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Mechanical property and biocompatibility of silk fibroin–collagen type II composite membrane
Materials Science & Engineering C 105 (2019) 110018
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Mechanical property and biocompatibility of silk fibroin–collagen type II composite membrane ⁎
T
⁎⁎
Xiang-Long Lina,b,d, Li-Lan Gaoa,b, , Rui-xin Lic, , Wei Chengd, Chun-Qiu Zhanga,b, Xi-zheng Zhangd a Tianjin Key Laboratory for Advanced Mechatronic System Design and Intelligent Control, School of Mechanical Engineering, Tianjin University of Technology, Tianjin, China b National Demonstration Center for Experimental Mechanical and Electrical Engineering Education (Tianjin University of Technology), Tianjin, China c Tianjin Stomatological Hospital, Tianjin, China d Institute of Medical Equipment, Academy of Military Medical Science, Tianjin 300161, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Silk fibroin–collagen type II Different ratio Composite membrane Mechanical behavior Theoretical model Biocompatibility
Osteoarthritis is caused by injuries and cartilage degeneration. Cartilage tissue engineering provides new ideas for the treatment of osteoarthritis. Herein, the different ratios composite membranes of silk fibroin/collagen type II were constructed (SF50-50:50, SF70-70:30, SF90-90:10). The surface properties of the composite membranes and chondrocyte morphology were observed by SEM (scanning electron microscopy). Physical functionality as well as stability of composite membranes was evaluated from tensile mechanical properties, the percentage of swelling and degradation. The tensile mechanical behavior of SF70 composite membranes was also predicted based on the constitutive model established in this study, and it is found that the experimental results and predictions were in good agreement. Biocompatibility was evaluated using chondrocytes (ADTC-5) culture. Cell proliferation was analyzed and the treatment of live/dead double staining was performed to assess the viability on chondrocytes. To sum up, SF70 showed the suitable morphology, physical stability, and biological functionality to promote proliferation of chondrocytes. This indicates that the mixing ratio of SF70 shows promise in the future as a scaffold material for cartilage repair.
1. Introduction More than two centuries ago, some scholars discovered that once articular cartilage is damaged, the ability to repair itself is extremely poor [1]. This is due to the avascular structure of articular cartilage, which has limited self-repairing ability and intrinsic healing ability [2–7]. Damage of articular cartilage causes joint pain, functional impairment and osteoarthritis (OA) [3,8]. Currently, many attempts are conducted to address the problem of articular cartilage repair such as autologous osteochondral graft transplantation, but the effects are not good [9]. Therefore, based on the method of tissue engineering, the construction of biomaterials at the defect area properly to regenerate new tissue is a promising approach for articular cartilage repair. The performance of biological materials directly affects the cartilage repair process. With the development of tissue engineering technology, a single material can no longer meet the ideal requirements for cartilage
defect repair. Physical and chemical modification and biomimetic treatment of two or more different materials, retaining the advantages of the material and improving its disadvantages, thereby form new materials with excellent properties. The are important for tissue regeneration. In the past 20 years, the research on the application of silk fibroin materials to tissue engineering has been carried out for the purpose of serving regenerative medicine at home and abroad [10–13]. The researches indicate that silk fibroin is a biocompatible, oxygen and water permeable, stable, low immunogenic and non-cytotoxic, versatile in processing with adequate tensile strength. These properties allow application of silk fibroin in tissue engineering [14,15]. The disadvantage of silk fibroin is the high brittleness, which makes it difficult to handle as a scaffold biomaterial, especially when implanted in load-bearing sites [16,17]. In addition, some findings show that some synthetic materials may decrease mechanical properties very early during
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Correspondence to: L.-L. Gao, Tianjin Key Laboratory for Advanced Mechatronic System Design and Intelligent Control, School of Mechanical Engineering, Tianjin University of Technology, Tianjin, China. ⁎⁎ Corresponding author. E-mail addresses: [email protected] (L.-L. Gao), [email protected] (R.-x. Li). https://doi.org/10.1016/j.msec.2019.110018 Received 27 September 2018; Received in revised form 9 February 2019; Accepted 25 July 2019 Available online 01 August 2019 0928-4931/ © 2019 Elsevier B.V. All rights reserved.
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degradation [18,19]. Retaining strength over a long time by many silk systems can be an advantage for silk, particularly in tissue engineering, where slow degradation is required. The degradation rate can be adjusted and the performance can be improved by making structural forms of silk fibroin and modifying with some biopolymers. Silk fibroin was selected as one of the building materials in this study. Articular cartilage is an avascular tissue composed of chondrocytes and extracellular matrix (ECM), which predominantly consists of collagen type II, aggrecan, and hyaluronic acid [20]. Collagen type II is one of the major components of ECM and plays a key role in maintaining chondrocyte function. According to previous reports of collagen type II as tissue engineering materials, collagen type II, which induces cell adhesion and proliferation, can initiate and maintain MSC chondrogenesis, maintain a differentiated chondrocyte phenotype, and enhance the effect of TGF β1 [21–25]. The concentration of type II collagen has an effect on the expression of TGF-β1 mRNA [26]. Matrix metalloproteinase (MMP) and A disintegrin and metallo proteinase with thrombospondin motifs (ADAMTS-4 and -5; also called aggrecanases-1 and -2) are known to play important roles in the degradation of ECM components [27–29]. During the repair process, type II collagen can be degraded by the enzyme secreted by the cells, and the product can be absorbed by the cells as a raw material for synthesizing a new matrix, and the chondrocytes are continuously stimulated to proliferate. Collagen type II, as a tissue engineering material, has a poor mechanical strength due to its rapid degradation rate, so it is often used in combination with other materials in order to exert its advantages. Collagen type II was selected as one of the building materials in this study. In summary, the two materials have their own advantages and disadvantages. The novelty of this work is to explore the possibility of repairing cartilage defects after mixing the two materials. The ultimate goal of this study was to find an effective ratio of silk fibroin/collagen II composites for post-production cartilage regeneration scaffolds. The composite membranes with different ratios of silk fibroin–collagen type II were prepared and their properties including tensile behavior, swelling and biodegradation property, biocompatibility, were investigated by tensile test, degradation test, SEM, Live/dead cell double staining, MTT assay etc.
Table 1 Experimental group.
SF50 SF70 SF90
Silk fibroin/collagen type II (50:50) Silk fibroin/collagen type II (70:30) Silk fibroin/collagen type II (90:10)
2.2. Preparation of collagen type II The fresh bladebone of bovine that was bought from supermarket (China) was cleaned carefully after removing musele tissue. The bladebone of bovine was crushed by a pulverizer and soaked in absolute ethanol for 24 h, and then was dissolved in acetic acid solution containing pepsin (Sigma-USA) for 72 h. The solution was centrifuged at 8000 r/min for 10 min at 4 °C, and the supernatant solution was neutralized with 0.5 M hydroxide sodium solution. The neutralizing solution was salted out by adding sodium chloride crystals for 2 h. The collagen floe was placed in a dialysis bag, dialyzed in deionized water for 4 days, and water was changed every 4 h. Finally the collagen after dialysis was stored at 4 °C. The mass fraction of type II collagen was measured to be 5.8% according to the above method 2.1. 2.3. Preparation of silk fibroin–collagen type II composite membrane According to the different mass ratio of dry weights of silk fibroin and type II collagen shown in Table 1, two materials were weighed and mixed at room temperature. The mixture was centrifuged at 2000 r/min for 15 min to remove the bubbles. About 2 mL of each solution was added into 6 well plates, and was freeze-dried for 36 h in a vacuum state and at −20 °C. The solution was added to a six-well plate cover at a thickness of 4 mm, and the plate covers were dried for 36 h at room temperature. The absolute ethanol was added to dried composite membrane so as to transform random coil to beta sheet structure. The freeze-dried composite membrane was sterilized by Co60 γ-ray irradiation and stored at −20 °C. The dried composite membrane at room temperature was used to test mechanical properties, so no sterilization was required.
2.1. Preparation of silk fibroin solution The 100 g natural silk that was purchased from supermarket (China), was immersed in the sodium carbonate solution (SolarbioBeijin) and boiled for 30 min. The turbid liquid was discarded after boiling, and the silk was cleaned. The silk fibroin was repeatedly boiled and cleaned twice, and then placed in a sixty degree oven to be dried at a constant temperature. A mixture of anhydrous calcium chloride, deionized water and absolute ethanol (Kermel-China) in a molar ratio of 1:8:2 was poured into a three-well bottle. The dried silk was cut into pieces and placed in a three-well bottle and stirred in a 60 °C water bath for 4 h. The silk fibroin solution was dialyzed in the dialysis bag with deionized water for 72 h. The silk fibroin solution after dialysis was concentrated with polyethylene glycol solution (Solarbio-Beijin). The supernatant of solution was collected by centrifugation at 8000 r/min for 10 min at 4 °C in a centrifuge. Finally the prepared silk fibroin solution was sealed and stored at 4 °C. Determination of silk fibroin solution concentration: The dry culture dish was weighed as M0; the appropriate amount of silk fibroin solution was added and weighed as M1. After drying in a constant temperature oven, the weight is recorded as M2. The concentration of silk fibroin solution can be calculated according to the following Eq. (1).
M2 − M0 × 100% M1 − M0
Detail
the mass fraction of silk fibroin solution,which was 7.8%.
2. Materials and methods
C=
Groups
2.4. Behavior of swelling and biodegradation The swelling behavior and biodegradation for composite membrane with different ratios were investigated. Each group of freeze-dried composite membrane was weighed as Wd and immersed in a PBS (phosphate-buffered saline) solution at room temperature for 24 h. The surface of the material was blotted with filter paper and weighed as Ww. The swelling ratios of composite freeze-dried membranes were calculated based on the Eq. (2). It is noted that three samples were tested in each group.
Rw (%) =
Ww − Wd × 100% Wd
(2)
Each group of freeze-dried composite membrane were immersed in 1 U/mL Type II collagenase buffer (pH = 7.4) at room temperature for 21 days. Then the composite membranes were washed with distilled water and freeze-dried for 36 h in a vacuum state. The freeze-dried composite membranes were weighed as Wr and the biodegradation ratios were calculated by Eq. (3). Three samples were tested in each group considering random error.
(1)
Rd (%) =
After repeating for three times, the average value was calculated as 2
Wd − Wr × 100% Wd
(3)
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were conducted at given stress levels for SF70 samples. Based on the obtained tensile strength, the creep stress levels of 1, 1.5 and 2 MPa were chosen and applied on SF70 samples at room temperature respectively. The creep time was 30 min. During the creep experiment, the loading test machine recorded the experimental data at intervals of 0.1 s. In order to ensure the wetness of the sample, the PBS was sprayed on the sample during the test with a watering.
2.6. The biocompatibility experiment of composite membrane The sterilized composite membrane was immersed in DMEM/F-12 (1:1) complete medium (containing 10% fetal bovine serum and 1% penicillin-Streptomycin Solution) for 24 h in GIBCO, USA. The moisture of the composite membrane was then blotted with sterile filter paper. The ADTC-5 chondrocytes (7.2 × 105) were seeded on each composite membrane. The composite membranes were incubated for 4 h at 37 °C in a humidified 5% CO2 and added DMEM/F-12 (1:1) complete medium. The composite membranes after being seeded with ADTC-5 chondrocytes were cultured for 1 day and 3 days.
Fig. 1. Tensile specimen.
2.6.1. Live/dead cell double staining Live/dead cell double staining can be utilized for simultaneous fluorescence staining of viable and dead cells. The composite membranes seeded with chondrocytes, which were statically cultured for 1 day and 3 days, were gently washed 2–3 times with PBS. Then, a sufficient amount of calcein working solution (2μM calcein AM, 8μM PI) was added to submerge the composite membrane. After incubation for 30–45 min at room temperature, the staining solution was aspirated to stop the treatment. The composite membrane was placed on a clean glass slide, covered with a cover glass, and the cells on the composite membrane were observed under a fluorescence microscope.
2.6.2. Surface properties of composite membranes and chondrocyte morphology under SEM Morphology and attachment of ADTC-5 chondrocyte on the composite membranes were observed by scanning electron microscopy (EM30 Plus, COXEM, Korea). Each composite was washed with PBS and fixed using 2.5% glutaraldehyde (Sigma-Aldrich, USA) and 1% osmium acid (H2(OSO4(OH)2)) at 4 °C before dehydration. Dehydration of membranes was processed by changing the graded series of ethanol (50, 60, 70, 80, 90, 100%) for 20 min each and all samples were dried at the room temperature. The membranes were sputtered with gold before SEM examination.
Fig. 2. The pneumatic chuck and sample.
Table 2 The fitting values of material constant. Material constant
α1
α2
α3
β1
β2
β3
Fitting value
−2.144
30.275
−10.467
6.921
−3.613
0.802
2.6.3. Cell proliferation assay (MTT) The proliferation and viability of cultured chondrocyte was assessed by using MTT (3-(4,-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide; thiazolyl blue, Sigma, USA) assay. The cultured media after 1 and 3 days of culture was removed and added 2 mL MTT solution (5 mg/mL in PBS). Samples were incubated for 4 h at 37 °C in a humidified 5% CO2 for formazan crystal formation. After incubation, the supernatant was removed and formazan crystals were dissolved by adding 2 mL of DMSO and dissolved for 10 min on a shaker. The dissolved solution was moved into 96-well plate and measured at the wavelength of 490 nm by using a microplate reader (Thermo Inc.).
2.5. Mechanical experiments of composite membrane The Instron S5800 universal experimental testing machine was applied to perform the mechanical experiments of composite membrane. The dried composite membranes were made in a dimension of about 30 mm in length, 10 mm in width and 0.2 mm in thickness by using the manual paper cutter, as shown in Fig. 1. The specimen was clamped by the pneumatic chuck of testing machine as shown in Fig. 2. The mechanical tests can be divided into three parts. Firstly the tensile fracture tests for the composite membrane specimens with different ratios were carried out with loading rate of 30 mm/min at room temperature. For each condition three samples were tested considering random error. According to the experimental data, the Young's modulus, tensile strength and ultimate strain of all samples were analyzed. Secondly, the tensile tests for SF70 specimens were carried out with different stress rates such as 0.01 N/s, 0.1 N/s, 0.5 N/s so as to investigate the rate-dependent tensile property. Finally, the creep tests
2.7. Statistical analysis SPSS18.0 was used to evaluate the significant differences among the three groups. Data were presented as mean ± standard error. In all cases, the results were considered statistically different at p < 0.05(*). 3
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Fig. 3. The macrostructure of freeze-dried membranes with different ratios.
Fig. 4. The surface structure of freeze-dried composite membranes under SEM.
Fig. 5. Swelling percentage of composite membranes(n = 3, P < 0.05(*)). t
3. Theoretical model
ε(t) =
∫ C1 (t − t1 ) σ˙ (t1 ) dt1 0
3.1. Derivation of constitutive model for composite membranes
t
+
The composite membrane was described by the following nonlinear viscoelastic constitutive equation for uniaxial tensile loading. In the 1965, Onaran and Findley [30] proposed a broadly representative onedimensional triple integral nonlinear constitutive relation for multiaxial loads, as shown in the following.
0 t
+
t
∫ ∫ C2 (t − t1, t − t2 ) σ˙ (t1 ) σ˙ (t2 ) dt1 dt2 0 t
t
∫ ∫ ∫ C3 (t − t1 , t − t2 , t − t3 )σ˙ (t1 ) σ˙ (t2 ) σ˙ (t3 ) dt1 dt2 dt3 0
0
0
(4)
where the C1, C2 and C3 are creep function of material, ε is the tensile ∂σ strain, t is the tensile time, σ is the tensile stress and σ̇ (t ) = ∂t . 4
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Fig. 6. Degradation percentage of composite membranes(n = 3, P < 0.05(*)).
Eq. (4), the constitutive equation is obtained under uniaxial tensile loading. t
ε(t) = ∫ [α1 + β1 (t − t1 )n] σ˙ (t1 ) dt1 0 t
t
+ ∫ ∫ [α2 + β2 (t − t1 )n /2 (t − t2 )n /2] σ˙ (t1 ) σ˙ (t2 ) dt1 dt2 0 0 t t t
+ ∫ ∫ ∫ [α3 + β3 (t − t1 )n /3 (t − t2 )n /3 (t − t3 )n /3] σ˙ (t1 ) σ˙ (t2 ) σ˙ (t3 ) dt1 dt2 dt3 0 0 0
(10) Based on the constitutive relation Eq. (10), considering that the uniaxial tensile test is controlled by the loading rate, the tensile stress can be expressed as
σ(t) = σ̇ ·t
(11)
By solving the integral of Eq. (10), we obtained
˙ ) + α2 (σt ˙ )2 + α3 (σt ˙ )3] + ε(t) = [α1 (σt Fig. 7. Uniaxial tensile curves of composite membranes with different ratios.
˙ )3 ⎤ 27β3 (σt ˙ ) ˙ )2 4β (σt ⎡ β1 (σt + 2 + × tn 3 ⎥ 2 ⎢n + 1 ( n + 2) ( n + 3) ⎣ ⎦
For creep deformation under constant stress, Eq. (4) can be described as follows.
ε(t) = C1 (t ) σ + C2 (t ) σ 2 + C3 (t ) σ 3
3.2. Determination of parameters
(5)
Based on Eq. (6), the parameters ε0,εt and n are evaluated by the creep tests of composite membranes with different constant stresses at room temperature. The parameter n is fitted as 0.13. The α1, α2, α3 and β1, β2, β3 are determined by Eqs. (8) and (9). The least squares regression is used to obtain the values of the parameters. The fitting values are shown in Table 2. Based on the built constitutive model, the creep behaviors and tensile properties of composite membrane can be predicted.
The Eq. (5) is the nonlinear creep constitutive relation. The creep strain ε(t) at constant stress σ is supposed to satisfy the power law relation [31], as follows in Eq. (6).
ε(t) = ε0 + εt t n
(6)
where the ε0 is the initial creep strain that is related to stress, εt is the nominal instantaneous creep strain that is related to stress and temperature, n is the creep parameter being related to temperature. In order to be consistent with the power law relation, the creep function C1, C2 and C3 can be expressed as:
C1 (t ) = α1 + β1
tn
C2 (t ) = α2 + β2
tn
C3 (t ) = α3 + β3
tn
4. Results and discussions
(7)
4.1. Physical property of composite membranes
By substituting Eq. (7) into Eq. (5) and comparing with Eq. (6), the following relationship is obtained
ε0 = α1 σ + α2 σ 2 + α3 σ 3
(8)
εt = β1 σ + β2 σ 2 + β3 σ 3
(9)
(12)
4.1.1. Characterization of composite membranes Fig. 3 shows the macrostructure of freeze-dried composite membranes with different ratios. It is found that the composite membranes present a white sponge structure. The sample is hard and brittle after freeze-dried. However the sample becomes soft and its elasticity is good after soaking. Fig. 4 shows the surface properties of the composite membranes
where α1, α2, α3 are the material constants, β1, β2, β3 are the material parameters that are related to temperature. By substituting Eq. (7) into 5
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Fig. 8. Effect of mix proportion on the tensile mechanical properties. (a) Young's modulus; (b) ultimate strain; (c) tensile strength (n = 3, P < 0.05(*)).
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4.1.3. Degradation of composite membranes The degradation of composite membranes with different ratios was evaluated in vitro and Fig. 6 shows the percentage of degradation. The results demonstrate that the degradation percentage of composite membrane reduces with content of type II collagen decreasing. It is noted that the degradation percentage of SF50 composite membrane reaches 46.2%, the degradation percentage of SF70 composite membrane reaches 25.3%, and however the degradation percentage of SF90 is only 11.6%. Type II collagenase degradation fluid mainly simulates the degradation of extracellular matrix by enzymes in the body. According to an experimental study of previous type II collagen repair of knee articular cartilage defects, neonatal cartilage tissue appeared four weeks after surgery [34]. In our degradation experiments for 21 days, it can be found that the type II collagen is degraded, and the silk fibroin powder appears around the composite membrane. The results show that SF50 composite membrane almost degraded half of itself. Its mechanical properties may be lost during the early stages of degradation. When the material ratio of SF90 is used in tissue engineering scaffolds, it may affect the repairing effect due to the slower degradation. The rate of cartilage scaffold degradation is very important for its application in cartilage repair.
Fig. 9. Creep strain of SF70 composite membrane with different stress levels.
under SEM. It is found that the fusion performance of silk fibroin and collagen type II for SF70 composite membrane is better than that of SF50 and SF90 composite membranes. There is no collagen fiber on the surface of SF70 composite membrane, and the surface is relatively flat. There are many collagen fibers on the surface of SF50 and SF90 composite membranes, and their surfaces are rough. This indicates that when the ratio of silk fibroin and collagen type II is 7:3, the fusion of the two materials is better.
4.2. Mechanical behaviors for composite membranes 4.2.1. Tensile mechanical properties of composite membranes with different ratios The tensile mechanical properties for composite membranes with different ratios were investigated with loading rate of 30 mm/min and Fig. 7 shows the stress-strain curves of composite membranes with different ratios. It is found that the stress-strain curves are not coincident, which means that there are much differences about the mechanical properties of composite membranes with different ratios. Fig. 8 shows the Young's modulus, ultimate strain and tensile strength of composite membranes with different ratios at loading rate of 30 mm/min. The Young's modulus of composite membranes increases obviously with decrease of the col-II and the nearly 2.5 times difference between the SF50 and SF90 is observed as shown in Fig. 8(a). In contrast, the ultimate strain and tensile strength of composite membranes decrease with decrease of the col. II. The ultimate strain and tensile strength value of SF90 are the smallest. According to Dong et al., [35] the tensile strength of articular cartilage was 4.75 MPa, and the ultimate strain was 1860 με. The tensile strength of cartilage is similar to that of composite membranes, but the ultimate strain varies greatly. This is because the composite material mainly plays the role of support when it is used as a scaffold. It is only the transition stage material. The mechanical properties of the repaired cartilage tissue will be compared with the mechanical properties of the host cartilage. It can be concluded
4.1.2. Swelling behavior of composite membranes The water adsorption ability of material is related to prevention of losing body fluid and nutrients after transplantation in vivo [32]. Swelling behavior is also selected to evaluate the physical stability of composite membrane to be similar to the hydrogel characteristics of cartilage tissue [33]. Fig. 5 shows the remarkable water adsorption capacity for all membranes. The swelling percentage of the composite membrane decreases as the percentage of type II collagen reduces (SF50:1447%, SF70:1235%, SF90: 967.6%). This comes from the hydrophilicity of type II collagen which can maintain a high amount of water. The SF90 composite membrane shows the lowest swelling percentage while SF50 composite membrane has the highest swelling percentage. In general, the greater the percentage of swelling of the material after soaking, the greater the deformation that occurs and the lower the stability of the material. The results also indicate that the composite membrane with a high amount of type II collagen has lower physical stability.
Fig. 10. The tensile mechanical properties of SF70 at different stress rates. (a) 0.01 N/s; (b) 0.1 N/s; (c) 0.5 N/s. 7
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Fig. 11. Live/dead cell double staining of composite membranes for 3 days of culture.
Fig. 12. Cell proliferation of chondrocytes on composite membranes (n = 3, P < 0.05(*)).
Fig. 13. The morphology and attachment of chondrocytes on SF70 composite membrane. (a) The morphology of multiple chondrocytes. (b) The morphology of single chondrocyte.
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4.3.3. The morphology and attachment of chondrocytes under SEM The morphology and attachment of chondrocytes on composite membrane with different ratios were observed by scanning electron microscopy. Surprisingly, it is found that chondrocytes are observed only on the SF70 composite membrane under scanning electron microscopy as shown in Fig. 13. No clear cell morphology is found on the surface of the SF50 and SF90 composite membranes. Fig. 13(a) shows the different morphologies of chondrocytes on the SF70 composite membranes, indicating that the proliferation, division and growth of chondrocytes on the SF70 composite membrane are better. Fig. 13(b) shows a single attachment chondrocyte on the SF70 composite membrane and the chondrocytes' antennae stretches sufficiently and the growth state is good.
that the tensile mechanical properties of SF70 are more stable. 4.2.2. Tensile mechanical behaviors of SF70 composite membrane Since SF70 composite membrane shows more stable tensile properties in 4.2.1, the tensile mechanical properties of SF70 with different stress levels and stress rates are further investigated. Fig. 9 shows the creep behaviors of SF70 composite membrane with different tensile stress levels. It is found that the initial strain and instantaneous strain increase with the increase of tensile stress. Within creep time of 100 s, the creep strain increases fast, and then increases slowly as the creep time goes by. In order to further probe the creep behavior of SF70, the creep strains with different stress levels have also been predicted by the built theoretical model. Fig. 9 demonstrates the comparisons of experimental results and predictions. It is noted that there are good agreements between them. The tensile mechanical behaviors for SF70 composite membrane were investigated with different stress rates and Fig. 10 shows the relationships of stress and strain with different stress rates. It is found that the stress-strain curves are not coincident with different stress rates, and stress rate makes very remarkable influence on the tensile mechanical behaviors of SF70. The reason is that the rate of submolecule motion within the sample is smaller than the rate of employing force during a certain interval considering the viscoelastic characteristic of collagen fiber. The stress-strain behaviors of SF70 with different stress rates have also been predicted by the constitutive model (Eq. (11)) and the predictions agree with the experimental results as shown in Fig. 10. Articular cartilage exhibits a viscoelastic response to load, and creep and rate-related behavior is a typical mechanical response of cartilage to load. The SF70 composite membrane also showed a similar mechanical response.
5. Conclusion In this research, the composite membranes of silk fibroin/collagen type II with different ratios were constructed, and their mechanical property and biocompatibility were investigated. The following conclusions can be obtained: 1. There is no dissociative collagen fiber on the surface of SF70 composite membrane, and the surface is relatively flat. The fusion of silk fibroin/collagen type II within SF70 composite membrane is better. The SF70 composite membrane has a suitable swelling ratio and degradation rate, and the its physical performance is more stable. 2. Tensile mechanical properties for composite membranes of different ratios were compared and it is found the tensile mechanical properties of SF70 are more stable. The tensile mechanical behavior of SF70 composite membrane is dependent on stress rate. Its creep strain increases with increase of stress level. The constitutive model was established and applied to predict the creep strain and tensile property of SF70 composite membrane. It is noted that the experimental results and predictions are in good agreement. 3. The biocompatibility of three kinds of composite membranes was investigated. All composite membranes are non-toxic and biocompatible. In particular, the SF70 composite membrane has better proliferation ability. To sum up, the mixing ratio of silk fibroin and collagen type II 7:3 shows promise in the future as a scaffold material for cartilage repair.
4.3. Biocompatibility testing of composite membranes 4.3.1. Live/dead cell double staining The treatment of live/dead double staining was performed to assess the viability of chondrocytes on the composite membranes (SF50, SF70, SF90) after 3 days of culture. Fig. 11 shows the staining results observed with a fluorescence microscope. It is found that the live chondrocytes abound on three kinds of membranes, and the red fluorescence of all composite membranes is almost absent, which means that all three kinds of composite membranes have good cell proliferation. It is also noted that the green fluorescence on SF50 and SF90 composite membranes is not clearly observed by adjusting the focal length of the microscope. This is because the surfaces of the SF50 and SF90 composite membranes are rough, as shown in Fig. 4. However the live chondrocytes are most and evenly distributed on the SF70 composite membrane.
Declaration of competing interest There are no conflicts of interest for either author. Acknowledgments This study was sponsored by the National Natural Science Foundation of China (Nos. 11572222, 51571150, 11672208, 11432016).
4.3.2. Proliferation of chondrocytes on composite membranes The cell proliferation ability on the scaffold is a critical factor to support and guide regeneration of new tissue after transplantation. MTT testing was performed to investigate the cell proliferation of composite membranes with different ratios. The chondrocytes proliferation was analyzed after 1 day and 3 days of culture to evaluate the biological performance of composite membranes with different ratios as shown in Fig. 12. The results show that the chondrocytes on all membranes increase over culture time by comparing the absorbance values with day 1 and 3. There is no obvious difference for chondrocytes proliferation on all composite membranes at day 1. However at day 3, there is higher proliferation on the SF70 composite membranes than the others. This is consistent with the results of live/dead cell double staining. The influence of material structural characteristics on cell proliferation and migration has been reported previously [36]. Proliferation results demonstrate that SF70 composite membrane has better structural characteristics.
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msc chondrogenesis, Biotechnol. Bioeng. 93 (2010) 1152-1163. [22] P.D. Benya, S.R. Padilla, M.E. Nimni, Independent regulation of collagen types by chondrocytes during the loss of differentiated function in culture, Cell. 15 (1978) 1313–1321. [23] D. Girotto, S. Urbani, P. Brun, D. Renier, R. Barbucci, G. Abatangelo, Tissue-specific gene expression in chondrocytes grown on three-dimensional hyaluronic acid scaffolds, Biomaterials. 24 (2003) 3265–3275. [24] L.A. Solchaga, J.S. Temenoff, J. Gao, A.G. Mikos, A.I. Caplan, V.M. Goldberg, Repair of osteochondral defects with hyaluronan- and polyester-based scaffolds, Osteoarthr. Cartilage. 13 (2005) 297–309. [25] Y.H. Jeon, J.H. Choi, J.K. Sung, T.K. Kim, B.C. Cho, H.Y. Chung, Different effects of PLGA and chitosan scaffolds on human cartilage tissue engineering, J. Craniofac. Surg. 18 (2007) 1249–1258. [26] W.N. Qi, S.P. Scully, Extracellular collagen regulates expression of transforming growth factor-β1 gene, J. Orthop. Res. 18 (2000) 928–932. [27] K. Sugimoto, T. Iizawa, H. Harada, K. Yamada, M. Katsumata, M. Takahashi, Cartilage degradation independent of MMP/aggrecanases, Osteoarthr. Cartilage. 12 (2004) 1006–1014. [28] J.S. Mort, C.J. Billington, Articular cartilage and changes in arthritis: matrix degradation, Arthritis. Res. Ther. 3 (2001) 337–341. [29] J.M. Milner, T.E. Cawston, Matrix metalloproteinase knockout studies and the potential use of matrix metalloproteinase inhibitors in the rheumatic diseases, Curr. Drug. Targets: Inflammation. Allergy. 4 (2005) 363–375. [30] K. Onaran, W.N. Findley, Combined stress-creep experiments on a nonlinear viscoelastic material to determine the kernel functions for a multiple integral representation of creep, J. Rheol. 9 (1965) 299–327. [31] W.N. Findley, J.S. Lai, K. Onaran, Creep and relaxation of nonlinear viscoelastic materials, Int. J. Appl. Electrom. 44 (1976) 505–509. [32] J. Liao, Y. Qu, B. Chu, X. Zhang, Z. Qian, Biodegradable CSMA/PECA/graphene porous hybrid scaffold for cartilage tissue engineering, Sci. Rep-UK. 5 (2015) 9879. [33] R. Jin, L.M. Teixeira, P.J. Dijkstra, M. Karperien, C.A. Van Blitterswijk, Z.Y. Zhong, et al., Injectable chitosan-based hydrogels for cartilage tissue engineering, Bionaterials. 30 (2009) 2544–2551. [34] S.M. Li, X.H. Yang, L. Fang, C.T. Ye, X.H. Li, P.H. Liang, Experimental study on repairing rabbit knee articular cartilage defects with high purity porcine cartilage type II collagen, Chin J Orthop Trauma 10 (2008) 844–849. [35] Q.Z. Dong, Y.J. Wang, Biomechanical properties of articular cartilage, Suzhou University Journal of Medical Science. 19 (1999) 244–246. [36] M. Chanasakulniyom, A. Glidle, J.M. Cooper, Cell proliferation and migration inside single cell arrays, Lab Chip 15 (2015) 208–215.
[7] L. Da, X. Zhang, X. Hu, C. Zhou, Y. Ao, Silencing of microRNA-101 prevents il-1βinduced extracellular matrix degradation in chondrocytes, Arthritis. Res. Ther. 14 (2012) R268. [8] R.J. Lories, F.P. Luyten, The bone–cartilage unit in osteoarthritis, Nat. Publ. Group 7 (2010) 43–49. [9] R.B. Jakobsen, L. Engebretsen, J.R. Slauterbeck, An analysis of the quality of cartilage repair studies, J. Bone Joint Surg. Am. 87 (2005) 2232–2239. [10] C. Foss, E. Merzari, C. Migliaresi, A. Motta, Silk fibroin/hyaluronic acid 3d matrices for cartilage tissue engineering, Biomacromolecules. 14 (2013) 38–47. [11] S. Aliramaji, A. Zamanian, M. Mozafari, Super-paramagnetic responsive silk fibroin/chitosan/magnetite scaffolds with tunable pore structures for bone tissue engineering applications, Mater. Sci. Eng., C-Mater. 70 (2017) 736–744. [12] D.W. Lia, X. Lei, F.L. He, J. He, Y.L. Liu, Y.J. Ye, et al., Silk fibroin/chitosan scaffold with tunable properties and low inflammatory response assists the differentiation of bone marrow mesenchymal stem cells, Int. J. Biol. Macomol. 105 (2017) 584–597. [13] D.W. Li, F.L. He, J. He, X. Deng, Y.L. Liu, Y.Y. Liu, From 2d to 3d: the morphology, proliferation and differentiation of mc3t3-e1 on silk fibroin/chitosan matrices, Carbohyd. Polym. 178 (2017) 69–77. [14] J. Wang, Y. Wei, H. Yi, Z. Liu, D. Sun, H. Zhao, Cytocompatibility of a silk fibroin tubular scaffold, Mater. Sci. Eng. C. 34 (2014) 429–436. [15] A.M. Ghaznavi, L.E. Kokai, M.L. Lovett, D.L. Kaplan, K.G. Marra, Silk fibroin conduits: a cellular and functional assessment of peripheral nerve repair, Aesthet. Plast. Surg. 66 (2011) 273–279. [16] W. Luangbudnark, J. Viyoch, W. Laupattarakasem, P. Surakunprapha, P. Laupattarakasem, Properties and biocompatibility of chitosan and silk fibroin blend films for application in skin tissue engineering, The. Scientific. World. J. 2012 (2012) 1–10. [17] N. Bhardwaj, S.C. Kundu, Silk fibroin protein and chitosan polyelectrolyte complex porous scaffolds for tissue engineering applications, Carbohyd. Polym. 85 (2011) 325–333. [18] L.S. Nair, C.T. Laurencin, Biodegradable polymers as biomaterials, Prog. Polym. Sci. 32 (2007) 762–798. [19] P.A. Gunatillake, R. Adhikari, Biodegradable synthetic polymers for tissue engineering, Eur. Cells. Mater. 5 (2003) 1–16. [20] K. Nakashima, K. Nakatsuka, K. Yamashita, K. Kenichi, H. aro, An in vitro model of cartilage degradation by chondrocytes in a three-dimensional culture system, Int. J. Biomed. Sci. 8 (2012) 249–257. [21] D. Bosnakovski, M. Mizuno, G. Kim, S. Takagi, M. Okumura, T. Fujinaga, Chondrogenic differentiation of bovine bone marrow mesenchymal stem cells (MSCs) in different hydrogels: influence of collagen type II extracellular matrix on
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Materials Science & Engineering C journal homepage: www.elsevier.com/locate/msec
Mechanical property and biocompatibility of silk fibroin–collagen type II composite membrane ⁎
T
⁎⁎
Xiang-Long Lina,b,d, Li-Lan Gaoa,b, , Rui-xin Lic, , Wei Chengd, Chun-Qiu Zhanga,b, Xi-zheng Zhangd a Tianjin Key Laboratory for Advanced Mechatronic System Design and Intelligent Control, School of Mechanical Engineering, Tianjin University of Technology, Tianjin, China b National Demonstration Center for Experimental Mechanical and Electrical Engineering Education (Tianjin University of Technology), Tianjin, China c Tianjin Stomatological Hospital, Tianjin, China d Institute of Medical Equipment, Academy of Military Medical Science, Tianjin 300161, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Silk fibroin–collagen type II Different ratio Composite membrane Mechanical behavior Theoretical model Biocompatibility
Osteoarthritis is caused by injuries and cartilage degeneration. Cartilage tissue engineering provides new ideas for the treatment of osteoarthritis. Herein, the different ratios composite membranes of silk fibroin/collagen type II were constructed (SF50-50:50, SF70-70:30, SF90-90:10). The surface properties of the composite membranes and chondrocyte morphology were observed by SEM (scanning electron microscopy). Physical functionality as well as stability of composite membranes was evaluated from tensile mechanical properties, the percentage of swelling and degradation. The tensile mechanical behavior of SF70 composite membranes was also predicted based on the constitutive model established in this study, and it is found that the experimental results and predictions were in good agreement. Biocompatibility was evaluated using chondrocytes (ADTC-5) culture. Cell proliferation was analyzed and the treatment of live/dead double staining was performed to assess the viability on chondrocytes. To sum up, SF70 showed the suitable morphology, physical stability, and biological functionality to promote proliferation of chondrocytes. This indicates that the mixing ratio of SF70 shows promise in the future as a scaffold material for cartilage repair.
1. Introduction More than two centuries ago, some scholars discovered that once articular cartilage is damaged, the ability to repair itself is extremely poor [1]. This is due to the avascular structure of articular cartilage, which has limited self-repairing ability and intrinsic healing ability [2–7]. Damage of articular cartilage causes joint pain, functional impairment and osteoarthritis (OA) [3,8]. Currently, many attempts are conducted to address the problem of articular cartilage repair such as autologous osteochondral graft transplantation, but the effects are not good [9]. Therefore, based on the method of tissue engineering, the construction of biomaterials at the defect area properly to regenerate new tissue is a promising approach for articular cartilage repair. The performance of biological materials directly affects the cartilage repair process. With the development of tissue engineering technology, a single material can no longer meet the ideal requirements for cartilage
defect repair. Physical and chemical modification and biomimetic treatment of two or more different materials, retaining the advantages of the material and improving its disadvantages, thereby form new materials with excellent properties. The are important for tissue regeneration. In the past 20 years, the research on the application of silk fibroin materials to tissue engineering has been carried out for the purpose of serving regenerative medicine at home and abroad [10–13]. The researches indicate that silk fibroin is a biocompatible, oxygen and water permeable, stable, low immunogenic and non-cytotoxic, versatile in processing with adequate tensile strength. These properties allow application of silk fibroin in tissue engineering [14,15]. The disadvantage of silk fibroin is the high brittleness, which makes it difficult to handle as a scaffold biomaterial, especially when implanted in load-bearing sites [16,17]. In addition, some findings show that some synthetic materials may decrease mechanical properties very early during
⁎
Correspondence to: L.-L. Gao, Tianjin Key Laboratory for Advanced Mechatronic System Design and Intelligent Control, School of Mechanical Engineering, Tianjin University of Technology, Tianjin, China. ⁎⁎ Corresponding author. E-mail addresses: [email protected] (L.-L. Gao), [email protected] (R.-x. Li). https://doi.org/10.1016/j.msec.2019.110018 Received 27 September 2018; Received in revised form 9 February 2019; Accepted 25 July 2019 Available online 01 August 2019 0928-4931/ © 2019 Elsevier B.V. All rights reserved.
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degradation [18,19]. Retaining strength over a long time by many silk systems can be an advantage for silk, particularly in tissue engineering, where slow degradation is required. The degradation rate can be adjusted and the performance can be improved by making structural forms of silk fibroin and modifying with some biopolymers. Silk fibroin was selected as one of the building materials in this study. Articular cartilage is an avascular tissue composed of chondrocytes and extracellular matrix (ECM), which predominantly consists of collagen type II, aggrecan, and hyaluronic acid [20]. Collagen type II is one of the major components of ECM and plays a key role in maintaining chondrocyte function. According to previous reports of collagen type II as tissue engineering materials, collagen type II, which induces cell adhesion and proliferation, can initiate and maintain MSC chondrogenesis, maintain a differentiated chondrocyte phenotype, and enhance the effect of TGF β1 [21–25]. The concentration of type II collagen has an effect on the expression of TGF-β1 mRNA [26]. Matrix metalloproteinase (MMP) and A disintegrin and metallo proteinase with thrombospondin motifs (ADAMTS-4 and -5; also called aggrecanases-1 and -2) are known to play important roles in the degradation of ECM components [27–29]. During the repair process, type II collagen can be degraded by the enzyme secreted by the cells, and the product can be absorbed by the cells as a raw material for synthesizing a new matrix, and the chondrocytes are continuously stimulated to proliferate. Collagen type II, as a tissue engineering material, has a poor mechanical strength due to its rapid degradation rate, so it is often used in combination with other materials in order to exert its advantages. Collagen type II was selected as one of the building materials in this study. In summary, the two materials have their own advantages and disadvantages. The novelty of this work is to explore the possibility of repairing cartilage defects after mixing the two materials. The ultimate goal of this study was to find an effective ratio of silk fibroin/collagen II composites for post-production cartilage regeneration scaffolds. The composite membranes with different ratios of silk fibroin–collagen type II were prepared and their properties including tensile behavior, swelling and biodegradation property, biocompatibility, were investigated by tensile test, degradation test, SEM, Live/dead cell double staining, MTT assay etc.
Table 1 Experimental group.
SF50 SF70 SF90
Silk fibroin/collagen type II (50:50) Silk fibroin/collagen type II (70:30) Silk fibroin/collagen type II (90:10)
2.2. Preparation of collagen type II The fresh bladebone of bovine that was bought from supermarket (China) was cleaned carefully after removing musele tissue. The bladebone of bovine was crushed by a pulverizer and soaked in absolute ethanol for 24 h, and then was dissolved in acetic acid solution containing pepsin (Sigma-USA) for 72 h. The solution was centrifuged at 8000 r/min for 10 min at 4 °C, and the supernatant solution was neutralized with 0.5 M hydroxide sodium solution. The neutralizing solution was salted out by adding sodium chloride crystals for 2 h. The collagen floe was placed in a dialysis bag, dialyzed in deionized water for 4 days, and water was changed every 4 h. Finally the collagen after dialysis was stored at 4 °C. The mass fraction of type II collagen was measured to be 5.8% according to the above method 2.1. 2.3. Preparation of silk fibroin–collagen type II composite membrane According to the different mass ratio of dry weights of silk fibroin and type II collagen shown in Table 1, two materials were weighed and mixed at room temperature. The mixture was centrifuged at 2000 r/min for 15 min to remove the bubbles. About 2 mL of each solution was added into 6 well plates, and was freeze-dried for 36 h in a vacuum state and at −20 °C. The solution was added to a six-well plate cover at a thickness of 4 mm, and the plate covers were dried for 36 h at room temperature. The absolute ethanol was added to dried composite membrane so as to transform random coil to beta sheet structure. The freeze-dried composite membrane was sterilized by Co60 γ-ray irradiation and stored at −20 °C. The dried composite membrane at room temperature was used to test mechanical properties, so no sterilization was required.
2.1. Preparation of silk fibroin solution The 100 g natural silk that was purchased from supermarket (China), was immersed in the sodium carbonate solution (SolarbioBeijin) and boiled for 30 min. The turbid liquid was discarded after boiling, and the silk was cleaned. The silk fibroin was repeatedly boiled and cleaned twice, and then placed in a sixty degree oven to be dried at a constant temperature. A mixture of anhydrous calcium chloride, deionized water and absolute ethanol (Kermel-China) in a molar ratio of 1:8:2 was poured into a three-well bottle. The dried silk was cut into pieces and placed in a three-well bottle and stirred in a 60 °C water bath for 4 h. The silk fibroin solution was dialyzed in the dialysis bag with deionized water for 72 h. The silk fibroin solution after dialysis was concentrated with polyethylene glycol solution (Solarbio-Beijin). The supernatant of solution was collected by centrifugation at 8000 r/min for 10 min at 4 °C in a centrifuge. Finally the prepared silk fibroin solution was sealed and stored at 4 °C. Determination of silk fibroin solution concentration: The dry culture dish was weighed as M0; the appropriate amount of silk fibroin solution was added and weighed as M1. After drying in a constant temperature oven, the weight is recorded as M2. The concentration of silk fibroin solution can be calculated according to the following Eq. (1).
M2 − M0 × 100% M1 − M0
Detail
the mass fraction of silk fibroin solution,which was 7.8%.
2. Materials and methods
C=
Groups
2.4. Behavior of swelling and biodegradation The swelling behavior and biodegradation for composite membrane with different ratios were investigated. Each group of freeze-dried composite membrane was weighed as Wd and immersed in a PBS (phosphate-buffered saline) solution at room temperature for 24 h. The surface of the material was blotted with filter paper and weighed as Ww. The swelling ratios of composite freeze-dried membranes were calculated based on the Eq. (2). It is noted that three samples were tested in each group.
Rw (%) =
Ww − Wd × 100% Wd
(2)
Each group of freeze-dried composite membrane were immersed in 1 U/mL Type II collagenase buffer (pH = 7.4) at room temperature for 21 days. Then the composite membranes were washed with distilled water and freeze-dried for 36 h in a vacuum state. The freeze-dried composite membranes were weighed as Wr and the biodegradation ratios were calculated by Eq. (3). Three samples were tested in each group considering random error.
(1)
Rd (%) =
After repeating for three times, the average value was calculated as 2
Wd − Wr × 100% Wd
(3)
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were conducted at given stress levels for SF70 samples. Based on the obtained tensile strength, the creep stress levels of 1, 1.5 and 2 MPa were chosen and applied on SF70 samples at room temperature respectively. The creep time was 30 min. During the creep experiment, the loading test machine recorded the experimental data at intervals of 0.1 s. In order to ensure the wetness of the sample, the PBS was sprayed on the sample during the test with a watering.
2.6. The biocompatibility experiment of composite membrane The sterilized composite membrane was immersed in DMEM/F-12 (1:1) complete medium (containing 10% fetal bovine serum and 1% penicillin-Streptomycin Solution) for 24 h in GIBCO, USA. The moisture of the composite membrane was then blotted with sterile filter paper. The ADTC-5 chondrocytes (7.2 × 105) were seeded on each composite membrane. The composite membranes were incubated for 4 h at 37 °C in a humidified 5% CO2 and added DMEM/F-12 (1:1) complete medium. The composite membranes after being seeded with ADTC-5 chondrocytes were cultured for 1 day and 3 days.
Fig. 1. Tensile specimen.
2.6.1. Live/dead cell double staining Live/dead cell double staining can be utilized for simultaneous fluorescence staining of viable and dead cells. The composite membranes seeded with chondrocytes, which were statically cultured for 1 day and 3 days, were gently washed 2–3 times with PBS. Then, a sufficient amount of calcein working solution (2μM calcein AM, 8μM PI) was added to submerge the composite membrane. After incubation for 30–45 min at room temperature, the staining solution was aspirated to stop the treatment. The composite membrane was placed on a clean glass slide, covered with a cover glass, and the cells on the composite membrane were observed under a fluorescence microscope.
2.6.2. Surface properties of composite membranes and chondrocyte morphology under SEM Morphology and attachment of ADTC-5 chondrocyte on the composite membranes were observed by scanning electron microscopy (EM30 Plus, COXEM, Korea). Each composite was washed with PBS and fixed using 2.5% glutaraldehyde (Sigma-Aldrich, USA) and 1% osmium acid (H2(OSO4(OH)2)) at 4 °C before dehydration. Dehydration of membranes was processed by changing the graded series of ethanol (50, 60, 70, 80, 90, 100%) for 20 min each and all samples were dried at the room temperature. The membranes were sputtered with gold before SEM examination.
Fig. 2. The pneumatic chuck and sample.
Table 2 The fitting values of material constant. Material constant
α1
α2
α3
β1
β2
β3
Fitting value
−2.144
30.275
−10.467
6.921
−3.613
0.802
2.6.3. Cell proliferation assay (MTT) The proliferation and viability of cultured chondrocyte was assessed by using MTT (3-(4,-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide; thiazolyl blue, Sigma, USA) assay. The cultured media after 1 and 3 days of culture was removed and added 2 mL MTT solution (5 mg/mL in PBS). Samples were incubated for 4 h at 37 °C in a humidified 5% CO2 for formazan crystal formation. After incubation, the supernatant was removed and formazan crystals were dissolved by adding 2 mL of DMSO and dissolved for 10 min on a shaker. The dissolved solution was moved into 96-well plate and measured at the wavelength of 490 nm by using a microplate reader (Thermo Inc.).
2.5. Mechanical experiments of composite membrane The Instron S5800 universal experimental testing machine was applied to perform the mechanical experiments of composite membrane. The dried composite membranes were made in a dimension of about 30 mm in length, 10 mm in width and 0.2 mm in thickness by using the manual paper cutter, as shown in Fig. 1. The specimen was clamped by the pneumatic chuck of testing machine as shown in Fig. 2. The mechanical tests can be divided into three parts. Firstly the tensile fracture tests for the composite membrane specimens with different ratios were carried out with loading rate of 30 mm/min at room temperature. For each condition three samples were tested considering random error. According to the experimental data, the Young's modulus, tensile strength and ultimate strain of all samples were analyzed. Secondly, the tensile tests for SF70 specimens were carried out with different stress rates such as 0.01 N/s, 0.1 N/s, 0.5 N/s so as to investigate the rate-dependent tensile property. Finally, the creep tests
2.7. Statistical analysis SPSS18.0 was used to evaluate the significant differences among the three groups. Data were presented as mean ± standard error. In all cases, the results were considered statistically different at p < 0.05(*). 3
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Fig. 3. The macrostructure of freeze-dried membranes with different ratios.
Fig. 4. The surface structure of freeze-dried composite membranes under SEM.
Fig. 5. Swelling percentage of composite membranes(n = 3, P < 0.05(*)). t
3. Theoretical model
ε(t) =
∫ C1 (t − t1 ) σ˙ (t1 ) dt1 0
3.1. Derivation of constitutive model for composite membranes
t
+
The composite membrane was described by the following nonlinear viscoelastic constitutive equation for uniaxial tensile loading. In the 1965, Onaran and Findley [30] proposed a broadly representative onedimensional triple integral nonlinear constitutive relation for multiaxial loads, as shown in the following.
0 t
+
t
∫ ∫ C2 (t − t1, t − t2 ) σ˙ (t1 ) σ˙ (t2 ) dt1 dt2 0 t
t
∫ ∫ ∫ C3 (t − t1 , t − t2 , t − t3 )σ˙ (t1 ) σ˙ (t2 ) σ˙ (t3 ) dt1 dt2 dt3 0
0
0
(4)
where the C1, C2 and C3 are creep function of material, ε is the tensile ∂σ strain, t is the tensile time, σ is the tensile stress and σ̇ (t ) = ∂t . 4
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Fig. 6. Degradation percentage of composite membranes(n = 3, P < 0.05(*)).
Eq. (4), the constitutive equation is obtained under uniaxial tensile loading. t
ε(t) = ∫ [α1 + β1 (t − t1 )n] σ˙ (t1 ) dt1 0 t
t
+ ∫ ∫ [α2 + β2 (t − t1 )n /2 (t − t2 )n /2] σ˙ (t1 ) σ˙ (t2 ) dt1 dt2 0 0 t t t
+ ∫ ∫ ∫ [α3 + β3 (t − t1 )n /3 (t − t2 )n /3 (t − t3 )n /3] σ˙ (t1 ) σ˙ (t2 ) σ˙ (t3 ) dt1 dt2 dt3 0 0 0
(10) Based on the constitutive relation Eq. (10), considering that the uniaxial tensile test is controlled by the loading rate, the tensile stress can be expressed as
σ(t) = σ̇ ·t
(11)
By solving the integral of Eq. (10), we obtained
˙ ) + α2 (σt ˙ )2 + α3 (σt ˙ )3] + ε(t) = [α1 (σt Fig. 7. Uniaxial tensile curves of composite membranes with different ratios.
˙ )3 ⎤ 27β3 (σt ˙ ) ˙ )2 4β (σt ⎡ β1 (σt + 2 + × tn 3 ⎥ 2 ⎢n + 1 ( n + 2) ( n + 3) ⎣ ⎦
For creep deformation under constant stress, Eq. (4) can be described as follows.
ε(t) = C1 (t ) σ + C2 (t ) σ 2 + C3 (t ) σ 3
3.2. Determination of parameters
(5)
Based on Eq. (6), the parameters ε0,εt and n are evaluated by the creep tests of composite membranes with different constant stresses at room temperature. The parameter n is fitted as 0.13. The α1, α2, α3 and β1, β2, β3 are determined by Eqs. (8) and (9). The least squares regression is used to obtain the values of the parameters. The fitting values are shown in Table 2. Based on the built constitutive model, the creep behaviors and tensile properties of composite membrane can be predicted.
The Eq. (5) is the nonlinear creep constitutive relation. The creep strain ε(t) at constant stress σ is supposed to satisfy the power law relation [31], as follows in Eq. (6).
ε(t) = ε0 + εt t n
(6)
where the ε0 is the initial creep strain that is related to stress, εt is the nominal instantaneous creep strain that is related to stress and temperature, n is the creep parameter being related to temperature. In order to be consistent with the power law relation, the creep function C1, C2 and C3 can be expressed as:
C1 (t ) = α1 + β1
tn
C2 (t ) = α2 + β2
tn
C3 (t ) = α3 + β3
tn
4. Results and discussions
(7)
4.1. Physical property of composite membranes
By substituting Eq. (7) into Eq. (5) and comparing with Eq. (6), the following relationship is obtained
ε0 = α1 σ + α2 σ 2 + α3 σ 3
(8)
εt = β1 σ + β2 σ 2 + β3 σ 3
(9)
(12)
4.1.1. Characterization of composite membranes Fig. 3 shows the macrostructure of freeze-dried composite membranes with different ratios. It is found that the composite membranes present a white sponge structure. The sample is hard and brittle after freeze-dried. However the sample becomes soft and its elasticity is good after soaking. Fig. 4 shows the surface properties of the composite membranes
where α1, α2, α3 are the material constants, β1, β2, β3 are the material parameters that are related to temperature. By substituting Eq. (7) into 5
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Fig. 8. Effect of mix proportion on the tensile mechanical properties. (a) Young's modulus; (b) ultimate strain; (c) tensile strength (n = 3, P < 0.05(*)).
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4.1.3. Degradation of composite membranes The degradation of composite membranes with different ratios was evaluated in vitro and Fig. 6 shows the percentage of degradation. The results demonstrate that the degradation percentage of composite membrane reduces with content of type II collagen decreasing. It is noted that the degradation percentage of SF50 composite membrane reaches 46.2%, the degradation percentage of SF70 composite membrane reaches 25.3%, and however the degradation percentage of SF90 is only 11.6%. Type II collagenase degradation fluid mainly simulates the degradation of extracellular matrix by enzymes in the body. According to an experimental study of previous type II collagen repair of knee articular cartilage defects, neonatal cartilage tissue appeared four weeks after surgery [34]. In our degradation experiments for 21 days, it can be found that the type II collagen is degraded, and the silk fibroin powder appears around the composite membrane. The results show that SF50 composite membrane almost degraded half of itself. Its mechanical properties may be lost during the early stages of degradation. When the material ratio of SF90 is used in tissue engineering scaffolds, it may affect the repairing effect due to the slower degradation. The rate of cartilage scaffold degradation is very important for its application in cartilage repair.
Fig. 9. Creep strain of SF70 composite membrane with different stress levels.
under SEM. It is found that the fusion performance of silk fibroin and collagen type II for SF70 composite membrane is better than that of SF50 and SF90 composite membranes. There is no collagen fiber on the surface of SF70 composite membrane, and the surface is relatively flat. There are many collagen fibers on the surface of SF50 and SF90 composite membranes, and their surfaces are rough. This indicates that when the ratio of silk fibroin and collagen type II is 7:3, the fusion of the two materials is better.
4.2. Mechanical behaviors for composite membranes 4.2.1. Tensile mechanical properties of composite membranes with different ratios The tensile mechanical properties for composite membranes with different ratios were investigated with loading rate of 30 mm/min and Fig. 7 shows the stress-strain curves of composite membranes with different ratios. It is found that the stress-strain curves are not coincident, which means that there are much differences about the mechanical properties of composite membranes with different ratios. Fig. 8 shows the Young's modulus, ultimate strain and tensile strength of composite membranes with different ratios at loading rate of 30 mm/min. The Young's modulus of composite membranes increases obviously with decrease of the col-II and the nearly 2.5 times difference between the SF50 and SF90 is observed as shown in Fig. 8(a). In contrast, the ultimate strain and tensile strength of composite membranes decrease with decrease of the col. II. The ultimate strain and tensile strength value of SF90 are the smallest. According to Dong et al., [35] the tensile strength of articular cartilage was 4.75 MPa, and the ultimate strain was 1860 με. The tensile strength of cartilage is similar to that of composite membranes, but the ultimate strain varies greatly. This is because the composite material mainly plays the role of support when it is used as a scaffold. It is only the transition stage material. The mechanical properties of the repaired cartilage tissue will be compared with the mechanical properties of the host cartilage. It can be concluded
4.1.2. Swelling behavior of composite membranes The water adsorption ability of material is related to prevention of losing body fluid and nutrients after transplantation in vivo [32]. Swelling behavior is also selected to evaluate the physical stability of composite membrane to be similar to the hydrogel characteristics of cartilage tissue [33]. Fig. 5 shows the remarkable water adsorption capacity for all membranes. The swelling percentage of the composite membrane decreases as the percentage of type II collagen reduces (SF50:1447%, SF70:1235%, SF90: 967.6%). This comes from the hydrophilicity of type II collagen which can maintain a high amount of water. The SF90 composite membrane shows the lowest swelling percentage while SF50 composite membrane has the highest swelling percentage. In general, the greater the percentage of swelling of the material after soaking, the greater the deformation that occurs and the lower the stability of the material. The results also indicate that the composite membrane with a high amount of type II collagen has lower physical stability.
Fig. 10. The tensile mechanical properties of SF70 at different stress rates. (a) 0.01 N/s; (b) 0.1 N/s; (c) 0.5 N/s. 7
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Fig. 11. Live/dead cell double staining of composite membranes for 3 days of culture.
Fig. 12. Cell proliferation of chondrocytes on composite membranes (n = 3, P < 0.05(*)).
Fig. 13. The morphology and attachment of chondrocytes on SF70 composite membrane. (a) The morphology of multiple chondrocytes. (b) The morphology of single chondrocyte.
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4.3.3. The morphology and attachment of chondrocytes under SEM The morphology and attachment of chondrocytes on composite membrane with different ratios were observed by scanning electron microscopy. Surprisingly, it is found that chondrocytes are observed only on the SF70 composite membrane under scanning electron microscopy as shown in Fig. 13. No clear cell morphology is found on the surface of the SF50 and SF90 composite membranes. Fig. 13(a) shows the different morphologies of chondrocytes on the SF70 composite membranes, indicating that the proliferation, division and growth of chondrocytes on the SF70 composite membrane are better. Fig. 13(b) shows a single attachment chondrocyte on the SF70 composite membrane and the chondrocytes' antennae stretches sufficiently and the growth state is good.
that the tensile mechanical properties of SF70 are more stable. 4.2.2. Tensile mechanical behaviors of SF70 composite membrane Since SF70 composite membrane shows more stable tensile properties in 4.2.1, the tensile mechanical properties of SF70 with different stress levels and stress rates are further investigated. Fig. 9 shows the creep behaviors of SF70 composite membrane with different tensile stress levels. It is found that the initial strain and instantaneous strain increase with the increase of tensile stress. Within creep time of 100 s, the creep strain increases fast, and then increases slowly as the creep time goes by. In order to further probe the creep behavior of SF70, the creep strains with different stress levels have also been predicted by the built theoretical model. Fig. 9 demonstrates the comparisons of experimental results and predictions. It is noted that there are good agreements between them. The tensile mechanical behaviors for SF70 composite membrane were investigated with different stress rates and Fig. 10 shows the relationships of stress and strain with different stress rates. It is found that the stress-strain curves are not coincident with different stress rates, and stress rate makes very remarkable influence on the tensile mechanical behaviors of SF70. The reason is that the rate of submolecule motion within the sample is smaller than the rate of employing force during a certain interval considering the viscoelastic characteristic of collagen fiber. The stress-strain behaviors of SF70 with different stress rates have also been predicted by the constitutive model (Eq. (11)) and the predictions agree with the experimental results as shown in Fig. 10. Articular cartilage exhibits a viscoelastic response to load, and creep and rate-related behavior is a typical mechanical response of cartilage to load. The SF70 composite membrane also showed a similar mechanical response.
5. Conclusion In this research, the composite membranes of silk fibroin/collagen type II with different ratios were constructed, and their mechanical property and biocompatibility were investigated. The following conclusions can be obtained: 1. There is no dissociative collagen fiber on the surface of SF70 composite membrane, and the surface is relatively flat. The fusion of silk fibroin/collagen type II within SF70 composite membrane is better. The SF70 composite membrane has a suitable swelling ratio and degradation rate, and the its physical performance is more stable. 2. Tensile mechanical properties for composite membranes of different ratios were compared and it is found the tensile mechanical properties of SF70 are more stable. The tensile mechanical behavior of SF70 composite membrane is dependent on stress rate. Its creep strain increases with increase of stress level. The constitutive model was established and applied to predict the creep strain and tensile property of SF70 composite membrane. It is noted that the experimental results and predictions are in good agreement. 3. The biocompatibility of three kinds of composite membranes was investigated. All composite membranes are non-toxic and biocompatible. In particular, the SF70 composite membrane has better proliferation ability. To sum up, the mixing ratio of silk fibroin and collagen type II 7:3 shows promise in the future as a scaffold material for cartilage repair.
4.3. Biocompatibility testing of composite membranes 4.3.1. Live/dead cell double staining The treatment of live/dead double staining was performed to assess the viability of chondrocytes on the composite membranes (SF50, SF70, SF90) after 3 days of culture. Fig. 11 shows the staining results observed with a fluorescence microscope. It is found that the live chondrocytes abound on three kinds of membranes, and the red fluorescence of all composite membranes is almost absent, which means that all three kinds of composite membranes have good cell proliferation. It is also noted that the green fluorescence on SF50 and SF90 composite membranes is not clearly observed by adjusting the focal length of the microscope. This is because the surfaces of the SF50 and SF90 composite membranes are rough, as shown in Fig. 4. However the live chondrocytes are most and evenly distributed on the SF70 composite membrane.
Declaration of competing interest There are no conflicts of interest for either author. Acknowledgments This study was sponsored by the National Natural Science Foundation of China (Nos. 11572222, 51571150, 11672208, 11432016).
4.3.2. Proliferation of chondrocytes on composite membranes The cell proliferation ability on the scaffold is a critical factor to support and guide regeneration of new tissue after transplantation. MTT testing was performed to investigate the cell proliferation of composite membranes with different ratios. The chondrocytes proliferation was analyzed after 1 day and 3 days of culture to evaluate the biological performance of composite membranes with different ratios as shown in Fig. 12. The results show that the chondrocytes on all membranes increase over culture time by comparing the absorbance values with day 1 and 3. There is no obvious difference for chondrocytes proliferation on all composite membranes at day 1. However at day 3, there is higher proliferation on the SF70 composite membranes than the others. This is consistent with the results of live/dead cell double staining. The influence of material structural characteristics on cell proliferation and migration has been reported previously [36]. Proliferation results demonstrate that SF70 composite membrane has better structural characteristics.
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