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Silk Biomaterials for Tissue Engineering and Regenerative Medicine
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Commencer à lireLongueur: 1,107 pages12 heures
- Éditeur:
- Elsevier Science
- Sortie:
- Mar 24, 2014
- ISBN:
- 9780857097064
- Format:
- Livre
Description
Silk is increasingly being used as a biomaterial for tissue engineering applications, as well as sutures, due to its unique mechanical and chemical properties. Silk Biomaterials for Tissue Engineering and Regenerative Medicine discusses the properties of silk that make it useful for medical purposes and its applications in this area.
Part one introduces silk biomaterials, discussing their fundamentals and how they are processed, and considering different types of silk biomaterials. Part two focuses on the properties and behavior of silk biomaterials and the implications of this for their applications in biomedicine. These chapters focus on topics including biodegradation, bio-response to silk sericin, and capillary growth behavior in porous silk films. Finally, part three discusses the applications of silk biomaterials for tissue engineering, regenerative medicine, and biomedicine, with chapters on the use of silk biomaterials for vertebral, dental, dermal, and cardiac tissue engineering.
Silk Biomaterials for Tissue Engineering and Regenerative Medicine is an important resource for materials and tissue engineering scientists, R&D departments in industry and academia, and academics with an interest in the fields of biomaterials and tissue engineering.
Discusses the properties and applications of silk for medical purposes Considers pharmaceutical and cosmeceutical applicationsInformations sur le livre
Silk Biomaterials for Tissue Engineering and Regenerative Medicine
Longueur: 1,107 pages12 heures
Description
Silk is increasingly being used as a biomaterial for tissue engineering applications, as well as sutures, due to its unique mechanical and chemical properties. Silk Biomaterials for Tissue Engineering and Regenerative Medicine discusses the properties of silk that make it useful for medical purposes and its applications in this area.
Part one introduces silk biomaterials, discussing their fundamentals and how they are processed, and considering different types of silk biomaterials. Part two focuses on the properties and behavior of silk biomaterials and the implications of this for their applications in biomedicine. These chapters focus on topics including biodegradation, bio-response to silk sericin, and capillary growth behavior in porous silk films. Finally, part three discusses the applications of silk biomaterials for tissue engineering, regenerative medicine, and biomedicine, with chapters on the use of silk biomaterials for vertebral, dental, dermal, and cardiac tissue engineering.
Silk Biomaterials for Tissue Engineering and Regenerative Medicine is an important resource for materials and tissue engineering scientists, R&D departments in industry and academia, and academics with an interest in the fields of biomaterials and tissue engineering.
Discusses the properties and applications of silk for medical purposes Considers pharmaceutical and cosmeceutical applications- Éditeur:
- Elsevier Science
- Sortie:
- Mar 24, 2014
- ISBN:
- 9780857097064
- Format:
- Livre
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Silk Biomaterials for Tissue Engineering and Regenerative Medicine
USA
Part I
Fundamentals, processing and types of silk biomaterials
Outline
Chapter 1: Introduction to silk biomaterials
Chapter 2: Applications of silk biomaterials in tissue engineering and regenerative medicine
Chapter 3: Processing of Bombyx mori silk for biomedical applications
Chapter 4: Silk nanostructures based on natural and engineered self-assembly
Chapter 5: Electrospun silk sericin nanofibers for biomedical applications
Chapter 6: Silk fibroin microfiber and nanofiber scaffolds for tissue engineering and regeneration
Chapter 7: Silk powder for regenerative medicine
1
Introduction to silk biomaterials
D. Naskar, R.R. Barua, A.K. Ghosh and S.C. Kundu, Indian Institute of Technology Kharagpur, India
Abstract:
The term ‘silk’ makes almost all of us think first of lustrous and shimmery fabrics. This chapter discusses in brief the history of the evolution of silk from China and how silks spread to the rest of the world. It describes the different types of mulberry and non-mulberry silkworm species, their habits and habitats. This chapter briefly describes silk protein sericins (glue proteins) and fibroins produced by different silkworms as well as silk protein encoding genes. It also outlines the different diseases of silkworms and, finally, different applications of silk proteins in the core fields of biomedical engineering.
Key words
silk history
mulberry
non-mulberry
sericin
fibroin
silkworm diseases
1.1 Introduction
It is a well-known fact that silk, commonly known as the ‘queen of all fabrics’, was first discovered in China (Columbia Encyclopedia, 2000). Silk production, or ‘sericulture’, has a long and colorful history. According to the Confucian testimonial, the use of Bombyx mori silkworm cocoons and their silk production can first be dated to around 2700 bce in China, although archeologists have speculated that the history of silk cultivation can be traced back to the Yangshao period (5000–3000 bce) (Barber, 1992). Chinese history popularly described Lady Hsi-Lin-Shih (wife of the Yellow Emperor, Shi Huang di) as having tea one day under a mulberry tree when a cocoon fell into her cup. She observed that the cocoon spun a strong continuous thread which could be reeled and used as weaving thread (The Silk Association of Great Britain, 2007). From this point onwards silk has been woven specifically for royal families and became a symbol of royalty and wealth, and for more than 2000 years China kept the secret of silk. During the Shang dynasty, the production and use of silk reached its peak in craftsmanship, displaying the brightness of dyes and the perfectly honed skill of embroidery (People’s Daily Online, 2007).
Silk culture later spread to Korea, first at around 200 bce, with the movement of migrants, and from there gradually extended to other parts of Asia and Europe, such as Japan, India and Persia at around 300 bce (Qin, 2006). Another story relates the first time silk crossed the border from China, as early as 552 bce (Maltretus, 1729). When Zhang Qian served as the Chinese ambassador, two Persian monks visited China as missionaries, and silkworm eggs were secretly transported by them to Constantinople inside their walking canes. Another similar story relates the first time silk had been smuggled through the border of Japan to reach Europe. In this case, a student brought the eggs to Europe. In this way Europe could be seen to profit from the silk industry through a case of fraud in ancient times (Wardle, 1881). During the seventh century, silk was spread to Arabia, Africa, Sicily and Spain and by the thirteenth century it finally reached Italy. Silk materials and other valuable fabrics were transported to the west along the famously prosperous ‘Silk Road’ (the term coined in 1877 by Ferdinand von Richthofen, a well-known German geographer); this was a 4000 mile-long road which linked China with the Roman Empire (Eliseeff, 1998). In this way the silk trade was promoted for the cultural as well as economic exchange between the East and West. Besides its use in cloths at that time, silk was also used in the production of various other luxury objects such as handkerchiefs, wallets and wall hangings, and also for other less decorative purposes such as papers, fishing cords, bowstrings and strings for musical instruments (Meyer, 2000).
Silkworms, their eggs and the technology of sericulture were first introduced to India by Buddhist monks, and by the princess who married the king of Khotan, in Tibet (Hill, 2009). About two and half centuries ago silk was introduced in Karnataka by the ruler Tipu Sultan, and then spread to Tamil Nadu in the early 1960s. This area is now considered one of the most important regions for silk production in India (R. T. I. act, Chapter 18, 2012). The archeological evidence found in Harappa and Mohenjo-Daro gave rise to some interesting discoveries. The evidence found in these regions suggests that sericulture was also being practiced in South Asia in the Indus Valley Civilization, which was almost contemporary with production in China (Good et al., 2009).
Analysis of silk production distribution worldwide has shown that China is the largest producer of silk, producing 79.1% of the total worldwide raw silk production. India is the second largest producer after China, and produces 17.5% of the total worldwide raw silk production. Japan, Brazil, Korean Republic, Uzbekistan, Thailand and Vietnam follow as other significant producers of raw silk materials (Antha, 2011). Major silk producing states in India are Karnataka, Tamil Nadu, Andhra Pradesh, West Bengal, Madhya Pradesh, Jammu and Kashmir, Maharashtra, Manipur, Mizoram and Assam, while other states in India also produce a little silk (TNAU Agritech Portal, 2012). This chapter deals with the various species of silkworms, their products and different uses.
1.2 General information about silkworms
A wide variety of natural silks from hundreds of different silkworm species are available throughout the world. Among these, the family Bombycoidea consists of eight families of which Bombycidae and Saturniidae are commercially important, as shown in Table 1.1. The family Bombycoidea silkworm silk is categorized as ‘mulberry’, while the Saturniidae fall under the category of ‘non-mulberry’.
Table 1.1
Main mulberry and non-mulberry silkworms found particularly in India
Sources: Ahmed and Rajan (2011) and Central Silk Board, Ministry of Textiles – Govt of India; web portal (2012).
1.2.1 Mulberry silkworms
Mulberry silkworms belong to the family Bombycidae and the silk is called mulberry silk. Commercially available mulberry silk is produced from one single species called Bombyx mori. Mulberry silkworms are entirely domesticated, and they do not occur naturally. They need human care for their growth and reproduction. It is believed that this species originates from its native wild ancestor species (which is of Chinese stock rather than Japanese or Korean stock), Bombyx mandarina by gene duplication and chromosomal fusion mechanism (Mahendran et al., 2006). Depending upon the geographical distribution, B. mori is classified as Chinese, Japanese, European, Indian, etc. The number of breeds/crops per year greatly depends upon the climatic conditions. According to climate, these species are also classified as univoltine, bivoltine or multivoltine (Rossiter, 1881). They are also classified as pure, monohybrid or polyhybrid, based on the crossings made between two pure strains or more than two pure strains (Central Silk Board, 2012b).
Bombyx mori
Scientific classification (according to Carolus Linnaeus, 1758): Kingdom:
Animalia; Phylum: Arthropoda; Class: Insecta; Order: Lepidoptera; Family: Bombycidae; Genus: Bombyx; Species: mori.
Bombyx mandarina
Scientific classification (according to Frederic Moore, 1872): Kingdom:
Animalia; Phylum: Arthropoda; Class: Insecta; Order: Lepidoptera; Family: Bombycidae; Genus: Bombyx; Species: mandarina.
Food habits
The larvae prefer to eat white mulberry (Morus alba), though they can also eat the leaves of other Morus species (see Table 1.1). Silkworms require a complete diet, containing four main constituents. The feeding leaves must contain fiber, saccharides, water and resin. The first three nourish the worm and fourth component helps in preparing silk (Capsadel, 1883). The nutritive value of the food is the most important part as it helps in the production of good cocoons. There are a wide range of improved mulberry varieties cultivated in India for this purpose (Tikader and Kamble, 2008).
Life cycle
A typical life cycle of the mulberry silkworm (B. mori) from the egg to the moth is shown in Fig. 1.1; it undergoes a complete metamorphosis process, consisting of a wide range of conspicuous and abrupt variations in terms of physiological, morphological and feeding parameters (Ahmed and Rajan, 2011).
1.1 Eggs, matured larvae, male and female moths, cocoons of B. mori and also mutant cocoons of Sericin-Hope.
Eggs
Female moths lay some 200–800 eggs in their lifetime. The number per laying varies across species. Newly laid eggs are yellow in color and the outside of the chorion contains a gummy layer, which helps them to attach to a surface. Eggs are very tiny and oval in shape, similar to the size of a pin head (1.1–1.4 mm × 0.89–1.02 mm) (Matei et al., 2009). During the next 12–15 days they gradually change color to a dark shade of gray. When the eggs are nearing the hatching stage they become lighter in color, before finally hatching very small larvae (Rossiter, 1881).
Larvae
Newly hatched larvae are less than one quarter of an inch, black or dark gray in color and are hairy. Gradually, as they grow, the hair and color both change. These larvae take 30–40 days to become mature enough to begin the spinning process. The time depends upon the amount and quality of food available to them, temperature, breed, etc. During this time the larvae eat a great deal, and as a result they burst their skin as a molt (Rossiter, 1881). In the larval stage they shed their skin a total of four times. The time period between molting is called an ‘age’ or ‘instar’.
The division of time per age/instar for bivoltine species (two crops in a year) is as follows (Winsted Silk Co., 1915):
• first instar: 5–6 days;
• second instar: 4–5 days;
• third instar: about 5 days;
• fourth instar: 5–6 days;
• fifth or last instar: 8–10 days (size of the worm: 75–80 mm).
Fully grown, fifth instar larvae are creamy white or creamy yellow in color and are totally hairless, with a horn-like appendage on the back end of the body. The head is relatively bigger than its size at the hatching stage, with a strong jaw to chew leaves. The body is made of several ring-like segments, with six forelegs and ten hind legs, and with a hook attached beneath the body (Rossiter, 1881).
Silk spinning organs
The silk spinning organ consists of two large glands, situated laterally along its sides, under the alimentary canal. The glands are made of glandular epithelium and are surrounded by two layers of cells. Each gland has three parts (see Fig. 1.2):
1.2 The morphology of B. mori silk glands.
• anterior (2 cm in length, consists of 250 secretory cells, secretes protein sericin);
• middle (7 cm in length, consists of 300 secretory cells, secretes protein sericin) and
• posterior (15 cm in length, consists of 500 secretory cells, secretes protein fibroin).
The anterior part opens into the spinnerets present in the lower jaw of the mouth. The liquid silk is synthesized in the silk glands and stored in the lumen of the silk glands (Mondal et al., 2007). The fluid content of the glands expels out through the openings of the spinnerets and becomes thread in the presence of air. The thread is called silk. Fully grown, fifth instar larvae stop eating and become focused on finding a suitable place to begin spinning a cocoon. They hold themselves tightly by ten hind legs and move their frontal body part freely for spinning (Winsted Silk Co., 1915).
Pupae/cocoons
It takes 2–5 days to complete the spinning process to form a cocoon, depending upon the season. Initially a loose shell form is wound by the silkworm around itself. The silkworms then start more consistent and compact spinning after covering the body inside the cocoon shell. After 3 days of complete spinning, the worms transform themselves into chrysalis form by shedding the skin for the last time and entering a hibernating stage. The pupa remains inside the cocoon depending upon the voltinism of the variety. Between 15 and 20 days later, the pupa wakes up and emerges from the cocoon in the form of a moth, with a totally different physiology and morphology (Winsted Silk Co., 1915). The size of B. mori cocoons is about 30–35 mm whereas in the case of B. mandarina, the cocoon size is around 20–25 mm (Emilio Wallet photographs, 2010). Cocoons of B. mori can be found in different colors, such as white, bright yellow, pale yellow, creamy white and of greenish hue (Winsted Silk Co., 1915).
Moths
In moth form, the worms lose their ten hind legs, retaining only six, and also lose the mouth. They gain two large compound eyes, two pairs of scaly wings and one pair of feather-like antenna (Winsted Silk Co., 1915). The full wing-span of B. mori is about 30–50 mm and of B. mandarina is about 32–45 mm. Wild adult moths are dark grayish brown in color and well built, with strong wide wings and a slender body. During the stage at which they emerge from the cocoon, moths secrete an alkaline enzyme called cocoonase, which can dissolve the cocoon at one pole (Kafatos et al., 1967).
This phase is the adult (imago) phase, when male and female moths are ready for breeding. They do not eat anything during this period. After copulation the females lay eggs within a few weeks, depending upon the optimum conditions (temperature and light) for the next generation to start.
Sericin-Hope
A new strain of B. mori has been developed artificially (Teramoto and Miyazawa, 2003) from two mutant species (Naked pupa (Nd, Stock No.804) and sericin cocoon (Nd-s, Stock No.805) mutants) and one normal species (high cocoon production strain, KCS83). These three strains have been developed in the laboratory of the National Institute of Agrobiological Sciences (NIAS), Tsukuba, Japan. The progeny obtained from the backcrossing between the female of KCS83 × Nd or KCS83 × Nd-s and male of KCS83 can produce very thin fragile cocoons made mostly of sericin threads. These strains can only secrete sericin (contains 98.5% protein sericin) in the form of thread without the protein fibroin. The sericin isolated from the cocoons of Sericin-Hope is known as Virgin Sericin. The strain is so named because it is hoped that a wide variety of materials can be produced from the cocoon sericin of the newly developed strain (Mase et al., 2006).
1.2.2 Non-mulberry silkworms
This classification of silkworms comes from the feeding habit of the silk producing insects from the members of the families Saturniidae and Lasiocampidae (Mahendran et al., 2006). Tropical (Antheraea mylitta) and temperate (A. pernyi, A. roylei, A. proylei and A. frithi) tasar, Eri (Philosamia ricini/Samia ricini), Muga (Antheraea assamensis) (see Fig. 1.3), Fagaria (Attacus atlas) and Shashe (Gonometa postica) are the principal non-mulberry silkworms (Jolly et al., 1974). These are mainly wild by habitat (with the exception of P. ricini, which is the only completely domesticated non-mulberry species (Nagaraju, 2008)) and have been found in polymorphic forms growing in a variety of host plants distributed in different geographical regions. For this reason, silk produced by them also varies with respect to its luster, color and tensile properties (see Table 1.1).
1.3 The moths and the cocoons produced by different species of non-mulberry silkworms. (a)–(c) The moths of Antheraea mylitta, Antheraea assama and Philosamia ricini, respectively. (d)–(j) The cocoons of Antherina suraka, Ceranchia appolina, Antheraea mylitta, Antheraea assama, Philosamia ricini, Antheraea pernyi and Cricula trifenestrata, respectively.
Non-mulberry silkworms mainly have Indo-Australian biographic origin (Singh and Debaraj, 2011) and have played an important role in the economy of many countries such as China, Japan, India and Indonesia since ancient times. Nearly 95% of the total global production of non-mulberry silk is reported to come from the tasar strains. India, being the second largest silk producing country in the world (Giridhar et al., 2011), had an annual production of 4050 MT of non-mulberry silk in the year 2010–2011, an increase of about 20% compared with previous year’s records (Central Silk Board, 2010–2011). Developed techniques and methodologies have contributed to increasing the yield in most of the countries in recent years. The USA has shown annual export earnings of Rs 3600 million for the year 2011–2012, with an increase of around 18% compared to the annual export earnings for the previous year (Central Silk Board, 2012a).
Scientific classification of Antheraea spp.
Kingdom: Animalia; Phylum: Arthropoda; Class: Insecta; Order: Lepidoptera; Family: Saturniidae; Genus: Antheraea.
Life cycle
The life cycle consists of four distinct stages, as with mulberry species, namely eggs, larvae, pupae and imago (Thangavelu and Sinha, 1992).
Eggs
Eggs are laid by adult female moths and are hatched usually after 3 or more days.
Larvae
The larval life cycle mainly lasts for about 26–28 days in the first crop, 42–45 days in the second crop and 4–5 months in the third crop. The larvae from the newly hatched eggs grow by eating the host plants and undergo four different molting stages, which give five larval stages. The first to fifth instar generally takes 4–5, 3–4, 7–9 and 10–15 days, respectively, to enter into the next phase of the life cycle (Thangavelu and Sinha, 1992).
Pupae
At the end of fifth instar, the larvae stop eating and start spinning, forming the hard shell covering called the cocoon and shortening its body by forming a pupa.
Imago
Further transformation occurs inside the cocoon before the adult moth emerges from its shell. The adult moths are now mature and have reached the reproduction stage.
The shape, size and color of the cocoon and larvae, including the duration of each cycle, varies from one species to another. Thus silk produced by them also varies in their texture within each species.
Different types of silks
Tasar
Tasar silk can be differentiated into two categories: tropical and oak (temperate) tasar. Tropical tasar produced by A. mylitta is copperish in color and is less lustrous than mulberry silk (Packard, 1914), whereas oak tasar is produced by A. frithi, A. pernyi, A. proylei and A. yamamai, which produce various shades of color (Central Silk Board, 2012a; Delport et al., 2006). The tropical wild species has the highest capacity to produce tasar silk because this species of silkworm is able to spin the largest cocoons among all other non-mulberry species (Akai, 2000).
Muga
Muga is a golden yellow silk produced by A. assamensis. Because of its fine quality, it is widely used for making high value products such as ‘saree’, ‘mekhela’ and ‘chaddar’ designated locally in the North-Eastern part of India, where this species originates. Annual production of muga in this country was recorded to be 124 MT for the year 2010-2011 (Delport et al., 2006).
Eri
Silk produced by S. ricini comes under this category. They mainly feed on castor plants, from which its name is derived (Delport et al., 2006). This silk is widely used for making traditional household cloths in the north-eastern states of India, called ‘chaddar’ locally. The total production of eri silk in the country was found to be 2760 MT in the year 2010–2011, an increase in the last 5 years (Central Silk Board, 2010–2011).
Fagaria
This category of silk is produced by several members of the genus Attacus, of which A. atlas is one of the major species. Silk produced by this species resembles tasar. It is polyphagous by nature, and feeds on different species of plants from the genus Atlantus, Ligustrum and Syringa (Mahendran et al., 2006). This species breeds twice a year (Tikader et al., 2011).
Shashe
A consistent problem in the Shashe silk industry is the lack of supply of cocoons (G. postica). They have six larval instars with hard cocoons, due to high sericin content. They are partly polyphagous in nature, and feed on different species of Acacia (Sinha et al., 1994).
Cricula silk
Cricula trifenestrata is a unique species producing golden yellow silk. It mainly feeds on high density plants like Mangifera indica and Anacardium occidentale. Cocoons produced by this species are yellow and orange in color and are used for spinning for silk yarn. This silk is known for its high luster, water and heat resistance, fine quality, porosity and non-allergic nature (Yadav et al., 1996).
Challenges related to non-mulberry silkworms
Collecting samples of silkworms can be highly challenging for the silk industry, since many varieties are confined only to specific regions. Most silkworms are reared outdoors. Seed production is also found to be a rate-limiting step in the production of wild silk. Many of the worms are infected by different diseases and hence the percentage yield is affected. It is also difficult to rear and save silkworms from predators as they grow in the natural environment. Large scale deforestation also lowers the cultivation of these non-mulberry silk species (Takasu et al., 2002 ).
1.3 Silk proteins
Silk is mainly produced by the two important groups of insects: the larvae of order Lepidoptera and the adult web spinners of the order of arthropods (spiders). There are several other species which also can make silk, such as wasps, crickets, fleas, bee larvae, beetle larvae, mites, pseudoscorpions, midges and several other arthropod taxa (Craig, 1997).
This chapter will briefly discuss the silk proteins produced by silkworm species. Silk is a strong and lustrous natural fiber which mainly contains protein polymer. Silk protein obtained from different silkworm species consists of two totally different families of proteins, namely fibroin and sericin (Dash et al., 2007).
1.3.1 Fibroins
Fibroin is secreted from the posterior gland of silkworms and sericin is secreted from the middle and anterior gland of the silkworms. During spinning, the larvae secretes two very thin (~ 10 μm diameter) fibroin twin strands from the two exocrine silk glands (aligned on both sides of the body) through the spinnerets, simultaneously gluing them together with sericin (Inoue et al., 2000). In the presence of air the protein fiber becomes stronger and harder.
Fibroin protein is the major constituent (around 72–81%) of the cocoon and the remaining 19–28% is sericin protein (Lee, 1999). Being a hydrophobic glycoprotein, fibroin is insoluble in water (Gamo et al., 1977). It contains a large amount of hydrogen bonds. The molecular composition and orientation makes this protein form a semi-crystalline structure which contains two phases: highly ordered crystalline antiparallel β-sheet separated by less ordered β-sheet spacers. The crystalline part contributes to the strength and toughness and the non-crystalline part contributes the flexibility and elasticity to the fiber (Hoa et al., 2012; Lotz and Colonna-Cesari, 1979).
1.3.2 Sericins
Sericin isolated from the cocoon has two subunits, namely α-sericin, found in the external layer, and β-sericin, found in the inner layer of the cocoon. Because of the presence of a lesser amount of C and H and a higher amount of N and O than β-sericin, α-sericin is more soluble than β-sericin (Bose et al., 1989). Sericin is a hydrophilic (soluble in hot water) protein, and can therefore be removed or separated from the fibroin by a simple thermochemical process known as ‘degumming’ (Zurovec et al., 1998). This protein is amorphous and glue-like in nature, which helps in adhering one fibroin to the next fibroin fiber and in maintaining the structural integrity of the cocoon. Another very rare and discrete silk protein seroin can be found in the cocoon, which is produced in the middle and posterior gland of the silkworm (Hoa et al., 2012).
1.3.3 Protein from Sericin-Hope
The improved mutant variant of B. mori is known as Sericin-Hope, which was developed by Yamamoto and colleagues. As the posterior gland is degenerated in this species, they are able to produce threads which contain almost 98.5% (Yamamoto et al., 2002) sericin protein and a negligible amount of fibroin protein. The sericin is known as virgin sericin (Mase et al., 2006). The protein contains eighteen kinds of hydrophilic polar amino acids with nucleophilic side groups and the presence of serine as a major amino acid (one third of the total amino acids present). The cocoon made from this thread is very thin, brittle and easily dissolvable in water with less hydrolysis (Lotz and Colonna-Cesari, 1979; Teramoto and Miyazawa, 2003). The molecular structure of virgin sericin is the same as the sericin protein isolated from a normal B. mori cocoon. It is produced in pure conditions in comparison with normal thermochemically degraded sericin, and it has better mechanical strength and other physicochemical properties (Teramoto and Miyazawa, 2005).
1.4 Genetics of silkworms
Bombyx mori silkworm has n = 28 chromosomes and the size of the haploid nuclear genome is estimated to be around 5 × 10⁸ bp (Mita et al., 2003), which encode different physiological, morphological and biochemical traits of the silkworms. Different mutants can be produced by altering respective nucleic acid sequence for genetic and genomic studies. Transgenic silkworms can be exploited as bioreactors for the production of different bioactive molecules and vaccines. The detailed molecular structure of silk proteins from both mulberry and non-mulberry origins, their encoding genes and available Expressed Sequence Tag (EST) databases are described.
1.4.1 Molecular structure of mulberry silk proteins and encoding genes
Fibroin
B. mori silk protein fibroin is a heterodimeric protein, which contains three subunits encoded by a single gene. Fibroin isolated from silk glands contains a heavy chain (H) of 325–395 kDa, a light chain (L) of 25–26 kDa and P25 (fibrohexamerin) of 30 kDa (Tanaka et al., 1999) whereas fibroin isolated from cocoons has one 325 kDa subunit and one 25 kDa subunit (Altman et al., 2003). For a single molecule of protein, the heavy and the light chain form the dimer. Six such dimers are loosely bound to a P25 sub-unit polypeptide which acts as a chaperone (H:L:P25 = 6:6:1 ratio) (Inoue et al., 2000). The fibroin protein, being a glycoprotein, has N terminal glycosylation and contains a very small quantity of mannose and glucosamine (Sinohara et al., 1971).
Genes encoding these three polypeptides are located on different chromosomes, however the interactions between H-chain and L-chain or P25 are essential for the secretion of fibroin. The fibroin heavy chain consists of two exons (67 and 15 750 bp) and one intron (971 bp). The coding region contains a highly repetitive and G-rich core flanked by non-repetitive 5΄ and 3΄ ends (Zhao et al., 2008). Secondary structure predictions suggest that the N-terminal of fibroin may fold to form a globular structure due to the presence of alpha helix and extended strands. The P25 component of B. mori silk is a glycoprotein, which contains an Asn-linked oligosaccharide chain and contributes to the solubility and assembly to form a quaternary complex (Tanaka et al., 1999). The fibroin light chain of B. mori is 13.4 kb long, containing seven exons of which the first exon occupies 60% of the gene (Kikuchi et al., 1992). cDNA consists of 1200 bp and encodes protein which is highly hydrophilic in nature, and helps to make the quaternary complex soluble (Yamaguchi et al., 1989).
The H-chain is mainly responsible for contributing the fibrous characteristics of the silk fiber. Glycine (46%), alanine (30%) and serine (12%) are the predominant amino acids found in this chain. The bulk of it contains the repeat motif of type β (1) containing twelve dipeptides as – (12 GX)n– (where X is Ala in 65%, Ser in 23% and Tyr in 10%) (Zhou et al., 2000). The nonfibrous L-chains mainly contain valine, isoleucine, leucine and other acidic amino acids (Shimura, 1983).
Sericin
The silk protein sericin isolated from B. mori contains several polypeptide chains whose molecular weight ranges from 40 to 400 kDa (in the case of cocoon sericin) and 80 to 310 kDa (in the case of gland sericin). Among those, only five polymers are reported, those are S1, S2, S3, S4 and S5. The first three are the main structural component, produced in the posterior, middle and middle to anterior, respectively. The remaining two proteins are less important and are produced in different parts of the middle gland (Gamo et al., 1977).
Three sericin genes named as Ser1, Ser2 and Ser3 are cloned. The Ser1 gene consists of nine exons and generates four mRNAs (2.8, 4.0, 9.0 and 10.5 kb) by alternative splicing (Garel et al., 1997). The Ser2 gene generates two mRNA variants, 3.1 kb and 5.0–6.4 kb. The mature protein contains 1740 and 882 amino acid residues and is believed to be responsible for the adhesiveness of Ser2 products (Kludkiewicz et al., 2009). The Ser3 gene contains only a single transcript of 4.5 kb having two serine-rich regions of an 86-amino acid motif and an 8-amino acid repeated sequence (Takasu et al., 2007). Predominant amino acids in mulberry sericin are throeonine, glutamic acid, cystine and phenyl amine whereas serine, proline, methionine, glucosamine, galactosamine and histidine are found in smaller amounts (Yamada, 1978). Being a glycoprotein, sericin contains either some glucose molecules in its polypeptide chain, such as monosaccharide N-acetylgalactosamine, or a disaccharide and several mannose residues and two N-acetylglucosamine residues (Sinohara, 1979).
1.4.2 Molecular structure of non-mulberry silk proteins and encoding genes
Fibroins
A. mylitta silk fibroin, isolated directly from the silk gland, is a homodimeric protein of molecular mass 395 kDa and each of the monomer sub-units has a molecular weight of about 197 kDa (Datta et al., 2001). Among other Saturniidae, A. assama fibroin consists of two fragments of 220 and 20 kDa (Kasoju, 2009) and P. ricini comprises two fractions of 97 and 45 kDa (Robson, 1998). It does not contain any L-chain or P25 polypeptide. Silk fibroin of non-mulberry silkworms (A. pernyi and A. yamamai) consists of only one polypeptide.
In A. pernyi the gene consists of a first exon encoding 14 amino acid residues, a short intron (120 bp) and a long second exon encoding 2625 amino acid residues, which are rich in alanine, glycine and serine residues (Sezutsu and Yukuhiro, 2000). No homolog of fibroin light chain and P25 has been found, suggesting the quaternary complex is not essential for the spinning of silk protein.
The highly ordered crystal structure of fibroin protein contains predominantly antiparallel β-sheets (type β 3a) of polyalanine –(Ala)n– repeat sequence with small amount of α-helix (Fu, 2011). Because the polypeptide containing –(Ala)n– is much more hydrophobic than the –(Gly-Ala)n– containing polypeptides, non-mulberry fibroin is more hydrophobic than the mulberry fibroin (Hayashi et al., 1999). The non-mulberry fibroin mostly contains glycine, alanine and serine amino acids. The protein is glycosylated by O-linked oligosaccharides joined to the backbone of the protein via N-acetylgalactosamine. In comparison with mulberry fibroin, non-mulberry fibroin specially contains a tripeptide motif called RGD (Arg-Gly-Asp) (Datta et al., 2001).
Sericins
Three major polypeptides are present in the sericin protein isolated from the A. mylitta cocoon. These are 70 kDa, nearly 200 kDa and greater than 200 kDa polypeptides and sericin isolated from the gland containing four different fractions ranging from 30 to 200 kDa (Dash et al., 2007). One 66 kDa protein from A. assama and S. ricini has also been reported (Ahmad et al., 2004). None of their genes has been cloned.
Non-mulberry sericin protein contains 35–36.7% β-sheet, 52.7–63% random coils and 10.6% turns with no α-helical content (Dash et al., 2006). Considering the molecular structure of sericin, non-mulberry sericin has some amino acids, such as serine, glycine and tyrosine in a lower percentage than mulberry sericin. In non-mulberry sericin serine, glycine, glutamic acid, threonine and tyrosine are present in a higher amount (Dash et al., 2006, 2007). Among these amino acids, serine is present in a greater amount at almost 39% (Takasu et al., 2002).
1.4.3 Expressed Sequence Tag (EST) databases
For complete genome analysis of B. mori, Mita et al. (2003) have constructed an EST database of this insect, comprising 35 000 ESTs from 36 cDNA libraries from different tissues. Arunkumar et al. (2008) have created a wild silk based database comprising 57 113 ESTs from different tissues of A. assama, A. mylitta and S. ricini. These ESTs have been clustered and assembled into 4019 contigs and 10 019 singletons. Maiti et al. (2010) have also established an EST database (Amybase) comprising 2476 good quality ESTs and grouped into 390 contigs and 648 singletons. These databases are providing valuable resources for the functional and evolutionary study of different genes, comparative genomics, functional genomics, gene discovery, genome organization and gene expression by microarray in various silk moths.
1.4.4 Silkworm as bioreactor
The development and improvement of protocol for the production of transgenic silkworms opens new areas of application both for fundamental research and for the applied field (Prudhomme and Couble, 2002). The expression of functional human granulocyte–macrophage-colony-stimulating factors in transgenic B. mori pupa (Chen et al., 2006) and in silk glands (Xue et al., 2011) have been carried out. Recombinant spider dragline silk has also been produced in transgenic B. mori cocoons (Wen et al., 2010).
1.5 Diseases of silkworms
Both mulberry and non-mulberry silkworms are susceptible to different kinds of diseases caused by various pathogens. Damage to the sericulture industry occurs mostly as a result of these diseases, rather than because of unfavorable weather conditions which can lead to poor availability of host plant leaves. The most common pathogens that cause silkworm diseases are viruses, bacteria, protozoa and fungi.
1.5.1 Viruses
Four types of virus are known to infect mulberry and non-mulberry silkworms and causes most economic damage to silk industry. These are the nuclear polyhedrosis virus (NPV), cytoplasmic polyhedrosis virus (CPV), infectious flacherie virus (IFV) and densonucleosis virus (DNV).
Nuclear polyhedrosis virus (NPV)
B. mori NPV (BmNPV) belongs to the baculovirus family. It infects various tissues and multiplies in the nucleus of infected cells. The virus is rod shaped (300 × 45 nm) and contains large circular double stranded DNA in its genome. During viral infection, two types of progeny viruses such as the budded virus (BV) and occlusion derived virus (ODV) are produced in different phases in host cells. In the early phase of infection, BV is produced and it spreads infection from cell to cell within the same insect (Rahman, 2004) whereas in the late phase of infection (24–48 h), large amounts of a proteinaceous matrix called polyhedrin is produced within which virus particles are occluded (ODV) and form polyhedra. Polyhedra help to protect embedded virion particles from high temperatures, pH, etc., and allow the virus to survive several years in soil (Slac and Arif, 2006). Polyhedra also help its transmission into fresh insect larvae. Upon ingestion of polyhedral contaminated leaves by a fresh larva, in the alkaline pH environment of the larval midgut, polyhedra dissolve and the released viruses infect midgut epithelial cells to spread infection in this uninfected host. Viral transmission from an infected mother moth to larvae via eggs has also been reported in B. mori (Khurad et al., 2004). Various promoters (p10, polyhedrin, etc.) have been cloned from this virus to create baculovirus expression vectors for the expression of foreign proteins in B. mori cells or in the larva itself (Sriram and Gopinathan, 1997). Recently Xiang et al. (2012) have reported that foreign protein could be immobilized into the BmNPV polyhedra for the expression of polyhedrin and foreign genes simultaneously. Conventional microscopy is used for the detection of BmNPV polyhedra after only 3–8 days of infection. Recently polymerase chain reaction (PCR)-based methods have been developed using polyhedrin specific primers for the detection of viral infection. The BmNPV genome has been sequenced and analyzed (Gomi et al., 1999) and transgenic silkworms resistant to BmNPV have been created by knocking down the pathogenic genes via transgenic RNAi (Kanginakudru et al., 2007) or by overexpressing endogenous (Jiang et al., 2012a) or exogenous genes (Jiang et al., 2012b). There are few reports regarding NPV infection in non-mulberry silkworms, in A. yamamai and A. pernyi (Herniou et al., 2003).
Cytoplasmic polyhedrosis virus (CPV)
CPV also belongs to the Baculoviridae family and infects the midgut columnar epithelial cells of insect larvae. Viruses multiply in the cytoplasm of infected cells and, similar to NPV, form non-occluded virus in early infection and occluded virus or polyhedra in late infection. The virus is icosahedral in shape (60 nm in diameter) and contains 10–11 double stranded segmented RNA and transmits horizontally. CPVs are self competent for transcription, possessing all the enzymes necessary for mRNA synthesis and processing.
B. mori CPV (BmCPV), the type Cypovirus, has a single layer capsid made up of 120 copies of the major capsid protein, VP1, which is decorated with 12 turrets on its icosahedral vertices (Hill et al., 1999). These hollow turrets are involved in post-transcriptional processing of viral mRNA and provide a channel through which newly synthesized 5 capped viral RNA is released from the capsid into the cytoplasm of infected cells (Reinisch et al., 2000). After translation of this mRNA into capsid, polymerase and other proteins assemble into viral procapsid. Within this, one copy of each genome segment plus polarity RNA, are packaged and replicated to form double-stranded RNA (dsRNA). Recently, a near atomic resolution density map of this virus by cryoelectron microscopy has been reported (Yang et al., 2012; Yu et al., 2008). CPV infecting non-mulberry silkworm, Antheraea sp. has also been reported (Qanungo et al., 2002). All of the genome segments of BmCPV and A. mylitta CPV have been cloned, sequenced and characterized (Chakrabarti et al., 2010; Ghorai et al., 2010; Rao et al., 2003). Sequence analysis has shown that different types of CPV infect B. mori and Antheraea spp., and are also different from other insect cypoviruses. Microarray analysis has also demonstrated different gene expression profiles in B. mori midgut cells infected with CPV (Wu et al., 2011).
Infectious flacherie virus (IFV)
Flacherie disease of the silkworm B. mori is a major factor causing serious loss of cocoon production in sericultural farms. Based on its pathological symptoms, the causative agent of this disease is labeled as IFV. The target of IFV is the goblet cells of the midgut epithelium, and the virus multiplies in the cytoplasm (Kawsae et al., 1980). The virus particles are icosahedral and measure about 27 nm in diameter. The virion contains a single-stranded RNA that is polyadenylated at its 3΄ end and covalently linked with a protein (VPg) at its 5΄ end (Hashimoto et al., 1986). It contains an internal ribosome entry site (IRES) that mediates cap-independent translation and is applied to simultaneously express several proteins (Li et al., 2012). Cloning of IFV cDNA shows that the whole genome consists of 9650 nt containing a large open reading frame of 9255 nts, flanked by the short 5΄ non-coding region (156 nts) and by a long 3΄ non-coding (239 nts) (Isawa et al., 1998).
Densonucleosis virus (DNV)
B. mori densovirus (BmDNV), a major pathogen of silkworms, causes significant losses to the silk industry. Two strains of BmDNV are detected serologically: BmDNV-1 and BmDNV-2, based on their genomic characteristics. BmDNV-1 contains single-stranded DNA molecules of 5.048 kb, and replicates only in the midgut to cause fatal disease (Ito, 2012). The structure of BmDNV-1 has been determined at 3.1 Angstrom resolution (Kaufmann et al., 2011). BmDNV-2 contains two ssDNA molecules of 6.542 and 6.032 kb in size, and is less pathogenic than BmDNV-1 (Bando et al., 1995). Resistance to DNV is conferred by two recessive genes nsd-1 and nsd-2 and one dominant gene Nid-1 (Kidokoro et al., 2010). A multiplex polymerase chain reaction has been developed for the detection of densovirus in infected silkworms (Ravikumar et al., 2011).
1.5.2 Fungal infections
B. mori suffers from infection by entomopathogenic fungi Beauveria bassiana and Aspergillus flavus and causes ‘muscardine disease’. These fungi kill the insect larvae in 4–5 days by direct penetration of the cuticle, followed by multiplication in the hemocoel (Kumar, 2004). Several host genes induced by B. bassiana have been identified by subtractive hybridization. Recently, an in vivo system has been developed to evaluate the therapeutic effects of antifungal drugs using the silkworm infection model of Cryptococcus neoformans (Matsumoto et al.,
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