CHAPTER 3 COMPARISON OF MULBERRY AND ERI SILK FIBROIN SCAFFOLDS FOR TISSUE ENGINEERING APPLICATIONS

Transcription

1 54 CHAPTER 3 COMPARISON OF MULBERRY AND ERI SILK FIBROIN SCAFFOLDS FOR TISSUE ENGINEERING APPLICATIONS 3.1 INTRODUCTION Silk is a naturally occurring biopolymer that has been used clinically as sutures for centuries. Silk fibroin has been used for biomedical applications due to its biocompatibility, slow degradability and remarkable mechanical properties of the material (Wang et al 2006). Silk fibroin consists of heavy (350 kda) and light chain (25kDa) polypeptides respectively, connected by a disulfide link (Tanaka et al 1999). The saliva from the gland of silk worms has Silk I, which gets converted to silk II structure after being spun into the form of filaments connected with anti parallel - sheet structure. The structural transformation from water-soluble silk I within the lumen of the gland, to the oriented and water insoluble silk II structure in the spun fibre, is one of nature s most remarkable feats in materials engineering (He et al 1999). The silk filament regenerated into the form of film, foam, sponge membrane and electrospun nanofibres are in helix, non-oriented structure silk I structure. The silk I structure gets converted to a -sheet structure when exposed to methanol or ethanol treatment (Huemmerich et al 2006, Ishida et al 1990). Silk fibroin proteins consist of repetitive protein sequences which form heterogeneous, semi-crystalline solids. The primary structure of mulberry fibroin can be divided into an insoluble -sheet forming [Gly Ala Gly Ala Gly Ser] domains, and a more hydrophilic tyrosine-rich

2 55 segment that constitutes the amorphous phase (Mai-ngam et al 2011). Silk fibroin provides an important set of material options for biomaterials and scaffolds for tissue engineering, because of the impressive mechanical properties, biocompatibility and biodegradability (Kim et al 2005). The silk fibroin has an RGD (arginine-glycine-aspartic acid) sequence, which enhances cell adhesion, cell proliferation and differentiation (Kardestuncer et al 2006, Chen et al 2003). The rate of cell proliferation was higher on silk films than on collagen. In-vivo studies were carried out using the films made of silk, collagen and PLA with rat MSCs, and there was no inflammatory reaction due to the degummed silk fibroin (Inouye et al 1998, Meinel et al 2005, Santin et al 1999, Sugihara et al 2000, Vepari et al 2007). The eri (Samia cynthia ricini), a type of wild silkworm extrudes silk fibre with a primary structure, that is considerably different from that of mulberry silk fibroin. The eri silk fibroin mainly comprises about 100 repeats of alternating polyalanine (Ala ) and glycine-rich domains (Nakazawa et al 2003). The glycine motifs are basically present in the random coil state structure, and it provides flexibility to the silk fibre, whereas the alanine rich motifs support to form a crystalline of sheet structure. The sum of Gly and Ala residues in eri silk is 82%, which is similar to mulberry silk fibroin, but the relative composition of Ala and Gly is reversed (Huemmerich et al 2006, Nakazawa et al 2003). Sericin is one of the factors, which induces inflammatory reactions during the tissue engineering application (Meinel et al 2005, Santin et al 1999). Sen and Babu (2004) found that the presence of sericin in wild silk (eri silk) is less than that of mulberry silk, and also it possesses a higher amount of moisture regain than mulberry silk. The higher moisture regain of non-mulberry silks suggests that they may consist of a higher ratio of hydrophilic to hydrophobic amino acid residues in their chemical architecture, compared to that of the mulberry varieties (Sen and

3 56 Babu 2004). Mai-ngam et al (2011) found that the eri silk fibroin has a higher content of hydrophilic and positively charged amino acids, which enhances the cell attachment and proliferation on the scaffold (Mai-ngam et al 2011). In this research, the scope of using eri silk fibroin as bio material was investigated. Comparison was made between the nano fibrous scaffolds produced from eri silk and mulberry silk fibroin. These scaffolds were produced using electrospinning method and were treated with ethanol to improve the structural stability. The physical and chemical characterization of the scaffold was carried out using Differential Scanning Calorimeter (DSC), Thermogravimetric Analyzer (TGA), Fourier Transform Infra Red (FTIR) spectroscope and X-ray diffractometer (XRD). The blood compatibility and platelet adhesion on the scaffolds were examined and the L6 rat fibroblast cell was used to assess the cell attachment and cell viability on the silk fibroin scaffolds. 3.2 MATERIALS AND METHODS Preparation of Eri Silk and Mulberry Silk Fibroin Scaffolds The eri silk ( Central Silk Board, Banglore, India) was degummed with sodium carbonate solution boiling at 75 C for 30 min, and at a ph level maintained at , to remove the sericin from the silk filament. The degummed silk (silk fibroin) was dissolved in trifluoro-acetic acid (99.5%). The fibres were produced using electrospinning setup as shown in figure 2.9. The rotating collector was used instead of stationary plate. The fibroin solution was taken in a 2 ml syringe having a diameter of 0.55 mm. The syringe was fixed on the infusion pump in a vertical position.intially spraying of solution and formation of beads occurred, while electrospinning from silk fibroin solution. Number of trails has been conducted to optimize the concentration of fibroin in the solution and electrospinning process

4 57 parameters such as distance between the syringe and collection drum, voltage and flow rate such that the fibres were formed in the nanoscale without the formation of beads. The optimum concentration of polymer in trifluoroacetic acid was found to be 13% (wt/ vol.). The distance between the syringe and the collecting drum was kept at 15 cm, and a 20 kv supply was applied between the syringe and the collecting drum. The flow rate of the solution was maintained at 1.0 ml per hour. The mulberry silk fibroin scaffold was also prepared under the same conditions as used for eri silk. The majority of the fibres, from both the silk fibroins, had the diameter in the range of 401 to 500 nm as shown in Figure 3.1(a-b). The scaffold had the problem of curling and shrinking, when it was treated with the solutions used for tissue culture. Amiraliyan et al (2010) treated the electrospun nanofibrous mat of mulberry silk with methanol and ethanol, to improve the structural stability and crystallinity. Hence, the eri silk fibroin scaffold was treated with ethanol at room temperature for 30 min to improve the dimensional stability. Figure 3.1(a) SEM image and fibre diameter distribution of mulberry silk fibroin scaffold

5 58 Figure 3.1(b) SEM image and fibre diameter distribution of eri silk fibroin scaffold Physical Characterization of Scaffolds The silk fibroin (degummed silk filament), untreated electrospun fibrous scaffold (without ethanol treatment) and ethanol treated electrospun scaffold of eri silk and mulberry silk were analyzed for functional groups using FTIR spectrometer (Bruker, USA) in the region of cm -1 with 4 cm -1 spectra resolution. The thermal stability was analyzed using TGA (TA Instruments, Q500) at temperatures ranging from 37 to 700 C in a nitrogen atmosphere at a heating rate of 20ºC/ min. The thermal properties of the eri silk and mulberry silk scaffolds were studied using the DSC. The temperature range of ºC was used, with a scan rate 10ºC/min in a nitrogen atmosphere. X- ray diffraction was carried out to study the crystalline size, structure and percentage of crystallinity of the silk fibroin scaffolds. The eri silk and mulberry silk scaffolds were evaluated under the XRD (Bruker, D8) with Cuk- radiation ( = 1.54 Aº). Scanning was carried out at a speed of 0.04º/sec with a measurement range of 1 to 70º. The area of scattering was

6 59 measured by fityk software; the crystal size and % of crystallinity were measured using Equations (3.1) and (3.2). K Crystal size A (3.1) cos Total area of crystalline peak Crystallnity % x100 (3.2) Total area of crystalline and amorphous The scaffold was tested for tensile properties under standard atmospheric conditions, using Universal (Instron, 3369) strength tester. The scaffold was cut into the specimen size of 10 mm 50 mm. Glue tapes were fixed at the top and bottom of the scaffold, where it was clamped on the jaw of the tester. The gauge length was maintained at 30 mm and the test speed was 20 mm/min. The thickness of both the eri silk and mulberry silk scaffolds was maintained the same for comparison; the mean thickness was 0.16mm ± 0.01mm and 0.15mm ±0.01mm for untreated and ethanol treated scaffolds respectively Biological Characterization of Scaffolds Blood compatibility Biocompatibility, especially of blood, is the most important property with regard to biomedical materials; hence the eri silk and mulberry scaffolds were subjected to a hemolytic test. Human blood collected from a healthy volunteer in a 3.8% sodium citrate coated tube was diluted with PBS (ph 7.4) at a ratio of 1:20 (vol. /vol.). The blood diluted with PBS was taken as a negative control, and the blood with tritonx was taken as a positive control. Eri and mulberry silk scaffolds were treated with ethanol and then autoclaved. The scaffolds were immersed in 100µl of blood and PBS solution followed by incubation at 37 C for 60 minutes. Then, the samples were spun

7 60 at 3000 rpm for 10 minutes. The optical density value (OD) of the supernatant was measured using spectrophotometer at 545 nm and the hemolytic rate was estimated using Equation (3.3). OD value of sample OD value of negative Hemolytic % x100 OD value of positive OD value of negative (3.3) Biomaterials anticipated for long-term contact with blood must be subjected to hemocompatibility, as it is the most important property of materials used for implant purposes. The materials in contact with blood must not induce thrombosis, thromboembolisms, antigenic responses, destruction of blood constituents and plasma proteins. A Platelet adhesion test was used to evaluate the interaction of human platelets with the surface of the eri and mulberry silk fibroin scaffolds. For this study, 5ml of fresh human blood was collected from a healthy person. The fresh blood was treated with 3.8% sodium citrate, and spun at 3000 rpm for 10 min at 4 C to obtain platelet-rich plasma (PRP), then it was placed on the eri silk and mulberry silk fibroin scaffolds. The platelet-attached eri silk and mulberry silk fibroin scaffolds were washed twice with PBS, and then immersed in PBS containing 2.5% glutaraldehyde (ph 7.4) overnight. They were subsequently dehydrated in gradient ethanol (20%, 40%, 60%, 80%, and 100%) for 15 min and then dried in vacuum. The morphology of the platelets those adhered on the scaffolds was characterized by the scanning electron microscope (SEM) analysis Rat L6 muscle cell culture Rat L6 muscle fibroblasts were seeded at a density of cells per silk fibroin scaffold. The cells were incubated at 37 C with 5% CO 2 for a period of 24 and 48 hrs. After the incubation, the silk fibroin scaffolds were removed from the well, and rinsed with PBS twice to remove non-adherent cells from the scaffold. Then the scaffolds were fixed with 2.5% phosphate-

8 61 buffered glutaraldehyde and kept at 4 C for 2 hours, and dehydrated with gradient ethanol solution (20%, 40% 60% 80% and 100%). The dried scaffolds were sputtered with iron and observed by SEM MTT assay Rat L6 muscle fibroblasts were seeded at a density of per well on a 96-well plate. After confluence, the samples were placed on the well plate and cells treated with Triton X-100 were used as the positive control. After the requisite incubation time, 5µl of MTT reagent (10mg/ml) was added to the medium, and incubated for 4 hrs at 37 C, 95% RH in incubator containing 5% CO 2. Subsequently, the medium was discarded and 200µl of Dimethyl sulfoxide (DMSO) was added and optical density (OD) was measured using spectrophotometer at 570 nm. The MTT assay varies linearly with the viable cell population. 3.3 RESULTS AND DISCUSSION Thermal Stability The thermogrametric curves of eri silk and mulberry silk scaffolds are shown in Figures Figure 3.2 (a-b) shows the percentage weight loss of the degummed mulberry silk and eri silk filament respectively. The initial weight loss of the degummed silk filament at around 100ºC is due to the evaporation of water from the silk fibroin (Simchuer et al 2010). The second weight loss takes place at the temperature of ºC and C respectively, for mulberry silk and eri silk, and the weight loss is 46% for both the silk filaments. Figure 3.3(a-b) shows the percentage weight loss of mulberry and eri silk fibrous scaffolds prepared by electrospinning. The Figure 3.3 shows that the initial weight loss at 100 C due to evaporation of water, which is similar to that obtained for degummed silk fibroin. The

9 62 second weight loss takes place at the temperature of C and C respectively, for mulberry and eri silk scaffolds and the weight loss is 48% for both the scaffolds. Figure 3.4 (a-b) shows the percentage weight loss of the mulberry and eri silk scaffolds treated with ethanol. The initial weight loss is similar to the earlier cases and the second weight loss takes place at the temperature of ºC and ºC for ethanol treated mulberry and eri silk scaffolds respectively, and the weight loss is 48% in both the cases. The second weight loss of the silk is due to the breakdown of the side chain of the amino group s residuals as well as the cleavage of the peptide bond (Freddi et al 1999). The results show that eri silk fibroin scaffolds have better thermal stability than those of mulberry silk fibroin. Figure 3.2 Thermograms of degummed (a) mulberry and (b) eri silk filaments

10 63 Figure 3.3 Thermograms of ethanol untreated (a) mulberry and (b) eri silk scaffolds Figure 3.4 Thermograms of ethanol treated (a) mulberry and (b) eri silk scaffolds

11 64 DSC curves in Figure 3.5( a-c) exhibit two peaks; the first one at 107.2, 92.1 and 87.7ºC, and the second at 369.7, and 355.9ºC respectively, for the degummed eri silk filament, ethanol untreated eri silk scaffold, and ethanol treated eri silk scaffold respectively. DSC curves in Figure 3.6 a-c exhibit two peaks; the first one at 77.2, 76.1 and 88.5ºC, and the second at 309.6, and 281.5ºC respectively, for the degummed mulberry silk filament, ethanol untreated mulberry silk scaffold and ethanol treated mulberry silk scaffold. The first peak below 110º in Figure 3. 5 and 3.6 is attributed to the loss of water during heating. The second endothermic peak in the range of ºC in Figure 3.5, and ºC in Figure 3.6 indicate the thermal degradation of the eri silk fibroin and mulberry silk fibroin respectively. The thermal decomposition of the silk fibroin is highly dependent on the native morphology and degree of molecular orientation. Freddi et al (1999) found that the decomposition of the silk fibroin below 300ºC is associated with the non oriented structure of the silk fibroin and decomposition above 300ºC is associated with the well oriented, crystalline silk fibroin materials. Both the TGA and DSC analyses show that the thermal stability of the eri silk is better than that of mulberry silk. From the findings of Freddi et al (1999) and Tsukada et al (1996) it can be argued that the eri silk fibroin, which decomposes above 350ºC, has a well-oriented crystallized structure, and hence, the thermal stability of the eri silk fibroin is better than that of the mulberry silk fibroin.

12 65 Figure 3.5 DSC curves of (a) degummed eri silk filament, (b) eri silk scaffold and (c) ethanol treated eri silk scaffold Figure 3.6 DSC curves of (a) degummed mulberry silk filament, (b) mulberry silk scaffold and (c) ethanol treated mulberry silk scaffold

13 FTIR Spectra Analysis To assess any changes in the functional groups of eri and mulberry silk before and after degumming, they were investigated using a FTIR spectroscope. The FTIR spectra in Figure 3.7(a-b) shows the amide I absorption band at 1617 and 1617 cm -1 ( C= O stretch) and amide II absorption band at 1510 and 1509 cm -1 ( N- H Bending), and amide III band absorption at 1221 and 1221cm -1 (C N stretching) respectively for undegummed mulberry and eri silk filament. The above absorption bands are attributed to the - sheet structure of the silk fibroin (Rajkhowa et al 2011). The spectra of the degummed mulberry silk and eri silk in Figure 3.8 (a-b) shows amide I absorption band at 1693 and 1618 cm -1 (C=O stretch), amide II absorption band at 1517 and 1518 cm -1 ( N- H bending) and amide III absorption at 1171 and 1225 cm -1 (C-N stretching) respectively. From the spectra 3.7(a) and 3.8(a), it can be seen that the wave number of amide I of mulberry silk has shifted from 1617 to 1693 cm -1 due to the degumming process. It may be attributed to the helix structure of the silk fibroin. However, the wave number for amide I band of eri silk is not much changed due to the degumming process, due to the - sheet structure arrangement of eri silk. Figure 3.9(a-b) shows the FTIR spectra of mulberry silk and eri silk scaffolds without ethanol treatment. The spectra show the amide I absorption band at 1691 and 1693 cm -1 (C =O stretching), amide II absorption band at 1518 and 1517 cm -1 (N-H bending) and amide III absorption band at 1171 cm -1 respectively, for mulberry and eri silk scaffolds. The absorption bands are attributed to the helix structure of silk fibroin scaffolds (Mai-ngam et al 2011). Figure 3.10 shows the absorption band of ethanol treated mulberry and eri silk nanofibrous scaffolds. The spectra show the amide I absorption band at 1624 and 1628 cm -1 (C=O stretching), amide II absorption at 1516 and 1520 cm -1 (N-H bending), and amide III adsorption at 1190 and 1232 cm -1 (C-N stretching) respectively. The amide I band has shifted from 1691 to 1624 cm -1 for

14 67 the mulberry scaffold, and 1693 to 1628 cm -1 for the eri silk scaffold due to ethanol treatment. This may be due to the change of the -helix structure to the -structure of the silk. The ethanol treatment process causes rearrangement of the hydrogen bonds in the silk fibroin nanofibrous scaffold (Cao et al 2009). Figure 3.7 FTIR spectra of un-degummed (a) mulberry and (b) eri silk filaments Figure 3.8 FTIR spectra of degummed (a) mulberry and (b) eri silk fibroins

15 68 Figure 3.9 FTIR spectra of (a) mulberry and (b) eri silk scaffolds without ethanol treatment Figure 3.10 FTIR spectra of ethanol treated (a) mulberry and (b) eri silk scaffolds

16 XRD Analysis Figure 3.11(a) shows the diffraction peaks at 20.5º, 29.4º and 40.5º (2 ), for the degummed mulberry silk fibroin and their corresponding spaces d are 4.32 Aº, 3.02 Aº and 2.22 Aº respectively. The Figure 3.11(b) shows the diffraction peaks at 18.3, and 40.10º (2 ) for mulberry silk scaffold (without ethanol treatment) and their corresponding spaces d are 4.8, 3.0 and 2.1Aº respectively. The Figure 3.11(c) shows the diffraction peaks at 20.20, and 40.44º (2 ) for ethanol treated mulberry silk scaffold and their corresponding spaces are 4.39, 3.03 and 2.27 Aº respectively. The degummed mulberry silk fibroin (Figure 3.11a) shows strong peak intensity at 20.5 (2 ) and 29.4º (2 ) for the corresponding space of 4.32Aº and 3.02 Aº. The strong intensity peaks are attributed to the crystalline structure. The weak intensity peak that appears at 40.5º (2 ) and its space d = 2.22Aº, is attributed to its non crystalline structure. The weak intensity of peak that appears at 18.3º (2 ) in Figure 3.11(b), and its space d=3.0 Aº is an indicative of amorphous content in the mulberry silk electrospun nanofibrous scaffold. The ethanol treated mulberry silk electrospun scaffold shows strong intensity peaks at 20.20º and 29.4º (2 ), with the corresponding space (d) of 4.32 and 3.02 Aº. This is attributed to the sheet crystalline structure formed due to the ethanol treatment of the scaffold and the peak is similar to the one obtained for degummed silk filament. The result shows that the electrospun scaffolds possessed a random coil (non oriented structure) structure, and is converted to the - sheet structure due to ethanol treatment. The average crystal size and percentage of crystallinity of the degummed silk fibroin, the untreated scaffold and the ethanol treated scaffold are given in Table 3.1. The result shows that the crystal size of the ethanol treated scaffold is equal to that of the degummed silk. The crystallinity of the ethanol treated scaffold is higher than that of the untreated scaffold due to the structural change from the random

17 70 coil to the - sheet structure. Figure 3.12(a) shows the diffraction peaks at 16.6º, 20.24º and º (2 ) for degummed eri silk filament and their corresponding spaces are 5.32, 4.38 and 3.68A respectively. Figure 3.12(b) shows the diffraction peaks at 11.23º, 18.4º and 29.6º (2 ) for electrospun eri silk scaffold without ethanol treatment and their corresponding spaces are 7.8, 4.8 and 3.0Aº respectively. Figure 3.12(c) shows the diffraction peaks at 11.0º, 19.3º and 28.5º (2 ) for eri silk nano fibrous scaffold after ethanol treatment and their corresponding spaces are of 7.1Aº, 4.5Aº and 3.1Aº respectively. The strong intensity peaks at 16.6º and 20.24º (2 ) with corresponding space of 5.32 and 4.38Aº for the degummed eri silk (Figure12a) are attributed to the - crystalline structure (Mai-ngam et al 2011, Rajkhowa et al 2011). The eri silk nanofibre (Figure 3.12b) shows a medium peak at 11.23º and 18.4º; its corresponding space of 7.8 and 4.8Aº indicate the amorphous content in the eri silk electrospun scaffold without ethanol treatment. The ethanol treated eri silk nanofibre (Figure 3.12c) shows a strong peak intensity of 19.3º (2 ) with corresponding space of 4.5Aº, which is attributed to the - sheet structure. The percentages of the crystallinity and average crystal sizes of the degummed eri silk filament, eri silk electrospun scaffold without ethanol treatment and ethanol treated eri silk scaffold are given in Table 3.1. It could be observed from Table 3.1 that the crystallinity % and crystalline sizes of the ethanol treated eri silk nanofibre are higher than those of the degummed eri silk filament and eri silk scaffold without ethanol treatment. This may be due to the - sheet structure. The crystalline size and % crystallinity of the eri silk fibroin are higher than those of the mulberry silk fibroin due to the presence of higher amount of alanine in the eri silk fibroin.

18 71 Table 3.1 Percentage of crystallinity and crystal size of mulberry silk and eri silk Materials Crystallinity % Crystal size (A ) Mulberry degummed silk Mulberry fibroin scaffold Ethanol treated mulberry scaffold Eri degummed silk Eri silk fibroin scaffold Eri silk ethanol treated scaffold Figure 3.11 XRD diffractograms of (a) degummed mulberry silk filament, (b) mulberry silk scaffold without ethanol treatment and (c) mulberry silk scaffold with ethanol treatment

19 72 Figure 3.12 XRD diffractograms of (a) degummed eri silk filament, (b) eri silk scaffold without ethanol treatment and (c) eri silk scaffold with ethanol treatment Tensile Strength Figures 3.13(a) and 3.13(c) respectively show the tensile stressstrain curve of the mulberry and eri silk scaffolds without ethanol treatment. The mean tensile stress and strain values are 0.993Mpa, 6.789% and, 1.075Mpa, 7.153% for the mulberry and eri silk scaffolds respectively. It can be seen that the eri silk scaffold has a marginally higher tensile strength and tensile strain than that of mulberry silk, though not significant. Figure 13.3 (b) and 13.3(d) show the stress strain curve of the ethanol treated scaffolds. The mean tensile stress and strain values are 1.6 Mpa, 14.7%, and 2.53 Mpa and 7.15% respectively, for the ethanol treated mulberry and eri silk scaffolds respectively. It can be noticed that the tensile stress and strain increases due to ethanol treatment in both the scaffolds. The increase is higher in the case of eri silk compared to that of the mulberry silk scaffold. This may be attributed to an increase in the content of the crystalline region, and presence of higher

20 73 amount of alanine in eri silk due to ethanol treatment. The trend matches with that of methanol treated mulberry silk fibroin experimented by Min et al (2006). Due to ethanol treatment, the scaffold shrinks, as mentioned by Min et al (2004), which increases the fibre to fibre friction and cohesive force between the fibres in the scaffold, and which in turn, increases the tensile stress. Figure 3.13 (a) Tensile stress strain curve of mulberry silk scaffold Figure 3.13(b) Tensile stress strain curve for ethanol treated mulberry silk scaffold

21 74 Figure 3.13(c) Tensile stress strain curve of eri silk scaffold Figure 3.13(d) Tensile stress strain curve of ethanol treated eri silk scaffold Hemolytic% The blood compatibility of the eri silk and mulberry silk scaffolds was estimated by their hemolytic ratio (HR). Excellent blood compatibility materials should have a low hemolysis ratio (Zobe1 et al 1999, Hou et al 2008), that is, lower than 5% (Yang et al 2005). Figure 3.14 shows the blood

22 75 compatibility of the eri silk and mulberry silk scaffolds. The hemolytic% of the eri silk and mulberry silk scaffolds is 1% and 3% respectively. It can be noticed that both the eri silk and the mulberry silk scaffolds have hemolytic rate values lower than 5% (Qu et al 2006),which is indicating that both the scaffolds exhibit good blood compatibility, and may be used for biomaterial applications. Figure 3.14 Hemolytic % of eri and mulberry silk scaffolds Platelet Adhesion Figure 3.15(a b) shows the platelet adhesion on the surface of the eri and mulberry silk fibroin scaffolds. From the SEM images, it is observed that the platelet adhesion on the mulberry silk fibroin scaffold is more than that of the eri silk fibroin scaffold, as the eri silk fibroin is more hydrophilic than mulberry silk. Since, platelet non adhesion material is essential for biomedical applications, eri silk fibroin is a better biomaterial compared to mulberry silk fibroin.

23 76 Figure 3.15(a) Platelet adhesion on the mulberry silk scaffold Figure 3.15(b) Platelet adhesion on the eri silk scaffold

24 L6 Rat Fibroblast Attachment Figure 3.16 show the SEM image of the cell attachment on the mulberry silk and eri silk fibroin scaffolds for 24 hrs and 48 hrs respectively. The SEM images 3.16 (a-b) respectively show the attachment and spread of the rat L6 fibroblast on the mulberry and eri silk scaffolds. The L6 fibroblast cell is not clearly attached and spread on the mulberry silk fibroin scaffold after 24 hrs of incubation; however, the attachment and spreading is better after 48 hrs of incubation. The SEM images 3.16(c-d) respectively show the cell attachment and spread of the rat L6 fibroblast on the eri silk scaffold after 24 hrs and 48 hrs of incubation. The cell attachment and spread on the eri silk fibroin scaffold is better than that on the mulberry silk fibroin scaffold after 24 hrs of incubation. The better performance of the eri silk may be due to the higher amount of positively charged amino acid and hydrophilic ratio (Maingam et al 2011) than those of mulberry silk fibroin. Figure 3.16(a) L6 fibroblast attachment on the mulberry silk scaffold after 24 hours of incubation

25 78 Figure 3.16(b) L6 fibroblast attachment on the mulberry silk scaffold after 48 hours of incubation Figure 3.16(c) L6 fibroblast attachment on the eri silk scaffold after 24 hours of incubation

26 79 Figure 3.16(d) L6 fibroblast attachment on the eri silk scaffold after 48 hours of incubation L6 Rat Fibroblast Cell Viability Viability of L6 rat cells on the mulberry and eri silk fibroin scaffolds was studied after 24 hrs and 48hrs of incubation. Figure 3.17 shows the viability percentage of the mulberry and eri silk fibroin scaffolds. The percentage cell viability of the eri silk fibroin scaffold is 95% and 81% respectively for the incubation period of 24 hrs and 48 hrs, whereas the cell viability of the mulberry silk fibroin scaffold is 86% and 70% respectively for the period of 24 hrs and 48 hrs. The difference is statistically significant at 95% confidence level based on the t test performed. The cell viability percentage of the eri silk fibroin scaffold is higher than that of the mulberry silk fibroin scaffold, because of higher hydrophilic nature of eri silk and the presence of positive charged amino acids (Mai-ngam et al 2011). From the results, it is seen that the eri silk fibroin scaffold may be a better candidate for biomedical applications.

27 80 Figure 3.17 Cell viability of eri silk and mulberry silk scaffolds 3.4 CONCLUSIONS The eri silk and mulberry silk fibroin scaffolds were produced by the electrospinning method. Majority of the fibres had the diameter in the range of 401 to 500 nm. Following conclusions are drawn from the physical characterization of the scaffolds: Thermal stability of the eri silk fibroin scaffold is higher than that of mulberry silk fibroin scaffold. The ethanol treatment of scaffold increases the crystallinity percentage and crystal size of both the eri and mulberry silk fibroin scaffolds. The eri silk scaffold has higher tensile stress than the mulberry silk scaffold.

28 81 The FTIR shows that ethanol treatment process causes arrangement of the hydrogen bonds in the silk fibroin nanofibrous scaffolds. following results: The biological studies carried out on the scaffolds showed the The hemolysis% of the eri silk scaffold is less than that of the mulberry silk scaffold; however, both the scaffolds have a hemolysis% less than 5% indicating that both the scaffolds have good biocompatibility. The platelet adhesion on the eri silk scaffold is lesser than that on the mulberry scaffold. The cell viability on the eri silk scaffold is higher than that of the mulberry. The cell attachment, binding and spreading on the eri silk fibroin scaffold is superior compared to the mulberry silk fibroin scaffold. Hence, it is concluded that eri fibroin scaffold, which shows better performance compared to that of mulberry scaffold, can be used for tissue engineering applications.