Silk Fibroin Scaffolds Were Prepared Biology Essay

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Silk fibroin scaffolds were prepared from aqueous silk fibroin solutions by combining salt-leaching and freeze drying Methodologies. However the mechanical properties of the scaffold exhibited concentration dependence. The scaffold showed great stability and maintained its properties after in vitro degradation for 30 days. It also shows that it is suitable for meniscus and cartilage tissue engineering [11].

SF in conjugation with poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) has been used to fabricate the tissue engineered cardiovascular scaffolds due to its controllable and suitable mechanical properties. However it was found that SF-modified PHBHHx scaffolds are highly biocompatible with cardiovascular-related cells, showing that it can be utilized for the extensive applications of PHBHHx in the cardiovascular regeneration. [12].

In another study it is found that silk fibroin has good biocompatibility with rat Dorsal Root Ganglia (DRG) and is also beneficial to the survival of Schwann cells without exerting any significant cytotoxic effects on their phenotype or functions, which can open a gateway for silk fibroin as a material for nerve tissue engineering [13].

Another study about a developed novel method for cross-linking a silk fibroin solution by gamma irradiation to form a hydrogel that might serve as a dermal scaffold. The silk fibroin hydrogel is not cytotoxic to human fibroblasts or human keratinocytes. The mechanical properties of the silk fibroin hydrogel formed by gamma irradiation method were superior to that of silk fibroin hydrogel created using a freeze-drying method. The silk fibroin hydrogel created using this novel method is suitable as a dermal scaffold in Skin Tissue Engineering [14].

Recently silk fibroin and elastin scaffolds were produced for the treatment of burn wounds SF & Elastin scaffolds were produced for the treatment of burn wounds. The excellent properties of SF were combined with elastin protein and resulted in a scaffold which mimics the extracellular matrix (ECM). By using genipin as a cross-linker they obtained scaffolds with smaller pore size and reduced swelling ratio degradation and release rates. The composition had a great effect on the scaffold's physical properties; composition can be easily controlled to make the scaffold suitable for biological applications. The cytocompatibility with human skin fibroblasts along with the healing improvements make these scaffolds suitable for wound dressing applications [15].

Silk fibroin has an exceptional and valuable blend of properties, including great biocompatibility and outstanding mechanical performance. These features provided key principles for the usefulness of scaffold/matrix made of regenerated silk fibroin for tissue engineering. The silk fibroin scaffolds utilized for the tissue engineering must degrade at a rate that matches the rate of growth of the tissue. The results confirm that a high content of β-sheet structure gives a low degradation rate in the scaffold. The results show that to in order to control and manipulate the degradation rate of the scaffold it is done by adjusting the content of β-sheet structure [16].The preparation of SF for applications in tissue engineering was studied, Pure SF was extracted from silk worm cocoons by degumming and solubilizing and then further purification was carried out. However the results indicate that the regenerated silk fibroin can be used for fabrication of porous silk fibroin scaffolds for various tissue engineering applications [17].

For tissue engineering, it is very important to design and control the pore architecture of three-dimensional (3D) polymeric scaffolds, which plays an important role in directing tissue formation and function. Different pore structures were formed according to the pH of silk fibroin because silk fibroin exhibits water-like behavior under basic conditions and gel-like behavior under acidic conditions [18].

The pore architecture of scaffolds plays a critical role in tissue engineering as it provides the vital framework for seeded cells to organize into a functioning tissue. By manipulating the concentration the pore sizes of the scaffolds decreased as the concentration of fibroin protein increased. Human bone marrow mesenchymal stromal cells (BMSC) transfected with the BMP7 gene were cultured in different pore sized scaffolds. The results showed that BMSC expressing BMP7 preferred a pore size between 100 and 300 microns in silk fibroin protein fabricated scaffolds, with better cell proliferation and ECM production. Furthermore, in vivo transplantation of the silk fibroin scaffolds combined with BMSC expressing BMP7 induced new bone formation. This means that optimized pore architecture of silk fibroin scaffolds can modulate the bioactivity of BMP7-transfected BMSC in bone formation [19].

Physical Properties

Mechanical Properties

Silk is a very versatile biomaterial with its significant crystallinity, high elasticity, strength and toughness, and resistance to failure in compression. The combination of the β-sheet crystals, the interphase between the crystals, the semi-crystalline regions and the shear alignment of the molecular chains are the basis for silk's unique mechanical properties. While the highly organized β-sheet regions of the protein provide the tensile integrity, the semi-crystalline regions are the basis for the protein's elasticity [22]. The β-sheet structure affects the tensile properties, degradation rate and elasticity of the scaffold, so the tailoring of these properties can be done in part with the cross-linking process. The transition depends on the length of time exposed to the solvent as well as the solvent concentration. Methanol treatment is a widely used process to induce β-sheet formation although it does not transform all molecular regions. Ethanol, 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide [23] hydrochloride (EDC), glutaraldehyde or genipin also cause the transition from random coils to β-sheet configurations [24, 25, 26, and 27]. Studies show that regenerated silk fibers can hold their initial tensile integrity for 21 days under immune deficient in vitro culture conditions [8]. Moreover, the solvent used for electrospinning can affect the β-sheet formation of the scaffold's secondary structure, which in turn can alter the mechanical properties. Formic acid, HFIP and water have been used to electrospin silk scaffolds, and of those, water and formic acid seem to enhance the mechanical properties of the scaffolds [28, 29].

Degradation

According to the US Pharmacopia, an absorbable biomaterial loses most of its tensile strength 60 days after implantation. Even though silk is considered non-degradable by this definition, it does in fact degrade but over a longer period of time. Silk will lose most of its tensile strength within a year in vivo, and will be unrecognizable at the implantation site within 2 years. However, the rate of degradation depends on the animal model and the tissue implantation site. Silk is considered biodegradable because it is vulnerable to bacterial and enzymatic degradation. Studies show that proteases will cleave the protein at the less-crystalline regions after which the resulting peptides can be phagocytized by the cell [22, 24]. The solvent used to electrospin the silk scaffold may affect the degradation of the scaffold in vivo and in vitro. It was studied that electrospinning from an aqueous solution instead of an organic solvent like HFIP can increase the degradation rate while promoting cell proliferation and penetration. The silk scaffold electrospun from an aqueous solution degraded between 2 and 6 months while those electrospun out of HFIP lasted over a year. Also methanol treatment can significantly decrease the degradation rate [24, 30]. There is a wide variety of biocompatible polymers used for tissue applications aside from silk fibroin. However, the degradation rates of other polymers cannot be tailored within such a high range as that of silk. Collagen, which is a widely used biomaterial, degrades between 1 to 4 weeks and sometimes longer depending on the cross-linking process [31]. Polycaprolactone (PCL) can last within the body for more than 2 years [32]. Another synthetic polymer, poly (lactic-co-glycolic acid) (PLGA) (85:15) usually degrades within 26 weeks, while PLGA (50:50) degrades between 6 and 8 weeks in vitro [33, 34, 35]. Silk scaffolds, however, can be modified to have similar degradation rates by changing the solvent for electrospinning [30].

Swelling properties

The degree of swelling depends on the ionization of the network, its degree of crosslinking and its hydrophilic/ hydrophobic balance [36] Changes in polymer compositions can influence the degree of swelling [37]. This can potentially increase the cumulative amount and rate of drug release. The swelling ratio of SF scaffolds has also decreased with an increase in Silk Fibroin concentration. Blending of Silk Fibroin with other materials such as chitosan [38, 39] and hyaluronic acid [40] led to increase the swelling when compared with pure Silk Fibroin.

Solubility

Crystalline SF is insoluble in most solvents as well as in Water, Commonly applied to dissolve SF are highly concentrated salt solutions of lithium bromide, lithium thiocyanate calcium thiocyanate or calcium chloride [41].These electrolyte solutions are able to disrupt the hydrogen bonds that stabilize β-sheets [42]. The possibility to control the solubility of SF not only allows for longer storage times for SF solutions but also for an increase in SF concentration without aggregation.

Biocompatibility

The foreign body response after the implantation of Silk Fibroin in vivo has been shown to be comparable to or even less than the other biomaterials in use today [22, 43, 44]. All-aqueous- and Hexafluoroisopropanol (HFIP)-derived scaffolds have been tested in a one-year implantation study in rats. Those scaffolds were accepted by the host animals and the host immune response to the implanted scaffolds was low and local [45]. This matches another study with SF films [41].

Scaffolds and Cell Culturing

The design of new scaffolds mimicking the natural environment during tissue formation is an important issue in biomaterials research. Silk fibroin and hyaluronan scaffolds were prepared with porous microstructures by freeze-drying silk fibroin and hyaluronan aqueous solutions also to induce water insolubility of silk fibroin they were incubated in methanol. For three weeks Mesenchymal stem cells were seeded and cultured on silk fibroin/hyaluronan scaffolds. After cell culture, enhanced ingrowth of the cells into silk fibroin/hyaluronan scaffolds was shown by the histology of the constructs as compared to pure silk fibroin scaffolds. However Silk fibroin/hyaluronan scaffolds are good as a biomimetic platform for mesenchymal stem cells in the field of tissue engineering. [47]

Scaffold composition, configuration and resulting properties critically affects tissue development. The influence of silk fibroin concentration and correspondent processing method (aqueous or HFIP-derived) and three-dimensional scaffold structure (lamellar or porous, with distinct pore size) on bone tissue formation by osteogenic differentiation of human adipose tissue derived stem cells (hASC) were studied. We observed that very similar bone tissue was formed in all silk fibroin scaffold groups, evaluated by alkaline phosphatase activity, calcium production, collagen type I deposition and scaffold bone volume fraction. [48]

Due to the high surface area of the fibers with nanoscaled diameters, biomaterial scaffolds are provided with a lot of benefits. A study was carried out concerning the adhesion, proliferation and spreading of human bone marrow stromal cells (BMSCs) on these matrices. Biocompatibility and biodegradability properties of the silk matrix along with the electrospun silk matrices ability of supporting BMSC attachment, spreading and growth in vitro, propose possible usage of these matrices as scaffolds for tissue engineering. [49]

Before lyophilization, freezing temperature and NaCl concentration of the silk fibroin solution were varied in order to modify the morphology of the lyophilized silk fibroin 3D-scaffolds. Sponge-like interconnected porous networks resulted after freezing at -22°C, however, stacked leaflet structures formed after fast freezing at -73°C. The usage of millimolar NaCl (50-250mM) increased the porosity of the scaffolds. The seeding of P19 embryonic carcinoma cells showed that the presence of salt and freezing conditions influenced the cell distribution into the scaffolds, with salt addition increasing the access of cells to deeper regions. [50]

Pore architecture of scaffolds is known to play a critical role in tissue engineering as it provides the vital framework for the seeded cells to organize into a functioning tissue, in this study different concentrations and pore sizes were investigated, The pore size of the scaffold decreases as the concentration of fibroin protein increases, Human bone marrow mesenchymal stromal cells (BMSCs) transfected with BMP7 gene were cultured in these scaffolds, The results showed that BMP7 expressing BMSCs preferred a pore size between 100 and 300 µm of silk fibroin protein fabricated scaffolds, with better cell proliferation and ECM production , in vivo transplantation of the silk fibroin scaffolds combined with BMP7-expressing BMSCs induced new bone formation. This study identified that optimized pore architecture of silk fibroin scaffolds could modulate the bioactivities of BMP7 transfected BMSCs in bone formation. [51]

Starting from a variety of cell sources including primary cell-based and stem cell based platforms cartilage tissue can be engineered. cellular responses of isolated human embryonic stem cells, human chondrocytes and mesenchymal stem cells (MSCs) derived out of these three sources adipose tissue, bone marrow and human embryonic stem cells, were used with two biomaterials chitosan and silk scaffolds, in the presence and lack of bone morphogenetic protein 6 (BMP6), well-preserved chondrogenic phenotype with unique characteristics in both scaffolds with regard to cartilage formation were shown by embryonic stem cells-derived MSCs, embryonic stem cells-derived MSCs were favorable for cartilage formation, especially in the silk scaffolds with BMP6. Results show that cell source differences are important for chondrogenic outcomes; however, human embryonic stem cells-derived MSCs were utmost favorable cell source. [52]

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