This essay has been submitted by a student. This is not an example of the work written by our professional essay writers.
In this paper Bombyx mori silk based hydrogels were prepared and their biorelevant properties like physical, chemical and thermal properties were studied. Firstly, silk fibroin aqueous solution were prepared and the molecular weight of fibroin protein was determined followed by paticle size analysis for the confirmation of study. Silk fibroin hydrogels were prepared by treating a 12% (w/v) silk fibroin aqueous solution at 4 °C (thermgel) and lyophilized. The rheological and swelling properties of fibroin hydrogels were studied. The morphology and crystalline structure of lyophilized hydrogels were investigated by scanning electron microscopy (SEM) and wide-angle diffractometry, respectively while the surface functional groups were analyzed by FT-IR. The thermal behavior was also studied by means of differential scanning calorimetry and gravimetric method. Lyophilized fibroin gel of high strength & high thermal stability were obtained. The Î²-crystelline structure of lyophilized fibroin hydrogel has shown excellent swelling capacity to mimic the living tissues. The surface of these hydrogels were found supporting to cell adherence and proliferation. These gels could be used as scaffolds able to promote in situ bone regeneration.
Millions of people every year worldwide suffer from injured and deficient tissues or damaged organ. In such cases only two therapeutic choices such as mechanical replacement and organ transplantation are employed. However, these approaches encounter several clinical issues such as poor biocompatibility of biomaterials used for making artificial organs, shortage of organ donors or the adverse effects of immunosuppressive agents. To break through the problems regenerative medical therapy has been considered as promising strategy to cure the disease based on the natural healing potential of patients. One such technique is tissue engineering which has been evolved as more advanced and promising approach to regenerate tissues and organs. One of the major aspect of this technique is the development of scaffold from biocompatible and biodegradable polymers .
Hydrogels are three-dimensional polymeric networks prepared by physical or chemical crosslinking and resistant to swelling in aqueous solution without dissolving in it [2, 3]. Since hydrogels are highly hydrated hydrophilic polymer networks that contain pores and void spaces between the polymer chains, it can be implanted for tissue restoration or local release of therapeutic factors. Such hydrogels provide many advantages over the common conventional solid scaffold materials, including an enhanced supply of nutrients and oxygen for the cells. Pores within the network provide space to cells for proliferation and expansion. Due to their high water content hydrogels thus are similar to some tissues and extracellular matrices (ECM) . Widespread research is under way on using hydrogels as scaffold materials for application in tissue engineering, where the spaces might be filled with stem cells, growth factors or both. Hydrogels prepared from variety of polymers such as alginates, chitosan, and collagen have been extensively studied for use as scaffold in tissue engineering [5, 6]. Hydrogels formed from synthetic polymers offer the benefit of gelation and gel properties that are controllable and reproducible through the use of specific molecular weights, block structures, and modes of crosslinking but the less biocompatibility and use of toxic reagents limits their application. Generally, gelation of naturally derived polymers is reported to be less controllable, although the hydrogels formed are more compatible for hosting cell and bioactive molecules [3, 7].
Bombyx mori silk, a member of Bombycidae family is composed of a filament core fibroin proteins cemented together with glue-like sericine proteins [8, 9]. Silk due to its unique combination of properties such as mechanical strength, biodegradability and biocompatibility is considered as a potential matrix for controlled release in various forms which include fibroin scaffolds, electrospun woven mats, silk microspheres and silk fibroin films [10-14]. Silk fibroin hydrogels are of great interest for drug delivery and tissue engineering applications. Recently, many applications suggest the potential of porous hydrogels for cell culture and regenerative medicine [15, 16]. Fini et al. found that low pH induced silk fibroin hydrogels shows better healing of cancellous rabbit distal femurs than the control material, poly(D, L lactide-glycolide) as a bone-filling biomaterial . For many cell-based applications, gelation must be induced under mild conditions to reserve its mechanical and biocompatibility properties which could potentially alter cell function and affect cell viability. The study of SF hydrogels formation is important for understanding the behavior of SF metastable solution. During the gelation process, SF experiences transition in structure from random coil to Î²-sheet due to enhanced hydrophobic interactions and hydrogen bond formation leading to form a more stable structure [15, 17-20].
The objective of present paper was to examine the processability of concentrated aqueous silk fibroin solutions into highly porous gel sponges and study of their various properties for possible tissue engineering applications.
2. Materials and Methods
2.1. Preparation of aqueous silk fibroin solution
Domesticated Bombyx mori silk cocoon used for the present study were obtained from Mulberry Farms of chitoor district, Hyderabad (India). Dried cocoon shells were cut into small pieces and treated with boiling aqueous solution of sodium carbonate of varying concentration with stirring. The whole mass was repeatedly washed with Milli-Q water to remove the glue-like sericine protein and kept in hot air oven for drying. Silk fibroin solution was prepared by dissolving 10gms of degummed silk in 9.3M LiBr solution at 70Â°C for 2Â½ hrs. The fibroin solution was dialyzed in a cellulose membrane based dialysis cassette (molecular cutoff 12,400.) against deionized water for 3 days, changing water every 6 hrs. in order to completely remove LiBr. After dialysis, silk fibroin solution was centrifuged at 5-10ËšC and 9000 rpm for 20 min. to remove impurities and precipitated matter. The concentration of the silk fibroin aqueous solution at the end was 12 wt%.
2.2. Preparation of Silk Fibroin Hydrogels & Sponges
The regenerated silk fibroin prepared as above, when kept at 20 °C for 3 days under humid environment to form translucent silk fibroin hydrogels (thermgels).The hydrogels such obtained when kept at -20 °C for 24 hrs. and lyophilized for next 24 hrs. forms fibroin sponges.
2.3. Swelling property of silk hydrogel
The swelling properties of the hydrogels were studied using conventional gravimetric method. The swelling behavior of dried hydrogels was monitored as a function and was determined by immersing the completely dried (60Â Â°C for 24Â h) hydrogel samples in double-distilled water at 37Â Â°C. Swollen gels were weighed by an electronic balance at pre-determined time intervals after wiping excess surface liquid by filter paper. The swelling ratio (SR) was calculated from the following equation:
where Mt is the mass of the swollen gel at time t, and M0 is the mass of the dry gel at time 0.
2.4. Scanning Electron Microscopy
The morphology of degummed silk fibers and lyophilized fibroin hydrogels were determined by SEM at different magnification. Samples were air-dried overnight and affixed via carbon tape to the SEM sample holders and vacuum-coated with a 20-nm layer of platinum. Specimens were observed on a JEOL JSM -6480LV SEM and photographed at a voltage of 15 kV and room temperature.
2.5. Measurement of molecular weight
The molecular weight of the regenerated silk fibroin was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) according to the method described by Laemmli (1970) using 12% acrylamide gel and 5% condensing gel, which was stained with the Easy Stain Commassie Blue Kit (Invitrogen, Carlsbad, CA).
2.6. Particle size analysis
Average particle size of regenerated silk fibroin in solution was measured by Zetasizer Nano ZS (Malvern, U.K) particle analyzer at 30Â°C after discarding precipitated impurities of centrifuged fibroin solution at 9000 rpm for 10 min. at 10 Â°C. Particle size measurement was based on laser beam scattering technique. The optic unit contained a 4 mW He-Ne (633 nm) laser.
2.7. Rheological properties
The Rheological property of the prepared silk fibroin solution was assayed by measuring the viscosity by a cone and plate viscometer (BOHLIN VISCO-88, Malvern, U.K.). The cone angle is 5.4o and diameter 30 mm. A gap of 0.15 mm was maintained between the cone and plate for all the measurements.Â The temperature wasÂ maintained using an external water circulator as 30Â°C,35Â°C,40Â°C,45Â°C,50Â°C Â±1Â°C.
2.8. X-ray diffraction
An X-ray diffractometer XRD (Phillips PW-1830) with Ni-filterd Cu-Ka radiation operating at 35 kV and 30mA was used to record the diffraction pattern of samples of the degummed silk fibre and silk fibroin scaffold. Crystallinity was determined by integration using KaleidaGraph (Synergy Software).
2.9. FT-IR spectroscopy
The degummed silk fibre and lyophilized fibroin hydrogels were analyzed by FTIR spectroscopy. Room-temperature FT-IR spectra were recorded on solid samples in KBr pellets by means of a Shimadzu FT-IR spectrometer (IRPrestige-21) with a resolution of 4 cm-1. The spectra were smoothened with constant smooth factor for comparison.
2.10. Differential scanning calorimetry
The thermal behavior of degummed silk fibre and lyophilized fibroin hydrogels were determined by means of differential scanning calorimetry (DSC) using a Mettler Toledo DSC822e Differential Scanning Calorimeter . The samples used weighed between 10 and 15 mg and were measured between the range of -20-300 Â°C at a scanning rate of 20Â°C/min under nitrogen atmosphere.
2.11. Thermal gravimetric Analysis
The thermal stability degummed silk fibre and lyophilized fibroin hydrogels were characterized using a DTG-6H (Simadzu). The amount of sample for each measurement was about 1Â mg, and all of the measurements were carried out under a nitrogen atmosphere and heated up to 700Â Â°C at a heating rate of 10Â Â°CÂ minâˆ’1.
3. Results & Discussion:
3.1. Morphology of Degummed silk Fiber:
The morphology of degummed silk fibroin were investigated by SEM (figure 1). The native undegummed silk fibre (figure 1(a)) clearly shows the smooth & stucking look due to presence of sericine. The figure 1(b) shows incomplete degumming while at 0.02 M conc. ,the complete removal of sericine was found . Surface with more rough look ,besides the complete removal of sericine was found with further increase in concentration(figure 1(c)).
Figure 1. Scanning electron micrographs illustrating morphologies of fibroin silk prepared by degumming of silk cocoon. a) Control silk. The sericin coating of two brins was clearly evident in control silk before degumming. b) Silk fibre degummed by 0.01 M Na2CO3 , showing comparatively incomplete removal of gum. c) Silk fibre degummed by 0.02 M Na2CO3 , appearing relatively smooth, and individual longitudinal strands were also clearly visible. d) Silk fibre degummed by 0.03 M Na2CO3 , individual longitudinal strands were clearly visible.
3.2. Characterization of silk fibroin
During dissolution of degummed silk fiber in LiBr, the amide bonds of fibroin molecular chain might be cleaved to different extent resulting in easy solubilization in water. The water soluble silk fibroin is called regenerated silk fibroin and are easily denatured & gelled by various factors. Despite of temperature & pH , the stability of silk fibroin solution largely depends on its molecular mass range. The silk fibroin aqueous solutions were kept at 4 Â°C and characterization is done within a month.
Figure 2. (a) SDS-PAGE (12% gel) of silk fibroin protein. Marker and fibroin lanes mean the standard protein ladder from 10 to 200 kDa (Gibco Co., USA) and the molecular mass range of silk fibroin, respectively. (b) Particle size distribution of silk fibroin by particle analyzer.
Figure 2(a) shows a smear band of silk fibroin corresponding to molecular marker ranging between 200 kDa to 30 kDa As the result reveals, the regenerated protein solution were composed of a mixture of polypeptides with several molecular weights. A broad dull band from 200 to 35 kDa and a clear sharp band at 25 kDa is obtained. The former band might be degradation products of heavy chain(350 kDa) obtained due to cleavage of amide bonds of raw silk protein formed by degumming and dissolution. While, the band at 25 kDa corresponds to the light chain of raw silk protein. Result was confirmed by particle size analysis of silk fibroin solution. The results of size distribution (figure 2(b)) were in good agreement with the results obtained from the molecular weight distributions. As shown in figure 2(b), particle size distribution profile is bimodal, with peaks at around 100nm and 5Âµm. Since particle size of the starting SF solution is disintegrated due to processing of extraction using salts , the results confirms the presence of light chain (25 kDa) with disintegrated heavy chain(350 kDa).
3.3. Morphological study of Silk fibroin hydrogels:
Morphologically, the hydrogels showed a sponge-like cross-linked structure produced by physical entanglement as well as chemical hydrogen and covalent bindings. Water-stable hydrogels were formed from SF aqueous solutions after 24 hrs, which leads to formation of porous matrices. This was due to induction of a sol-gel transition in the concentrated solution sample.
Figure 3. Silk fibroin hydrogel when kept at 20 Â°C for 3 days with conc. 12% (w/v) of silk fibroin. Simple eye view(a) and Optical micrographs,10X (b) & 20X (c)
Figure 4. Scanning electron micrographs showing the pororus lyophilized hydrogel leaf-like structure prepared from 4 wt % silk fibroin solution (a) and hydrogel sponge prepared from â€º 12 wt % silk fibroin solution (b).
Regenerated silk fibroin solution when kept at 20 °C in humid environment for more than 72 hrs., the silk fibroin aqueous solution was converted into gel and the hydrogel (thermgel) is formed. The process was induced by adding few drops of methanol as crystellinity inducing solvent. The silk hydrogel intact in shape is formed and stabilized due to internetworking. The gel doesn't lose its integrity when kept vertically as shown in figure 3(a). Figure 3(b, c) is a representative optical micrograph of prepared silk fibroin hydrogel sample as prepared. Morphological study of silk fibroin porous matrices were observed by SEM (figure 4(a,b) after lyophilizing the hydrogel samples at âˆ’80Â° C. Lyophilized hydrogels prepared from silk fibroin solutions of 4-12 wt % concentration showed leaf-like morphologies while concentration more than 12 wt % exhibited sponge-like structures .
3.3.1. Swelling property of silk hydrogel:
The swelling state of the polymer was reported to be important for its bioadhesive property [24, 25]. It was found that the molecular structure of fibroin was not changed by the change in water content of the gel, while the physical properties of the gel, however, changes significantly. It can be observed from figure 5 that water uptake by hydrogels increased with time until they attained equilibrium.
Figure 5. (a) Dehydrated Gel (b) Swollen Hydrogel (after 36 hrs. kept in deionized water at 37Â°C)
Table 1. Gain in water content (w/w %) of dehydrated hydrogel kept in deionized water at different time(hrs) interval.
Weight of gel (mg)
Swelling ratio (%)
Figure 6. Gain in water content of dehydrated fibroin hydrogel as a function of time.
Figure 6 shows the swelling behaviour of dehydrated hydrogel kept in deionized water at 37 °C for different time intervals. The complete removal of water from hydrogel during dehydration was assumed when no further change in weight was found. The dehydrated gel was non-brittle showing presence of water molecule to maintain equilibrium with respect to physical properties of the gel. The brittle gel could be found when dehydrated in the oven. The swelling rate was higher during the initial stage indicating the sucking of water molecules into the collapsed gel and adsorption of water over the dried surface of gel. Within 2 hrs. the gel gains weight of 75.05% and 216.28% after 4 hrs. indicating decline in swelling rate. After 24 hrs. the change was found 290.1 % which was increased upto 290.15 % at 32 hrs..No further change was observed after this period. After this period the water molecules goes inside the collapsed gel network at a much slower rate.
3.5.2. Thermorheological Behavior of the Silk fibroin hydrogel:
It was reported that the aggregation of fibroin molecules through formation of a Î²-structure in the gel produced the network structure . Three dimensional molecular network of fibroin depends on the remaining ratio of a Î²-structure in the fibroin solution. Generally, the gelling properties of fibroin have been studied at temperature 20 Â°C or less [19, 27]. The present study revealed that an increase in temperature enhanced the gelation of fibroin as evidenced by the increase in the viscosity of the fluid upto 45 Â°C. The sudden break in the intermolecular bonding above 45 Â°C resulting in the decrease in viscosity.
Table 2. Temperature dependence of viscosity for silk fibroin gel
Figure 7. Newtonian behavior showing the linear relation between shear stress and shear rate studied between the temperature range of 30-50 Â°C.
The thermorheological behavior of the silk fibroin hydrogel prepared by incubating the fibroin solution at 20 Â°C for 7 days was examined with initial fibroin concentration of 12 % (w/v) in the Temperature range from 30 to 50 Â°C measuring shear stress and viscosity at 5 degrees interval. Figure 7 shows the changes in viscosity as temperature was increased. Recent DSC results of the silk fibroin samples reveals a broad endotherm with a peak near 45 Â°C indicating gradual structural change with increase in temperature and a new structure is formed above 45 Â°C. This thermal behavior shows consistency with the viscoelastic behavior. Preliminary ATR FT-IR study have indicated that the fraction of antiparallel Î² -sheet conformation appreciably increased at 45 Â°C . Irreversible structural change could be considered as a reason to the thermoreheological behavior observed.
The graphs plotted between Viscosity and Shear Rate at different temperature showed the linearly proportionality at the low shear rate region of less than 100 s-1.Rheopectic fluids increase their viscosity over time with application of shear forces, while thixotropic fluids have a reversible time-dependent loss of viscosity accompanying the application of shear force . If the time-dependent loss of viscosity is nonreversible, the fluid is considered to have rheodestructive properties. Study at different temperature shows , gel exhibits rheopectic properties and either thixotropic or rheodestructive properties. Repeated observation reveals that continuous swirling of the mixture leads to an irreversible decrease in the viscosity. Therefore, the time-dependent reduction in the gel's viscosity is rheodestructive rather than thixotropic.
3.3.3. Structural Analysis:
Structural characterization of silk fibroin fibre and lyophilized hydrogels were performed by X-ray diffraction and FTIR analysis. The XRD patterns of degummed silk fiber and lyophilized RSF hydrogel are shown in figure 8 & figure 9. Figure 8 shows X-ray profiles of lyophilized hydrogels prepared from SF aqueous solutions. When silk fibroin solutions freeze at low temperature, below the glass transition from âˆ’34Â° C to -20Â° C, the structure doesn't change significantly . The amorphous structure of lyophilized silk fibroin hydogel samples were indicated by exhibition of a broad peak at around 20Â° . Silk fibroin hydrogels showed a distinct peak at 20.6Â° and two minor peaks at around 9Â° and 24Â°, very similar to Î²-sheet crystalline silk fibroin structure (Silk I) [26, 31]. X-ray diffraction results showed that the gelation of silk fibroin solutions induced a transition from random coil to Î²-sheet conformation as reported previously. [15, 26, 32].
Figure 8.Â X-ray diffraction of (a) degummed silk fiber and (b) Lyophilized silk fibroin hydrogel
The RSF hydrogel (line B in figure 8) shows more amorphous state, while the degummed silk fiber (line A in figure 8) shows a typical X-ray diffractogram of Î² -sheet crystalline structure, which has four diffraction peaks at 18.9Â°, 20.6Â°, 24.3Â° and 28.1Â°â-¦, corresponding to Î²-sheet crystalline spacing of 4.69Â°, 4.31Â°, 3.66Â°, 3.17 AÂ° , respectively [26, 31] . While the lyophilized fibroin hydrogel shows the characteristic peak at 20.6Â° while other peaks corresponding to degummed silk fibre is not visible indicating the more a-helical secondary structure. In conclusion, the processing of silk fibroin results into loss of Î² -sheet structure thus liable to be more biodegradability and loss in mechanical strength. The functional groups over the surface were analyzed by FT-IR for more detail.
Figure 9. FT-IR spectra of degummed silk fibers (a) and Lyophilized silk fibroin hydrogel (b).
Since the IR spectrum represents typical absorption bands sensitive to the molecular conformation of SF, scientists oftenly investigate the conformation of SF and its blend using IR spectroscopy. In order to confirm the conformational changes of SF, FTIR spectroscopy was performed and the results for degummed silk fibroin and lyophilized silk fibroin hydrogel have been shown in figure 9. Silk protein exists in three conformations, namely, random coil, silk I (R form), and silk II (Î²- sheet) .The absorption bands at 1657, 1525, 1245 & 651 cm-1 which correspond to amide I, amide II ,amide III & amide V bands, respectively, confirms that the degummed silk fiber was mainly in Î²-sheet conformation similar to the native silk fibre [34, 35]. The corresponding peaks of silk fibroin lyophilized hydrogel were found 1625, 1509, 1245, 667 cm-1 respectively indicating the Î²-sheet conformation at the end of processing through regeneration, gelation and lyophilization. Besides, few more peaks were found in lyophilized fibroin powder at frequencies corresponding to 1327-1393 cm-1 indicating stretched C-O bonding and the relatively unstable state. All the peaks observed in the three amide band regions varied in their width and intensities.
3.3.4. Thermal Analysis:
B. mori degummed silk and lyophilized fibroin hydrogel showed different thermogravimetric curves as in figure 10. As shown in thermogravimetric(TG) curves, the initial weight loss below 100 °C was due to the water evaporation[ 33]. At temperature above 200 °C, the weight loss was occurred again. However, the silk did not completely decompose even at 700 °C . The result shows that silk fibre underwent of at least three thermal decomposition stages, which are 200-300 °C, 300-350 °C and 350-400 °C. A similar decomposition pattern is observed with lyophilized hydrogel,However the rate of degradation is comparatively faster in this case.
Figure 10. Thermogravimetric(TGA) curves of degummed silk fibers (a) and Lyophilized silk fibroin hydrogel (b).
The decomposition at approx. 300° C is attributed to a disintegration of the intermolecular side chains during the crystalline melting process, while that at around 400 °C is attributed to a main chain disintegration, coupled with simultaneous carbon atom rearrangements. It was reported that the decomposition at 300 °C indicated the low crystellinity of the unoriented Î²-type configuration and, therefore it can be said that there is less possibility of obtaining a crystalline Î²-structure, which occurs in the temperature range 325-330 °C . It was found that the weight losses at 400 °C were low for the gels due to low water content. This suggests that the fibroin molecules come into close contact with each other during the lyophilization process and form a dense aggregate which could mechanically resist carbon atom rearrangements, thus resulting in low weight losses. This phenomenon of the fibroin molecules could be a possible reason for the lyophilized fibroin gel.
Figure 11. DSC heating curves of degummed B.mori silk fibre (a) and lyophilized silk fibroin hydrogel (b)
Thermal analysis of degummed silk fibroin and lyophilized fibroin hydrogel have been shown as different thermal calorimetric curve (figure 11). In the DSC curve of degummed silk of B. mori , an endothermic peak starts at 56.8 °C and has the maximum at 76.3 °C without any trace of exothermic transition due to Î²-sheet structure of degummed fibroin sample whereas the DSC curve for lyophilized fibroin hydrogel starts at 35.4 °C and has maximum at 67.7 °C .This difference in results is due to change in Î²-sheet conformation. A conformational change through the crystallization of SF from random coil to Î²-sheet causes an exotherm curve to exist. In the DSC curves of two exothermic peaks were observed at around 166 °C and 255 °C which is attributed to conformational change to Î²-form while two endothermic peaks observed near 220 °C & 286 °C is attributed to the conformational change of Î²-form into random coils. The exotherm at 220 Â°C indicates the crystallization of the fibroin random coils to Î±-form of crystals. The decomposition peaks of all the regenerated fibroin materials (endotherms around 290 Â°C) were shifted down compared with the original silk fiber , indicating the lower thermal stability of regenerated samples. The low thermal stability may be due to lower crystallinity as well as molecular weight decrease during the degumming process of the regenerated fibroin materials compared with the original silk fiber.
The main aim of the present study is the preparation & characterization of silk fibroin hydrogel.
Degumming of silk fiber was better at more than 0.02 M Na2CO3 at the optimized temperature and time. SEM clearly depicts the complete removal of sericine over the surface. Gel electrophoresis indicated a decreasing amount of the silk 25kDa light chain and a shift in the molecular weight of the heavy chain. The particle size distribution curve confirmed the result by showing binodal curves around 100 nm and 5 Âµm. A high strength and a high thermal stability to the dehydrated fibroin gels were obtained by close contact of fibroin molecules. Thermal transition in aqueous solution is a green method in order to obtain a gel with a required volume and shape without incorporating toxic chemicals or other gel inducing solvents. The gel, once formed, has a Î²-oriented network structure, as a result of intermolecular hydrogen bonding between the molecular components. This stabilizes the confirmation with an additional rotational stability, thus holding the water molecules in the said structure. Rheological and swelling behaviors of fibroin hydrogel were appreciable at 37 °C to mimic the body tissue. Results from study of secondary structure and crystellinity favour the stability and strength of lyophilized hydrogen as supportive biomaterial. Thermal analysis support that silk as biomaterial with predictable long-term degradation characteristics. The regenerated fibroin solution can also be used for thin films or 3D scaffold fabrication. An improved understanding of the in vivo environment and their role in the degradation of silk fibroin will provide the next logical step in modeling an appropriate long-term degradable scaffold for various tissue engineering applications.