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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

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 12 13) 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

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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

3.2.1 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 8.5–9.0, 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

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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 trifluoro-

acetic 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

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Figure 3.1(b) SEM image and fibre diameter distribution of eri silk

fibroin scaffold

3.2.2 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 4000- 400cm-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 30 – 400ºC 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

59

measured by fityk software; the crystal size and % of crystallinity were

measured using Equations (3.1) and (3.2).

KCrystal size A

cos (3.1)

Total area of crystalline peakCrystallnity % x100

Total area of crystalline and amorphous (3.2)

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.

3.2.3 Biological Characterization of Scaffolds

3.2.3.1 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

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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 negativeHemolytic % 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.

3.2.3.2 Rat L6 muscle cell culture

Rat L6 muscle fibroblasts were seeded at a density of 1 × 104 cells

per silk fibroin scaffold. The cells were incubated at 37 C with 5% CO2 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-

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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.

3.2.3.3 MTT assay

Rat L6 muscle fibroblasts were seeded at a density of 1 ×104

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% CO2. 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

3.3.1 Thermal Stability

The thermogrametric curves of eri silk and mulberry silk scaffolds

are shown in Figures 3.2- 3.4. 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 280-350ºC and 320-

390°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

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second weight loss takes place at the temperature of 250–350°C and 320-

390°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 300-320ºC and 280-360ºC 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

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

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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, 361.4 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, 278.9 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 350-370ºC in Figure 3.5, and 278- 310ºC 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.

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

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3.3.2 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

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

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

69

3.3.3 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, 29.40 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,

29.41 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

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coil to the - sheet structure. Figure 3.12(a) shows the diffraction peaks at

16.6º, 20.24º and 24. 19º (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.

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Table 3.1 Percentage of crystallinity and crystal size of mulberry silk and

eri silk

Materials Crystallinity % Crystal size (A )

Mulberry degummed silk 51 16.7

Mulberry fibroin scaffold 50 15.1

Ethanol treated mulberry scaffold 53 16.2

Eri degummed silk 48 40

Eri silk fibroin scaffold 45 26

Eri silk ethanol treated scaffold 54.5 55

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

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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

3.3.4 Tensile Strength

Figures 3.13(a) and 3.13(c) respectively show the tensile stress-

strain 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

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

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

3.3.5 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

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

3.3.6 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.

76

Figure 3.15(a) Platelet adhesion on the mulberry silk scaffold

Figure 3.15(b) Platelet adhesion on the eri silk scaffold

77

3.3.7 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 (Mai-

ngam 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

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

79

Figure 3.16(d) L6 fibroblast attachment on the eri silk scaffold after

48 hours of incubation

3.3.8 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.

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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.

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The FTIR shows that ethanol treatment process causes

arrangement of the hydrogen bonds in the silk fibroin

nanofibrous scaffolds.

The biological studies carried out on the scaffolds showed the

following results:

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.