1. Kanyaluk Kaewprasit1, Amornrat Promboon2, Siriporn Damrongsakkul1*
Characteristics and cell responses on silk fibroin film prepared from three the silkworms, Bombyx Mori
Kanyaluk Kaewprasit1, Amornrat Promboon2, Siriporn Damrongsakkul1*
1 Department of Chemical Engineering, Faculty of Engineering, Chulalongkorn University, Bangkok, Thailand
2 Department of Biochemistry, Faculty of Science, Kasetsart University, Bangkok, Thailand
Good afternoon ladies and gentlemen. My name is Kanyaluk Kaewprasit from Department of chemical engineering, Faculty of engineering, Chulalongkorn university.
Today I would like to present part of my work with the topic “Characteristics and cell responses on silk fibroin film prepared from three the silkworms, Bombyx Mori “.

2. Introduction
OK.!!! Let’s start with introduction.

3. Silk & Silk fibroin Silk Silk fibroin An insoluble fibrous protein
Beta sheet structure of silk fibroin
An insoluble fibrous protein
Secondary structure is betasheet
G - X sequence (G = Glycine , X = Alanine ,Serine, Tyrosine, Valine)
Silk from the silkworm is a natural protein that has been used as material in biomedical applications. It composed of two proteins.
1. The core structural protein is called silk fibroin which found around wt% of raw silk fiber. and
2. The glue-like protein that holds fibroin fiber together is called sericin.
In this study, we interest in the silk fibroin component.
The silk fibroin is an insoluble fibrous protein. The fibroin protein consists of layers of beta sheets.
Its secondary structure mainly consists of amino acid sequence G-X repeats with X being Alanine, Serine, Tyrosine and Valine.
Attractive properties of silk fibroin for in biomedical applications are biocompatibility, good mechanical properties and slow biodegradability.
Silk is consisted of two main proteins:
Silk fibroin (75-80 wt% of raw silk fiber)
Silk sericin (20-25 wt% of raw silk fiber)
Attractive properties in biomedical applications:
Good mechanical properties
Biocompatibility and biodegradability
Ref: Vepari. C., Kaplan. D.L. Silk as a biomaterial, Prog. Ploym. Sci. 32(2007):

4. Silk fibroin races derived from “the silkworms, Bombyx mori”
Bombyx mori silk
Several Bombyx mori silkworm races:
- Thai race
- Japanese race
- Chinese race
- Indian race
etc.
The applicati ons in tissue engineeri ng field:
- Bone & cartilage tissue
- Blood vessel
- Wound dressing
- Drug delivery
Silk fibroin can be derived from wild silkworms and domesticated silkworms. In this case, we are interested in domesticated silkworms generally known the silkworms, Bombyx mori that is one of the most widely studied silk species in tissue engineering but there are several silkworm races such as Thai race, Japanese race, Chinese race, Indian race. That has been used in a wide variety of biomedical applications, for example Bone & cartilage tissue, Blood vessel, Wound dressing and Drug delivery.
Ref: Vepari. C., Kaplan. D.L. Silk as a biomaterial, Prog. Ploym. Sci. 32(2007):

5. Objectives
To characterize silk fibroin from three Bombyx mori silkworm races including:
Thai race (Nangnoi-Srisaket 1; NN)
Japanese race (K1)
Chinese race (K8)
To compare cell responses on all silk fibroin films.
So, The first objective of this study were to characterize silk fibroin from three Bombyx mori silkworm races including:
- Thai race
- Japanese race and
- Chinese race
And the second one is to compare cell responses on all silk fibroin films.

6. Experiments
For the experiments of this study.

7. Thai race (Nangnoi-Srisaket 1; NN)
Silk cocoons
The Bombyx mori cocoons were kindly given by Queen Sirikit Sericulture Center, Nakornratchasima province, Thailand.
Thai race (Nangnoi-Srisaket 1; NN)
Japanese race (K1)
Chinese race (K8)
The Bombyx mori cocoons were kindly given by Queen Sirikit Sericulture Center, Nakornratchasima province, Thailand.
- Thai race (Nangnoi-Srisaket 1) or NN, this cocoon
- Japanese race or K1
- and Chinese race or K8.
The most obvious characteristic of Thaisilk cocoon, it’s yellow colour while the others are white.
The shape of Thai race look like capsule. The shape of Japanese like capsule but with waist line and the last, Chinese is more sperical shape.

8. Preparation of silk fibroin solution
Silk cocoons
Silk fiber degummed in Na2CO3
Wash DI water
Fibroin dissolution in LiBr
Air dry silk fibroin
Dialysis against DI water for 3 days
Silk fibroin solution
For the preparation of silk fibroin solution.
Firstly, Silk cocoons were boiled in Na2CO3
and then rinsed thoroughly with DI water to get rid of sericin.
Dried Thai silk fibroin was dissolved in LiBr solutions.
The solution was dialyzed against DI water for 3 days.
Finally, The concentration of silk fibroin solution was % by weight.
Ref : Kim, U.J. Park, J., Kim, H.J., and Kaplan, D.L. Effects of biodegradation of gelatin/Thai silk fibroin scaffolds. Biomaterials 26, (2005):

9. Preparation of silk fibroin films
Air dried overnight
Silk fibroin solution ( wt%)
Diluted silk fibroin solution and casted films
Immersed in 70 vol% methanol for 30 min
To prepared silk fibroin films.
Start from, diluted silk fibroin solution with DI water and casted film on the mold. After air drying overnight, silk fibroin films were obtained. The films were then immersed in 70 % by volume of methanol for 30 minutes to induce the conformation change of silk fibroin from random coil to β-sheet and air dried overnight again.

10. Physicochemical characterizations
Degradation temperature (Td) Thermogravimetric analyzer (TGA)
- Temperature range 30 – 700 ºC
- Heating rate = 10 °C/min
Conformations of silk fibroin ATR-FTIR spectra
- Wave number range 2500 – 650 cm-1
The Physicochemical characterizations have 3 parts:
- The degradation temperature of different silk fibroins was analyzed by thermogravimetric analyzer
- Molecular conformation of the silk fibroin films were analyzed by ATR-FTIR
and
- The amino acid compositions were analyzed using HPLC.
Amino acid compositions High performance liquid chromatography (HPLC)

11. In vitro attachment and proliferation test
Isolation of rat-bone marrow derived mesenchymal stem cells (MSCs)
Cultured in TCP containing α-MEM at 37 ºC in a 5% CO2
Seed cells onto the silk fibroin films at 2×104 cells/film
Evaluate the number of MSCs attached and proliferated on films by MTT assay
Cultured for 6, 24, 72, 120 h
To test the In vitro biological properties of silk fibroin films, the attachment and proliferation of rat bone marrow-derived mesenchymal stem cells cultured on the films were evaluated.
- First, rat bone marrow-derived stem cells or MSCs were isolated from the bone shaft of femurs of 3 week old female Wistar rat.
- Then, cultured this cells in α-MEM supplemented with 15% fetal bovine serum (FBS) at 37ºC, 5% CO2.
- MSCs were seeded on the sterilized films and Tissue culture plate (TCP) as a control at the density of 20,000 cells/film.
- Cultured for 6, 24, 72, 120 h
- After that, the number of cells attached and proliferated on silk fibroin films were evaluated by MTT assay.

12. Results and discussions
Now, I can show you the results and dicussion.

13. Degradation temperature
Table 1: The degradation temperature (Td) of Bombyx mori silk fibroin films from three silkworm races.
Silkworm races
Td (ºC)
Thai race (Nangnoi-Srisaket 1; NN)
264.8
Japanese race (K1)
264.2
Chinese race (K8)
265.0
This table shows the degradation temperature of silk fibroin films from three silkworm races
All silk fibroin films showed similar degradation temperatures at around ºC (degree of celcese)

14. FTIR spectra of silk fibroin film
Amide I
( cm-1)
Amide II
( cm-1)
Amide III
( cm-1)
Absorbance (a.u.)
Wavenumber (cm-1)
Amide I
Amide II
Amide III
This figure show FTIR spectra of silk fibroin films. It could be seen that three peak positions including:
- Amide I were found at 1610–1630 cm-1
- Amide II (N-H deformation and C-N stretching) were found at 1510–1520 cm-1
- and Amide III (C-N stretching and N-H deformation) were found at 1230–1270 cm-1
These amide bands were attributed to the β-sheet structure. The FTIR results revealed that the structure and conformation of silk fibroin films from three silkworm races were similar.

15. Amino acid compositions
Amino acid group NN K1 K8
Acidic
2.98
3.60
3.69
Basic
1.33
1.38
1.76
Polar
14.22
17.95
17.38
Total (% by mole)
18.33
22.93
22.83
Hydrophilic groups
Amino acid group NN K1 K8
Non- polar
74.73
67.39
68.17
Aromatic
6.94
9.68
9.00
Total (% by mole)
81.67
77.07
77.17
The amino acid compositions of Bombyx mori silk fibroin from three silkworm races shows in this slide.
The amino acids were classified into 2 groups: hydrophilic and hydrophobic groups.
The percentage of hydrophilic amino acid groups in K1 was slightly higher than K8 while NN was the lowest. On the other hand, NN contained the highest percentage of hydrophobic amino acid groups at 81.67%, comparing to other two silkworm races. This indicated that NN was the most hydrophobicity.
Hydrophobic groups

16. In vitro attachment and proliferation tests
Here is the results of in vitro attachment and proliferation of MSC cultured on each silk fibroin film.
- There was no significant difference in the number of cells attached on different silk fibroin films after 6 h of culture.
- At 24, 72 and 120 h of culture, cells cultured on TCP showed the greatest number along culture period.
- The number of cells proliferated on K1 films was slightly greater than other silk fibroin films, however, the significant difference was not found.
Remark : Tissue culture plate (TCP) as a control
a - g represented significant difference among samples at p < 0.05.

17. Substrate Cell attachment (%)
Table 2: The percentage of cell attachment on Bombyx mori silk fibroin films prepared from three silkworm races.
Substrate
Cell attachment (%)
Tissue culture plate (TCP)
95.6±5.1
Thai race (NN)
68.9±1.9
Japanese race (K1)
81.1±5.1
Chinese race (K8)
78.9±6.9
The percentage of cell attachment on Bombyx mori silk fibroin films at 6 h was calculated compared to the number of initial seeding.
The percentage of cell attachment on TCP was the highest while that of K1 film was slightly higher than other two silk fibroin films.
The percentage of cell attachment on NN film was the lowest.
The hydrophilic surface promotes the adsorption of many proteins such as fibronectin, vitronectin and albumin which are necessary for cell attachment and proliferation.

18. Conclusions
All silkworm races had the similar physicochemical properties :
Degradation temperature
Molecular conformation
Japanese silk fibroin film (K1) promoted cell attached and proliferated slightly better than the others posssibly due to the most hydrophilic amino acid groups.
From the result, it can be concluded that:
- All silkworm races had the similar physicochemical properties including thermal properties and molecular comfprmation.
- Japanese or K1 silk fibroin film promoted cell attached and proliferated slightly better than the others posssibly due to the most hydrophilicity amino acid groups.

19. Acknowledgements
Financial supports from The National Research University Project of Commission on Higher Education (CHE) and the Ratchadaphiseksomphot Endowment Fund (AS615A-55).
We thank Tanom Bunaprasert, M.D. for the use of cell culture facilities at i-Tissue Laboratory, Faculty of Medicine, Chulalongkorn University.

20. Thank you for your attention

23. Heavy chain structure in fibroin proteins
GAGAGS - 49%
Silk has 12 crystalline domains which are linked by 11 amorphous chains
Crystalline domains are in β-sheet forms
The crystalline domains consist of Gly-X repeats, with X being Ala, Ser, Thr &Val
Each domain consists of sub-domain hexapeptides including: GAGAGS, GAGAGY, GAGAGA or GAGYGA
Ref: Vepari & Kaplan, Prog. Polym. Sci. 32 (2007) 991–1007
GAGAGY - 14%
GAGYGA - 4%
Amorphous domains
are the linkers between each crystalline domains
has almost identical 25 amino acid residue (non-repetitive sequence), which is composed of charged amino acids not found in the crystalline regions
GAGAGAGAGAGTGSSGFGPYVANGGYSGYEYAWSSESDFGTGS
Ref: Zhou et. al., Proteins 2001;44(2):119–22
GAGAGA - 3%

24. Amino acid compositions
Hydrophilic groups
Hydrophobic groups
Amino acid group NN K1 K8
Acidic
Aspartic acid
1.83
2.08
2.18
Glutamic acid
1.15
1.52
1.51
Basic
Arginine
0.30
0.31
0.44
Lysine
0.20
0.26
Histidine
0.83
0.81
1.02
Polar
Serine
13.42
16.87
16.30
Threonine
0.80
1.08
Cysteine -
Total
18.33
22.93
22.83
Amino acid group NN K1 K8
Non- polar
Glycine
38.32
33.00
35.76
Alanine
34.29
31.26
29.39
Proline
0.42
0.64
Valine
1.15
1.67
1.53
Leucine
0.27
0.38
0.43
Isoleucine
0.20
0.31
0.32
Methionine
0.08
0.13
0.10
Aromatic
Tyrosine
5.75
7.66
7.16
Phenylalanine
0.98
1.69
1.39
Trytophan
0.21
0.33
0.45
Total
81.67
77.07
77.17

25. REF : http://www.learners.in.th/blogs/posts/264172

26. Tetrazolium (yellow colour) Formazan (purple colour)
MTT Assay
MTT or 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide.
Objective
To evaluate the number of cell viability.
Principle
Enzymes (e.g. mitochondrial reductase) of metabolic cells reduce MTT to a purple formazan crystals. Absorbance indicates the enzyme activity, representing cell activity and viability.
Tetrazolium (yellow colour)
Formazan (purple colour)
Ref: T. Mosmann, J. Immunol. Methods.65 (1983)

27. Method of MTT assay
Prepare 0.5 mg/ml of MTT in DMEM without phenol red or PBS. PROTECT FROM LIGHT!!
Remove old medium, wash with PBS, add 350 µl/well of MTT solution and incubate at 37 ºC, 5% CO2 for 30 min (1-4 h for hydrophobic scaffolds or scaffolds with mass transfer limit)
Remove MTT solution, add 1 ml/scaffold of DMSO to elute the purple ice crystals of MTT and homogenize the purple solution
Measure the absorbance of the solution at 570 nm using a microplate reader

28. HPLC conditions
High Performance Liquid Chromatography (HPLC)
Column : Shim-pack ISC-07/S1504Na
Mobile phase:
1.) 0.2N sodium citrate pH 3.2 (containing 7% Ethanol)
2.) 0.6N sodium citrate + 0.2M boric acid pH 10.0
3.) 0.2M sodium hydroxide

29. Natural materials in tissue engineering
Silks from silkworms
Gelatin
Chitosan
Collagen
Keratin
Polymer materials both synthetic and natural are widely used as cell scaffolds for tissue engineering. There are many interesting natural materials such as silks from silkworms, gelatin, chitosan, collagen and keratin. In this work, we focus on silks from silkworms.