However, a multitude of parameters directly affect the electrospinning process thus requiring a fundamental understanding of how various parameters affect the process and resulting fiber
Introduction
Introduction to Electrospinning
Electrospinning is a fiber processing technique that involves applying a high voltage to a polymeric solution, which results in nanometer to micron diameter scale fibers The interconnected porous network, high surface area, and tailorable surface morphology has made electrospinning a popular fiber processing technique ideal for a variety of applications The electrospinning process was first patented in 1934 (1) but a revival of this technology has come about only in the past decade, which can be seen in the number of electrospinning publications over the past few years In 1999, there were only 11 publications dealing with the topic of electrospinning However, to date, there are approximately 400 electrospinning publications The increased interest in this technology is almost certainly due to the ease of fabrication of nanometer to micron diameter fibers that is difficult to achieve using conventional fiber spinning techniques (2)
The small fiber diameters and high surface area make electrospun fibers ideal for a variety of applications such as filtration (3, 4), electronic and fuel cell applications (5-7), biological/chemical resistant clothing (3, 4), and biomedical applications (8-23) Table 1.1 lists examples of the variety of polymers that have been successfully electrospun Although much of this research has been done at the bench top scale, there are companies utilizing the electrospinning process for commercial filtration and biomedical applications (24, 25) This is indicative of the fact that with proper processing protocols, it is possible to scale up the electrospinning process to produce large, commercially viable membranes
Table 1.1 Examples of Polymers Fabricated into Fibers via Electrospinning
Table 1.1 Examples of Polymers Fabricated into Fibers via Electrospinning
Polymers Dextran Water, DMF/Dimethyl
Acetone, Water, Carbon Tetrachloride (CHCl3)
Poly(lactic acid) Dimethyl formamide (DMF) (34)
Poly(lactide-co- glycolide) (PLGA)
Polystyrene (PS) Carbon Disulfide (CS 2 ), DMF,
Polymer Blends Chitosan/PEO Acetic Acid (41)
Silk/PEO Silk aqueous solutions (43)
The Electrospinning Process
The electrospinning process employs an electrostatic potential difference to form nanometer to micron diameter scale fibers A polymeric solution is contained in a syringe attached to a syringe pump The syringe pump allows for a constant flow of polymer solution to the tip of the needle A drop of solution is suspended from the needle tip due to surface tension of the polymeric solution The increasing electric charge that accumulates on the droplet, due to the high voltage power supply, causes the protruding droplet of polymer solution to deform This causes the hemispherical droplet to become conical in shape and it is then referred to as the Taylor Cone (44) Eventually, the charge accumulated on the surface of the droplet overcomes the solution surface tension and a fibrous jet is ejected from the needle and travels towards a target The target generally consists of a grounded metal sheet, but a rotating cylinder has also been used to align the fibers (33, 45, 46) As the fibrous jet travels towards the target, evaporation of the solvent occurs and the fibers are typically dry when arriving at the target
There has been a great deal of research dedicated to the study of the trajectory of the electrospinning jet (2, 47-56) This is because after initiation, the path of the jet follows a straight line for a short distance At this time, charge begins to accumulate on the surface of jet as the solvent evaporates, leaving a smaller surface area for the charges to reside Then, due to electrostatic repulsion, the jet undergoes a bending instability represented by a chaotic whipping of the jet Initially, it was believed that this bending instability was due to a process called splaying, in which accumulation of charges on the surface of the jet overcomes the cohesive forces of the jet and results in the division of the jet into multiple, smaller jets to reduce the charge (57, 58) Although this phenomena is not completely understood; further investigations, with the assistance of high frame-rate, short exposure time cameras, determined that the jet was not being divided into multiple, smaller jets but rather one jet was rapidly bending and whipping in a confined space (2, 55) There is still much research being completed in this area to clarify the motion and elongation of the electrospinning jet.
Effects of Experimental Parameters on the Electrospinning Process
The main complexity of the electrospinning process is the multitude of parameters that directly affect the process and the resulting fibers Both processing parameters and solution characteristics play an integral role in the electrospinning process Applied voltage (58), working distance (36), electric field (59), and atmospheric conditions (36, 38) are processing parameters that directly affect the outcome of the electrospinning process However, solution characteristics are equally important when considering their effect on fiber formation and properties Solution surface tension (5), molecular weight (38, 60), concentration (33, 58, 61), solution viscosity (5, 33), and choice of solvent (33, 37) can determine whether electrospinning occurs and can have a direct affect on fiber properties
The above-mentioned parameters can affect the formation of fibers (27, 62), fiber diameter (57, 58, 61, 63), fiber shape (59, 61), and surface morphology (38, 64) This is mainly attributed to the fact that chain entanglements (62, 65) and/or intermolecular associations are required for fiber formation and affect fiber properties (66) Reneker et al (61) showed the effect of solution concentration and viscosity on the morphology of electrospun PEO fibers The research indicated that beaded fibers were produced from solutions with a viscosity of less than 527 centipoise The diameter of the beads increased with increasing viscosity changing shape from spherical to a more elongated appearance The beads disappeared after a certain viscosity was reached for this system Deitzel et al (58) showed that the diameter of PEO fibers increased with increasing solution concentration Baumgarten (67) reported a quantitative correlation between fiber diameter (d) and viscosity (η): d η 0.5 ; thus showing the significant impact that viscosity has on fiber diameters Applied voltage also has been reported to affect fiber diameter; in that, increased voltage can decrease fiber diameters by as much as 50% in electrospun polystyrene fibers (36)
1.3.2 Factors Affecting Fiber Shape and Surface Morphology
Electrospun fiber morphology has been shown to vary depending upon the experimental parameters employed Fibers can be ribbon-like, circular, or flat in their cross-sectional shape (59) Reneker et al (59) suggested that fiber shape is directly related to the formation of skin on the surface of the jet As the electrospinning jet is ejected from the needle and travels towards the target, solvent is evaporated quickly and causes the formation of a thin layer of dried polymer, referred to as the skin The interior of the jet still contains solvent and as this interior solvent evaporates, the skin can collapse and result in various shapes of electrospun fibers Therefore, polymer concentration, polymer molecular weight, solvent evaporation rate, and working distance directly affect fiber shape (27, 33, 59, 60)
The rapid evaporation of the solvent in the jet not only affects fiber shape, but also directly affects surface morphology Bognitzki et al (64) reported the presence of pores on the surface of poly(lactic acid) fibers electrospun from dichloromethane The formation of these pores was attributed to rapid phase separation due to the high evaporation rate of the solvent Research completed in our lab shows that porous features are found on the surface of electrospun polystyrene fibers when electrospun in a humid atmosphere (>30% relative humidity) (36, 38) This suggests that not only is the electrospinning process susceptible to experimental parameters, but also to environmental conditions This illustrates the complexity that is involved when trying to control the electrospinning process; many variables directly affect the outcome of this fiber formation technique.
Application Areas
As previously mentioned, electrospun fibers have been useful in a variety of applications such as filtration (3, 4), electronic and fuel cell applications (5-7) However, much of the current research in the field has focused on using electrospun fibers for biomedical applications such as wound dressings (68), drug delivery vehicles (13, 15, 16), and tissue engineering scaffolds (8-10, 14, 18, 20, 21, 69-72) Electrospun fibers are ideal for tissue engineering constructs due to the three- dimensional, porous membrane that can be produced This is an important characteristic of a tissue engineered scaffold due to the fact that cells need to be able to move throughout the membrane in order to grow and proliferate The porosity also allows for the uptake of nutrients and excretion of cellular waste products (73) Also, the nanometer diameter electrospun fibers mimic the size scale of fibrous proteins found in the extracellular matrix (ECM) (18) For these reasons, a variety of natural and synthetic polymers such as: fibrinogen, collagen, poly(glycolic acid), poly(ethylene-co-vinylacetate), and poly(ethylene-co-vinyl alcohol) have been electrospun for tissue engineering and other related applications (9, 10, 16, 30, 68)
Xu et al (21) employed aligned, electrospun poly(L-lactide-co-caprolactone) to serve as a scaffold for human coronary artery smooth muscles cells (SMCs) to be used in blood vessel engineering The researchers showed that SMCs attached to the electrospun fibers and migrated along the axis of the aligned fibers The SMCs also showed a spindle-like contractile phenotype, which is characteristic of this cell type (21) Shin et al (31) cultured cardiomyoctes on electrospun poly(caprolactone) fibers for use as a cardiac graft Findings showed that the cardiomyoctes began synchronized beating after only 3 days in culture and the cells secreted cardiac- specific and ECM proteins onto the electrospun scaffold (31) These are just a few of the studies that prove electrospinning to be successful in creating fibers for biomedical applications However, these studies focus on recapitulating the structural aspects of the ECM only and lack the incorporation of biological elements, such as growth factors and other ECM proteins In order for a scaffold to be truly ECM-mimetic, the scaffold must mimic the ECM in both structure and function
Recently, Chew et al (74) successfully mixed human nerve growth factor (NGF) into a copolymer solution of poly(caprolactone) and poly(ethyl ethylene phosphate) (PCLEEP) to produce electrospun fibers containing NGF Findings suggested that the growth factor was present in aggregate form throughout the fiber matrix at low loading levels (3.10 ± 0.53 x 10 -4 %) The authors believe that the presence of these aggregates, along with the low loading levels, may be attributed to the phase separation that was observed during electrospinning due to the different charge densities of the aqueous growth factor solution and the polymer solution However, despite the phase separation, NGF was released from the electrospun matrix The NGF showed a burst release of 20%, then a steady release over a period of 3 months The authors indicated that it was likely that the electrospinning process may have denatured the NGF, however, they were unable to quantify the NGF bioactivity after release This study shows that electrospun fibers can be successfully used in drug delivery applications The goal of our work was to incorporate heparin into the electrospun matrix in order to allow for the binding of growth factors The addition of growth factors after electrospinning offers advantages such as control over the amount of growth factor present in the matrix and the ability to circumvent any phase separation issues Incorporating biological molecules into the electrospun matrix would allow for a structurally and biologically relevant scaffold.
Research Motivation
Given the multitude of parameters that are involved in the electrospinning process, it is crucial to understand how these parameters affect the process and the resulting fibers A fundamental understanding of how to control the formation, shape, texture, and morphology of these fibers is essential Determining the link between electrospinning parameters and electrospun fiber morphology will allow for the design of polymer fibers to meet specific application needs The initial goal of this work is to investigate how processing parameters and solution characteristics affect the electrospinning process This knowledge will then be applied in the fabrication of electrospun fibers for biomedical applications The specific areas of research discussed in this dissertation are as follows:
1 Understanding the effects of humidity, molecular weight, and viscosity on the electrospinning process and resulting fiber properties: Chapter 2 (38)
2 Functionalization of electrospun fibers with biologically relevant molecules for biomaterial applications: Chapter 3 (23)
3 Attachment of proteins to electrospun collagen and gelatin membranes for use as tissue engineering scaffolds: Chapter 4 (27)
Experimental Design
The electrospinning apparatus consisted of a syringe pump (Orion Sage TM , Fisher, Fair Lawn, NJ), high voltage supply (12-30 kV, Glassman Series EH, High Bridge, NJ), syringe (1-5 mL, Popper & Sons, New Hyde Park, NY) and a needle (Hamilton, Reno, NV) with an inner diameter varying from 0.26–1.60 mm A grounded aluminum sheet was used as the target and was positioned 15-35 cm from the tip of the needle for sample collection Figure 1.1 shows a schematic of the electrospinning apparatus used The electrospinning setup was encased in a vented Plexiglas box (2.5 ft x 2.5 ft x 3.5 ft) The concentration of the polymeric solutions varied depending upon the type of polymer, molecular weight, and solvent employed Specific formulation of each polymer solution is detailed in the Experimental Section of each chapter
Figure 1.1 Schematic representation of the electrospinning apparatus
The electrospun fibers were analyzed for both their materials and biological properties Materials characterization was done to confirm and report the structural characteristics of the fibers Analytical techniques such as field emission scanning electron microscopy (FESEM), atomic force microscopy (AFM), multiphoton and laser scanning confocal microscopy were employed for materials characterization The function of the electrospun materials with respect to biological applications was also analyzed Assays such as toluidine blue, enzyme-linked immunosorbent assay (ELISA), dot blot, growth factor binding and release were employed to test the biological properties of these materials Each of the above techniques will be described in detail in the appropriate chapter.
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INFLUENCE OF HUMIDITY AND MOLECULAR WEIGHT ON FIBER
Introduction
Understanding how to control the surface morphology of electrospun fibers is essential to gain a fundamental understanding of the electrospinning process Parameters such as voltage, working distance, solution concentration, molecular weight, and humidity all play an integral role in the electrospinning process Our efforts have focused on understanding how molecular weight and humidity directly affect this fiber formation process Learning how these parameters affect both fiber formation and surface morphology is the first step in being able to control and understand the electrospinning process The ability to control fiber formation and introduce porous surface features of a known size is crucial for tailoring electrospun fibers for specific end-use applications
One important issue in the electrospinning process is the effect of polymer molecular weight on fiber formation The literature shows that electrospun fibers can vary in diameter from nanometer to micron (1-7), but little is known about what causes these diameter differences Studies also show that electrospinning equipment can be used to produce freestanding beads, which would make this an electrospray process (8) We believe that molecular weight plays a major role in controlling fiber formation and fiber diameter Understanding how to utilize polymer molecular weight to manipulate fiber formation and fiber diameters is necessary in order to learn more about the fiber formation technique and how to control it in order to make it a commercially viable process
In addition to the polymer molecular weight, atmospheric conditions, such as humidity, have been found to directly affect the surface morphology of electrospun fibers Fibers electrospun from polystyrene (PS), polycarbonate (PC), and poly (methyl methacrylate) (PMMA) were found to contain submicron surface features when the polymers were dissolved in a volatile solvent and electrospun in the presence of humidity (2) These surface features or “pores” resemble those seen in thin films (9-12) Srinivasarao et al (9) observed the formation of a three-dimensionally ordered array of pores in a thin PS film These structures were not observed when the films were formed in the absence of humidity Since pore formation was dependent upon a moist atmosphere and the use of a volatile solvent, the structures were attributed to be breath figures Phase separation techniques have also been used to produce submicron structures in thin films as described by Van de Witte et al (13)
The presence of pores on the surface of electrospun fibers can serve to increase the surface area for filtration applications, act as a cradle for enzymes, or be used to capture nanoparticles Previous work has confirmed the existence of pores on the surface of electrospun fibers and the ability to fill these pores with silver nanoparticles (2) The goal of this research is to study the effect of increasing humidity and varying molecular weight on pore size and distribution Understanding the link between humidity and fiber morphology will allow for better control over the properties of electrospun fibers An in-depth investigation of humidity and molecular weight effects on the electrospinning process will provide a greater fundamental understanding of how to the control the electrospinning process.
Experimental Procedures
Polystyrene fibers were electrospun using a 35 wt% concentration of PS in THF (Fisher Scientific, Atlanta, GA), unless otherwise indicated Molecular weights of the
PS samples were 31,600, 44,100, 75,700, 171,000, 190,000, 560,900, and 1,800,000 g/mol (Scientific Polymer Products, Ontario, NY) PEO (300,000 g/mol MW, Scientific Polymer Products, Ontario, NY) was dissolved in water, at various concentrations, to make PEO electrospun fibers All polymers dissolved at room temperature with up to 2 h of stirring
The electrospinning apparatus, as described in Chapter 1, consisted of a 5 mL syringe connected to a syringe pump with a flow rate of 0.07 mL/min A voltage of +10 kV was applied to the tip of the syringe needle The inner diameters of the needles used were 0.26 mm for the 31,600 g/mol solution, 0.34 mm for the 44,100 g/mol solution, 0.51 mm for the 75,700, 171,000 and 190,000 g/mol solutions, and 1.60 mm for both the 560,900 and 1,800,000 g/mol solutions The target consisted of a grounded aluminum sheet placed 35 cm from the needle tip to ensure that the fibers were dry upon collection The relative humidity (RH) was varied using a humidifier (Holmes HM-1700, TruValue Hardware, Newark, DE) placed inside of the electrospinning box The temperature was kept at a constant 75-76 °F
The electrospun PS fibers were characterized using optical microscopy and FESEM Fibers were directly electrospun onto SEM mounts then sputter coated with gold for analysis Pore size and distribution information was obtained from FESEM images using Scion Image Software The micrographs used for these studies were acquired under the same magnification, electron beam density, and working distance AFM investigations were performed under ambient conditions in tapping mode using TESP (tapping mode etched, silicon probe) cantilevers Electrospun fibers were analyzed on a double-sided carbon adhesive and scanned along the fiber length Pore depth and surface area were calculated using manufacturer provided software The 3 àm x 3 àm images were inverted and particle size analysis was used to determine average pore depth In order for the values to have statistical significance, more than
40 pores were measured per sample
The molecular weight of the 1,800,000 g/mol PS was determined before and after the electrospinning process using gel permeation chromatography (GPC) THF was used as the solvent for GPC analysis and the detectors used were differential refractive index (Waters 410), viscometry (Viscotek, Houston, TX), and multi-angle light scattering (Wyatt Technology, Santa Barbara, CA) All GPC data was acquired using
Wyatt software and was calculated directly from the scattering intensity of the eluting polymer.
Results
2.3.1 Influence of Polymer Molecular Weight on Fiber Formation
PS molecular weights from 31,600 g/mol to 1,800,000 g/mol were investigated as outlined in Table 2.1 The solutions were electrospun under the following experimental conditions: 35 wt% PS dissolved in THF, +10 kV, and 25% relative humidity The needle diameter increased with increased molecular weight due to changes in viscosity FESEM was used to characterize the samples Figures 2.1, parts a and b, show that the lower molecular weights PS yielded beads, thus electrospraying had occurred Fiber formation began at the 75,700 g/mol molecular weight and was apparent by the presence of fiber ‘tails’ on the ends of the beads, Figure 2.1c However, it was not until a molecular weight of 171,000 g/mol that typical electrospun fibers began to form, Figure 2.1d At this increased molecular weight, the PS fibers were approximately 3-10 àm in diameter The normal ribbon-like shape of PS electrospun fibers was not observed when electrospinning the 560,900 and 1,800,000 g/mol molecular weight solutions Instead, these fibers appeared to have a rippled surface as seen in Figures 2.1, parts e and f These fibers, produced from high molecular weight PS, also had larger fiber diameters of up to 40 àm Thus, molecular weight affects both fiber formation and fiber diameter
Table 2.1 Electrospinning Various PS Molecular Weights in THF
75,700 0.51 Beads (15-20 àm), fiber ‘tails’ (0.5 àm)
Figure 2.1 FESEM micrographs of (a) 31,600 g/mol, (b) 44,100 g/mol, (c) 75,700 g/mol, (d) 171,000 g/mol, (e) 560,900 g/mol, and (f) 1,800,000 g/mol PS/THF electrospun fibers
We believe that fiber formation is dependent upon the presence of chain entanglements and/or intermolecular associations Parameters such as solution concentration, polymer molecular weight, and viscosity are directly related to chain entanglements The Mark-Houwink equation allows for the calculation of intrinsic viscosity ([η]) as a function of molecular weight (Mw): [η]= KMw a
“a” are dependent on the polymer/solvent system When the value of [η]C is between
1 and 10, the solution is considered to be in the semi-dilute regime where coil overlap dominates (14) When [η]C > 10, the solution is concentrated and chain entanglements are present (14) To determine if chain entanglements are present in the PS solutions investigated, the Mark-Houwink equation was employed For PS in THF, K= 11x10 -3 and a= 0.725 Applying this equation to the various molecular weights of PS studied, it is evident that fiber formation occurs when chain entanglements are present, [η]C > 10 (Table 2.2) This indicates that chain entanglements play a role in the formation of PS electrospun fibers To further test this idea, the 31,600 g/mol PS/THF was electrospun again, but this time the concentration was increased to 80 wt% At this concentration and molecular weight, [η]C= 16 which is in the entangled regime that should produce fibers Figure 2.2 shows that increasing the concentration of this low molecular weight PS results in micron diameter fibers This leads us to believe that sufficient chain entanglements ([η]C > 10) are necessary for fiber formation
Recently, Koski et al (4) showed that solution concentration plays a major role in the formation of electrospun polyvinyl alcohol (PVA) fibers PVA fiber formation was observed when [η]C values were between 5 and 12, suggesting that chain entanglements were necessary for formation of PVA fibers However, in the case of electrospinning PVA, other forces such as inter- and intra-chain bonding between the polar hydroxyl groups in the PVA molecules also affect chain entanglement This may be why PVA fibers can be formed when 5 < [η]C < 12 and PS fibers require [η]C
> 10 Although slight differences between the two polymer/solvent systems exist, the results indicate that chain entanglements play a direct role in fiber formation
Table 2.2 Applying the Mark-Houwink Equation to Various PS Molecular
PS Molecular Weight (g/mol) [η]C Electrospinning Observations
171,000 24 Fibers 560,900 57 Fibers 1,800,000 132 Fibers a35 wt% concentration
Figure 2.2 FESEM micrograph of 31,600 g/mol PS electrospun from an 80 wt% solution of PS/THF
To test if this hypothesis, i.e., [η]C > 10 in order for fibers to form, is valid for aqueous systems, a series of PEO samples dissolved in water was also studied For PEO in water, K= 1.25x10 -2 and a= 0.78 The molecular weight, concentration, and [η]C values are listed in Table 2.3 The first set of experiments consisted of electrospinning 100,000 g/mol PEO (Scientific Polymer Products, Ontario, NY) in water at the following concentrations (wt%): 0.5%, 3%, 10%, 15%, and 20% Figure 2.3, parts a and b, show that only micron diameter beads or microspheres were produced at the 0.5% and 3% concentrations As evident in Figure 2.3c, a mixture of fibers and beads were present at the 10 wt% solution concentration Above the 10 wt% concentration, mostly fibers were produced (Figure 2.3d)
Table 2.3 Applying the Mark-Houwink Equation to Various PEO Molecular
10% 10 Fibers (100-200 nm), beads (1-4 àm) 15% 15 Fibers (100-200 nm), beads (1-8 àm) 20% 20 Fibers (500 nm), few beads (10-15 àm) 300,000 5% 12 Beads (
10 for the PEO/water system Thus confirming that using the Mark-Houwink equation to determine chain entanglements in flexible polymers is useful for predicting fiber formation as applied to the electrospinning process
2.3.2 Effect of Humidity on Surface Morphology
A series of experiments were conducted to determine the effect of humidity on pore formation in PS/THF electrospun fibers Relative humidity in the atmosphere surrounding the electrospinning apparatus was varied from 25-72% in order to determine the effects of atmospheric conditions on the electrospinning process and fiber characteristics Since molecular weight was proven to play an integral role in fiber formation, molecular weight was also studied to determine if it had an effect on pore formation under humid atmospheric conditions
A 35 wt% solution of PS (190,000 g/mol) dissolved in THF was electrospun under five different humidity ranges: 10) in order for fibers to be produced The Mark-Houwink equation proved useful for flexible polymers in both organic and aqueous systems The use of this approximation is a great tool because it saves time and material by providing a rough estimate of the polymer solution characteristics required to produce electrospun fibers
In Chapter 2, not only was the importance of solution characteristics illustrated, but the effect of processing parameters proved to be equally critical to the fiber spinning process It was determined that electrospinning in an atmosphere of less than 25% relative humidity produced smooth fibers However, when the humidity reached 30% and above, the formation of nanopores (~85 nm in diameter) on the surface of the fibers was observed Detailed experiments revealed that increasing the humidity during the electrospinning process resulted in increased pore diameters AFM studies showed that pore depth and diameter increased as the humidity in the atmosphere increased Understanding how parameters, such as relative humidity, affect fiber properties is crucial for obtaining a better understanding of the electrospinning process In order for electrospun fibers to be successfully used in commercial applications, the processing parameters and solution characteristics that affect the fiber formation process must be fully understood and able to be controlled This way the electrospinning process could be used to tailor fibers to meet specific application requirements
The effect of molecular weight and concentration on the electrospinning process became very important when producing electrospun fibers for drug delivery and tissue engineering applications In Chapter 3, we discussed the functionalization of fibers with biologically relevant macromolecules The molecules of interest were LMWH and PEG-LMWH Studies revealed that the heparin-based molecules could be incorporated into PEO or PLGA electrospun fibers at concentrations ranging from 3.5- 9.5 àg per milligram of electrospun fibers Although both PEG-LMWH and LMWH were incorporated into electrospun fibers, the presence of PEG-LMWH resulted in functional advantages such as improved growth factor binding The increased growth factor binding of the PEG-LMWH/PLGA fibers is most likely due to improvements in retention of the PEG-LMWH in the fibers over LMWH alone PEG-LMWH shows retention in the fiber membranes for up to 14 days, in contrast to LMWH, which is almost completely released after 1 day The ability for a system to slowly release heparin and growth factors would allow for drug delivery over an extended period of time that would more closely match the time scales needed for tissue repair
Along these same lines, Chapter 4 focused on the attachment of proteins to collagen and gelatin electrospun fibers for use in bone regeneration applications Initial work on this project consisted of determining proper solution characteristics and processing parameters to obtain electrospun collagen and gelatin fibers The focus then shifted to optimizing crosslinking and sterilization protocols Once the fiber production, crosslinking, and sterilization were successful, proteins were then attached to the fiber surface EDC/NHS coupling was used to crosslink PlnDI and heparin-BSA proteins to the fibers The presence of these proteins was then confirmed via NeutrAvidin and their growth factor binding ability was determined The results suggested that although heparin-BSA and PlnDI can both be attached to the fiber surface, PlnDI exhibited higher growth factor binding at significantly lower concentrations This research has provided the necessary protocols for preparing electrospun membranes for use as tissue engineering scaffolds for bone regeneration.
Future Work
The research presented in this dissertation has provided an understanding of how solution characteristics and processing parameters affect fiber properties and established the necessary processing protocols required to prepare electrospun fibers for use in biomaterial applications Although a great deal of work has been accomplished, much work is still needed to determine how electrospun fibers function in vivo The PEG-LMWH results require more attention on the controlled release studies As illustrated in Chapter 3, the release of bFGF from the LMWH and PEG-LMWH membranes is below 1% However, it is not clear as to whether the release is actually this low or if the bFGF is denaturing upon release from the electrospun matrix Performing another release assay using radiolabeled bFGF would be useful in addressing this issue The radioactively labeled bFGF would allow for the counting of bFGF regardless of whether it is denatured or not This would indicate if the 1% release seen in the ELISA assays is correct If the radiolabeled bFGF studies reveal that the 1% release is correct, then the release may be able to be increased by tuning the degradation rate of PLGA This could be accomplished by changing the ratio of lactide to glycolide in the PLGA backbone so that, with increased polymer degradation, an increase in bFGF release should be observed This would also allow for the control of growth factor release to meet specific application requirements The addition of other growth factors could also be investigated If other heparin-binding growth factors can be released from these electrospun membranes, then the membranes can be used for a wide variety of biomedical applications The final stage of this project would consist of using animal models to test the ability of the membranes to function as expected within the body
The studies discussed in Chapter 4 illustrate the ability to couple proteins to electrospun collagen and gelatin membranes In these studies, heparin-BSA was used a model system for comparison with PlnDI PlnDI showed higher growth factor binding, although at lower concentrations than heparin-BSA The next step of this research is to complete a cell proliferation assay to determine if the presence of PlnDI