International Journal of Biological Macromolecules 65 (2014) 516–523
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Effect of hyaluronan molecular weight on structure and biocompatibility of silk fibroin/hyaluronan scaffolds Zhihai Fan a,1 , Feng Zhang b,1 , Tao Liu c , Bao Q. Zuo c,∗ a b c
Department of Orthopedics, The Second Affiliated Hospital, Soochow University, Suzhou, Jiangsu, PR China Jiangsu Province Key Laboratory of Stem Cell Research, Medical College, Soochow University, Suzhou, Jiangsu, PR China College of Textile and Clothing Engineering, National Engineering Laboratory for Modern Silk, Soochow University, Suzhou, Jiangsu, PR China
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Article history: Received 27 November 2013 Received in revised form 10 January 2014 Accepted 24 January 2014 Available online 1 February 2014 Keywords: Silk fibroin Hyaluronic acid Molecular weight Morphology Structural conformation Biocompatibility
a b s t r a c t The structure of scaffolds is known to play a key role in tissue engineering as it provides structural support and physical environment allowing cells to reside and rebuild the target tissue. In this work we investigated the effects of hyaluronan (HA) molecular weight (MW : 0.6, 1.6 and 2.6 × 106 Da) on the pore structure, secondary structure, and biocompatibility of lyophilized silk fibroin (SF)/HA composite scaffolds. The results showed that HA promoted the pore structure formation and restrained the formation of separate sheet like structures in the SF/HA blend scaffolds, which was dependent on HA MW . The 3D pore structure maintained the scaffold shape during the process of 75% ethanol annealing. Structural studies indicated that HA did not induce but hinder SF conformation transition from random coil to sheet before and after treatment. In addition, SF/HA scaffolds showed an increase in cell proliferation compared to pure SF scaffold. These findings demonstrated the important role of HA MW in preparing SF/HA blend scaffolds suitable for application in tissue engineering. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Tissue engineering has been emerged as a promising alternative strategy to treat patients with tissue loss or organ failure, which has the potential to revolutionize the treatment of many diseases [1]. The design of scaffold is important for tissue engineering [2]. Ideally scaffolds should have a proper structure similar to natural extracellular matrices (ECM) which induce regenerative processed by interacting with relevant cell populations. Many materials have been employed to construct tissue engineering scaffolds, in particular, lots of attention has been focused on naturally derived polymers, such as collagen, silk, chitosan, hyaluronic acid (HA), alginic acid, etc. [3]. Among these native materials, silk has been given much attention due its biocompatibility, slow degradability, remarkable mechanical properties, and excellent processing ability [4]. Silk fibroin has been processed into a variety of shapes including hydrogels, sponges, films and nanofibers, for new biomedical applications [5–7]. Compared to other material formats, porous sponge scaffolds exhibit more important role in providing a versatile 3D porous structure for cell attachment, proliferation, and migration, as well as for nutrient
∗ Corresponding author. Tel.: +86 512 67061157. E-mail address: [email protected] (B.Q. Zuo). 1 These authors contributed equally to this work. 0141-8130/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2014.01.058
and waste transport [8]. A wide variety of techniques, salt leaching, lyophilization, gas foaming, have been reported to design and fabricate porous scaffolds [4,8]. The lyophilized porous silk sponges from aqueous solution demonstrated excellent interconnectivity between the pores and improved cell attachment compared to the solvent-based porous sponges, likely due to the rougher surfaces and the benefit of not using organic solvents [4,8]. Recently a number of reports demonstrate that the combination of SF and other synthetic or natural polymers, such as poly (vinyl alcohol), collagen, chitosan, polylactic acid, HA, and so forth, has displayed advantages over the use of either material alone for tissue engineering applications [9–11]. SF/HA blend scaffolds could significantly promote cell proliferation, cell infiltration, and tissue formation as compared to plain SF scaffolds, suggesting the important role of HA in manipulating cell behavior [12–14]. Hyaluronic acid (HA), a naturally occurring linear polysaccharide distributed in the ECM of mammalian soft tissues, has been implicated in diverse biological processes such as angiogenesis and migration, tissue development, as well as the proliferation and differentiation of progenitor cells [15]. Scaffolds derived from natural polysaccharides are very promising in tissue engineering applications and regenerative medicine, as they resemble glycosaminoglycans in the ECM [16]. SF and HA have been made into SF/HA blend nanofibers [12], hydrogel [17], film [18], and porous scaffold [19] for application in regeneration medicine. The increased use of these materials will require finely tuned and
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controllable material characteristics, such as HA molecular weight. The function of HA based on the specific interaction with cells and ECM components in the body highly relies on the molecular weight of HA [20]. However, the effect of HA molecular weight on the structure and property of this system (SF/HA blend scaffolds) has not been characterized. The goal of the present study was to develop an optimum SF/HA blend scaffolds used for tissue engineering. HA with different MW was blended with SF to prepare SF/HA combined scaffold through freeze-drying. The morphology, structure, and biological properties of these scaffolds were investigated. More importantly, the effect of HA MW was discussed in detail.
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medium. The culture medium was changed every 3 days up to the indicated time points. 2.4. Scanning electron microscopy The cross-sections of the silk-based scaffolds were platinumcoated and examined using scanning electron microscope (SEM, Hitach S-4800, Tokyo, Japan). Cross-sections were prepared by cutting the dried silk-based scaffolds with a razor blade in liquid nitrogen. 2.5. Fourier transform infrared spectroscopy
2. Experimental 2.1. Preparation of SF solution Bombyx mori fibroin solutions were prepared according to our previously published procedures [21]. Cocoons were boiled for 20 min in an aqueous solution of 0.02 M Na2 CO3 and then rinsed thoroughly with distilled water to extract the sericin proteins. After drying, the extracted silk fibroin was dissolved in 9.3 M LiBr solution at 60 ◦ C for 4 h, yielding a 20% (w/v) solution. This solution was dialyzed against distilled water using Slide-a-Lyzer dialysis cassettes (Pierce, molecular weight cut-off 3500) for 72 h to remove the salt. The solution was optically clear after dialysis and was centrifuged to remove the small amount of silk aggregates that formed during the process. The final concentration of aqueous silk solution was ∼7 wt.%, determined by weighing the remaining solid after drying. The silk fibroin solution was then diluted to 6 wt.% with deionized water. 2.2. Preparation of SF–HA scaffolds Pure 0.6 wt.% HA solutions with molecular weight (MW ) of 0.6, 1.6, and 2.6 × 106 Da were prepared by dissolving HA powder in deionized water. The above 6 wt.% SF solution was mixed with HA solutions of different MW at room temperature for 2 h. Finally, mixed solution containing 3 wt.% SF and 0.3 wt.% HA with different MW was prepared. The prepared SF and SF–HA suspensions were then frozen at −20 ◦ C for about 24 h to freeze and then lyophilized for about 72 h. Lyophilized SF and SF–HA scaffolds were placed on a removable platform under which 75% ethanol was filled in a desiccator with a 25 in. Hg vacuum for 2 h to produce water-insoluble scaffold. 2.3. Cell seeding and culture Primary cultures of OECs were prepared from 1-month male Sprague-Dawley rats (Experimental Animal Center of Soochow University) as reported previously [22]. All experiment procedures were carried out in accordance with the regulations for the administration of affairs concerning experimental animals of Soochow University. The complete culture medium for OECs consists of DMEM/F-12 (Gibco, Grand Island, NY, USA), with 10% fetal bovine serum (Gibco, Grand Island, NY, USA), 1% glutamine (Sigma, St. Louis, MA, USA), and 2% penicillin–streptomycin (Hayao, Haerbin, China). After 10 days in culture, OECs were used for biocompatibility evaluation with SF/HA blend scaffolds. Scaffolds were cut into disks with diameter 8 mm and thickness 2 mm, transferred to 96-well plates and then sterilized by radiation. The scaffolds were incubated with the culture medium overnight, then seeded with OECs at a density of 1.0 × 105 OECs. The cells were allowed to adhere to the scaffolds for 3 h and then the cell-scaffold complexes were covered with 150 l of culture
Fourier transform infrared (FTIR) spectra were obtained using a Magna spectrometer (NicoLET5700, America) in the spectral region of 400–4000 cm−1 , the powdered SF and SF–HA scaffolds were pressed into potassium bromide (KBr) pellets prior to data collection. 2.6. X-ray diffraction X-ray diffraction (X’PERT PRO MPD, PANalytical Company, Holland) was operated at 40 kV tube voltage and 40 mA tube current, CuK␣ radiation was used with diffraction angle 2 = 2◦ –45◦ , the scanning rate is 2◦ /min with powdered SF and SF–HA scaffolds. 2.7. Thermogravimetry Thermogravimetry analysis was performed in a TG-DTA, PE-SII (America) in the temperature range of 40–600 ◦ C with a ramp rate of 10 ◦ C/min, and nitrogen flux of 50 ml/min. 2.8. Differential scanning calorimetry Differential scanning calorimetry (DSC) measurements were performed in TA instruments Q100 DSC (TA Instruments, New Castle DE) scanning from 40 to 300 ◦ C. The sample was weighed and placed into aluminum pans. The pans were heated at a constant rate of 2 ◦ C/min and a nitrogen gas flow rate of 50 ml/min. 2.9. Cell morphology The cell morphology on the scaffolds was observed by SEM. OECs were cultured for 10 days on the scaffolds, then fixed with 2.5% glutaraldehyde for 3 h at room temperature, rinsed three times with PBS and dehydrated in a gradient of alcohol (50%, 70%, 80%, 90%, 100%, 100%). Samples were then lyophilized, coated with gold and observed by SEM (Hitach S-4800, Tokyo, Japan). 2.10. DNA content To study cell proliferation on the scaffolds, samples were harvested at the indicated time point (from day 1 to day 15), and digested with proteinase K buffer solution for 16 h at 56 ◦ C. The DNA content was determined using the Quant-iTTM PicoGreen dsDNA assay, following the protocols of the manufacturer (Invitrogen, Carlsbad, CA). Samples (n = 3) were measured at an excitation wavelength of 480 nm and emission wavelength of 530 nm, using a BioTec Synergy 4 spectrofluorometer (BioTec, Winooski, UK). The amount of DNA was calculated by interpolation from a standard curve prepared with lambda DNA in 10 × 10−3 M Tris–HCl (pH 7.4), 5 × 10−3 M NaCl, 0.1 × 10−3 M EDTA over a range of concentrations.
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Fig. 1. Photographs of SF/HA blend scaffolds before and after 75% aqueous ethanol annealing: (a) SF/HA scaffold without annealing; (b) pure SF scaffold after annealing; (c) SF/HA (MW : 0.6 × 106 Da) scaffold after annealing; (d) SF/HA (MW : 1.6 × 106 Da) scaffold after annealing; (e) SF/HA (MW : 2.6 × 106 Da) scaffold after annealing.
2.11. Statistical analysis All experiments were carried out in triplicate. Means and standard deviations (SD) were calculated by the statistical significance of differences among each group examined by two-way ANOVA. The significance was set at p < 0.05. 3. Results and discussion 3.1. Morphological characteristics Porous SF and SF/HA scaffolds were formed in 24-pore plate using freeze-drying method. Fig. 1 showed the photographs of SF and SF/HA blend scaffolds before and after 75% ethanol annealing. We observed that SF scaffolds displayed a visible distortion and shrinkage. The change in morphological appearance of scaffolds could be suppressed by adding HA, the higher the HA MW , the clearer this inhibiting effect. The pore architecture of scaffolds is known to play an important role in tissue engineering as it provides the structural support and physical environment allowing cells to organize into a functioning target tissue. We further studied the morphology of the SF/HA blend scaffolds by scanning electron microscope (SEM). Fig. 2 showed
the influence of HA MW on scaffold porous structure. SF spontaneously formed leaf or sheet structure instead of 3D pore structure in the freezing-dry process (Fig. 2(a)) in accordance with previous reports [10,11,19]. Following the addition of HA with increasing MW , the SF/HA scaffolds showed increasing pore structure. In previous report, the increase of HA content decreased separate sheets and increased porous structures in the SF/HA blend scaffolds [23]. It demonstrated that the MW and content of HA all show significant effect in the porosity of SF scaffold. Interestingly, the formation of pore structure in SF/HA scaffolds was closely related to HA MW , more pore structure formed when higher MW of HA was employed. The observed difference in scaffold shape after 75% ethanol annealing was likely due to the different scaffold morphology (leaf or sheet, and pore). SF blended with other polymers, such as collagen [11], gelatin [10], chitosan [24], had been proved useful to prepare porous scaffold for tissue regeneration. Here, we revealed that the formation of pore structure and stability improvement of SF scaffold could be readily achieved by adding high MW HA. The results also indicated that the addition of HA to silk solutions resulted in the formation of specific nanostructure in the macropore walls of SF/HA scaffolds, as shown in Fig. 3. When HA with different MW was added into SF solution, the regular nanofibrous structures smaller than 100 nm in diameter formed. HA chains interact with SF molecules via the hydrogen bonding, and this may break the hydrophobicity property of fibroin molecules resulting in a different self-assembly behavior from molecules to nanofibers. Then the SF nanofilament further restrained the formation of lamellar structure, and facilitated the pore structure formation during the lyophilization process [11]. 3.2. Structural analysis Aside from the results that HA significantly altered the morphology of SF/HA scaffolds, further insight into the structural changes were sought. By analyzing the IR spectra, the structural conformation of SF was determined according to the wavenumber location of the absorption bands of amides I (1700–1600 cm−1 ),
Fig. 2. SEM images of SF–HA scaffolds with different MW HA: (a) pure SF; (b) HA 0.6 × 106 Da; (c) HA 1.6 × 106 Da; (d) HA 2.6 × 106 Da.
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Fig. 3. Cross-section images of macropore wall of SF–HA scaffolds with different MW HA: (a) pure SF; (b) HA 0.6 × 106 Da; (c) HA 1.6 × 106 Da; (d) HA 2.6 × 106 Da.
amides II (1600–1500 cm−1 ), amides III (1350–1200 cm−1 ) and amides V (700–600 cm−1 ) [25,26]. The IR spectra of SF/HA scaffolds before and after 75% ethanol annealing treatment were shown in Figs. 4 and 5. Pure SF scaffolds (Fig. 4(a)) showed absorption bands at 1654 cm−1 , 1540 cm−1 , 1240 cm−1 , and 666 cm−1 , attributed to the random coil or ␣-helix conformation. The IR spectra of HA with different MW were almost similar, so only the spectra of HA (MW 1.6 × 106 Da) were provided as control. In SF/HA blend scaffolds, no new peaks or obvious peak shift was observed, indicating their similar structure. The results indicated the predominance of random coil or ␣-helix conformation in SF scaffolds before post-treatment, and this structure was not changed significantly by adding HA with different MW .
Fig. 4. FTIR spectra of SF–HA scaffolds with different MW HA: (a) pure SF; (b) HA 0.6 × 106 Da; (c) HA 1.6 × 106 Da; (d) HA 2.6 × 106 Da; (e) pure 1.6 × 106 Da HA powder (without annealing). The inset image is the FTIR spectra of HA with different MW .
To increase the stability of SF/HA scaffolds in water, and avoid the loss of HA content, the SF/HA scaffolds were annealed by 75% ethanol. After treatment, FTIR spectra of SF/HA scaffolds showed peaks typical for significant -sheet conformation: 1632 cm−1 , 1522 cm−1 , and 683 cm−1 , and a shift from 1240 cm−1 to 1230 cm−1 . Those adsorption peaks are similar to that of native silk (Fig. 5(e)). The results revealed that SF conformation converted from random coil to -sheet after being treated with 75% ethanol vapor. Because the amide III region of SF was the only one that is free from any interference with HA [18,19], we choose it to quantitatively analyze the effect of HA on the crystallinity degree of silk
Fig. 5. FTIR spectra of SF–HA scaffolds with different MW HA: (a) pure SF; (b) HA 0.6 × 106 Da; (c) HA 1.6 × 106 Da; (d) HA 2.6 × 106 Da and (e) degummed native silk (after 75% ethanol annealing).
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Fig. 6. Crystallinity degree of SF/HA scaffolds before and after annealing.
fibroin by comparing the intensities of the pair component bands at 1260 cm−1 and 1230 cm−1 . The crystallinity of these SF/HA scaffolds before and after annealing was shown in Fig. 6, which was calculated by the ratio of the peak area at 1260 cm−1 and the sum of peak areas at 1260 cm−1 and 1230 cm−1 . The crystallinity degree of SF/HA scaffolds was 32.9%, 32.2%, and 31.9% when the HA molecular weight was 0.6 × 106 Da, 1.6 × 106 Da and 2.6 × 106 Da, respectively, which were slight lower than pure SF scaffolds without HA prepared with the same lyophilization process (33%). After 75% ethanol annealing, the crystallinity degree of pure SF and SF/HA scaffolds increased to 38.8, 36.6, 36.8, and 36.6%, respectively, resulting in the water stability of the SF/HA scaffolds. The crystallinity of scaffolds containing HA was lower than that of pure SF scaffolds, suggesting that HA decreased the formation of -sheet conformation, especially when the scaffolds were annealed with 75% ethanol. This result could be due to the fact that SF molecules interact with HA chains via the hydrogen bonding [13]. The formed hydrogen bonding restricted the molecular motion of SF to form well-organized -sheet structure. The results were consistent with analyses by Ren and Zhang [12,19], but conflict with that of GarciaFuentes’s [18]. The origin of the difference was likely due to the different preparation conditions, such as solution concentration and freeze temperature which had significant effect on SF structure [18,27]. It is well-known that the FTIR spectroscopy is sensitive to short-range order while the XRD technique is generally used to investigate the long-range order of polymers, including silk fibroin [26,28,29]. X-ray diffraction measurements were conducted on the SF/HA scaffold to double-check the crystalline structure in the samples, as shown in Fig. 7. The pure SF and SF/HA blend scaffolds before 75% ethanol annealing demonstrated an amorphous state, characterized by the presence of a broad peak in the 2 scattering angle range from 12.5◦ to 35◦ . After annealing treatment, all those scaffolds were crystalline, and presented a main reflection at 2 at 20.4◦ similar to that of degummed native silk (Fig. 7), typical for SF in -sheet conformation. No other new distinctive diffraction peaks were observed. 3.3. Thermal properties Differential scanning calorimetry (DSC) is one of the techniques that have been used extensively in the study of physical and structural characteristics of silk-based materials. DSC and TG measurements were performed in order to detect the temperature of denaturation of pure SF and SF/HA scaffolds and to assess their thermal stability. Fig. 8 showed the DSC thermograms for the SF/HA
Fig. 7. XRD data of SF–HA scaffolds with different MW HA: before annealing (a) pure SF; (b) HA 0.6 × 106 Da; (c) HA 1.6 × 106 Da; (d) HA 2.6 × 106 Da and after 75% ethanol annealing (e) pure SF; (f) HA 0.6 × 106 Da; (g) HA 1.6 × 106 Da; (h) HA 2.6 × 106 Da and (o) native silk fibers.
scaffolds before and after 75% ethanol annealing. All of the samples tested showed an endothermic peak at around 75 ◦ C, a glass transition region at around 177 ◦ C, a nonisothermal crystallization peak at around 220 ◦ C and a degradation peak at around 257 ◦ C. The endothermic peak at around 75 ◦ C was related to the evaporation of the adsorbed water in the silk-based materials [17,30]. Then the glass transition regions were observed at around 181 ◦ C. During the glass transition process, the mobility of silk fibroin chain has started to increase, and the Tg decreased with the addition of higher MW HA. After the glass transition temperature, a strong nonisothermal crystallization peak appeared around 210–230 ◦ C and shifted from 220 ◦ C (before annealing) to 225 ◦ C (after annealing) similar to the thermal transition of silk films studied previously [31,32]. The crystallization peak of SF/HA scaffolds was more obvious than that for pure SF scaffolds, suggesting that HA retarded the SF crystalline and decreased the crystallinity of SF during 75% ethanol annealing process, as shown in Fig. 8(B), which was consistent with the FTIR result. After crystallization, a strong endothermic peak at around 257 ◦ C was attributed to the decomposition of SF. TGA was employed to assess the thermal degradation of the SF/HA scaffolds, as shown in Fig. 9. The initial weight loss below 120 ◦ C was due to the evaporation of silk-bound water [30]. The addition of HA decreased the residual weight of SF/HA scaffolds at 120 ◦ C (Table 1), indicating the higher water content of SF/HA scaffolds than pure SF scaffolds. The main decomposition of pure HA was in a range between 200 ◦ C and 300 ◦ C with a center at 230 ◦ C [18]. The lower residual quantity of SF/HA blend scaffolds at 290 ◦ C and 600 ◦ C (Table 1) indicated that HA accelerated the decomposition of SF/HA scaffolds due to the lower decomposition temperature of HA [18] and crystallinity of SF. In order to examine the thermal behavior of degradation in detail, differential thermogravimetric (DTG) curves were provided. Fig. 10 revealed the degradation rates and maximum degradation temperatures of SF/HA scaffolds. During the initial heating between room temperature and 120 ◦ C, bound water was removed from all samples. The decomposition temperature for SF appeared at around 308 ◦ C (Fig. 10A and B), and a decomposition of HA with different MW centered at around 260 ◦ C (Fig. 10C), which were attributed to the disintegration of intermolecular interaction and the partial breakage of the molecular structure [6]. Scaffolds containing 10% HA showed the lower thermal-degradation
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Fig. 8. Standard differential scanning calorimetry scans of SF/HA scaffolds with different MW HA: (a) pure SF; (b) HA 0.6 × 106 Da; (c) HA 1.6 × 106 Da; (d) HA 2.6 × 106 Da. (A) Before and (B) after 75% ethanol annealing.
Fig. 9. Thermogravimetric analysis of SF/HA scaffolds with different MW HA: (a) pure SF; (b) HA 0.6 × 106 Da; (c) HA 1.6 × 106 Da; (d) HA 2.6 × 106 Da. (A) Before and (B) after 75% ethanol annealing.
temperature at around 304 ◦ C. The thermal degradation of HA mainly occurred before 300 ◦ C, resulting in about 6% more weight loss of blend scaffolds at 300 ◦ C (Table 1). The small shoulder decomposition temperature at 260 ◦ C for HA was also observed in the blend scaffolds (Fig. 10), in agreement with the previous
Table 1 Weight remaining of SF/HA scaffolds and HA at indicated temperature point. The value listed in bold are related to the pure silk scaffold before (B) and after (A) 75% ethanol annealing. Sample
Weight remaining at 120 ◦ C (%)
Weight remaining at 300 ◦ C (%)
Weight remaining at 600 ◦ C (%)
SF scaffold-B SF/HA-0.6-B SF/HA-1.6-B SF/HA-2.6-B SF scaffold-A SF/HA-0.6-A SF/HA-1.6-A SF/HA-2.6-A HA-0.6 HA-1.6 HA-2.6
89.4 89.3 86.8 86.9 90.4 89.1 89.6 89.4 79.5 77.8 75.4
80.6 78.2 74.5 74.9 82.5 76.7 78.2 77.9 38.3 37.7 36.6
33.2 33.6 24 29.2 33.4 29.2 30.3 29.6 22.3 23.5 18.3
B represent before 75% ethanol annealing; A represent after 75% ethanol annealing.
report in which distinct HA peaks could be observed when blending 40% HA [18]. The biphasic decomposition for blend scaffolds was observed: one at 260 ◦ C and another at 300 ◦ C. The SF/HA blend scaffolds showed degradation behavior in separate steps of weight loss with according to the characteristic of each component, suggesting the phase separation structure of these composite materials [6,18].
3.4. Biocompatibility of SF/HA scaffolds Fig. 11 showed the growth state of OECs on SF/HA blend scaffolds. The OECs adhered and grew very well on the surface of the scaffolds at day 10, and the cell increased and formed a monolayer on the surface of the porous walls. The cell proliferation on scaffolds was evaluated by DNA content. Fig. 12 showed a significant increase in DNA content on the four kinds of scaffolds during the testing time. The data indicated that the percentage of viable cells on the SF/HA scaffolds was significantly higher than that on the pure SF scaffolds at day 10 and 15, suggesting that OECs proliferation decreased on the SF scaffolds compared with the SF scaffolds containing HA. It has been widely reported that HA has a marked effect on cell behavior on scaffolds, such as increased cell proliferation and penetration. HA regulated cell behavior probably through changing the physical properties of scaffolds (microstructure, swelling
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Fig. 10. Differential thermogravimetric (DTG) curves of SF/HA scaffolds with different MW HA: (a) pure SF; (b) HA 0.6 × 106 Da; (c) HA 1.6 × 106 Da; (d) HA 2.6 × 106 Da. Before (A) and after (B) 75% ethanol annealing, and HA with different MW (C).
Fig. 11. SEM images of OECs cultivated on SF/HA scaffolds with different molecular weights at day 10: (a) pure SF; (b) HA 0.6 × 106 Da; (c) HA 1.6 × 106 Da; (d) HA 2.6 × 106 Da.
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the SF/HA blend scaffolds, make this blend scaffolds an interesting platform for biomedical applications. Acknowledgements We thank the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) for support of this work. We also thank the National Natural Science Foundation of China (81271723) for support of this work. References
Fig. 12. Proliferation of OECs on the four scaffold groups was determined by DNA content. Error bars represent mean ± standard deviation with n = 3. The lowest cell proliferation rate was in pure SF scaffold, which was statistically significantly different copaired with the other groups (*p < 0.05).
property), and the functional groups presented in HA capable interacting with cells and other macromolecules of the extracellular matrix [23]. The molecular weight of HA has a marked effect on its biological function and mitogenic properties, such as regulating matrix synthesis. [20]. The highest cell proliferation was observed on scaffolds containing HA with MW 2.6 × 106 Da, although there was no significant difference in cell proliferation between this scaffold and the others. The preliminary results of cell compatibility encourage further exploration of SF/HA scaffold with different MW HA as biomimetic scaffolds for application in tissue engineering. 4. Conclusions In the present study, we prepared SF/HA blend scaffolds with different MW HA in a green process, avoiding the use of organic solvents and harsh chemical processes. HA exhibited important ability to induce the formation of pore structure in lyophilized SF-based scaffolds, which relied on HA molecular weight. The structural transition of SF from random coil to -sheet during lyophilization and 75% ethanol annealing process was not promoted but prevented by adding HA. Cell compatibility test exhibited that SF/HA blend scaffolds were suitable for OECs’ adhesion, survival and proliferation. These properties, together with the improved cell proliferation on
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Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Effect of hyaluronan molecular weight on structure and biocompatibility of silk fibroin/hyaluronan scaffolds Zhihai Fan a,1 , Feng Zhang b,1 , Tao Liu c , Bao Q. Zuo c,∗ a b c
Department of Orthopedics, The Second Affiliated Hospital, Soochow University, Suzhou, Jiangsu, PR China Jiangsu Province Key Laboratory of Stem Cell Research, Medical College, Soochow University, Suzhou, Jiangsu, PR China College of Textile and Clothing Engineering, National Engineering Laboratory for Modern Silk, Soochow University, Suzhou, Jiangsu, PR China
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Article history: Received 27 November 2013 Received in revised form 10 January 2014 Accepted 24 January 2014 Available online 1 February 2014 Keywords: Silk fibroin Hyaluronic acid Molecular weight Morphology Structural conformation Biocompatibility
a b s t r a c t The structure of scaffolds is known to play a key role in tissue engineering as it provides structural support and physical environment allowing cells to reside and rebuild the target tissue. In this work we investigated the effects of hyaluronan (HA) molecular weight (MW : 0.6, 1.6 and 2.6 × 106 Da) on the pore structure, secondary structure, and biocompatibility of lyophilized silk fibroin (SF)/HA composite scaffolds. The results showed that HA promoted the pore structure formation and restrained the formation of separate sheet like structures in the SF/HA blend scaffolds, which was dependent on HA MW . The 3D pore structure maintained the scaffold shape during the process of 75% ethanol annealing. Structural studies indicated that HA did not induce but hinder SF conformation transition from random coil to sheet before and after treatment. In addition, SF/HA scaffolds showed an increase in cell proliferation compared to pure SF scaffold. These findings demonstrated the important role of HA MW in preparing SF/HA blend scaffolds suitable for application in tissue engineering. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Tissue engineering has been emerged as a promising alternative strategy to treat patients with tissue loss or organ failure, which has the potential to revolutionize the treatment of many diseases [1]. The design of scaffold is important for tissue engineering [2]. Ideally scaffolds should have a proper structure similar to natural extracellular matrices (ECM) which induce regenerative processed by interacting with relevant cell populations. Many materials have been employed to construct tissue engineering scaffolds, in particular, lots of attention has been focused on naturally derived polymers, such as collagen, silk, chitosan, hyaluronic acid (HA), alginic acid, etc. [3]. Among these native materials, silk has been given much attention due its biocompatibility, slow degradability, remarkable mechanical properties, and excellent processing ability [4]. Silk fibroin has been processed into a variety of shapes including hydrogels, sponges, films and nanofibers, for new biomedical applications [5–7]. Compared to other material formats, porous sponge scaffolds exhibit more important role in providing a versatile 3D porous structure for cell attachment, proliferation, and migration, as well as for nutrient
∗ Corresponding author. Tel.: +86 512 67061157. E-mail address: [email protected] (B.Q. Zuo). 1 These authors contributed equally to this work. 0141-8130/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2014.01.058
and waste transport [8]. A wide variety of techniques, salt leaching, lyophilization, gas foaming, have been reported to design and fabricate porous scaffolds [4,8]. The lyophilized porous silk sponges from aqueous solution demonstrated excellent interconnectivity between the pores and improved cell attachment compared to the solvent-based porous sponges, likely due to the rougher surfaces and the benefit of not using organic solvents [4,8]. Recently a number of reports demonstrate that the combination of SF and other synthetic or natural polymers, such as poly (vinyl alcohol), collagen, chitosan, polylactic acid, HA, and so forth, has displayed advantages over the use of either material alone for tissue engineering applications [9–11]. SF/HA blend scaffolds could significantly promote cell proliferation, cell infiltration, and tissue formation as compared to plain SF scaffolds, suggesting the important role of HA in manipulating cell behavior [12–14]. Hyaluronic acid (HA), a naturally occurring linear polysaccharide distributed in the ECM of mammalian soft tissues, has been implicated in diverse biological processes such as angiogenesis and migration, tissue development, as well as the proliferation and differentiation of progenitor cells [15]. Scaffolds derived from natural polysaccharides are very promising in tissue engineering applications and regenerative medicine, as they resemble glycosaminoglycans in the ECM [16]. SF and HA have been made into SF/HA blend nanofibers [12], hydrogel [17], film [18], and porous scaffold [19] for application in regeneration medicine. The increased use of these materials will require finely tuned and
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controllable material characteristics, such as HA molecular weight. The function of HA based on the specific interaction with cells and ECM components in the body highly relies on the molecular weight of HA [20]. However, the effect of HA molecular weight on the structure and property of this system (SF/HA blend scaffolds) has not been characterized. The goal of the present study was to develop an optimum SF/HA blend scaffolds used for tissue engineering. HA with different MW was blended with SF to prepare SF/HA combined scaffold through freeze-drying. The morphology, structure, and biological properties of these scaffolds were investigated. More importantly, the effect of HA MW was discussed in detail.
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medium. The culture medium was changed every 3 days up to the indicated time points. 2.4. Scanning electron microscopy The cross-sections of the silk-based scaffolds were platinumcoated and examined using scanning electron microscope (SEM, Hitach S-4800, Tokyo, Japan). Cross-sections were prepared by cutting the dried silk-based scaffolds with a razor blade in liquid nitrogen. 2.5. Fourier transform infrared spectroscopy
2. Experimental 2.1. Preparation of SF solution Bombyx mori fibroin solutions were prepared according to our previously published procedures [21]. Cocoons were boiled for 20 min in an aqueous solution of 0.02 M Na2 CO3 and then rinsed thoroughly with distilled water to extract the sericin proteins. After drying, the extracted silk fibroin was dissolved in 9.3 M LiBr solution at 60 ◦ C for 4 h, yielding a 20% (w/v) solution. This solution was dialyzed against distilled water using Slide-a-Lyzer dialysis cassettes (Pierce, molecular weight cut-off 3500) for 72 h to remove the salt. The solution was optically clear after dialysis and was centrifuged to remove the small amount of silk aggregates that formed during the process. The final concentration of aqueous silk solution was ∼7 wt.%, determined by weighing the remaining solid after drying. The silk fibroin solution was then diluted to 6 wt.% with deionized water. 2.2. Preparation of SF–HA scaffolds Pure 0.6 wt.% HA solutions with molecular weight (MW ) of 0.6, 1.6, and 2.6 × 106 Da were prepared by dissolving HA powder in deionized water. The above 6 wt.% SF solution was mixed with HA solutions of different MW at room temperature for 2 h. Finally, mixed solution containing 3 wt.% SF and 0.3 wt.% HA with different MW was prepared. The prepared SF and SF–HA suspensions were then frozen at −20 ◦ C for about 24 h to freeze and then lyophilized for about 72 h. Lyophilized SF and SF–HA scaffolds were placed on a removable platform under which 75% ethanol was filled in a desiccator with a 25 in. Hg vacuum for 2 h to produce water-insoluble scaffold. 2.3. Cell seeding and culture Primary cultures of OECs were prepared from 1-month male Sprague-Dawley rats (Experimental Animal Center of Soochow University) as reported previously [22]. All experiment procedures were carried out in accordance with the regulations for the administration of affairs concerning experimental animals of Soochow University. The complete culture medium for OECs consists of DMEM/F-12 (Gibco, Grand Island, NY, USA), with 10% fetal bovine serum (Gibco, Grand Island, NY, USA), 1% glutamine (Sigma, St. Louis, MA, USA), and 2% penicillin–streptomycin (Hayao, Haerbin, China). After 10 days in culture, OECs were used for biocompatibility evaluation with SF/HA blend scaffolds. Scaffolds were cut into disks with diameter 8 mm and thickness 2 mm, transferred to 96-well plates and then sterilized by radiation. The scaffolds were incubated with the culture medium overnight, then seeded with OECs at a density of 1.0 × 105 OECs. The cells were allowed to adhere to the scaffolds for 3 h and then the cell-scaffold complexes were covered with 150 l of culture
Fourier transform infrared (FTIR) spectra were obtained using a Magna spectrometer (NicoLET5700, America) in the spectral region of 400–4000 cm−1 , the powdered SF and SF–HA scaffolds were pressed into potassium bromide (KBr) pellets prior to data collection. 2.6. X-ray diffraction X-ray diffraction (X’PERT PRO MPD, PANalytical Company, Holland) was operated at 40 kV tube voltage and 40 mA tube current, CuK␣ radiation was used with diffraction angle 2 = 2◦ –45◦ , the scanning rate is 2◦ /min with powdered SF and SF–HA scaffolds. 2.7. Thermogravimetry Thermogravimetry analysis was performed in a TG-DTA, PE-SII (America) in the temperature range of 40–600 ◦ C with a ramp rate of 10 ◦ C/min, and nitrogen flux of 50 ml/min. 2.8. Differential scanning calorimetry Differential scanning calorimetry (DSC) measurements were performed in TA instruments Q100 DSC (TA Instruments, New Castle DE) scanning from 40 to 300 ◦ C. The sample was weighed and placed into aluminum pans. The pans were heated at a constant rate of 2 ◦ C/min and a nitrogen gas flow rate of 50 ml/min. 2.9. Cell morphology The cell morphology on the scaffolds was observed by SEM. OECs were cultured for 10 days on the scaffolds, then fixed with 2.5% glutaraldehyde for 3 h at room temperature, rinsed three times with PBS and dehydrated in a gradient of alcohol (50%, 70%, 80%, 90%, 100%, 100%). Samples were then lyophilized, coated with gold and observed by SEM (Hitach S-4800, Tokyo, Japan). 2.10. DNA content To study cell proliferation on the scaffolds, samples were harvested at the indicated time point (from day 1 to day 15), and digested with proteinase K buffer solution for 16 h at 56 ◦ C. The DNA content was determined using the Quant-iTTM PicoGreen dsDNA assay, following the protocols of the manufacturer (Invitrogen, Carlsbad, CA). Samples (n = 3) were measured at an excitation wavelength of 480 nm and emission wavelength of 530 nm, using a BioTec Synergy 4 spectrofluorometer (BioTec, Winooski, UK). The amount of DNA was calculated by interpolation from a standard curve prepared with lambda DNA in 10 × 10−3 M Tris–HCl (pH 7.4), 5 × 10−3 M NaCl, 0.1 × 10−3 M EDTA over a range of concentrations.
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Fig. 1. Photographs of SF/HA blend scaffolds before and after 75% aqueous ethanol annealing: (a) SF/HA scaffold without annealing; (b) pure SF scaffold after annealing; (c) SF/HA (MW : 0.6 × 106 Da) scaffold after annealing; (d) SF/HA (MW : 1.6 × 106 Da) scaffold after annealing; (e) SF/HA (MW : 2.6 × 106 Da) scaffold after annealing.
2.11. Statistical analysis All experiments were carried out in triplicate. Means and standard deviations (SD) were calculated by the statistical significance of differences among each group examined by two-way ANOVA. The significance was set at p < 0.05. 3. Results and discussion 3.1. Morphological characteristics Porous SF and SF/HA scaffolds were formed in 24-pore plate using freeze-drying method. Fig. 1 showed the photographs of SF and SF/HA blend scaffolds before and after 75% ethanol annealing. We observed that SF scaffolds displayed a visible distortion and shrinkage. The change in morphological appearance of scaffolds could be suppressed by adding HA, the higher the HA MW , the clearer this inhibiting effect. The pore architecture of scaffolds is known to play an important role in tissue engineering as it provides the structural support and physical environment allowing cells to organize into a functioning target tissue. We further studied the morphology of the SF/HA blend scaffolds by scanning electron microscope (SEM). Fig. 2 showed
the influence of HA MW on scaffold porous structure. SF spontaneously formed leaf or sheet structure instead of 3D pore structure in the freezing-dry process (Fig. 2(a)) in accordance with previous reports [10,11,19]. Following the addition of HA with increasing MW , the SF/HA scaffolds showed increasing pore structure. In previous report, the increase of HA content decreased separate sheets and increased porous structures in the SF/HA blend scaffolds [23]. It demonstrated that the MW and content of HA all show significant effect in the porosity of SF scaffold. Interestingly, the formation of pore structure in SF/HA scaffolds was closely related to HA MW , more pore structure formed when higher MW of HA was employed. The observed difference in scaffold shape after 75% ethanol annealing was likely due to the different scaffold morphology (leaf or sheet, and pore). SF blended with other polymers, such as collagen [11], gelatin [10], chitosan [24], had been proved useful to prepare porous scaffold for tissue regeneration. Here, we revealed that the formation of pore structure and stability improvement of SF scaffold could be readily achieved by adding high MW HA. The results also indicated that the addition of HA to silk solutions resulted in the formation of specific nanostructure in the macropore walls of SF/HA scaffolds, as shown in Fig. 3. When HA with different MW was added into SF solution, the regular nanofibrous structures smaller than 100 nm in diameter formed. HA chains interact with SF molecules via the hydrogen bonding, and this may break the hydrophobicity property of fibroin molecules resulting in a different self-assembly behavior from molecules to nanofibers. Then the SF nanofilament further restrained the formation of lamellar structure, and facilitated the pore structure formation during the lyophilization process [11]. 3.2. Structural analysis Aside from the results that HA significantly altered the morphology of SF/HA scaffolds, further insight into the structural changes were sought. By analyzing the IR spectra, the structural conformation of SF was determined according to the wavenumber location of the absorption bands of amides I (1700–1600 cm−1 ),
Fig. 2. SEM images of SF–HA scaffolds with different MW HA: (a) pure SF; (b) HA 0.6 × 106 Da; (c) HA 1.6 × 106 Da; (d) HA 2.6 × 106 Da.
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Fig. 3. Cross-section images of macropore wall of SF–HA scaffolds with different MW HA: (a) pure SF; (b) HA 0.6 × 106 Da; (c) HA 1.6 × 106 Da; (d) HA 2.6 × 106 Da.
amides II (1600–1500 cm−1 ), amides III (1350–1200 cm−1 ) and amides V (700–600 cm−1 ) [25,26]. The IR spectra of SF/HA scaffolds before and after 75% ethanol annealing treatment were shown in Figs. 4 and 5. Pure SF scaffolds (Fig. 4(a)) showed absorption bands at 1654 cm−1 , 1540 cm−1 , 1240 cm−1 , and 666 cm−1 , attributed to the random coil or ␣-helix conformation. The IR spectra of HA with different MW were almost similar, so only the spectra of HA (MW 1.6 × 106 Da) were provided as control. In SF/HA blend scaffolds, no new peaks or obvious peak shift was observed, indicating their similar structure. The results indicated the predominance of random coil or ␣-helix conformation in SF scaffolds before post-treatment, and this structure was not changed significantly by adding HA with different MW .
Fig. 4. FTIR spectra of SF–HA scaffolds with different MW HA: (a) pure SF; (b) HA 0.6 × 106 Da; (c) HA 1.6 × 106 Da; (d) HA 2.6 × 106 Da; (e) pure 1.6 × 106 Da HA powder (without annealing). The inset image is the FTIR spectra of HA with different MW .
To increase the stability of SF/HA scaffolds in water, and avoid the loss of HA content, the SF/HA scaffolds were annealed by 75% ethanol. After treatment, FTIR spectra of SF/HA scaffolds showed peaks typical for significant -sheet conformation: 1632 cm−1 , 1522 cm−1 , and 683 cm−1 , and a shift from 1240 cm−1 to 1230 cm−1 . Those adsorption peaks are similar to that of native silk (Fig. 5(e)). The results revealed that SF conformation converted from random coil to -sheet after being treated with 75% ethanol vapor. Because the amide III region of SF was the only one that is free from any interference with HA [18,19], we choose it to quantitatively analyze the effect of HA on the crystallinity degree of silk
Fig. 5. FTIR spectra of SF–HA scaffolds with different MW HA: (a) pure SF; (b) HA 0.6 × 106 Da; (c) HA 1.6 × 106 Da; (d) HA 2.6 × 106 Da and (e) degummed native silk (after 75% ethanol annealing).
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Fig. 6. Crystallinity degree of SF/HA scaffolds before and after annealing.
fibroin by comparing the intensities of the pair component bands at 1260 cm−1 and 1230 cm−1 . The crystallinity of these SF/HA scaffolds before and after annealing was shown in Fig. 6, which was calculated by the ratio of the peak area at 1260 cm−1 and the sum of peak areas at 1260 cm−1 and 1230 cm−1 . The crystallinity degree of SF/HA scaffolds was 32.9%, 32.2%, and 31.9% when the HA molecular weight was 0.6 × 106 Da, 1.6 × 106 Da and 2.6 × 106 Da, respectively, which were slight lower than pure SF scaffolds without HA prepared with the same lyophilization process (33%). After 75% ethanol annealing, the crystallinity degree of pure SF and SF/HA scaffolds increased to 38.8, 36.6, 36.8, and 36.6%, respectively, resulting in the water stability of the SF/HA scaffolds. The crystallinity of scaffolds containing HA was lower than that of pure SF scaffolds, suggesting that HA decreased the formation of -sheet conformation, especially when the scaffolds were annealed with 75% ethanol. This result could be due to the fact that SF molecules interact with HA chains via the hydrogen bonding [13]. The formed hydrogen bonding restricted the molecular motion of SF to form well-organized -sheet structure. The results were consistent with analyses by Ren and Zhang [12,19], but conflict with that of GarciaFuentes’s [18]. The origin of the difference was likely due to the different preparation conditions, such as solution concentration and freeze temperature which had significant effect on SF structure [18,27]. It is well-known that the FTIR spectroscopy is sensitive to short-range order while the XRD technique is generally used to investigate the long-range order of polymers, including silk fibroin [26,28,29]. X-ray diffraction measurements were conducted on the SF/HA scaffold to double-check the crystalline structure in the samples, as shown in Fig. 7. The pure SF and SF/HA blend scaffolds before 75% ethanol annealing demonstrated an amorphous state, characterized by the presence of a broad peak in the 2 scattering angle range from 12.5◦ to 35◦ . After annealing treatment, all those scaffolds were crystalline, and presented a main reflection at 2 at 20.4◦ similar to that of degummed native silk (Fig. 7), typical for SF in -sheet conformation. No other new distinctive diffraction peaks were observed. 3.3. Thermal properties Differential scanning calorimetry (DSC) is one of the techniques that have been used extensively in the study of physical and structural characteristics of silk-based materials. DSC and TG measurements were performed in order to detect the temperature of denaturation of pure SF and SF/HA scaffolds and to assess their thermal stability. Fig. 8 showed the DSC thermograms for the SF/HA
Fig. 7. XRD data of SF–HA scaffolds with different MW HA: before annealing (a) pure SF; (b) HA 0.6 × 106 Da; (c) HA 1.6 × 106 Da; (d) HA 2.6 × 106 Da and after 75% ethanol annealing (e) pure SF; (f) HA 0.6 × 106 Da; (g) HA 1.6 × 106 Da; (h) HA 2.6 × 106 Da and (o) native silk fibers.
scaffolds before and after 75% ethanol annealing. All of the samples tested showed an endothermic peak at around 75 ◦ C, a glass transition region at around 177 ◦ C, a nonisothermal crystallization peak at around 220 ◦ C and a degradation peak at around 257 ◦ C. The endothermic peak at around 75 ◦ C was related to the evaporation of the adsorbed water in the silk-based materials [17,30]. Then the glass transition regions were observed at around 181 ◦ C. During the glass transition process, the mobility of silk fibroin chain has started to increase, and the Tg decreased with the addition of higher MW HA. After the glass transition temperature, a strong nonisothermal crystallization peak appeared around 210–230 ◦ C and shifted from 220 ◦ C (before annealing) to 225 ◦ C (after annealing) similar to the thermal transition of silk films studied previously [31,32]. The crystallization peak of SF/HA scaffolds was more obvious than that for pure SF scaffolds, suggesting that HA retarded the SF crystalline and decreased the crystallinity of SF during 75% ethanol annealing process, as shown in Fig. 8(B), which was consistent with the FTIR result. After crystallization, a strong endothermic peak at around 257 ◦ C was attributed to the decomposition of SF. TGA was employed to assess the thermal degradation of the SF/HA scaffolds, as shown in Fig. 9. The initial weight loss below 120 ◦ C was due to the evaporation of silk-bound water [30]. The addition of HA decreased the residual weight of SF/HA scaffolds at 120 ◦ C (Table 1), indicating the higher water content of SF/HA scaffolds than pure SF scaffolds. The main decomposition of pure HA was in a range between 200 ◦ C and 300 ◦ C with a center at 230 ◦ C [18]. The lower residual quantity of SF/HA blend scaffolds at 290 ◦ C and 600 ◦ C (Table 1) indicated that HA accelerated the decomposition of SF/HA scaffolds due to the lower decomposition temperature of HA [18] and crystallinity of SF. In order to examine the thermal behavior of degradation in detail, differential thermogravimetric (DTG) curves were provided. Fig. 10 revealed the degradation rates and maximum degradation temperatures of SF/HA scaffolds. During the initial heating between room temperature and 120 ◦ C, bound water was removed from all samples. The decomposition temperature for SF appeared at around 308 ◦ C (Fig. 10A and B), and a decomposition of HA with different MW centered at around 260 ◦ C (Fig. 10C), which were attributed to the disintegration of intermolecular interaction and the partial breakage of the molecular structure [6]. Scaffolds containing 10% HA showed the lower thermal-degradation
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Fig. 8. Standard differential scanning calorimetry scans of SF/HA scaffolds with different MW HA: (a) pure SF; (b) HA 0.6 × 106 Da; (c) HA 1.6 × 106 Da; (d) HA 2.6 × 106 Da. (A) Before and (B) after 75% ethanol annealing.
Fig. 9. Thermogravimetric analysis of SF/HA scaffolds with different MW HA: (a) pure SF; (b) HA 0.6 × 106 Da; (c) HA 1.6 × 106 Da; (d) HA 2.6 × 106 Da. (A) Before and (B) after 75% ethanol annealing.
temperature at around 304 ◦ C. The thermal degradation of HA mainly occurred before 300 ◦ C, resulting in about 6% more weight loss of blend scaffolds at 300 ◦ C (Table 1). The small shoulder decomposition temperature at 260 ◦ C for HA was also observed in the blend scaffolds (Fig. 10), in agreement with the previous
Table 1 Weight remaining of SF/HA scaffolds and HA at indicated temperature point. The value listed in bold are related to the pure silk scaffold before (B) and after (A) 75% ethanol annealing. Sample
Weight remaining at 120 ◦ C (%)
Weight remaining at 300 ◦ C (%)
Weight remaining at 600 ◦ C (%)
SF scaffold-B SF/HA-0.6-B SF/HA-1.6-B SF/HA-2.6-B SF scaffold-A SF/HA-0.6-A SF/HA-1.6-A SF/HA-2.6-A HA-0.6 HA-1.6 HA-2.6
89.4 89.3 86.8 86.9 90.4 89.1 89.6 89.4 79.5 77.8 75.4
80.6 78.2 74.5 74.9 82.5 76.7 78.2 77.9 38.3 37.7 36.6
33.2 33.6 24 29.2 33.4 29.2 30.3 29.6 22.3 23.5 18.3
B represent before 75% ethanol annealing; A represent after 75% ethanol annealing.
report in which distinct HA peaks could be observed when blending 40% HA [18]. The biphasic decomposition for blend scaffolds was observed: one at 260 ◦ C and another at 300 ◦ C. The SF/HA blend scaffolds showed degradation behavior in separate steps of weight loss with according to the characteristic of each component, suggesting the phase separation structure of these composite materials [6,18].
3.4. Biocompatibility of SF/HA scaffolds Fig. 11 showed the growth state of OECs on SF/HA blend scaffolds. The OECs adhered and grew very well on the surface of the scaffolds at day 10, and the cell increased and formed a monolayer on the surface of the porous walls. The cell proliferation on scaffolds was evaluated by DNA content. Fig. 12 showed a significant increase in DNA content on the four kinds of scaffolds during the testing time. The data indicated that the percentage of viable cells on the SF/HA scaffolds was significantly higher than that on the pure SF scaffolds at day 10 and 15, suggesting that OECs proliferation decreased on the SF scaffolds compared with the SF scaffolds containing HA. It has been widely reported that HA has a marked effect on cell behavior on scaffolds, such as increased cell proliferation and penetration. HA regulated cell behavior probably through changing the physical properties of scaffolds (microstructure, swelling
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Fig. 10. Differential thermogravimetric (DTG) curves of SF/HA scaffolds with different MW HA: (a) pure SF; (b) HA 0.6 × 106 Da; (c) HA 1.6 × 106 Da; (d) HA 2.6 × 106 Da. Before (A) and after (B) 75% ethanol annealing, and HA with different MW (C).
Fig. 11. SEM images of OECs cultivated on SF/HA scaffolds with different molecular weights at day 10: (a) pure SF; (b) HA 0.6 × 106 Da; (c) HA 1.6 × 106 Da; (d) HA 2.6 × 106 Da.
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the SF/HA blend scaffolds, make this blend scaffolds an interesting platform for biomedical applications. Acknowledgements We thank the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) for support of this work. We also thank the National Natural Science Foundation of China (81271723) for support of this work. References
Fig. 12. Proliferation of OECs on the four scaffold groups was determined by DNA content. Error bars represent mean ± standard deviation with n = 3. The lowest cell proliferation rate was in pure SF scaffold, which was statistically significantly different copaired with the other groups (*p < 0.05).
property), and the functional groups presented in HA capable interacting with cells and other macromolecules of the extracellular matrix [23]. The molecular weight of HA has a marked effect on its biological function and mitogenic properties, such as regulating matrix synthesis. [20]. The highest cell proliferation was observed on scaffolds containing HA with MW 2.6 × 106 Da, although there was no significant difference in cell proliferation between this scaffold and the others. The preliminary results of cell compatibility encourage further exploration of SF/HA scaffold with different MW HA as biomimetic scaffolds for application in tissue engineering. 4. Conclusions In the present study, we prepared SF/HA blend scaffolds with different MW HA in a green process, avoiding the use of organic solvents and harsh chemical processes. HA exhibited important ability to induce the formation of pore structure in lyophilized SF-based scaffolds, which relied on HA molecular weight. The structural transition of SF from random coil to -sheet during lyophilization and 75% ethanol annealing process was not promoted but prevented by adding HA. Cell compatibility test exhibited that SF/HA blend scaffolds were suitable for OECs’ adhesion, survival and proliferation. These properties, together with the improved cell proliferation on
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