New Route in Degumming of Bombyx Mory Silkworm Cocon for Biomaterial.

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CONTENTS

Volume

4,

Number

4,

August

2015

Physiology

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Maximal Rowing on Fixed and Slides Ergomeier

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Shctzlin Shahatudin and Sunil Agrawal

study of the Effect

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Salt Stress on Biometric characteristics of Barley: Hordeum \rulgare

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(Poaceae)

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Abdetkader Hacli Seltd and Malika Ben Abderahmane

Force Plate Balance Response of Seafarers during Still and Rough Sea conditions

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F

Gildenhul's and R' Dobson

Bioinformatics

veritication

of GIvl2 Activator Protein for Potential Application as Lung cancer Biomarker'-Laclclawatt. Potpromma,ee, Kongsctk. Boonyapranai' Padchanee' Sangthong' Busayamass'

Chewaskulyong, Shui-Tein. Chen, and

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Premalignant Pancreatic Cancer Diagnosis Using Proteomic Pattem

Analysis""'

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KiranGeorge,R-lvlatathi,J'Krishnatt'andNishaSuscutThoruas

Medical Device

hnmobilization of Glucose Oxidase on Gold Surface for Applications in hnplantable Biosensors Kuo-Yuan Hwa and Boopathi Subramani

Feasibitity of the Fabrication of the Silicone carotid Model

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peng cao, Qttan Yttan, Guillaume olympe, Bntno Ramond, and Franqois Langevit

Microcantilever based Biosensor for Disease Detection

Applications""""""""'

P. G. Gopinath, V R- Anilhcl, and S' Armta Mastctnr

Pharmacology

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Vitro

Anti-Inflammatory Activities of Red Gemor Q{othaphoebe cf Umbellitlora) Yttdi Firmanul Ari,fin, siti Hamidah, sudin Paniaitan, (lnd Eko suhartono

Antidiabetic and Antioxidant

Activity

of Jackfruit (Artocarpus Heterophyllus) Extract'

Agung Biw'oro,

Efrilia

Tanjung, Iskandar' Khqirina' and Eko Suhartono

Effect of Centella Asiatica on Oxidative Stress in Rat Lung after Formalin Exposure

H. I,htr.Hidayah, M. S. Nttr-Khairah-Izzati, T. I, Rasyidah, M. A. Koswandi, and R. M. i{oah

Nlesenchymal Stem Ce1l tnfusion in chronic RenalFailure Patients Halq Gabr and Rania

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rct of Heavy Metal on Malondialdehyde and Advanced Oxidation Protein Produtcs Concentuation:

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Focus Arsenic, Cadmiurn, and

Mercury

...332

Iwan Aflanie, Ruslan Muhyi, and Eko Suhartono ,material

v Route in Degurnming of Bombyx

Mory

Silkwonn Cocon for

8iomateria1...

...338

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New Route in Degumming of Bombyx Mory

Silkworm Cocon for Biomaterial

Tjokorda Gde Tirta Nindhia and I Wayan Surata

Department of Mechanical Engineering, Engineering Faculty, Udayana University, Jimbaran, Bali, Indonesia, 80361 E-mail: nindhia@yahoo.com, iwassura@gmail.com

Zdenek Knejzlik and Tomas Ruml

Faculty of Food and Biochemical Technology, Institute of Chemical Technology of Prague , Technicka 5, 166 28 Pragua Czech Republic

E-mail: {Zdenek.Knejzlik, Tomas.Ruml}@vscht.cz

Tjokorda Sari Nindhia

Faculty of veterinary Madicine, Udayana University, Jl. P.B. Sudirman, Denpasar, Bali, Indonesia E-mail: snindhia@yahoo.com

Abstract—This research focused on silkworm cocoon of

Bombyx mori grown in Indonesia. In this study the degumming process to eliminate the sericin from the fiber was explored and the result is a separated fiber that is tested for its biocompatibility. The silk can be prepared by degumming method of boiling in 0.01 M NaOH for 1 hour. Observation under microsccope indicate that The human osteosarcoma cell line (U2OS) able to attach and grow during following two days. This is an indication that the fiber having good biocompatibility by degumming process that is introduced in this report.

Index Terms—bombyx mori, silkworm, cocoon, degumming, biocompatibility

I. INTRODUCTION

Some Lepidoptera larvae such as silkworm, spiders produce fibers that are recognized as a silk [1]. Bombyx mori (Fig. 1) is the most famous silk and have been domesticated from ancient time. The mulberry leave (Fig. 2 ) is the preferred food for the Bombyx mori larvae. The silkworm then will produce cocoon as depicted in Fig 3. The appearance of Bombyx mori silk moth can be seen in Fig. 4.

Beside well-known in textile production, Bombyx mori of silkworm silk has been used as medical suture. Sutures require the characteristics such as: Free from infection, non-irritant, possible to be metabolized once its repair function has been completed, the stiffness is change over time, the ability to conform to the current stage of wound repair, Knot strength, tensile strength to match the clinical repair [1]. From just for medical suture, silk then become point of interest by the researcher to provide biomaterial for other purpose.

_{Manuscript received June 10, 2014; revised September 4, 2014.}

It was proofed that Bombyx mori is suitable for 3-D scaffolding material which is make possible for the cell spread along the fiber and after that covered the entire surface and grow to fill the gap to form the structure of the tissue [2]. Silk also possible to be used as implants for the healing of critical size of bone defects which demonstrate the feasibility of silk-based implants with engineered bone for the regeneration of bone tissues with a mechanically stable and durable option [3].

Figure 1. Silkworm larvae of Bombyx mori

Figure 2. Mulberry tree

Two major proteins contains in the silk that are sericin (which is the coat and encased of the fiber) that hold

Journal of Medical and Bioengineering Vol. 4, No. 4, August 2015

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fibroin fiber together to form cocoon case in protect the worm inside [1]. Sericin is antigenic gum-like protein surrounding the fibers and fibroin is the core filaments of silk comprised of b-sheet crystal regions and

semi-crystalline regions which is responsible for silk’s

elasticity [1].

The sericin induces hypersensitivity in patients, causing a Type I allergic reaction. Exposure to silk debris may sensitize patients to silk causing adverse allergic reactions when silk is used as a suture material. Sericin identified as the antigenic agent of silk and should be removed through process of degumming [1]

Before further processing for biomaterial, the silk usually should be degummed first in order to fine fiber will be obtained. It is common for Bombyx morri cocoon to be degummed by boiling in hot water bath. samples were placed in beaker, and sufficient distilled water was added to completely immerse the sample. The beaker was heated in a hot water bath, and the samples were then washed with heated distilled water [4]. But in practice this technique will not eliminate the sericin in total as can be seen in Fig. 5

Figure 3. Silkworm cocoon of Bombyx mori

Figure 4. Silkworm moth Bombyx mori

Other protocols for degumming of the silk also already established such as by using Na2CO3. Cocoons from

Bombyx mori were boiled for 1 h in an aqueous solution Na2CO3, and rinsed with water to extract sericin and

other contaminating [3]. Other modification from this technique is

Dried Bombyx mori silk cocoons were cut into small pieces and treated with boiling aqueous solution of sodium carbonate with stirring. The mass was repeatedly

washed with distilled water to remove the sericine protein and dried in hot air oven [5]. Usualy after this process it is continued by dissolved in LiBr [6] or formic acid [7]. It was also informed that degumming also possible by using urea urea [8]. All degumming methods above mentioned having advantages and disadvantages that the reader can find it in the reference cited.

Degumming is also possible for removal of sericin by using enzymes protease and lipase Enzyme degumming involves the proteolytic degradation of sericin, using the specific proteins with minimum effect on fibroin. When the substrate molecule fits into the active sites of the

enzyme’s molecular structure to form an enzyme

substrate complex which is yields an end product and the original enzyme molecule is reproduced. Enzymes treatment operates under low temperatures and mild conditions which reduce the energy consumption. The disadvantage of this method is lower performance of enzyme degummed silk including difficult to handle and high cost. This condition limited the application of enzymes on the silk industry [8]

The action of organic acids is generally less aggressive than that of an action by alkali solution. Acidic agents (tartaric acid or citric acid) for degumming was approved for physical property enhancement. The action of organic acids is generally less aggressive than that of an action by alkali solution. the high performance on degumming is achieved by tartaric acid in terms of sericin removal efficiency and of intrinsic physico-mechanical characteristics of silk fibers. In the case of critic acid for degumming yield dry and wet resiliency of silk which was remarkably increased with citric acid treatment but

acid causes the damage on the fibroin surface [8]

Figure 5. result by degumming in boiling water. The sericin was not totally eliminated

This research introduce new route in degumming of silkworm cocoon of Bombyx mori as complement of our previous work [9], [10], that can be used as alternative in order best choice can be achieved with limited disadvantages.

II. EXPERIMENTAL

Bombyx mori cocoon obtained from local collector from Indonesian source (fig.3) was incubated in water and NaOH solution (two concentration were prepared: 0.01 M and 0.1 M) and boiled for 1 hour. The samples

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then were shaken for about 20 seconds. The insoluble fiber then was washed in warm water (70oC) intensively. Scanning Electron Microscope (SEM) was utilized to observe the structure of the fiber before and after degumming. The results fibers were sterilized with 70% ethanol for 1 day at room temperature. The samples were washed by PBS and suspension of human osteosarcoma cell line (U2OS) cell in Dulbecco’s Modiﬁed Eagle's cultivation medium and then weere soaked (in the atmosphere (5% CO2 at 37oC and 95% humidity) for 2

hours. The cultivated medium was supplemented with 10% FBS. The cells were cultivated for 2 days. For observation of the cell, the sample was taken out from cultivation medium and transferred to PBS and washed. Fluorescence microscope was utilized to observe the cell growth (IX-71, Olympus Japan) as characteristic of biocompatibility.

III. RESULT AND DISCUSSION

The efficient removal of the sericin coat was observed after treatment by 0.01 M NaOH as presented in Fig. 6a and Fig 7b. By using 0.1 M NaOH resulting crashed fiber which is indication of strong hydrolysis of fibers, there fore not recommended (Fig. 6b).

(a) (b)

Figure 6. (a).After treatment by 0.01 M NaOH, (b). By using 0.1 M NaOH resulting crashed fiber which is indication of strong hydrolysis of

fibers

It was found that the cells are able to attach and grow on Bombyx mori fiber released from cocoon by 0.01 M NaOH during following two days base on observation under microscope. This is an indication that the fiber having good biocompatibility (Fig. 8). Our previous report [10] also informed that deguming by using 0.01 M NaOH also resulting good deguming result to other type of cocoon (Cricula trifenestrata) and resulting positive indication for biocompatibility.

(a) (b)

Figure 7. Scanning Electron Micrograph (SEM) of the Bombyx mori silk fiber (a) condition before degumming as obtained from our previous

publication [9]. (b) after degumming with 0.01m NaOH

(a) (b)

Figure 8. Cell growth on Bombyx mori fibers obtained by degumming of cocoon by using 0.01 M NaOH. (a) bright field. (b) After cell fixation, nuclei were stained using DAPI and analyzed by fluorescence

microscopy

As it is found by using only boiling water [4] for degumming, then the sericin is not totally removed (fig. 4). Other effect is the tensile strength is found low [4]. This research should be continued to prove that by using degumming method introduced in this report will also increase the tensile strength.

Comparing with other method of degumming, as for example by using Na2CO3 [3], [5], formic acid [7] and

urea [8], the method that is introduced in this research is also possible to be introduced to other types of silk cocoon such as from the species of Cricula trifenestrata [9], [10] and also Attacus atlas [9] meanwhile in our experience degumming by using Na2CO3, formic acid or

urea are not successful for degumming both cocoon of Cricula trifenestrata and Attacus atlas. Other negative side by using Na2CO3 is, sericin is emulsified by the soap

and finally, removed from the fibre. but, the presence of soap and alkalis in the wastewater of degumming process raise an issue of pollution. Besides, the degumming cycles of the soap ash bath is limited because of acidity of sericin hydrolysis products accumulating in the bath [8]

Other advantages of degumming method that is introduced in this research is, it can shorten the process. Other technique should initiate by boiling in water first and then continued by the next process, meanwhile in the technique which is introduced in this research is possible without initiate by boiling water. So that it can shorten the process for the benefit of production cost.

IV. CONCLUSION

The new route in degumming silk of Bombyx mori is established in this research. The silk is prepared by degumming method of boiling in 0.01 M NaOH in 1 hour. The human osteosarcoma cell line (U2OS) able to attach and grow during following two days. This is an indication that the fiber having good biocompatibility by degumming process that is introduced in this research.

ACKNOWLEDGMENT

The authors acknowledge financial support received from Directorate General of Higher Education of The Ministry Education and Culture The Republic of Indonesia trough Scheme of International research collaboration (Penelitian Kerjasama Luar Negri) under

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contract number: 238-40/UN14.2/PNI.01.03.00/2014. This work also supported by Czech Science Foundation project P108/12/G108 trough Institute of Chemical Technology of Prague which is also very much acknowledged. Special thank addressed to Prof. Ing. Katerina Demnerova, CSc. Together with Ing. Jan Lipov, Ph.D For the permission to use all research facility in the Department of Biochemical and Microbiology Institute technology of Prague, Czech Republic

REFERENCES

[1] G. H. Altman, F. Diaz, C. Jakuba, T. Calabro, R. L. Horan, et al., “Silk-based biomaterials,”Biomaterials, vol. 24, no. 3, pp. 401– 416, 2003.

[2] R. E. Unger, K. Petersa, M. Wolf, A. Motta, C. Migliaresi, C. J. Kirkpatrick, “Endothelialization of a non-woven silk fibroin net for use in tissue engineering: Growth and gene regulation of human endothelial cells,”Biomaterials, vol. 25, pp. 5137–5146, 2004.

[3] L. Meinel, R. Fajardo, S. Hofmann, R. Langer, et al., “Silk implant for the healing of critical bone defects,”Bone, vol. 37, pp. 688-698, 2005.

[4] P. Jiang, H. Liu, C. Wang, L. Wu, J. Huang, and C. Guo, “Tensile behavior and morphology of differently degummed silkworm (Bombyx mori) cocoon silk fibres,”Material Letters, vol. 60, pp. 919-925, November 2005.

[5] M. K. Sah and K. Pramanik, “Regenerated silk fibroin from B. mori silk cocoon for tissue engineering application,”International Journal of Environmental Science and Development, vol.1, no. 5, pp. 404-408, December 2010

[6] L. Uebersax, S. Hofmann, H. Hagenmuller, R. Muller, H. P. M erkle, and L. Meinel, “Highly porous silk scaffold for bone defect repair,”European Cells and Materials, vol. 10, no. 1, pp. 35, June 2005.

[7] C. Meechaisue, P. Wuttich Aroenmongkol, R. Waraput, T. Huangjing, et al., “Preparation of electrospun silk fibroin fiber mats as bone scaffolds: A preliminary study,” Biomedical Materials, vol. 2, pp. 181-188, August 2007

[8] M. P. Ho, H. Wang, K. T. Lau, J. H. Lee, and D. Hui, “Interfacial bonding and degumming effecr on silk/polymer biocomposites,”

Composites: Part B, vol. 43, pp. 2801-2812, May 2012.

[9] T. G. T. Nindhia, Z. Knejzlik, and T. Ruml, “Morphology, chemical elements composition, and biocompatibility of indonesia

wild silkworm cocoon,” in Proc. Seminar Tahunan Teknik mesin XI (SNTTM XI) & Thermofluid IV, Universitas Gadjah Mada (UGM) Yogyakarta, 16-17 Oktober 2012

[10] T. G. T. Nindhia, Z. Knejzlik, T. Ruml, T. S. Nindhia, “Tensile properties and biocompatibility of indonesian wild silk cricula trifenestrata: A preliminary study,” Journal of Medical and Bioengineering, vol. 3, no.2, June 2014

Tjokorda Gde Tirta Nindhia Was born in Denpasar, Bali, Indonesia on January 16th, 1972. Received Doctor Degree in Mechanical Engineering from Gadjah Mada University (UGM) Yogyakarta, Indonesia on August 2003, with major field of study was Material Engineering.

He participated in various international research collaboration such as with Muroran Institute of Technology Japan (2004), Toyohashi University of Technology Japan (2006), Leoben Mining University Austria (2008-2009), Technical University of Vienna Austria (2010) and Recently with Institute Chemical technology of Prague Czech Republic (2012-now). His current job is as Full Professor in the field of Material Engineering at Department of Mechanical Engineering, Engineering Faculty, Udayana University, Jimbaran, Bali, Indonesia. His research interest covering subjects such as, biomaterial, waste recycle, failure analyses, ceramic, metallurgy, composite, renewable energy, and environmental friendly manufacturing.

Tomas Ruml . is full professor at Faculty of Food and Biochemical Technology, Technicka 5, 166 28 Pragua Czech Republic

Zdenek Knejzlik. Is senior researcher at Faculty of Food and Biochemical Technology, Technicka 5, 166 28 Pragua Czech Republic

I Wayan Surata was born in Nusa Penida, Bali, Indonesia on July 5, 1958. Received Doctor Degree in the field of Ergonomic from Udayana University in 2011. His research interest very much related in process of manufacture. His Current job is researcher and lecturer at Department of Mechanical Engineering, Engineering Faculty, Udayana University, Jimbaran, Bali, Indonesia.

Tjokorda Sari Nindhia is Doctor of Veterinary Medicine (DVM) at faculty of Veterinary Medicine Udayana University, Bali, Indonesia since 1999. Finishing her master degree at biotechnology and biomolecular at postgraduate studies Udayana University in 2010