Development of Silk Fibroin Based Scaffolds for Tissue Engineering and Regenerative Medicine

Normal and disrupted fibers are seen side by side in (a) and a high magnification image of disrupted fibers is seen in (b). The sulfation reaction did not affect the scaffold architecture and fiber texture, as shown by SEM analysis: a,b) low and high magnification views respectively, and c) – high magnification image showing the texture of a fiber. The inset shows a pie chart showing the relative atomic percentages of each element (Au is omitted). The FTIR spectra of a) pure fibroin, b) pristine and c) sulfated micro–.

PART - II DEVELOPMENT OF BIOACTIVE SCAFFOLDS BY SURFACE OR BULK MODIFICATION, INCLUDING INCORPORATION OF BIOACTIVE MOLECULES.

Silk fibroin based curcumin releasing multi-purpose tissue engineering scaffold

Silk fibroin based artificial extracellular matrix for hepatic tissue engineering

Silk fibroin based composite matrix for bioartificial liver applications

TISSUE ENGINEERING

Cells
Biochemical and biomechanical cues
Scaffold
Bioreactors

Tissue or organ transplantation is a gold standard therapy to treat these patients (Brasile et al., 2002). Human embryonic stem cells (derived from discarded human embryos), adult stem cells and progenitor cells (derived from a variety of adult tissues) are considered potential alternative sources (Berthiaume et al., 2011).

SCAFFOLD

Scaffold: Design
Scaffold: Fabrication

While carbohydrate polymers include chitosan, chitosan derivatives, alginate, hyaluronic acid, etc. (Dhandayuthapani et al., 2011). Biomaterials classified by type of material with examples of their use in medical devices (Binyamin et al., 2006).

SILKWORM SILK FIBROIN

Properties
Various articles made from fibroin
Biomedical applications

Similarly, silk fibroin was found to support the growth and differentiation of various human stem cells (Wang et al., 2006). Vortex-induced beta-sheet-rich silk hydrogels were composed of permanent, physical, intermolecular bonds ( Yucel et al., 2009 ).

SILK FIBROIN IN TISSUE ENGINEERING

Bone tissue engineering
Cartilage tissue engineering
Ligament / tendon tissue engineering
Skin tissue engineering
Vascular tissue engineering
Neural tissue engineering
Spinal cord tissue engineering
Hepatic tissue engineering
Inter-vertebral disc tissue engineering
Tracheal tissue engineering
Muscle tissue engineering
Bladder tissue engineering
Ocular tissue engineering
Eardrum tissue engineering
Dental tissue engineering

Yang et al (2007a) studied the biocompatibility of SF material with peripheral nerve cells and tissues, finding that liquid SF extract did not show significant cytotoxicity to rat sciatic nerve Schwann cells, and the SF fiber substrate showed good biocompatibility with dorsal rat ganglia (DRG). The fabrication of composite scaffolds composed of SF and other naturally derived polymers was investigated by Ren et al (2009). Zhang et al (2007) demonstrated the use of a SF modified poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) scaffold for smooth muscle TE.

Recently, Bray et al (2011) compared the attachment, morphology and phenotype of primary cultures of HLE cells grown in SF with that in donor amniotic membrane (AM, the current standard clinical substrate for HLE transplantation).

SILK FIBRIN FROM NONMULBERRY SILKWORM

Antheraea pernyi Guerin-Meneville (Chinese Oak Tasar Silk moth)
Antheraea mylitta Drury (Indian Tropical Tasar Silk moth)
Antheraea assama or Antheraea assamensis Helfer (Indian Muga Silk moth) A.assama (Muga silkworm) (n= 15) is a wild, semi-domesticated, multivoltine silk moth
Samia cynthia ricini or Philosamia ricini Wardle (Indian Eri Silkmoth)

The proportion of Gly residues is greater in BmSF, while the content of Ala residues is greater in ApSF (Nakazawa and Asakura, 2002). The structure and properties of ApSF regenerated in aqueous solution have been studied by Tao et al (2007) and Kweon and Park (2001). Recently, He et al (2011) evaluated the effect of processing parameters on the morphology, structure and mechanical properties of e-spun nanofibers. Recently, Talukdar et al (2011) described their studies on the analysis of AmSF scaffolds seeded at different initial densities (25, 50 and 100 million cells/ml).

The name "eri" is derived from the Assamese word "era" meaning castor bean (Ricinus communis L.), the primary food plant of this omnivorous insect.

INTRODUCTION

However, conventional scaffold fabrication techniques, such as solvent casting of particle leaching, gas foaming, etc., are unable to precisely control pore size, pore geometry, the spatial distribution of pores, and the construction of internal channels within the scaffold (Edwards et al., 2004; Sachlos and Czernuszka, 2003). Fiber bonding methods, such as textile-based non-woven technologies, are known to be simple, efficient and clean and could enable precisely engineered structures. Furthermore, non-woven structures are usually characterized by their high porosity, large fiber surfaces, absorbency for other substances and possession of well-defined biomimetic microarchitecture similar to natural ECM, which is itself a non-woven structure.

Such biomimetic non-woven scaffolds have great potential in various TERM applications (Edwards et al., 2004).

MATERIALS AND METHODS .1 Materials

Processing and degumming of cocoons
Fabrication of nonwoven scaffold
Characterization studies .1 Morphological properties
Blood compatibility studies
Cyto-compatibility studies
Assessment of angiogenic properties
Biodegradation properties
Statistical analysis

The surface and cross-sectional morphology of the scaffold was examined under a scanning electron microscope (SEM, LEO 1430 VP and JSM 6380 LA, JEOL). The blood compatibility of the scaffold was evaluated in vitro and the following categories of blood interactions were studied: thrombogenicity and hemolytic potential. The amount of platelets adhered to the scaffold was determined according to a previous report (Singh et al., 1990).

A-549 (lung-derived), KB (oral epidermis-derived; HeLa contaminant), HepG2 (liver-derived), and HeLa (cervix-derived) obtained from the National Center for Cell Science (NCCS , Pune, India) - national repository for cell lines in India.

RESULTS

Blood compatibility studies .1 Thrombogenecity
Cyto-compatibility studies
Assessment of angiogenic properties .1 CAM assay
Biodegradation properties

Evaporation of the moisture content in the scaffold resulted in a weight loss during initial heating. SEM examination images of the one-week cell scaffold constructs revealed the presence of cells on and within the scaffold (Figure 2.6 and Figure 2.7). SEM images of cell scaffold constructs seeded with A-549 (A), KB (B), HepG2 (C), and HeLa (D) reveal successful cell attachment and spreading on the scaffold.

However, the penetration of new ships through the jetty was more clearly visible than in the captured images.

DISCUSSION

We performed in vitro thrombogenicity, hemolysis, platelet and leukocyte counts, protein adsorption and platelet adhesion experiments to evaluate the blood compatibility of the scaffold. The SEM examination of the cell-scaffold construct revealed the successful attachment and spreading of cells on and within the scaffold (Figures 2.6 and 2.7). Therefore, the results of cell attachment and spreading, cell migration, viability and growth, and collagen content collectively suggest very good cellular compatibility of the scaffold.

We pursued a simple, rapid, reproducible and efficient ex ovo CAM assay to evaluate the angiogenesis properties of the scaffold.

CONCLUSIONS

Scaffold degradation was relatively more than that of pure fibers, which may be due to changes caused by fibroin after treatment with formic acid/lithium bromide during the manufacturing process (Dal Pra et al., 2006). In addition, the results of SEM examination of trypsin-digested scaffold revealed decrease in fiber diameter, increase in fiber roughness and loss of microarchitecture, thus suggesting scaffold degradation (Figure 2.12b).

INTRODUCTION

Therefore, a nonwoven matrix composed of both nano- and microscale fibers could be an ideal architecture for TERM, where the advantages of both components can be exploited synergistically (Santos et al., 2008; Bondar et al., 2008). Success is far more certain with natural polymers such as proteins and polysaccharides such as silk fibroin (Mano et al., 2007). Moreover, the biological synthesis and processes involved in fibroin synthesis and assembly can offer important information about fundamental interactions involved in the formation of complex material architectures, as well as practical knowledge about new and important materials related to biomaterial applications and tissue engineering needs (Dobb et al., 1967; Valluzzi et al., 2002).

The self-assembly pathway involves a series of material length scales leading to liquid crystalline mesophases and ultimately to macroscopic structures (Dobb et al., 1967; Valluzzi et al., 2002).

MATERIALS AND METHODS .1 Materials

Fabrication of micro – nano fibrous nonwoven scaffold
Cyto-compatibility

The temperature of the mixture was gradually increased to 60 oC and the mixture was homogenized (T-25digital Ultra-Turrax®, IKA®) at 5000 revolutions per minute for 10 minutes. The compatibility of the scaffold with blood was evaluated in vitro and the following categories of blood interactions were studied: hemolytic potential. Protein adsorption and platelet adhesion studies were also performed to obtain detailed knowledge of the blood compatibility of the scaffold.

A-549 (derived from lung) obtained from National Center for Cell Science (NCCS, Pune, India) - National Repository of India.

RESULTS

Blood compatibility studies .1 Hemolytic assay
Cyto-compatibility studies 1 Cytotoxicity assay

A typical SEM image showing the adhesion of platelets to the scaffold is presented in Figure 3.7. A typical SEM image of 2 d old cell scaffold constructs together with a magnified image of a selected section clearly shows cell adhesion on the scaffold. In addition, an increase in cell spreading and growth across the scaffold was clearly seen in the SEM image of 7 d old cell-scaffold constructs (Figure 3.9).

SEM images 2 d (a) and 7 d (b) of cell scaffold constructs seeded with A-549 reveal successful cell attachment and spreading throughout the scaffold.

DISCUSSION

However, from the protein adsorption studies, the average amount of proteins adsorbed on the scaffold was found to be µg/cm. Therefore, it is preferred that the scaffold should facilitate the attachment, migration and growth of cells, resulting in the formation of a functional tissue. The qualitative assessment of cell adhesion and spreading of cells over the scaffold was achieved by SEM examination of cell-scaffold constructs.

Also, the comparative analysis of SEM images of 2 d and 7 d old cell framework constructs shows a marked increase in cell proliferation and growth in the framework (Figure 3.9).

CONCLUSIONS

INTRODUCTION

The blood compatibility of a skeleton is critical in determining the fate of an implant, as it is the blood that first comes into contact with the surface of the skeleton (Sharma, 2001; Tirrell et al., 2002). The foreign body reaction also results in capsule formation (characterized by unwanted growth of fibroitic tissue around the implant), loss of function and pain, often requiring additional surgeries for subsequent removal of the implant, thus pushing the patient back to original state of suffering with additional pain. complications (Courtney et al., 1994). The blood compatibility of a biomaterial can be significantly improved by various surface engineering methods, without altering the bulk properties of the material, including functionality with heparin, oligoethylene glycol groups, sulfate and sulfonate groups, etc. (Piskin, 1992; Nair, et al., 1998 Jandt, 2007; Vasita et al., 2008; Ratner and Bryant, 2004; Tamada.

There were only a few reports by Tamada et al in which they investigated the sulfation method of B.

MATERIALS AND METHODS .1 Materials

Fabrication of micro-nano fibrous nonwoven scaffold
Sulfation of biomimetic scaffold
Cyto-compatibility

It was finally dried overnight under vacuum at 37 oC and stored in a moisture-free bag until use. The scaffolds were then thoroughly washed with distilled water to remove traces of residual reagents. Finally they were dried overnight under vacuum at 37 oC and stored in a moisture-free bag until use.

Sterile scaffolds were extracted in serum-free DMEM for 24 hours at 37 oC and the extracts were used within 24 hours of preparation.

RESULTS

Blood compatibility studies .1 Hemolytic assay
Cyto-compatibility studies 1 Cytotoxicity assay

The thermal properties of the unsulfated and sulfated scaffolds were determined by TGA analysis (Figure 4.7). The amount of proteins adsorbed on unsulfated and sulfated scaffolds was estimated by SEM analysis. Typical SEM images showing the adhesion of platelets over the unsulfated and sulfated scaffolds are presented in Figure 4.9.

Typical SEM images of 1-week cultures of A549 cells growing on unsulfated and sulfated scaffolds are shown in Figure 4.11.

DISCUSSION

Thus, it was found that the increase in the overall water uptake capacity of the scaffold after sulfation reaction was about 29.26. Consequently, the reduction in hemolysis percentage, after sulfation of scaffolds, was found to be approximately 14.705. The overall decrease in the platelet adhesion percentage, after sulphation, was calculated to be approximately 60.

There was also no significant qualitative difference in the cytocompatibility between unsulfated and sulfated scaffolds.

CONCLUSIONS

INTRODUCTION

Among the various biomedically relevant polymers that have been studied for sustained and controlled drug release, silk fibroin has been advantageously exploited as a polymeric vehicle due to its unique properties (Altman et al., 2003). Fibroin matrices have been used as carriers for the encapsulation of various therapeutic agents, including insulin-like growth factor I, fibroblast growth factor 2, nerve growth factor, theophylline, adenosine, etc. (Bayraktar et al., 2005; Uebersax et al. Wenk et al. , 2010; Pritchard et al., 2010). During the spinning process at the cocoon formation stage, several such motifs interact with hydrogen bonds and hydrophobic interactions to form β-sheet stacks (Altman et al., 2003; Vepari and Kaplan, 2007; Numata and Kaplan, 2010).

We hypothesized that such hydrophobic environments could be readily used for encapsulating hydrophobic drugs such as.

MATERIALS AND METHODS .1 Materials

Aqueous regeneration of fibroin from cocoons
Fabrication of curcumin loaded and curcumin free fibroin scaffold
Fluorescence spectroscopy studies
Evaluation of drug release kinetics .1 Preparation of releasing medium
In vitro bioactivity of encapsulated curcumin .1 Anti cancer activity

The degree of swelling (Q) and water uptake (%) were calculated using the following equations (Kasoju et al., 2009). To analyze the release kinetics and mechanism, the data were adjusted to the following four mathematical models (equations 6-9), where Mt/M∞ is the fraction of drug released at time t, and k0, k1, kH and k represent the zero-order release constant. , the first-order relaxation constant, the Higuchi constant, or the Korsemeyer–Peppas constant (Siepmann et al., 2001; Peppas et al., 1985; Higuchi et al., 1963; Sahu et al., 2011; Korsmeyer et al., 1983). The overall radical scavenging capacity of free curcumin and curcumin from the loaded framework was determined using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging method (Ali et al., 2008).

The antimicrobial activity of free curcumin and curcumin from the loaded framework was tested by the good diffusion method against Staphylococcus aureus (G+), Bacillus subtilis (G+), Escherichia coli (G−) and Pseudomonas aeruginosa (G−) (Bhawana et al., 2011) .

RESULTS

Fluorescence spectroscopy studies

NMR spectroscopy: The NMR spectrum of curcumin extracted from the CU-SF scaffold was presented in Figure 5.5. The UV-Vis and fluorescence spectra of curcumin in PBS and fibroin solutions are shown in Figure 5.6. The effect of curcumin on intrinsic fluorescence of Tyr and Trp residues of fibroin is shown in Figures 5.8A and 5.8B, respectively.

Addition of curcumin also caused a gradual shift in the emission maximum from 354 nm to lower wavelengths.