Senan Ebrahim
Harvard College 2012

The silk macromolecule fibroin is used extensively in biomedicine in the silk I and II conformations, which have well characterized structures and properties. It was hypothesized that the fibroin crystalline structure silk III, which forms exclusively as a nanofilm at certain interfaces, could have similarly applicable properties, such as high tensile strength, biocompatibility, and liquid crystallinity. This research qualitatively and quantitatively assessed key properties of silk III fibroin at the air-water interface to facilitate future manufacture and biomedical applications. Mechanical strength and viscoelastic properties were studied. Silk III films of varied concentrations were subject to deformation experiments in which the time-varying moduli were analyzed. Both the time at which the crystallization began and the time at which the tan-delta value of the film reached unity were discovered to approximately follow the diffusion model, dependent on surface concentration. Additional data was collected regarding conditions requisite to silk III crystallization. A preliminary biocompatibility assessment, in which human fibroblast cell cultures were grown to confluence on silk III nanofilms, affirmed possible future use of silk III in human tissue.

Introduction

The unique properties of silkworm (Bombyx mori) silk give the material tremendous promise in modern applications such as regenerative medicine. Its biocompatibility, mechanical strength, and natural occurrence are all increasingly valuable features for a host of applications. In July 2007, scientists at Serica Technologies were recognized for the repair of anterior cruciate ligaments (ACL) in animals using silk fibroin grafts. Their tests successfully demonstrated that the fibroin graft met the “demanding mechanical and biological requirements of a functional ACL” (Serica Technologies Inc., 2007).  Silk fibroin has been the subject of many scientific inquiries from biochemistry to material science, but one particular fibroin structure, silk III, has several properties that have not yet been examined (Valluzzi et al., 1999a). The viscoelastic properties of silk III during formation have not yet been the subject of conclusive study, and its hypothesized crystal structure remains unconfirmed. Applications of silk III in the biomedical and textile industries involving the full capabilities of the molecule would be contingent upon an understanding of the fundamental science behind its crystal structure, biocompatibility, and tensile strength.

At the air-water interface of aqueous fibroin solutions, a thin film crystal structure forms with unique conformational properties (Valluzzi et al., 1999a). However, the precise nature of the conditions required to form this crystal structure (silk III) remains unknown. Modulating concentrations and conditions so as to alter the surfactant behavior of the fibroin effects a change in the formation of silk III. In this project, the uncharacterized properties of the silk III film were examined using a variety of interfacial techniques. The physical properties of silk III were studied using an Interfacial Stress Rheometer and a Langmuir Trough at various concentrations of fibroin.  Human fibroblast cells were cultured on a thin film of fibroin to test the biocompatibility of the surface. X-ray diffraction experiments were used to accurately study the crystal structure of fibroin molecules assembled at an air-water interface. These data were used to characterize the crystalline silk III film and assess its optimal conditions of formation.

Background

Fibroin is a fibrous protein from the silk of silkworms (Bombyx mori) that is studied intensively for its desirable mechanical properties such as tensile strength and biocompatibility. The silk emitted from the glands of Bombyx mori in pure form consists of fibroin strands coated in serecin, a globular protein which gives natural silk its sheen and texture. Fibroin is largely responsible for the unusual strength of natural silk (silk I) due to its secondary structure, which is made of several crystalline antiparallel β-pleated sheets (Figure 1). The alanine and glycine rich polymers with amino acid sequences similar to those of collagen pack closely because of their β-pleated sheet conformation, greatly increasing the material’s tensile strength.

The molecular mass of fibroin is essential to determining the mean molecular area (MMA) of fibroin when spread at the interface. Recent SDS page testing has indicated that it is approximately 300,000 Daltons, the mass used throughout this project (Terry et al., 2004).

Fibroin polymers arrange themselves in various solid-state configurations. Crystalline structures of silk I and II (silk I being the natural unprocessed silk fiber, and silk II being spun silk fiber) have been studied in great detail since these relatively simple structures were identified (Jin et al., 2003). A new silk structure, now called silk III, was first observed at the air-water interface and later at organic-water interfaces (Valluzzi et al., 1999a). The theoretical model proposed for the structure of silk III based on preliminary X-ray diffraction studies lacks the traditional pleated sheet structure between polymers, as is typically seen in other crystalline silk structures. Valluzzi hypothesized that an asymmetric three-fold helical conformation with axially aligned helices, as shown in Fig. 2, was forming in the film (Valluzzi et al., 1999b).

Fibroin structures silk I and silk II have numerous applications in the textile, cosmetics, and biomaterials industries. Silk fibroin in its natural form (silk I) has been used as a thread for suturing wounds and incisions for years. The greatest potential of silk III lies in its use as a biomaterial in skin, internal tissue, bone, or cartilage, if it retains the tensile strength of silk I and II.  It might also present valuable bulk fluid properties such as liquid crystallinity with many applications. The work of Altman et al. (2007) with silk II in ligament construction is one of many promising applications of silk in medicine. This work by Altman et al. (2007) regarding fibroin-based ligament grafts and the work by Meinel et al. (2006) that involved fibroin patches for human femur defects indicate the possibility of using silk-based biomaterials in medicine. Biocompatibility is a critical factor that determines whether silk III can ever be used in a similar manner. Human cells are known to be compatible with many fibroin configurations both in vitro and in some cases such as the silk in suture threads, in vivo. However, because medical complications could arise from variances in crystallinity, the biocompatibility of fibroin silk III specifically warrants further study.  As the femoral fortification proposal of Meinel et al. (2006) also relies on high tensile strength of the selected biomaterial, an assessment of the physical characteristics of silk III would also be helpful in ensuring its reliable use in human bone, cartilage, ligaments, and any other load-bearing applications.

In addition to tissue engineering applications, the work of Hofmann et al. (2006) points to the possibility of using silk fibroin in drug delivery. Fibroin crystalline casings were prepared from thin films to contain drugs intended to enter the human bloodstream. It was concluded that silk structures were desirable in pharmaceutical applications not only for their typical biocompatibility, but also because of their crystalline variability. Studying the crystalline properties of silk III could be helpful in determining its possible use in drug delivery capsules.

This investigation into the material properties of silk III at an air-water interface relied on established rheological properties. The viscous modulus and the elastic modulus can both be calculated by simple deformation experiments. Elasticity is an index of how much energy of deformation is stored, and viscosity measures how much energy is lost. The complex modulus is a collective measure of the dynamic mechanical properties of a material. The shear viscous modulus (G”) represents the resistance to irrecoverable deformation of a material in the complex modulus (G). The shear elastic modulus (G’) is the portion of the complex modulus resisting recoverable deformation.  They are related by the equation in the complex plane:

G = G’ + iG”

Changes in the two moduli were used as a mode of quantifying the rheometric properties of the viscoelastic crystalline film.

Figure 1
Figure 1. Secondary structure of fibroin in silk I. This image shows the secondary structure of fibroin, with interdigitation of the alanine and glycine side chains, a property which contributes to tensile strength. The β-pleated sheets of fibroin, such as the one above, are integral parts of the protein, vital to its structure and strength.
Figure 2. Theorized structural arrangement of fibroin in silk III. The imaging studies conducted by Professor Regina Valluzzi (Tufts University) have led to this hypothesized structure in crystalline silk III. The fibroin bands in this image are formed from parallel helices of fibroin strands, leading to a regular structure of silk III, detectable by techniques such as X-ray diffraction.

Materials and Methods

Preliminary Procedures

Silk fibroin was obtained in aqueous solution, about 10% by weight. The heterogeneity of the solution initially presented difficulties when assessing the film rheology, so the fibroin solution was subsequently centrifuged at 13000 rpm for 30 minutes. Varying amounts of fibroin and de-ionized water (Millipore), or fibroin and 0.1 M acetic acid, were then pipetted into vials in solutions of definite concentration and refrigerated in storage. Concentrations of fibroin in water were prepared daily ranging from 1.0 mg/mL to 20 mg/mL. The millipore water used had a resistivity of 18.2 Ωcm, and was generally devoid of confounding impurities and ions.

Isothermal and Imaging Experimentation

Numerous isothermal compressions of the fibroin film were conducted as a preliminary investigation of the crystallization process of fibroin. A basic compression experiment was set up with a Teflon interfacial Langmuir-Blodgett trough (172.0  cm2) and two equally sized Delrin barriers. Delrin surface-compressing barriers were used instead of Teflon ones, to optimize the compression of the surface film since the relatively hydrophilic Delrin would allow less leakage than a Teflon contact. A sterilized platinum Wilhelmy plate was suspended from a force detection device (KSV Instruments, Helsinki, FI) to measure the surface pressure at the center of the film on the trough during the compression (see Fig. 3a).

The trough was filled until a large convex meniscus formed, using about 120 mL of Phosphate Buffer Saline solution (PBS). PBS solution at ten times standard saline concentration (10× PBS) was prepared as a substitute for water as the supporting subphase in all interfacial experiments to amplify the amphiphilic nature of fibroin. After 45 minutes, the PBS surface was vacuumed to clean the interface for the fibroin film. Thirty microliters of fibroin solution was then spread evenly on top of the subphase with a syringe. Thirty microliters was calculated to be the approximate amount needed to form a nanofilm of single molecule thickness on the surface of the trough. After a further 45 minutes, the barriers were closed inwards to a displacement of 135 mm, each at a rate of 1.5 mm/min, uniformly diminishing the trough area and increasing the surface pressure at a variable rate. Several isothermal compressions were performed, varying PBS and fibroin concentrations, depositing centrifuged and uncentrifuged fibroin, depositing dilute fibroin dissolved in acetic acid instead of water.

Multiple imaging techniques were attempted in order to visually characterize the films, but the only one that rendered useful information was Atomic Force Microscopy (AFM). Brewster’s Angle Microscopy (BAM) was unsuccessful because of the large nature of the domains of orientation, and a dichroism setup failed because the dye (Sirius red) exhibited poor adhesion to the fibroin molecules. However, AFM conducted on glass slides of vertically deposited silk III films produced accurate visual representations of the structure under study.

Figure 3a. Schematic diagram of Langmuir trough for isotherm experiments. This diagram displays the elements of the Langmuir trough setup used to procure surface pressure curves for isothermal compression processes. KSV software sampled the surface pressure through the Wilhelmy plate approximately ever y twenty seconds. The delrin barriers were used to compress the film during crystallization.
Figure 3b. Interfacial Stress Rheometer (ISR) setup (LOT-Oriel Group, Europe ). At right: Schematic diagram of ISR with labeled components. The ISR was used to sample the moduli of a film at the air-water interface over time. The magnetic field generated by the Helmholtz coils moved the magnetic rod (a needle) which deformed the film. The film’s response to the deformation was measured and quantitatively reported by KSV software as the viscous and elastic moduli of the film.

Biocompatibility Assessment

In order to determine whether or not use of silk III is viable in humans as a biomaterial, a preliminary in vitro study of human cell growth on a silk III film was conducted. A silk III film was transferred from the air-water interface onto a thin glass slide using 5 mg/mL aqueous fibroin and the Langmuir Blodgett deposition technique. After drying in a desiccator for 24 hours, the films were seeded with primary adult Homo sapiens dermal fibroblast cells. The cells were grown to confluence in media before being transferred to the plates with the silk III films.

The following chemical components from Invitrogen (Gibco) were mixed in solution to make the cell culture media: 5 mL 100× penicillin/streptomycin, 5 mL 100× glutamine, 5 mL MEM (methoxyethoxymethyl) Non-Essential Amino Acids, 5 mL 100× sodium pyruvate, 500  mL D-MEM (high glucose) without dye, 50 mL FBS (fetal bovine serum).

The fibroblast cell concentration was estimated via a random count procedure, and the seeding density of the cultures on the two plates was 46 cells/mm2. The fibroblast cells were grown to confluence on both plates in a humidified 95% air, 5% CO2 incubator at 37° C.

Interfacial Stress Rheometer

To make a detailed assessment of the formation process of the silk III crystalline structure at the air-water interface, an Interfacial Stress Rheometer (ISR) was employed in multiple experiments. The ISR measures the fluid properties of the film, specifically the viscous and elastic moduli (G’s and G”s respectively). The kinetics of film formation was monitored by varying the initial concentration. Both centrifuged and uncentrifuged fibroin samples were used to test the effects of varying concentration on silk III film formation.

The ISR consists of two magnets in a Helmholtz condition creating a uniform magnetic field with a gradient of zero in the center set up to analyze the rheology of an interface. The objective is to measure viscosity and elasticity by moving a needle resting on the film with a controlled magnetic field and observing the response of the film to the deformation.

A cleaned Langmuir-Blodgett trough filled with 10× PBS was situated centrally between the Helmholtz coils (see Fig. 3b at left). A glass channel (8 mm internal width) was inserted centrally between the coils, resting on the trough, allowing the PBS to flow through it freely. A high-intensity focused white light shone on the channel (see Fig. 3b at right). A platinum Wilhelmy plate was also used to measure the surface pressure of the film simultaneously.

The air-water interface was swept with a vacuum and prepared for the fibroin film. A magnetized Teflon-coated ferromagnetic needle was placed on the surface of the PBS in the channel, held in place by the constant magnetic field of the Helmholtz coils. A magnifying camera (A601I, Edmund Industrial Optics) situated above the trough was focused on the needle edge and connected to a PCI card frame-grabber. The entire setup was enclosed in a Plexiglas casing (Thor Labs Inc., Newton, NJ) to prevent air currents from disrupting the interface.

A sterilized syringe was used to procure 55 μL of prepared fibroin from a vial, which was spread on the vacuumed interface. The elastic and viscous moduli of the film were sampled every 20 seconds by the software. The experiment was stopped when the elastic and viscous moduli had clearly stabilized. Varying the concentration of fibroin spread was hypothesized to affect the time needed for silk III crystallization to proceed. Several concentrations from 2.5 mg/mL to 20 mg/mL were tested multiple times to ensure reproducibility.

Results

Tests with fibroin prepared via centrifugation revealed greater reproducibility than those using uncentrifuged fibroin. The homogeneity of the centrifuged fibroin solution revealed surface pressure plots indicative of greater crystallization during isothermal compression. In comparison to the aqueous fibroin isotherm curves generated in conjunction with positive X-ray diffraction results, surface pressure curves of fibroin dissolved in acetic acid seemed characteristic of gelation instead of crystallization (Terry et al., 2004). The curves as displayed in Fig. 4 allow a comparison to determine the effects of centrifugation and acetic acid dissolution.

The smooth surface pressure curves of uncentrifuged fibroin and fibroin in acetic acid, contrast with those of centrifuged fibroin in water.  The procedure most conducive to crystallization was deduced via Grazing Incidence X-ray Diffraction (GIXD).

The GIXD images displayed in Fig. 5 indicated the optimal procedure to produce crystallization at an air-water interface (Kirkwood, personal communication). Structural peaks in the crystalline film as shown in Fig. 5 taken from one of the experimental silk III production procedures matched results of Valluzzi et al. (1999a) (not pictured in the figure). This diffraction pattern resulted exclusively from the procedure of using water as the solvent and centrifuging the fibroin solution. The centrifuged fibroin isotherm also matched the isotherms corresponding to the positive diffraction images most closely (see Fig. 4, Fig. 5). This information helped create a new procedure for fibroin experimentation: centrifuged fibroin in water was subsequently used in all experiments.

The AFM conducted on the slides with vertically deposited films of fibroin yielded positive results as well. The images displayed (see Fig. 6) are of a bundle of fibroin fibers in a silk III film. The coaxial helix theory proposed for the structure of silk III supports the aligment of the peaks on the micron scale displayed (Valluzzi et al., 1999b)

The biocompatibility assessment indicated that silk III is compatible with human fibroblasts, and does not inherently inhibit cell growth and function. The fibroblasts on both plates with silk III films successfully reproduced multiple times and grew to confluence after five days (see Fig. 7).

The ISR tests indicated a clear correlation between time of crystallization and varying concentration. The software’s measurements of the elastic and viscous moduli as time-dependent functions revealed curves of typical conformation (see Fig. 8).

The takeoff time, indicated by the black circle in Fig. 8, is an estimation of the starting point for the crystallization of the film at the interface.  This takeoff time can be approximated at the initial rise of the viscous modulus from a constant value.  The change in viscosity is a clear indication of a change in the film’s material properties. Fig. 8 demonstrates a clear enough curve (with very little noise) that the takeoff time could be extracted by determining when the first significant (greater than 1%) deviation from the constant modulus value occurred.

The approach taken here, intended to model the bulk properties of an interface, does not have the sensitivity required to create a detailed model of the kinetics. The crossover time was the second characteristic studied in silk III crystallization. The crossover time is a mathematically definitive point, analogous to the rheometric quantity of crossover frequency, at which point, the two moduli intersect (see white circle of Fig. 8).

The crossover and takeoff times were recorded for each experiment with different concentrations of fibroin. The takeoff times of viscous moduli with runs of different concentrations of fibroin were used to determine the effect of initial concentration on the time until crystallization begins. The values of both the takeoff time and crossover time were characterized by an apparent decrease with an increase in concentration.  The takeoff time variation closely fit an inverse relationship as shown in Fig. 9.

The trend of a generally inverse correlation is corroborated by the plot in Fig. 10 as the values of the takeoff time multiplied by the concentration remain roughly constant.

A similar analysis conducted for the crossover time indicates a roughly inverse relationship between concentration and crossover time as well (see Fig. 11). However, the correlation appears weaker as indicated by the gradual upward trend in Fig. 12.

Figure 4. Isotherms of silk III fibroin under various conditions. Above are surface pressure curves for fibroin films made using fibroin at the air-water interface. Scores of films were constructed and subjected to isothermal compression, under various conditions and using procedures varying parameters, in order to assess which procedure best effects silk III crystallization. Three such isotherm curves are shown above.
Figure 5. At left: Diffraction results of silk III with corresponding isotherm (Kirkwood, J.) The isotherm for the diffraction image displayed at right was compared to the isotherms of all procedures attempted, in order to optimize the procedure for silk III formation. Grazing Incidence X-ray Diffraction results are shown at left (original) and center (integrated), properly indicated characteristic peaks of the silk III structure (using the peaks defined by Valluzzi et al.). The integrated diffraction peaks shown at center – obtained experimentally – were compared to the definitive peaks, confirming that the structure formed using one particular procedure was in fact silk III, in turn identified by comparison of the isotherms for all procedures.
Figure 6. AFM images of silk III. At left: top-down view. At right: 3-D representation. These AFM images provide a detailed view of the structure of silk III as it forms at the air-water interface. The ridges in the 3-D representation support Valluzzi’s hypothesized structure.
Figure 7. Images of fibroblast growth on two plates with silk III films. These two magnified images of human fibroblast cells growing on silk III films imply an intrinsic biocompatibility of silk III. Cells reached confluence in five days.
Figure 8. Logarithmic vs. linear view of viscous and elastic moduli versus time. These moduli are from a film of 5 mg/mL fibroin. The viscous modulus (G") is shown in red and elastic modulus (G') in blue. The white circle indicates crossover time, black circle takeoff time.
Figure 9. Scatter plot of takeoff time as a function of concentration. On the scatter plot above, the inverse function (k/x) fit included, with the optimal coefficient determined by Mathematica. The plot shows the approximately inverse relationship between the takeoff time and fibroin concentration for silk III at the air-water interface. Error bars represent one standard deviation with four runs at one concentration.
Figure 10. Scatter plot of takeoff time multiplied by concentration as a function of concentration. This plot maintains a visibly near-constant y-value for any x-value. This feature demonstrates the constant nature of the takeoff time multiplied by the concentration of fibroin used. Error bars represent one standard deviation with four runs at one concentration.
Figure 11. Scatter plot of crossover time as a function of con- centration. On the scatterplot above, the inverse function (k/x) fit is included with the optimal coefficient determined by Mathematica. The plot shows the approximately inverse relationship between the crossover time and fibroin concentration for silk III at the air-water interface. Error bars represent one standard deviation with four runs at one concentration.
Figure 12. Scatter plot of crossover time multiplied by con- centration as a function of concentration. This plot maintains a visibly near-constant y-value for any x-value. This feature demonstrates the constant nature of the crossover time multiplied by the concentration of fibroin used. Error bars represent one standard deviation with four runs at one concentration.

Discussion

The crystallization of fibroin at the air-water interface, and the formation of silk III were confirmed by the GIXD results. It was also determined in the preliminary experimentation with isotherms that acetic acid is not an ideal solvent when working with fibroin at the air-water interface. Methanol as used by Meinel et al. (2006) could help the fibroin spread and reduce clumping, an improvement over acetic acid as a solvent. The AFM indicated general similarities of silk III to the coaxial model, but more detailed imaging is necessary to validate the theory.

The fibroblast cells on both plates grew to confluence in exactly five days—a time scale consistent with many biocompatible materials. As observed by Karamichos et al. (2009), fibroblast cells grow to confluence within three days in Fetal Bovine Serum (FBS) media cultures and within five to seven days in structures of natural human materials like collagen matrices. However, it is vital to note that while the fibroblast cells grew successfully on the silk III films, their growth only proves a rudimentary biocompatibility of silk III. Fibroblasts are relatively durable cells in the human body, since they function as sturdy connective tissues, and can handle a broader range of conditions than more advanced cells and tissues. Therefore, the histotoxicity of silk III to human tissue is not categorically disproved by these experiments. However, the fibroblast biocompatibility was still an important determination, as fibroblasts are critical to wound healing, and silk III could one day be used in various wound treatments.

From the interfacial rheology experiments at varying concentrations, the takeoff times and crossover times are defined by an inverse function. The time for the crystallization of silk III to begin (the takeoff time) can be characterized by the diffusive model, where the dependent condition is how long it takes the fibroin molecules to arrange (“diffuse”) themselves into an alignment conducive to silk III formation. This two-dimensional diffusion relationship between the time and length scale of the problem is written:

The variable L is the length scale of the problem, squared to represent the surface area available to the molecules. The diffusion constant D is a system dependent value, and tt is the takeoff time. To simplify the relationship, a new constant A can be defined as k/D. Substituting k/C for L2, as concentration and surface area are inversely proportional, and rearranging variables algebraically, a function can be constructed describing the takeoff time as depending on concentration:

The diffusive law is reproduced in the above simplified form so that the relationship between time and concentration becomes clear, so that time scale effects of concentration on silk III production may be analyzed. The above derived inverse proportionality between the takeoff time and fibroin concentration matches the inverse function fit in Fig. 9. This relationship also accounts for the fact that the quantity C × tt equals a constant At approximately equal to 12,500 mg-s/mL (see Fig. 10. A similar relation is evident for the crossover time, as deduced from an identical derivation, simply substituting tc (crossover time) for tt (takeoff time). The graph in Fig. 12 indicates that the constant Ac equals about 24,000 mg-s/mL.

The application of the diffusive model to the formation of silk III is significant for any future manufacturing process or other procedure requiring time-critical formation of silk III. It is quite possible that higher than a certain concentration the diffusive model breaks down, due to formation of a thicker, denser multilayer at the surface and more complex interactions between fibroin molecules. However, concentrations higher than 20 mg/mL could not be accurately tested because of the physical limitations of the instruments. The film would be unusually thick at higher concentrations, and the elasticity was beyond the range of the instrument.

While the data closely fit the diffusive model, there are other possible factors in the formation of silk III at the air-water interface. During late stage aggregation of fibroin, Lifshitz-Slyzov coarsening may be responsible for the behavior of the film as silk III forms in domains. Since according to Lifshitz-Slyzov coarsening, diffusion and coalescence of droplets follows the mathematical relation that the domain size is directly proportional to the cube-root of the time, it would have been necessary to measure the film rheology at later times for the coarsening to be a significant factor. Silk III exhibited such high moduli at these later times that they could not be properly measured by lab equipment in the late stage of film formation. Therefore, while the diffusive model aptly describes the formation of silk III films, the possibility of coarsening and other effects cannot be precluded.

Errors in the rheological experimentation may have been caused by changes in the fibroin solution over weeks of experimentation, despite storage at low temperature. Additionally, the Teflon coating on the magnetic needle began to wear off at the edge, which slightly affected the needle’s interaction with the film as well as the ability of the software to detect the needle edge. However, the same needle remained usable for all measurements, so this was not a confounding error. Errors applicable to fibroin films other than those made in the ISR trough include contamination from dust particles in the air and disturbance from convective air currents, as the ISR was encased in a Plexiglas container, whereas the Langmuir trough and trough used for vertical deposition were not. Future experimentation should be conducted in a space where environmental factors can be controlled with higher degree of sensitivity.

Conclusions

While experimentation served as a preliminary exploration of the behavior of fibroin at the air-water interface, much remains to be studied about silk III. The biocompatibility of silk III was only assessed using fibroblasts, which are resistant to various pathological conditions to which other human cells would succumb. Further tests on silk III films need to be conducted to determine whether various kinds of human cells, particularly more delicate tissues and cells with specialized functions are compatible with silk III films.

The study of the effect of concentration on the formation of silk III revealed that the crystallization process follows the diffusive model. The crystallization of silk III is primarily dependent on the initial concentration of fibroin, with time having an inverse relationship to concentration. The strong correlation, particularly of the takeoff times, indicates that the time is dependent on few other factors, and that the chief determinant is concentration.

Further research on silk III crystallization should be carried out using different concentrations of fibroin in water and organic solvents of different strengths. Crystallization might proceed more rapidly, and perhaps with more identifiable domains of orientation, because of newly introduced intermolecular forces between fibroin and additional molecules in the organic solvent, keeping the fibroin spread at the air-water interface and preventing the fibroin from clumping, gelling, or forming fibers. Research involving organic solvents such as hexane or decane as a replacement for air at the interface may also effect faster crystallization of silk III. Further imaging studies of silk III must also be conducted to view the exact structure of silk III, in order to validate the coaxial helix theory or propose an alternative model. Additionally, experimental measurements of domain size over time would be important to assess the early stages of silk III structural formation (before the takeoff time). Optical techniques such as imaging of fluorescently tagged fibroin proteins would have to be used to achieve this end in further study.

Silk III is a relevantly recent discovery by comparison to silk I and II but it holds promise as a material with industrial and biomedical application. The alternative structure of silk III could solve biocompatibility issues that have plagued studies of drug delivery where interference of the casing material with bodily function is a serious concern (Hofmann et al., 2006). Using silk III-based scaffolds in tissue reconstruction is also a possibility, given its biocompatibility and the incipient success of silk II-based scaffolds in animals (Altman et al., 2007). Multilayers of silk III could also eventually be used as textiles, or components of textiles, due to the high elasticity of the crystalline film observed during rheometric experimentation.

Silk fibroin already has many unique and vital qualities, and silk III may make fibroin even more valuable. Fibroin polymers are large and uniform enough to generate varied stable intermolecular structures, often with very high tensile strength. Fibroin is also usually biocompatible at the chemical level, as it originates as a natural biomolecule and is very similar to the protein collagen which is the most abundant in human body. Since a better understanding of silk III is necessary before applying it to practical uses such as those in which silk I and II are involved, the goal of this research was to better describe the nature of silk III through experimentation. Its basic properties in a nanofilm and its manner of formation were analyzed in order to characterize it for future study and application to industry.

The determinations of this study serve as a starting point for future experimentation on silk III. Since procedures are not yet established for working with silk III the same way they are for silk I and II, a procedure was developed to optimize crystallization. The biocompatibility assessment ensured that there is a distinct possibility of using silk III in biomedicine. The rheological analyses provide quantitative information about time needed for the crystalline film to form depending on the initial amount of fibroin added, which can be used both as a basis for testing other parameters of silk III crystallization in future research and in manufacture. Before silk III can enter the realm of applications, it is imperative that knowledge about it as a material be advanced. This research successfully assessed various quantitative and qualitative characteristics of silk III fibroin that remain essential to future study and use of this new material in potentially revolutionary biomedical applications.

Acknowledgements

Many thanks to Dr. Gerald Fuller of Stanford University for giving me the opportunity to conduct research in his laboratory, as well as to An Goffin and John Kirkwood of Stanford University, and Dr. Kate Schafer of The Harker School for their invaluable guidance throughout the duration of the study.

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