for a scaffold with a diameter of 3.5 cm and a thickness of 2 mm, there are only 94 μg GA left in the scaffold. In our previous work, it has been proved that the toxicity of residual GA in S1 can be ignored by the results of cell culture test and animal test. In Fig. 5B, the residual GA of S2 with the oven drying time was studied. The residual GA decreased sharply with the prolongation of drying time, and finally only 7 mg/g scaffold could be detected after dried for 5 h, which indicates less cytotoxicity than S1. All these results proved that oven drying is a feasible and effective method to remove the residual GA in the scaffolds. 3.6. Cytotoxicity Fibroblasts exist widely in the dermal layer and play key roles in the wound healing process [28,29]. To evaluate the effect of the fabricating method on the cytotoxicity of the scaffold, the morphology and viability of the fibroblasts seeded into the scaffold were compared. The CLSM images of the fibroblasts in the scaffolds at day 1 and day 7 are shown in Fig. 6. With the culture time prolongation, more cells were observed in both kinds of scaffolds, demonstrating the proliferation of fibroblasts. However, compared to S1, a higher cell density was obtained
1
2
3
4
5
Oven drying time (h) Fig. 5. The amount of residual GA in S1 (A) and S2 (B).
at day 7 in S2-0.04%, indicating better biocompatibility of S2. Fig. 7 shows the cell viability in the scaffolds with the culture time. From day 1 to day 10, the viability of the fibroblasts in both scaffolds increased with the prolongation of culture time. Except for day 1, no significant difference was observed between the cell viability of these two kinds of scaffold. All these results indicate that the facile method has no obvious effect on deteriorating the biocompatibility of the scaffold and is feasible for the preparation of the GA cross-linked collagen/chitosan scaffold.
4. Conclusions A facile method to prepare the glutaraldehyde (GA) cross-linked collagen/chitosan porous scaffold was developed. The results of macroscopic shapes and microstructures showed that S2 can overcome the shortcomings of S1 such as rough surface and inner structure collapse, resulting in a smooth surface and controlled size. At the same GA concentration, S1 and S2 showed the similar swelling ratios, which are big enough to ensure the nutrient supply in the early stage of wound healing. The fabrication method has an obvious effect on the cross-linking degree and in vitro degradation degree of the scaffolds. When the concentration of the treating GA is the same, the crosslinking degree of S2-0.25% is much higher than that of S1. Investigation of the tensile and compression properties of the scaffolds found that the mechanical property of S2-0.04% is closest to that of S1. The results of HPLC analysis proved that oven drying is a feasible and effective method to remove the residual GA of S2. By compared the morphology and viability of the fibroblasts in S1 and S2, it is proved that S2-0.04% could retain the original good cytocompatibility of S1 to accelerate cell infiltration and proliferation effectively. All these results indicate that the facile method is feasible for preparation of the GA cross-linked collagen/chitosan scaffold for skin tissue engineering.
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Day 7
Day 1
A
China (2011CB606203) and the Science Technology Program of Zhejiang Province (2009C14003).
B
References
S1
[1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11]
100µm
C
D
S2-0.04%
[12] [13] [14] [15] [16]
Fig. 6. CLSM images of fibroblasts cultured in S1 (A, B) and S2 (C, D) for 1 and 7 days. The scaffolds and the cell nucleus are labeled by FITC (green) and DAPI (blue), respectively.
[17] [18] [19] [20]
Optical density570nm
2.8
[21] [22] [23] [24] [25]
S2-0.04% S1
2.4 2.0
[26] [27] [28]
1.6
[29]
1.2 *
0.8 0.4 1
3
7
10
Culture time (d) Fig. 7. The viability of fibroblasts cultured in S1 and S2 for different days. * denotes statistically significant difference (p b 0.05).
Acknowledgments This study is financially supported by the Natural Science Foundation of China (20934003), the National Basic Research Program of
A.D. Metcalfe, M.W.J. Ferguson, Biomaterials 28 (2007) 5100. J.J. Marler, J. Upton, R. Langer, J.P. Vacanti, Adv. Drug Deliv. Rev. 33 (1998) 165. S.T. Boyce, Burns 27 (2001) 523. I.V. Yannas, In: CRC Press, Boca Raton, 1995, p. 1619. J.T. Schul III, R.G. Tompkins, J.F. Burks, Annu. Rev. Med. 51 (2000) 231. I.V. Yannas, J.F. Burke, J. Biomed. Mater. Res. 14 (1980) 65. E. Bell, H.P. Ehrlich, D.J. Battle, T. Nakatsuji, Science 211 (1981) 1052. S. Bottcher-Haberzeth, T. Biedermann, E. Reichmann, Burns 36 (2010) 450. Q.A. Dai, T.S. Potokar, P. Price, Burns 36 (2010) 23. D. Stiefel, S. Clemens, M. Martin, Burns 36 (2010) 114. M. Carlson, K. Faria, Y. Shamis, J. Leman, V. Ronfard, J. Garlick, Tissue Eng. Part A 17 (2011) 487. M.P. Lutolf, J.A. Hubbell, Nat. Biotechnol. 23 (2005) 47. S.G. Priya, H. Jungvid, A. Kumar, Tissue Eng. Part B Rev. 14 (2008) 105. F. Groeber, M. Holeiter, M. Hampel, S. Hinderer, K. Schenke, Adv. Drug Deliv. Rev. 63 (2011) 352. S. MacNeil, Mater. Today 11 (2008) 26. R. Jayakumar, M. Prabaharan, P.T. Sudheesh Kumar, S.V. Nair, H. Tamura, Biotechnol. Adv. 29 (2011) 322. R. Jayakumar, M. Prabaharan, S.V. Nair, H. Tamura, Biotechnol. Adv. 28 (2010) 142. A.B. Huang, R. Guo, S.J. Xu, L. Ma, C.Y. Gao, Acta Polym. Sin. 2 (2009) 111. M. Peter, N. Ganesh, N. Selvamurugan, S.V. Nair, T. Furuike, H. Tamura, R. Jayakumar, Carbohydr. Polym. 80 (2010) 687. L. Ma, C.Y. Gao, Z.W. Mao, J. Zhou, J.C. Shen, X.Q. Hu, C.M. Hang, Biomaterials 24 (2003) 4833. Y.C. Shi, L. Ma, J. Zhou, Z.W. Mao, C.Y. Gao, Polym. Adv. Technol. 16 (2005) 789. R. Guo, S.J. Xu, L. Ma, A.B. Huang, C.Y. Gao, Biomaterials 32 (2011) 1019. R. Guo, S. J. X., L. Ma, A.B. Huang, C.Y. Gao, Biomaterials 31 (2010) 7308. C.M. Ofner, W.A. Bubins, Pharm. Res. 13 (1996) 1821. J.S. Pieper, A. Oosterhof, P.J. Dijkstra, J.H. Veerkamp, T.H. van Kuppevelt, Biomaterials 20 (1999) 847. T.M. Freyman, I.V. Yannas, L.J. Gibson, Prog. Mater. Sci. 46 (2001) 273. Y.H. Gong, Q.L. Zhou, C.Y. Gao, J.C. Shen, Acta Biomater. 3 (2007) 531. A.E. Ghalbzouri, S. Commandeur, M.H. Rietveld, A.A. Mulder, R. Willemze, Biomaterials 30 (2009) 71. M. Kempf, Y. Miyamura, P.Y. Liu, C.H. Chen, H. Nakamura, H. Shimizu, Y. Tabata, R.M. Kimble, J.R. McMillan, Biomaterials 32 (2011) 478.
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Facile fabrication of the glutaraldehyde cross-linked collagen/chitosan porous scaffold for skin tissue engineering Yunyun Liu, Lie Ma ⁎, Changyou Gao MOE Key Laboratory of Macromolecular Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China
a r t i c l e
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Article history: Received 11 January 2012 Received in revised form 20 June 2012 Accepted 4 July 2012 Available online 10 July 2012 Keywords: Collagen Chitosan Scaffold Cross-linking Tissue engineering
a b s t r a c t Porous scaffold is one of the key factors in skin tissue engineering. In this study, a facile method was developed to prepare the glutaraldehyde (GA) cross-linked collagen/chitosan porous scaffold (S2). The properties of S2 were compared with the scaffolds prepared by the traditional method (S1). Compared to the rough surface and collapsed inner structure of S1, S2 showed a smooth surface and controlled size. After treated by GA with same concentration, S1 and S2 showed the similar swelling ratios, which are big enough to ensure the nutrient supply in the early stage of wound healing. The effects of the fabrication methods as well as the GA concentration on the cross-linking degree and in vitro degradation degree of the scaffolds were studied. It was found that the cross-linking degree of S2-0.25% was much higher than that of S1. Investigation of the tensile and compression properties of the scaffolds found that the mechanical property of S2-0.04% is closest to that of S1. High performance liquid chromatography (HPLC) was applied to determine the residual GA. The results proved that, compared to water rinse, oven drying is a feasible and effective method to remove the residual GA. Finally, the cytocompatibility of S2 was evaluated by in vitro culture of fibroblasts. The results of cell morphology and cell viability proved that S2-0.04% could retain the original good cytocompatibility of S1 to accelerate cell infiltration and proliferation effectively. All these results indicate that it is a feasible method to prepare the GA cross-linked collagen/chitosan scaffold. © 2012 Elsevier B.V. All rights reserved.
1. Introduction As the largest and highly complex organ of human body, skin, which is mainly composed of epidermis and dermis, plays a crucial role in thermoregulation, microbial defense and sensing [1]. However, because of trauma, burn and chronic disease, skin lose has become one of the most serious problems in clinic and millions of people are suffering from skin lose annually. Many treatments including skin transplantation, reconstructive surgery and wound dressing are applied for skin repair [2]. However, given the problems such as donor limitation and immune response [3–7], none of these approaches is ideal to fully accomplish the purpose of skin repair. Recently, the advances of tissue engineering and regenerative medicine make it possible and bring a bright future for clinicians to restore skin defects [8]. In the past decades, many kinds of skin substitutes such as Integra, Apligraf and Dermagraf, have been developed and applied for the treatment of full thickness skin defects in hospital [9–11]. However, many problems such as the limited effect, lack of appendixes and scarring are still the main hurdles to realize the full repair of the damaged skin. As one of the crucial factors determining the properties of the skin substitutes, porous scaffold offers an extracellular matrix analog which functions as the artificial dermis template for host cell ⁎ Corresponding author. Tel./fax: +86 571 87951108. E-mail address: [email protected] (L. Ma). 0928-4931/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2012.07.008
infiltration, proliferation and differentiation. Nowadays, many kinds of materials obtained from natural feedstocks such as collagen, chitosan and hyaluronic acid, or from synthetic feedstocks such as poly (lactic-co-glycolic acid) (PLGA), poly (glycolic acid) (PGA), are used in the scaffold fabrication [12–15]. Among them, collagen and chitosan are widely used as the main materials for tissue engineering scaffold. Collagen is one of the major constituents of extracellular matrix and is well known for the low antigenicity, excellent biocompatibility and biodegradability. In the development or healing of tissues and organs, collagen is involved in controlling the cell behaviors such as adhesion, proliferation and differentiation and plays an important role in the formation of extracellular matrix. Chitosan, an amino polysaccharide (poly-1,4-D-glucoamine) derived from chitin by deacetylation, has been widely used in a variety of biomedical fields such as wound dressings and drug delivery systems because of its good biocompatibility and biodegradability [16–19]. Furthermore, it was reported that chitosan can function as a bridge to increase the cross-linking efficiency of glutaraldehyde in the collagen/chitosan scaffolds because of its large number of amino groups in molecular chain [20]. Therefore, the scaffold fabricated by collagen and chitosan has been widely used in the repair of many kinds of tissue and organ such as vessel, skin, tendon and cartilage. In our previous work, a bilayer dermal equivalent consisting of a collagen/chitosan porous scaffold and silicone membrane was reported [21]. In vivo animal tests proved that the collagen/chitosan scaffold
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has no obvious cytotoxicity and has the property of reconstructing the damaged dermis within 4 weeks [22,23]. However, the previous fabricating method is quite complicated, which consists of many steps such as freeze-drying, cross-linking and refreeze-drying. Mainly due to the complicated process, problems such as surface collapse, crack formation and size deformation frequently occurred. To overcome the problems of previous method and enhance the quality of collagen/chitosan scaffolds, a facile method to fabricate the GA cross-linked collagen/chitosan scaffold was reported in this study. The effect of the different fabricating methods as well as the GA concentration on the macro and microstructure, cross-linking degree and degradation behavior, and the mechanical properties are investigated, and then the influence on the residual GA and the corresponding cytotoxicity are evaluated.
The mean pore size of the scaffolds was determined by analysis of the corresponding SEM images. The porosity of each kind of scaffold was measured by immersing the scaffolds (initial weight, W0) into ethanol at room temperature for 24 h (wet weight, W) and calculated by the formulas below.
2. Materials and methods
The swelling ratios of scaffolds were measured as the previous report [21]. Briefly, the scaffold was incubated in Milli-Q water at 37 °C and then the wet weight (W) was measured at different time points. The swelling ratio is defined as the ratio of weight increase (W − W0) to the initial weight (W0). Each value was averaged from three parallel measurements and expressed as mean ± standard deviation (SD).
2.1. Materials Collagen type Ι was isolated from fresh bovine tendon with the method of trypsin digestion and acetic acid dissolution as described previously [20]. Chitosan (Mη: 5×105, 85% deacetylation degree) was purchased from Jinan Haidebei Marine Bioengineering CO., LTD. Glutaraldehyde (GA, 25% water solution) was purchased from Sinopharm Chemical Reagent Co., Ltd. All other reagents and solvents were of analytical grade and used as received. 2.2. Preparation of the GA cross-linked collagen/chitosan porous scaffolds The GA cross-linked collagen/chitosan porous scaffolds were fabricated by two routes. The first route has been described in previous studies [20]. Briefly, collagen and chitosan were dissolved in 0.5 M acetic acid solution respectively, and then were homogenized to form a 0.5% (w/v) collagen/chitosan blend in a mass ratio of 9:1. The collagen/ chitosan blend was injected into a mold and freeze-dried to form the uncross-linked scaffold. Then the scaffold was rehydrated and further treated with 0.25% (w/v) GA at 37 °C for 4 h. After rinsed with Milli-Q water for six times to remove the excess GA, the scaffold was freeze-dried again to obtain the GA cross-linked collagen/chitosan scaffold (S1). In this study, a facile route was adopted to fabricate the GA cross-linked collagen/chitosan scaffold. The collagen/chitosan blend was prepared as the same way of S1. Then, the blend was injected into a container, and 2.5% GA solution were added to ensure the final concentration of GA in a setting value. The volume of the added 2.5% GA can be calculated from the below formula:
Porosity ð%Þ ¼
ðW−W0 Þρ1 100% ρ1 W þ ðρ2 −ρ1 ÞW0
where ρ1 and ρ2 represent density of collagen (1.21 g/ml) and ethanol (0.79 g/ml) respectively. 2.4. Swelling ratio
2.5. Cross-linking degree and in vitro degradation Since amino groups are consumed with the GA cross-linking treatment, the cross-linking degree of S1 and S2 can be defined as the amino group ratio of the GA treated scaffold to the uncross-linked one. Briefly, a weighed amount of scaffold (11 mg) was incubated in 1.0 ml 4% (w/v) NaHCO3 solution and 1.0 ml 0.5% (w/v) 2, 4, 6-trinitrobenzene sulfonic acid (TNBS) at 40 °C. After 4 h, 3.0 ml 6 M HCl was added and the reaction was carried out at 120 °C for 1 h. The resulting solution was diluted with 5.0 ml Milli-Q water and then extracted by 20 ml ethyl ether. Decanted 5.0 ml solution from aqueous phase and heated for 15 min at 37 °C. After cooled to room temperature, the solution was diluted again with 15 ml water and the absorbance was measured at 346 nm by UV–vis (Shimadzu, UV-2550) [24]. In vitro biodegradation test of S1 and S2 was performed by collagenase digestion as the previous studies [20]. The specimen was immersed in phosphate buffered saline (PBS, pH 7.4) containing 0.1 mg/ml (265 u/mg) collagenase (type I, Sigma) at 37 °C for 24 h. Following centrifugation at 1500 rpm for 10 min, the clear supernatant was hydrolyzed with 6 M HCl at 120 °C for 12 h. The content of hydroxyproline released from scaffold was measured with UV–vis. The biodegradation degree is defined as a percentage of the released hydroxyproline of the specimen to the uncross-linked one (%). 2.6. Mechanical performance
2:5% VGA ¼ C ðVGA þ Vblend Þ where VGA is the volume of the added 2.5% GA, Vblend is the volume of collagen/chitosan blend, and C is the final GA concentration in the blend. After stirred at 37 °C for 4 h, the collagen/chitosan blend was injected into a mold and freeze-dried. To remove the excess GA, the freeze-dried scaffold was treated under vacuum condition for 5 h at 37 °C to form the GA cross-linked collagen/chitosan porous scaffold (S2).
The mechanical performances of the scaffolds in both dry and wet conditions were tested with Instron Materials Test System (Instron 5543A) at room temperature. For the tensile test, the rectangular specimens (60 mm× 20 mm, with the thickness of 2 mm) were used. The distance of two air clamps was 20 mm, and the speed of crosshead was set as 6 mm/min. The round specimens with diameter of 15 mm and the thickness of 10 mm were applied in the compressive test. The compressive velocity was set as 2 mm/min. The wet samples were prepared by immersing in PBS at 37 °C for 24 h. 2.7. Analysis of residual GA
2.3. Macroscopic shape and microstructure observation Macroscopic images of S1 and S2 were taken by a digital camera (Canon, A480). The corresponding surface and cross section microstructure were observed by scanning electron microscopy (SEM, FEI SIRION-100) with an accelerating voltage of 25 kV after the samples were sputter-coated with a thin gold layer.
To detect the residual GA in scaffolds, S1 and S2 were immersed in 100 ml Milli-Q water at 37 °C for 96 h, and then the supernatants were analyzed by high performance liquid chromatography (HPLC, Agilent 1100 Series) with 0.25% (w/v) GA standard sample as control. Column temperature of HPLC was maintained at 30 °C. The volume of injected sample was 20 μl with acetonitrile/water (60:40) as the mobile
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phase and the flow rate was set as 0.14 ml/min. UV detection wavelength was 235 nm. 2.8. Cytotoxicity evaluation The cytotoxicity of scaffolds was evaluated by CLSM observation and cell viability test. Before cell culture, scaffolds were immersed in 75% ethanol for 12 h for sterilization, followed by solvent exchange with PBS for 6 times. Then the sterilized scaffold was placed on 48-well polystyrene plate and seeded with 100 μl human dermal fibroblast suspension at a density of 3 × 106 cells/ml. The fibroblast-seeded scaffold was cultured in a 5% CO2 incubator at 37 °C and the culture medium was changed every two days. At day 1 and day 7, cells seeded in the scaffold were observed by confocal laser scanning microscopy (CLSM, Leica TCS SP5). The specimen was washed with PBS for 2 times and then fixed with 4% formalin for 48 h. Following with the removal of the residual formalin in PBS, the specimen was stained with 5 mg/ml 4′,6-diamidino-2-phenylindole (DAPI) solution at 37 °C for 30 min. Therefore, the fibroblasts existed in the scaffold were distinct from the FITC labeled scaffold under CLSM. Cell viability as a function of culture time was evaluated by MTT assay according to the methods of Mosmann with minor modification [25]. Briefly, at each culture interval, the media in the scaffolds were removed and 200 μl MTT (5 mg/mL, Sigma) solution was injected into the scaffolds. After being incubated for 4 h, the residual MTT in the scaffolds was removed and 200 μl dimethyl sulfoxide (DMSO) was added for each scaffold and incubated at 37 °C for 15 min. Finally, the absorbance of violate substance was measured at 570 nm by using a microplate reader (Biorad 680). 3. Results and discussion 3.1. Macroscopic shape and microstructure To investigate the effects of the different fabrication methods on the structure of the collagen/chitosan scaffolds, which plays a crucial role in determining the infiltration and proliferation of cells, the macroscopic shape and microstructure of S1 and S2 are compared in Fig. 1. As shown in Fig. 1A, S1 shows a rough surface with several cracks. Comparably, the S2 scaffold shows a round and smooth surface (Fig. 1D). The corresponding microstructures from both surface and cross section view were observed by scanning electron microscopy (SEM) (Fig. 1B, C, E and F) and the structure parameters of the scaffolds were summarized in Table 1. From the surface view, the microstructures of S1 and S2 are quite different (Fig. 1B, E). A sheet-like
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structure with a pore size of 82 ± 19 μm is observed in the case of S1. However, for S2, a structure with more fibers is found and the slightly larger pores with an average size of 87 ± 16 μm were obtained. From the cross-section view, porous structures were mostly preserved in both kinds of scaffolds (Fig. 1B, F). However, an obvious collapse could be found in the case of S1, the pore size of which decreased to 66 ± 11 μm. For S2, the size of the pores in the cross-section image, i.e. 83 ± 8 μm on average, was similar to those on the surface, which is more suitable for the infiltration of fibroblasts [26]. As shown in Table 1, the porosity of S1was about 98.9%, whereas a slightly bigger porosity (with a value of 99.4%) was obtained for S2, indicating that the effect of the fabrication methods on the scaffold porosity is limited. Indeed, the collapse of S1 has been observed in our previous study. The explanation of this phenomenon is thought mainly due to the fabrication route, especially the refreeze-drying process. As described in the preparation of S1, the collagen/chitosan porous scaffolds were freeze-dried again after the GA treatment. During the refreeze-drying process, the newly-formed ice crystals will break the original structure of the scaffold. However, because of the GA cross-linking treatment, the biomacromolecules are hard to reorganize during the refreeze-drying treatment and therefore a collapse structure is obtained by the S1 method. 3.2. Swelling ratio In the early stage of wound healing, the nutrients that cells need will entirely depend on tissue fluids surrounding the wound. Therefore the ability of the scaffold to absorb water is one of the important factors in determining the biological activity of a dermal equivalent. The swelling ratios of S1 and S2 with the immersing time were compared in Fig. 2. For all the samples, the swelling ratios increase with the immersing time and reach to a platform after immersed for about 12 h. It is shown that S1 has the similar swelling ratio to that of S2-0.25%. However, in the S2 group, the swelling ratios changed with the concentration of GA. For S2 cross-linked by 0.1% GA (S2-0.1%), the highest swelling ratio was obtained. However, by increasing the GA concentration to 0.25% or decreasing to 0.04%, both resulted in the decrease of the swelling ratios. The swelling ratio of the scaffold was largely dependent on the hydrophilicity of the materials and the property to maintain the three-dimensional structure. In general, with the increase of GA concentration, the hydrophilicity of the scaffold became worse because of the consumption of the amino group. However, the cross-linking treatment can increase the ability of scaffold to maintain the 3D structure. Therefore, at the same GA
A
B
C
D
E
F
Fig. 1. Macroscopic shape of S1 (A) and S2 (D), and the corresponding microstructure from the surface (B, E) and cross-section view (C, F).
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A
Table 1 Comparison of the structure parameters of S1 and S2. S2
Freeze-drying times Macroscopic shapes Pore size (surface, μm) Pore size (cross-section, μm) Porosity (%)
2 Rough and shrinkage 82 ± 19 66 ± 11 98.9 ± 0.2
1 Smooth and controllable size 87 ± 16 83 ± 8 99.4 ± 0.1
concentration, S1 showed lower swelling ratio than S2 because of the structure collapse. Compared to S2-0.1%, the decrease of GA concentration in the case of S2-0.04% will result in an increase of hydrophilicity, but a decrease of the ability to maintain the 3D structure, the balance of these two effects finally determines a lower swelling ratio. Nevertheless, the swelling ratios of all the scaffolds are big enough to absorb a large amount of nutrients to support the cell growth in the early stage of wound healing. 3.3. Cross-linking degree and in vitro biodegradation It is known that the ideal scaffold should have a proper degradation behavior to match the regenerating process of the damaged skin [27]. Therefore, the cross-linking method, as well as the cross-linking degree is regarded as one of the most important parameters for the scaffold fabrication. In this study, the scaffolds were cross-linked by GA with two different methods. In Fig. 3A, the effects of the different treating methods, as well as the GA concentration, on the cross-linking degree of the scaffold were investigated. When treated with 0.25% GA, the scaffolds fabricated with different ways, i.e. S1 and S2-0.25%, possessed different cross-linking degree. The cross-linking degree of S2 is significantly higher than S1. It may be attributed to the instability of the formed Schiff base structure, which may be decomposed when S1 was washed with water for many times. With the increase of GA concentration, the cross-linking degree of S2 increased. However, no significant difference was observed between the cross-linking degrees of S2-0.1% and S2-0.25%. It is probably because of the saturation of the cross-linking degree when the GA concentration reached to 0.1%. The biological stability of the cross-linked scaffolds was evaluated by in vitro collagenase degradation test (Fig. 3B). After incubated in collagenase solution for 24 h, the uncross-linked collagen/chitosan scaffold had been thoroughly biodegraded, which indicates 100% degradation. For all of S2 scaffolds, with the increase of GA concentration, the ability to resist collagenase digestion gradually strengthens, which has a good consistence with the results of cross-linking degree in Fig. 3A. However, although the cross-linking degree of S1 is obviously lower than that of S2, no significant differences of biodegradation degree between S1
Swelling ratio (100%)
S2-0.04% S2-0.1% S2-0.25% S1
120
80
40
Cross-linking degree (%)
S1
160
45
*
40
Comparison
*
35 30 25 20 15 10 5 0 S1
B Degradation degree (%)
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S2-0.04%
S2-0.1%
S2-0.25%
90
*
75 60 45 30 15 0 S1
S2-0.04%
S2-0.1%
S2-0.25%
Fig. 3. The cross-linking degree (A) and in vitro degradation degree (B) of S1 and S2 treated by GA with different concentration.* denotes statistically significant difference (pb 0.05).
and S2 are found. It is probably because of the collapse structure of S1, which affects the infiltration of the collagenase solution into the S1 scaffold. Moreover, the concentration of collagenase may be another reason, which is probably too high to distinguish the difference of cross-linking degree between these two kinds of scaffolds. 3.4. Mechanical properties To evaluate the mechanical properties of the scaffolds, which are quite important for the clinical operation, the tensile and compressive tests of the different scaffolds are compared in Fig. 4. For the tensile test under dry state (Fig. 4A), the modulus of S1 is much higher than S2-0.25% although they are treated by GA with the same concentration. In the S2 groups, the modulus decreased when the GA concentration increased from 0.04% to 0.1%, which can be contributed to the increased cross-linking degree of the scaffold (Fig. 3A). However, there is no significant difference between S2-0.1% and S2-0.25%. One can notice that at dry state, the moduli of the scaffolds are higher than that at wet state. Here, water is thought as a kind of plasticizer of the scaffold. In the compressive test (Fig. 4B), the compression modulus of S1 at dry state is similar to that of S2 treated by 0.04% and 0.1% GA. When the GA concentration increased to 0.25%, a significant higher modulus was detected. In contrast to the results of tensile test, the compression modulus of the scaffold under wet condition was higher than that under dry state. It may be attributed to the absorbed water in the scaffolds. At this state, a higher compressive stress is needed to reach the same compressive ratio. 3.5. The amount of residual GA in scaffold
0 0
5
10
15
20
25
Time (d) Fig. 2. The swelling ratios of S1 and S2 with the immersing time. S2-0.04% indicates S2 scaffolds cross-linked by 0.04% GA. Values are mean ± SD (n = 3).
The previous results showed that S2-0.04% has the similar swelling ratio, biodegradation degree and mechanical property to S1, the healing property of which has been proved in a porcine model. Therefore, the GA concentration was set as 0.04% in the following tests.
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A
2.0
* *
1.6
Residual GA (mg/g scaffold)
1.8
Young's modulus (MPa)
500
* *
A
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*
1.4
dry
1.2
wet
1.0 0.8 0.6 0.4
400
300
200
100
0.2 0
0.0 S1
S2-0.1%
2
S2-0.25%
B
4.5 4.0
* dry
3.5
wet
3.0 2.5 2.0 1.5 1.0 0.5
3
4
5
6
Rinsing times
Residual GA (mg/g scaffold)
2
Compression modulus(×10- MPa)
B
S2-0.04%
350 300 250 200 150 100 50 0
0.0 S1
S2-0.04%
S2-0.1%
0
S2-0.25%
Fig. 4. The tensile (A) and compression (B) moduli of S1 and S2 in dry and wet states. * denotes statistically significant difference (p b 0.05). Values are mean± SD (n = 3).
It is known that the residual GA is the main reason for the cytotoxicity of the GA treated scaffold. Therefore, the analysis of the residual GA of the scaffold should be indispensable when comparing these two fabrication routes. Herein, HPLC was applied to detect the residual GA of the scaffolds under different treatments. For S1, the residual GA was removed by rinsing with a large amount of water. Therefore, the amount of the residual GA with the rinsing times was plotted in Fig. 5A. It is shown that with the increase of the rinsing times, the amount of residual GA in S1 decreased gradually. After rinsed for 6 times, the residual GA is low than 9.4 mg/g scaffold. It means that for a scaffold with a diameter of 3.5 cm and a thickness of 2 mm, there are only 94 μg GA left in the scaffold. In our previous work, it has been proved that the toxicity of residual GA in S1 can be ignored by the results of cell culture test and animal test. In Fig. 5B, the residual GA of S2 with the oven drying time was studied. The residual GA decreased sharply with the prolongation of drying time, and finally only 7 mg/g scaffold could be detected after dried for 5 h, which indicates less cytotoxicity than S1. All these results proved that oven drying is a feasible and effective method to remove the residual GA in the scaffolds. 3.6. Cytotoxicity Fibroblasts exist widely in the dermal layer and play key roles in the wound healing process [28,29]. To evaluate the effect of the fabricating method on the cytotoxicity of the scaffold, the morphology and viability of the fibroblasts seeded into the scaffold were compared. The CLSM images of the fibroblasts in the scaffolds at day 1 and day 7 are shown in Fig. 6. With the culture time prolongation, more cells were observed in both kinds of scaffolds, demonstrating the proliferation of fibroblasts. However, compared to S1, a higher cell density was obtained
1
2
3
4
5
Oven drying time (h) Fig. 5. The amount of residual GA in S1 (A) and S2 (B).
at day 7 in S2-0.04%, indicating better biocompatibility of S2. Fig. 7 shows the cell viability in the scaffolds with the culture time. From day 1 to day 10, the viability of the fibroblasts in both scaffolds increased with the prolongation of culture time. Except for day 1, no significant difference was observed between the cell viability of these two kinds of scaffold. All these results indicate that the facile method has no obvious effect on deteriorating the biocompatibility of the scaffold and is feasible for the preparation of the GA cross-linked collagen/chitosan scaffold.
4. Conclusions A facile method to prepare the glutaraldehyde (GA) cross-linked collagen/chitosan porous scaffold was developed. The results of macroscopic shapes and microstructures showed that S2 can overcome the shortcomings of S1 such as rough surface and inner structure collapse, resulting in a smooth surface and controlled size. At the same GA concentration, S1 and S2 showed the similar swelling ratios, which are big enough to ensure the nutrient supply in the early stage of wound healing. The fabrication method has an obvious effect on the cross-linking degree and in vitro degradation degree of the scaffolds. When the concentration of the treating GA is the same, the crosslinking degree of S2-0.25% is much higher than that of S1. Investigation of the tensile and compression properties of the scaffolds found that the mechanical property of S2-0.04% is closest to that of S1. The results of HPLC analysis proved that oven drying is a feasible and effective method to remove the residual GA of S2. By compared the morphology and viability of the fibroblasts in S1 and S2, it is proved that S2-0.04% could retain the original good cytocompatibility of S1 to accelerate cell infiltration and proliferation effectively. All these results indicate that the facile method is feasible for preparation of the GA cross-linked collagen/chitosan scaffold for skin tissue engineering.
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Day 7
Day 1
A
China (2011CB606203) and the Science Technology Program of Zhejiang Province (2009C14003).
B
References
S1
[1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11]
100µm
C
D
S2-0.04%
[12] [13] [14] [15] [16]
Fig. 6. CLSM images of fibroblasts cultured in S1 (A, B) and S2 (C, D) for 1 and 7 days. The scaffolds and the cell nucleus are labeled by FITC (green) and DAPI (blue), respectively.
[17] [18] [19] [20]
Optical density570nm
2.8
[21] [22] [23] [24] [25]
S2-0.04% S1
2.4 2.0
[26] [27] [28]
1.6
[29]
1.2 *
0.8 0.4 1
3
7
10
Culture time (d) Fig. 7. The viability of fibroblasts cultured in S1 and S2 for different days. * denotes statistically significant difference (p b 0.05).
Acknowledgments This study is financially supported by the Natural Science Foundation of China (20934003), the National Basic Research Program of
A.D. Metcalfe, M.W.J. Ferguson, Biomaterials 28 (2007) 5100. J.J. Marler, J. Upton, R. Langer, J.P. Vacanti, Adv. Drug Deliv. Rev. 33 (1998) 165. S.T. Boyce, Burns 27 (2001) 523. I.V. Yannas, In: CRC Press, Boca Raton, 1995, p. 1619. J.T. Schul III, R.G. Tompkins, J.F. Burks, Annu. Rev. Med. 51 (2000) 231. I.V. Yannas, J.F. Burke, J. Biomed. Mater. Res. 14 (1980) 65. E. Bell, H.P. Ehrlich, D.J. Battle, T. Nakatsuji, Science 211 (1981) 1052. S. Bottcher-Haberzeth, T. Biedermann, E. Reichmann, Burns 36 (2010) 450. Q.A. Dai, T.S. Potokar, P. Price, Burns 36 (2010) 23. D. Stiefel, S. Clemens, M. Martin, Burns 36 (2010) 114. M. Carlson, K. Faria, Y. Shamis, J. Leman, V. Ronfard, J. Garlick, Tissue Eng. Part A 17 (2011) 487. M.P. Lutolf, J.A. Hubbell, Nat. Biotechnol. 23 (2005) 47. S.G. Priya, H. Jungvid, A. Kumar, Tissue Eng. Part B Rev. 14 (2008) 105. F. Groeber, M. Holeiter, M. Hampel, S. Hinderer, K. Schenke, Adv. Drug Deliv. Rev. 63 (2011) 352. S. MacNeil, Mater. Today 11 (2008) 26. R. Jayakumar, M. Prabaharan, P.T. Sudheesh Kumar, S.V. Nair, H. Tamura, Biotechnol. Adv. 29 (2011) 322. R. Jayakumar, M. Prabaharan, S.V. Nair, H. Tamura, Biotechnol. Adv. 28 (2010) 142. A.B. Huang, R. Guo, S.J. Xu, L. Ma, C.Y. Gao, Acta Polym. Sin. 2 (2009) 111. M. Peter, N. Ganesh, N. Selvamurugan, S.V. Nair, T. Furuike, H. Tamura, R. Jayakumar, Carbohydr. Polym. 80 (2010) 687. L. Ma, C.Y. Gao, Z.W. Mao, J. Zhou, J.C. Shen, X.Q. Hu, C.M. Hang, Biomaterials 24 (2003) 4833. Y.C. Shi, L. Ma, J. Zhou, Z.W. Mao, C.Y. Gao, Polym. Adv. Technol. 16 (2005) 789. R. Guo, S.J. Xu, L. Ma, A.B. Huang, C.Y. Gao, Biomaterials 32 (2011) 1019. R. Guo, S. J. X., L. Ma, A.B. Huang, C.Y. Gao, Biomaterials 31 (2010) 7308. C.M. Ofner, W.A. Bubins, Pharm. Res. 13 (1996) 1821. J.S. Pieper, A. Oosterhof, P.J. Dijkstra, J.H. Veerkamp, T.H. van Kuppevelt, Biomaterials 20 (1999) 847. T.M. Freyman, I.V. Yannas, L.J. Gibson, Prog. Mater. Sci. 46 (2001) 273. Y.H. Gong, Q.L. Zhou, C.Y. Gao, J.C. Shen, Acta Biomater. 3 (2007) 531. A.E. Ghalbzouri, S. Commandeur, M.H. Rietveld, A.A. Mulder, R. Willemze, Biomaterials 30 (2009) 71. M. Kempf, Y. Miyamura, P.Y. Liu, C.H. Chen, H. Nakamura, H. Shimizu, Y. Tabata, R.M. Kimble, J.R. McMillan, Biomaterials 32 (2011) 478.