Novel Associated PVA/PVP Hydrogels for Nucleus Pulposus Replacement

A Thesis Submitted to the Faculty of Drexel University by Jonathan D. Thomas in partial fulfillment of the requirements for the degree of Master of Science in Materials Engineering September 2001

ii Acknowledgments This work represents two years of research. During this time, I have been blessed with two advisors: Michele Marcolongo and Anthony Lowman. Their confidence in my abilities and encouragement with my research has helped me grow as a researcher. I would like to also acknowledge and thank Thomas Dziubla, Xinyin Liu, Robert Murray, and Arvind Sivasubramanian for their friendship and the knowledge I gained from many hours of discussing experiments and results. Lastly, I would like to thank my Mother, Father, and Brother for their love and support they have always shown me.

iii Table of Contents List of Tables....................................................................................................................... v List of Figures .................................................................................................................... vi Abstract ............................................................................................................................... x 1 Introduction ................................................................................................................. 1 2 Background ................................................................................................................. 3 2.1 Intervertebral Disc............................................................................................... 3 2.1.1 Structure ...................................................................................................... 3 2.1.2 Mechanics.................................................................................................... 5 2.1.2.1 Disc Kinematics ...................................................................................... 5 2.1.2.2 Static Material Properties ........................................................................ 6 2.1.2.3 Viscoelastic Behavior.............................................................................. 8 2.2 Degenerative Disc Disease................................................................................ 15 2.3 Treatment Options............................................................................................. 19 2.3.1 Conservative Treatments........................................................................... 19 2.3.2 Surgical Procedures................................................................................... 19 2.4 Intervertebral Disc Prostheses........................................................................... 22 2.4.1 Total Disc Replacements........................................................................... 23 2.4.2 Nucleus Pulposus Replacement ................................................................ 28 2.4.3 Tissue Engineering Approach ................................................................... 34 2.5 PVA/PVP Hydrogels......................................................................................... 34 3 Experimental - Materials Synthesis and Testing Methods........................................ 38 3.1 Hydrogel Synthesis ........................................................................................... 38 3.2 Equilibrium Swelling Analysis ......................................................................... 39 3.3 Chemical Analysis............................................................................................. 40 3.4 Network Characterization with Rubber Elasticity Theory................................ 40 3.5 Thermal Analysis .............................................................................................. 42 3.6 Statistical Analysis ............................................................................................ 42 4 Results ....................................................................................................................... 44 4.1 Swelling Characterization ................................................................................. 44 4.2 Dissolution of Hydrogels .................................................................................. 47 4.2.1 Mass Loss Analysis................................................................................... 47 4.2.2 Chemical Analysis..................................................................................... 49 4.3 Mechanical Analysis ......................................................................................... 55 4.3.1 Youngs Modulus...................................................................................... 55 4.3.2 Network Characterization with Rubber Elasticity Theory........................ 58 4.4 Thermal Analysis .............................................................................................. 63 4.5 Effects of M w on Hydrogel Properties............................................................. 68 4.5.1 Equilibrium Swelling Characterization..................................................... 68 4.5.2 Mass Loss Analysis................................................................................... 69 4.5.3 Youngs Modulus...................................................................................... 69 4.5.4 Network Characterization with Rubber Elasticity Theory........................ 70 4.5.5 Thermal Analysis ...................................................................................... 71 5 Discussion ................................................................................................................. 87 5.1 Equilibrium Swelling ........................................................................................ 87

iv 5.2 Dissolution of Hydrogels .................................................................................. 87 5.2.1 Mass Loss Analysis................................................................................... 87 5.2.2 Chemical Analysis..................................................................................... 88 5.3 Mechanical Analysis ......................................................................................... 88 5.3.1 Youngs Modulus...................................................................................... 88 5.3.2 Network Characterization with Rubber Elasticity Theory........................ 89 5.4 Thermal Analysis .............................................................................................. 89 5.5 Effects of M w on Hydrogel Properties.............................................................. 90 5.6 Summary ........................................................................................................... 93 6 Conclusions ............................................................................................................... 95 7 Recommendations for Further Research ................................................................... 97 7.1 Processing of Hydrogels.................................................................................... 97 7.2 Quantitative Analysis of Hydrogen Bonding .................................................... 97 7.3 New Criteria Needed for Nucleus Pulposus Replacements .............................. 98 7.4 Drug Delivery Capabilities of PVA/PVP Hydrogel Blends ............................. 99 List of References............................................................................................................ 100

v List of Tables

Table 1. Projections for discectomy and fusion surgeries in the United States for the year 2000 [32] .............................................................................................. 22 Table 2. PVA/PVP polymer blends that were prepared................................................... 38 Table 3. Molecular weight series ..................................................................................... 68 Table 4. Comparison of the properties of blends prepared with 1% PVP ....................... 92 Table 5. Design chart for PVA/PVP hydrogel blends...................................................... 94

vi
List of Figures

Figure 1. The anatomy of the lumbar spine. Each of the intervertebral discs are spaced between 2 vertebral bodies. The discs are named accordingly [34]. ................................................................................................................. 10 Figure 2. A lateral cross section of a lumbar segment (vertebrae / disc / vertebrae) shows the position of the nucleus pulposus and the annulus layers [34]. ....... 11 Figure 3. An anterior / posterior cross section of an intervertebral disc shows the regions of the annulus fibrosus and nucleus pulposus [13]............................. 12 Figure 4. Axial compression causes hydrostatic pressure within the nucleus to place the annulus layers under tension [15]. ................................................... 13 Figure 5. The position of the nucleus pulposus changes under lateral bending and flexion/extension motions. Note the opposing tensile/compressive forces present in each case [15]....................................................................... 14 Figure 6. The nerve roots exit the spinal cord through the neuroforamina which are openings between the facet joints of 2 adjacent vertebral bodies [34]. ................................................................................................................. 17 Figure 7. The nucleus depicted in this cross section of an intervertebral disc has migrated into the annulus layers posteriorlaterally resulting in an annular bulge and nerve irritation [34]............................................................ 18 Figure 8. (a) Vertebral spinal unit (b) with detail of intervertebral disc. Prosthetic disc design by: (c) Fernstrom, (d) Hedman and Kostuik, (e) Patil, (f) Buttner-Janz, (g) Steffee et al., (h) Lee and Parsons, (i) Froning, (j) Ray [32]. ...................................................................................... 32

Figure 9. Radiographic images of SB Charite disc designed by Buttner-Janz show the position of the polyethylene core between 2 Co-Cr-Mo endplates. Core position can be determined by the radiographically opaque metal ring that surrounds the core [15]. .................................................................... 33 Figure 10. Interchain Hydrogen bonding within a PVA/PVP blend occurs between carbonyl groups on PVP and hydroxyl groups on PVA. .................. 37

Figure 11. The device that was used to cut PVA/PVP films into tensile strips was formed from four Feather Disposable Scalpels that were glued together. A natural rubber cutting surface was used. ..................................... 43

vii Figure 12. Mass swelling coefficient as a function of time of immersion. Blends prepared with higher portions of PVP swelled to a greater extent however blends prepared with excess PVP suffered dissolution and failed to reach a stable equilibrium swelling................................................... 46 Figure 13. Polymer mass loss after 120 days swelling as a function of PVP composition. Polymer mass loss reached a minimum at 0.75% and 1% PVP.................................................................................................................. 48 Figure 14. Typical ATR-FTIR spectra for PVA/PVP blend. The spectra shown was prepared with 95% PVA / 5% PVP. The four noted absorbance peaks are labeled in cm-1. ................................................................................ 51 Figure 15. ATR-FTIR Spectra of PVA/PVP blends (143K PVA / 10K PVP) prior to swelling. Intensity of the free C=O peak increases with PVP%. The hydrogen bonded C=O peak is not detectable above 5% PVP due to its relative intensity to the larger and broader free C=O peak. ................... 52 Figure 16. Change in peak height ratio of blend spectra over 56 days immersion in vitro. PVP not incorporated within the polymer network is free to dissolve into solution. An equilibrium value of free C=O in each gel is reached after 56 days immersion..................................................................... 53 Figure 17. Fractional loss of free carbonyl bonds as a function of PVP composition. The fraction loss was determined by the change in free C=O peak height ratio with ATR-FTIR spectra analysis................................ 54

Figure 18. Youngs Modulus (E) was calculated by determining the slope for the plot of tensile stress as a function of tensile strain.......................................... 56 Figure 19. Youngs Modulus of PVA/PVP polymer blend thin films as a function of PVP composition after various times of immersion in vitro....................... 57 Figure 20. Sample plot showing how tensile modulus (G) was determined. The slope was measured between 0 and 8.3% strain.............................................. 60 Figure 21. Effective molecular weight between crosslinks ( M c ) after 14, 28, and 56 days of immersion for 143K PVA / 10K PVP blend series. ...................... 61 Figure 22. Crosslinking density (X) after 14, 28, and 56 days of immersion for 143K PVA / 10K PVP blend series................................................................. 62 Figure 23. DSC scans of PVA/PVP blends (143K PVA / 10K PVP) showing changes in Tm with PVP composition. ............................................................ 64

viii Figure 24. Typical analysis of melting in a DSC scan with integration performed with a changing baseline using Universal Analysis software provided by TA Instruments. ...................................................................................... 65 Figure 25. Melting points (Tm) of PVA/PVP blends (143K PVA / 10K PVP) prior to immersion as a function of PVP composition............................................. 66 Figure 26. Changes of enthalpy at melting (Hmelt) of PVA/PVP blends (143K PVA / 10K PVP) prior to immersion as a function of PVP composition. .................................................................................................... 67 Figure 27. Mass swelling coefficient (q) for pure PVA hydrogels as a function of immersion time................................................................................................ 72 Figure 28. Mass swelling coefficient (q) for 99% PVA / 1% PVP hydrogel blends as a function of immersion time...................................................................... 73 Figure 29. Mass swelling coefficient (q) for 95% PVA / 5% PVP hydrogel blends as a function of immersion time...................................................................... 74 Figure 30. Mass swelling coefficient (q) for 90% PVA / 10% PVP hydrogel blends as a function of immersion time. ......................................................... 75 Figure 31. Mass swelling coefficient (q) for 75% PVA / 25% PVP hydrogel blends as a function of immersion time. ......................................................... 76 Figure 32. Mass swelling coefficient (q) for 50% PVA / 50% PVP hydrogel blends as a function of immersion time. ......................................................... 77 Figure 33. Polymer mass loss after 120 days swelling as a function of PVP composition. Polymer mass loss was not dependent on PVP M w . ............... 78 Figure 34. Influence of PVP M w on Youngs Modulus for 143,000 M w PVA after 56 days immersion. ................................................................................. 79 Figure 35. Influence of PVP M w on Youngs Modulus for 95,000 M w PVA after 56 days immersion. ................................................................................. 80 Figure 36. Influence of PVA M w on Youngs Modulus for 10,000 M w PVP after 56 days immersion. ................................................................................. 81 Figure 37. Influence of PVA M w on Youngs Modulus for 40,000 M w PVP after 56 days immersion. ................................................................................. 82

ix Figure 38. Effective molecular weight between crosslinks ( M c ) after 56 days of immersion as a function of PVP composition................................................. 83 Figure 39. Crosslinking density (X) after 56 days of immersion as a function of PVP composition............................................................................................. 84 Figure 40. Melting Point (Tm) for PVA/PVP blends as a function of PVP composition. .................................................................................................... 85

Figure 41. Enthalpy change due to melting (Hmelt) for PVA/PVP blends as a function of PVP composition. ......................................................................... 86

x
Abstract Novel Associated PVA/PVP Hydrogels for Nucleus Pulposus Replacement Jonathan D. Thomas Michele Marcolongo Anthony Lowman

Degenerative disc disease in the lumbar spine is marked by a dehydration of the intervertebral disc and loss of biomechanical function of the spinal unit. Since the current surgical procedures are ineffective in restoring natural biomechanical function back to the diseased disc, researchers have looked to replace the intervertebral disc. These designs are flawed in that they either dont restore natural movement back to the spinal unit, require surgeries that are highly invasive, or they further promote disc degeneration of adjacent spinal levels. Recently, researchers have sought to only replace the central portion of the disc called the nucleus pulposus. A potential nucleus

replacement could mimic a healthy nucleus pulposus in restoring healthy biomechanical function to the spinal unit. A hydrogel, poly(vinyl alcohol) (PVA), has been investigated to serve as a nucleus replacement. However, semicrystalline PVA suffers dissolution under physiological conditions. Blends of PVA and poly(vinyl pyrrolidone) (PVP) may provide a material that is a suitable nucleus pulposus replacement. Through interchain hydrogen bonding, they possess greater stability than pure PVA hydrogels. This research is an examination of the stability of these gels under physiological conditions. Polymer dissolution and stability were studied with equilibrium swelling analysis over 120 days immersion, ATR-FTIR analysis over 56 days immersion, and mechanical tensile testing over 56 days immersion. Rubber elasticity theory was used to combine mechanical results with swelling data to

xi calculate network characteristics such as the molecular weight between crosslinks and density of crosslinks. DSC studies after polymer blend preparation were used to illustrate relative degrees of physical crosslinking. Properties were examined as a function of PVA/PVP composition as well as PVA M w and PVP M w . Results indicate that PVA/PVP blends prepared with moderate amounts of PVP (0.5-5%) result in a polymer network stabilized through interchain hydrogen bonding between hydroxyl groups on PVA chains and carbonyl groups on PVP chains. Most notably, a significant decrease in percentage of polymer mass loss was seen for blends prepared with 143K M w PVA. Additions of larger amounts of PVP to the blends resulted in weaker gels that had more open network structures suffering from higher amounts of polymer dissolution. ATR-FTIR results indicate that PVP unincorporated in the network structure through hydrogen bonding suffers significant dissolution out of the polymer network and into solution. M w of PVA and PVP are shown to have a

significant influence on the blends network properties. Lower M w PVA resulted in a more stable blend containing a higher density of crosslinks. However blends prepared with a higher M w PVA showed superior polymer network stability in dissolution studies. Higher PVP M w was shown to have a better stabilizing influence on the higher M w PVA that was tested. The blend that had the best combination of network stability under physiological conditions and a relatively tight, stable, and crosslinked network was prepared with 99% PVA (143K) and 1% PVP (40K).

xii

1
1 Introduction

Lower back pain is the most common and costly disabling musculoskeletal ailment in the United States and is the second leading cause of lost work days next to upper respiratory tract illness [1-3]. Studies have estimated that between 50-90% of the adult population suffers from lower back pain [4, 5]. Treatment and compensation of the ailment have been estimated to cost more than $50 billion per year in the United States [6]. The impact of lower back pain on quality of life has also been considered

extensively. In many cases, pain usually subsides in a matter of weeks or months. However, chronic lower back pain can become a permanent disabling symptom for some patients. While the causes of lower back pain often remain unclear, it is estimated that 75% of the cases are associated with degenerative disc disease [7]. Each of the intervertebral discs, which are located between the spinal vertebrae, are composed of a fluid-like region called the nucleus pulposus surrounded by oriented, concentric layers of tissue collectively called the annulus fibrosus. The process of disc degeneration is marked by a dehydration of the nucleus pulposus. The ability of the nucleus to handle applied loads is decreased with dehydration causing the load supported by the annulus to increase. Further damage can result in the nucleus migrating through and delaminate the annulus layers. Complete penetration through the annulus fibrosus is referred to as a herniation. Pain can originate from degenerative disc material that stimulates the nerves exiting the spine. There are many conservative treatment options for lower back pain. They share the common goal of reducing the pain that stems from inflammation and nerve impingement. Conservative options do not aim to restore biomechanical function to the

2 disc. Surgical options, including discectomy and lumbar fusion, can be performed when lower back pain is not successfully relieved with more conservative treatments. These procedures can offer pain relief but they do not restore natural biomechanical function to the spine. Both of the surgical procedures can result in accelerated degeneration of adjacent discs. The goal of this project is to investigate a PVA/PVP blend as a potential material for a nucleus pulposus substitute. The effects of PVA / PVP composition and M w on the hydrogel structure and stability will be studied in vitro. A device designed with these blends has the potential to return biomechanical function to the degenerated spinal unit and relive pain for the patient. Under compression, the hydrogel nucleus substitute is designed to expand radially, placing the annulus layers under tension, similar to the way a healthy nucleus mechanically functions. These hydrogel blends are stable within the physiological environment due to physical crosslinks consisting of intermolecular hydrogen bonds between PVA and PVP and intramolecular hydrogen bonds within PVA crystals. Both PVA and PVP have proven biocompatibility. A hydrogel design allows for the arthroscopic insertion of the dehydrated nucleus substitute and subsequent rehydration, once in the body.

3
2 2.1 Background Intervertebral Disc

2.1.1 Structure

The human spine consists of vertebral bodies separated by intervertebral discs. Figure 1 shows the lumbar section of the spine. Discs are labeled with respect to the two vertebral levels which they are located between. For example, the L4/L5 disc is located between the 4th and 5th lumbar vertebrae. S1 represents the sacrum, which is the vertebral body located most inferior along the spinal column. The sacrum is located below the lumbar section of the spine. The discs allow motion of the vertebral bodies and distribute applied loads over the total area of the vertebral bodys cartilaginous endplates. Intervertebral discs have a fluid-like region called the nucleus pulposus that is surrounded by the slightly more fibrous annulus fibrosus as shown in Figure 2. The nucleus, in a young healthy spine, is a gelatinous material with a consistency similar to toothpaste [8]. A healthy nucleus contains between 70-90% water by weight which is responsible for its fluid-like properties [8, 9]. Proteoglycans make up for 65% of the dry mass of the nucleus [8]. These macromolecules consist of sulfated glycosaminoglycan side-chains covalently bonded to core proteins. Proteoglycan units can link together and form larger aggregate molecules. These molecules have the ability to attract and retain water due to ionic carbonyl and sulphate groups on the glycosaminoglycan chains [8, 10, 11]. Collagen, which constitutes 5-20% of the dry mass of the nucleus, holds the proteoglycan aggregates together [8, 9]. The remaining dry portion of the nucleus consists of elastic fibers and non-collagenous proteins.

4 The annulus fibrosus is an arrangement of concentric layers of oriented fibrous tissue. Depending on location within the disc, the fibers are connected to the vertebral endplates or directly to the vertebral body. Water accounts for 60-70% of the mass of the annulus [8]. Within each annular layer, fibers lie parallel to each other at an orientation that is 60-70 with respect to the spine axis (20-30 with respect to radial direction) as shown in Figure 3 [9, 10, 12]. Fibers in adjacent layers in the annulus are oriented in alternating directions, e.g. (...+65/-65/+65/-65...) [13]. Collagen accounts for 50-60% of the dry mass of the annulus [8]. Proteoglycans, contributing about 20% to the dry mass in the annulus, are located between collagen fibers in adjacent annular layers and serve to bind them together [8]. Other components of the annulus, elastin fibers,

contribute about 10% to the dry mass of the annulus. They are concentrated towards the superior and inferior portions of the annulus that contact the vertebral end plates [8, 9]. While the nucleus pulposus and annulus fibrosus appear to be very similar chemically, differences are found in their structural organization. Collagen in the annulus is mostly type I while the majority of collagen found in the nucleus is type II. Additionally, the microstructure of the annulus and nucleus is dependent on location within the disc. For example, the annulus layers adjacent to the nucleus and near the edge of the disc exhibit different tensile properties. Also, the water content of the nucleus varies as a function of distance from the center of the disc. The vertebral endplates serve to separate the nucleus and annulus from the vertebral bodies. The cartilage found on these endplates resembles the chemical structure of the adjacent portion of disc [8]. The center portions of the endplates have a higher water and proteoglycan content but less collagen content than the outer regions of the

5 endplates. The permeability of the central portion of the endplates plays a major role in the diffusional transport of water and nutrients to the nucleus pulposus [14]. Water and nutrients can reach the nucleus through the annulus as well but to a much lesser extent than the endplate route [14].
2.1.2 Mechanics 2.1.2.1 Disc Kinematics

In a functional spinal unit, the nucleus, annulus, and vertebral bodies work together to withstand the different load conditions that are placed on the spine. The basic modes of movement of the spine are axial compression, flexion / extension, lateral bending, and torsion. When the spinal unit is axially compressed as shown in Figure 4, hydrostatic stresses cause the nucleus to expand in the radial direction, increasing the tensile stress in the annular fibers. The tensile stresses in the annulus are greatest adjacent to the nucleus and decrease gradually away from the nucleus. Axial

compression causes discs to bulge in the radial direction. Stress states within the disc are more complex for bending or twisting movements. The annulus is subjected to

compressive stresses in the direction of the applied stress and tensile stresses in the opposite direction. The nucleus will actually move to the direction of the tensile forces (extension) in order to distribute the load across the disc as shown in Figure 5 [15]. Annular bulge preferentially occurs in the region of the disc that is under compressive stresses [15]. Torsion affects each region of the annulus fibrosus differently. Anterior and lateral regions undergo tensile stresses in annular layers in which the fibers are in the direction of the axial torque while layers in the opposite direction are subjected to no stress [15]. Annular layers in the posterior spine undergo tensile stresses when the disc is

6 subjected to torsion regardless of fiber orientation [12, 15]. Loading of the spine is often a coupling of different movements.
2.1.2.2 Static Material Properties

Biomechanical research has focused on determining the material properties of the intervertebral disc. Research has also concentrated on quantifying the stresses the disc is subject to in vivo and ex vivo. The wide range of reported values for disc properties and stress states are due in part to variations in analytical techniques and variations within disc structure that are dependent on location within the disc [16-18]. Furthermore, disc properties are dependent on patient age, disc location within the spine, and the degree of disc degeneration [12, 15, 19, 20]. Variations in tensile modulus of the annulus have been reported to be as low as 0.2 MPa and as high as 645 MPa [18, 21]. Ebara et al. [18] found values ranging from 5 MPa to 50 MPa in their study observing location dependent variations in tensile properties when they tested single annular layers from different radial and circumferential positions within the disc. Similarly, they saw variations in failure stress and strain with sample position. Keller et al. [16] found a similar pattern of dispersity in ultimate compressive strength and compressive modulus when they tested the properties of vertebral endplates. Umehara, et al. [22] used an indentation technique to find

compressive moduli for regions of the annulus and nucleus within the intervertebral disc. The indentation results correlated to modulus values that ranged from 5 kPa to 210 kPa [22]. However, indentation tests on the fibrous annular layers and fluid-like nucleus are ineffective ways to determine modulus values. Variations in modulus seen with their indentation test are more likely due to variations in the surfaces of the nucleus and the

7 annulus fibers. Indentation testing of fibrous tissue can leads to results that are errorprone and cannot be correlated to a compressive modulus. Previous researchers [12, 19, 23] have made attempts to characterize the mechanical properties of the complete disc. The compressive strength of lumbar

intervertebral discs has been measured to be from 5,000 N to 8,000 N, however the strength is a function of patient weight, disc position, and level of degeneration [12, 19]. One has to cautiously use these data because they are also dependent on a discs crosssectional area which varies with disc position and among a population of patients. Using these values to determine the stress state within the nucleus is not advised because the nucleus has been reported to take up anywhere between 30-60% of the cross-sectional area of the disc [24]. Researchers have also measured a value called the stiffness

coefficient to characterize discs. It is defined as the load per instantaneous displacement of the disc [23]. Stiffness coefficient values are subject to variation with disc geometry and are not a complete representation of the disc material. Intradiscal pressure is the measure of the hydrostatic pressure that is present in the nucleus. Under a simple movement like compression of the intervertebral disc, a

hydrostatic pressure is created in the nucleus resulting in tension in the annulus layers. It is an important measurement of the state of stress within a disc. Nachemson [25] was the first to measure the intradiscal pressure within an intervertebral disc. Pressures were measured with a transducer design of a needle equipped with a miniature pressure gauge at its tip. He found that most discs have an internal hydrostatic pressure that is 1.3-1.5 times axial stress applied to the spinal unit [25]. When he used this pressure transducer
in vivo, Nachemson was able to determine the dependence that posture had on intradiscal

8 pressure [26]. Normal intranuclear hydrostatic pressure for a healthy L5/S1 disc has been measured to be about 0.5 MPa [9, 27]. With physical exertion, the pressure was shown to almost triple [9, 27]. The pressure within the disc is estimated to be 50% higher for degenerated discs [28]. In biomechanical mathematical models, it has been assumed that the nucleus is an incompressible, inviscid fluid that has a bulk modulus value of 2,200 MPa [29, 30]. However some have assumed that the nucleus is a poroelastic solid with a Youngs modulus between 4.5-1,500 kPa and a Poissons ratio between 0.1 and 0.45 [21, 30]. Models based on inaccurate material property assumptions will only yield unreliable results. Proper determination of properties of the materials within a function spinal unit is important because it gives mathematical models their validity. Tadano et al. made the observation that stress and strain states in the intervertebral disc calculated with a mathematical model like finite element analysis are highly dependent on the chosen properties of the materials [31].
2.1.2.3 Viscoelastic Behavior

There has long been an uncertainty in whether the nucleus is a solid or a fluid. This debate stems from the viscoelastic nature of the disc. The nucleus of the disc acts as a compressible fluid having a 2-4 hour time constant [32]. This time period is used for the transport of fluid into and out of the disc [33]. Viscoelasticity allows the spinal unit to be highly flexible under low loads and more rigid under higher loads [15]. It has been suggested that collagen fibers take up an instantaneous load but if a load is applied over a period of time, fluid transport will change the content of the nucleus [15]. The time

9 dependent creep properties of the disc are crucial to withstanding prolonged loads. Under slow creep, the disc will eventually reach a final deformation under constant load.

10

Figure 1. The anatomy of the lumbar spine. Each of the intervertebral discs are spaced between 2 vertebral bodies. The discs are named accordingly [34].

11

Figure 2. A lateral cross section of a lumbar segment (vertebrae / disc / vertebrae) shows the position of the nucleus pulposus and the annulus layers [34].

12

Figure 3. An anterior / posterior cross section of an intervertebral disc shows the regions of the annulus fibrosus and nucleus pulposus [13].

13

Figure 4. Axial compression causes hydrostatic pressure within the nucleus to place the annulus layers under tension [15].

14

Figure 5. The position of the nucleus pulposus changes under lateral bending and flexion/extension motions. Note the opposing tensile/compressive forces present in each case [15].

15
2.2 Degenerative Disc Disease

The efficient transfer of stress from the nucleus to the annulus begins to change around the age of 30 with a change in the concentration and types of proteoglycans in the disc as well as a loss in the discs overall water content [11, 20, 35-37]. The normal aging process causes the water content in the nucleus to fall below 70% [13]. The distinction between the nucleus and annulus becomes unclear, most likely as a result of the increase in collagen content and decreases in the number of proteoglycan side chains and proteoglycan aggregates within the nucleus [13, 27]. The vertebral endplates become sclerotic and loose their permeability reducing water and nutrient transport to and from the disc. Metabolic products like lactic acid have no route to leave the disc leading to a decrease in pH [20, 35]. Frymoyer and Moskowitz [20] put forth that loss of the

proteoglycan matrix in degenerative disc disease stems from cell death due to lower pH levels within the disc. Nucleus dehydration results in a decrease in the load carried by the nucleus and an increase in load on the annulus [35]. In a degenerated spinal unit, stresses are transferred to the outer regions of the vertebral endplates and intervertebral disc [15]. Tears, cracks, and fissures can occur within the annular layers allowing the migration of the nucleus through the annular layers and causing pain by stimulating the sinu-vertebral nerve [15, 35]. The spinal canal and spinal cord are located posterior to the intervertebral disc. Nerves exit the spinal canal through openings called neuroforamina and travel to the extremities as shown in Figure 6. Nuclear migration can also lead to disc bulge reducing the height of the disc. Loss of disc height is a significant problem because it leads to a narrowing of the nerve root openings and potential buckling in the surrounding ligaments

16 [13, 20, 35, 38]. Pain also originates from the increased motion stemming from laxity that accompanies a reduction in disc height. With continued degeneration, a portion of the nucleus can penetrate through the annular layer in what is referred to as a disc herniation. This nuclear material or the bulge created by the nuclear material can impinge on sensitive nerves surrounding the disc as shown in Figure 7 and cause pain in the back or lower extremities. Furthermore, the herniated material elicits an inflammatory response because the nucleus of a healthy intervertebral disc is avascular [35]. The nuclei of intervertebral discs are the largest avascular structures within the body. This means they receive nutrients solely from diffusion transport mainly through the surrounding vertebral endplates rather than through vascularized tissue [32]. The body attacks the herniated material because it has become part of the vascular system. The reduction in disc volume leads to instability and as a result other parts of the spine including the bones, ligaments and endplates thicken and grow to compensate for this instability [35]. The reduction in space surrounding the nerves is referred to as spinal stenosis [35]. Inflammations can also occur at the facet joints, which are located posterior to the discs, because of wear generated by increased compressive stresses [35]. Approximately 95% of nuclear protrusions occur either at the L4/L5 and L5/S1 levels due to the high stress states found in these discs [20, 39]. Water loss is also highest in these two levels [11]. Degeneration has been described as a cascade process in which vertebral segments adjacent to the diseased disc are placed under increased stresses and as a result undergo an accelerated degenerative process [15, 35].

17

Figure 6. The nerve roots exit the spinal cord through the neuroforamina which are openings between the facet joints of 2 adjacent vertebral bodies [34].

18

Figure 7. The nucleus depicted in this cross section of an intervertebral disc has migrated into the annulus layers posteriorlaterally resulting in an annular bulge and nerve irritation [34].

19
2.3 Treatment Options

2.3.1 Conservative Treatments

Pain can occur at any point during the degeneration process.

A commonly

recommended initial treatment for lower back pain is bed rest. Bed rest is used to treat lower back pain because the prone position naturally reduces intradiscal pressure. Also, prolonged activity can cause inflammation to worsen. However, in acute conditions, bed rest resulted in no significant pain relief when compared to a control group having no bed rest [15]. Furthermore, prolonged bed rest can lead to muscle atrophy and cardiovascular deconditioning. Analgesics, anti-inflammatory drugs, muscle relaxants, sedatives, and

neurotrophic medications have been used to ease lower back pain. These drugs are therapeutic in that they reduce inflammation and relieve pain, but they do not address the deterioration of disc function. Other conservative treatments that have been used include electrotherapy, magnetic fields, ultrasound, chiropractic manipulation, acupuncture, and traction. However, the effectiveness of these treatments is still in question. Conservative treatments cannot reverse the degenerative process within the disc. Their aim is to reduce inflammation stemming from disc material pressing on the nearby nerves. Conservative treatment options do not sufficiently provide pain relief for 33% of lower back pain sufferers [15]. The only remaining option for these patients suffering from debilitating chronic lower back pain is surgery.
2.3.2 Surgical Procedures

Surgery is considered when conservative options fail to relieve pain or when a risk of nerve damage exists. Decompression of the intervertebral disc can involve cutting

20 away disc, bone, and ligament material that is applying pressure to spinal nerves. The traditional discectomy surgery involves removing herniated disc material posteriorly. This surgery may require removal of a part of the annulus and surrounding ligaments [5, 15, 35]. Endoscopic discectomy procedures, which became popular in the 1980s, involve the use of X-Ray fluoroscopy imaging to remove herniated disc material through posteriorlaterally placed cannulas [40, 41]. These minimally invasive procedures can be performed on an outpatient basis without general anesthesia [41]. Operation through a small cannula (4.5-6.0 mm) lessens the damage to the annulus, bone, and ligaments during surgery [35]. Discectomy has been very successful in reducing pain attributed to disc herniation because it involves removal of the disc material that is causing nerve root compression [15, 35, 40]. However, pain relief in discectomy does not come from any biomechanical improvements to the spine. In fact, it has been found that discectomy leads to a high occurrence of disc height reduction [42]. Success rates have been found to be very unpredictable. Hanley et al. [42] found that 14% of patients experienced disabling lower back pain after discectomy surgery. Lee et al. [43] found that as many as 50% of patients have continued lower back pain after disc excision. The offending portion of the disc is removed but biomechanical instability is introduced as well [44]. Discectomy does not restore function to the vertebral joint. The degeneration process continues, often making further surgery necessary. Lumbar interbody fusion is a procedure that is performed for sufferers of chronic lower back pain stemming from an advanced instability of the intervertebral disc joint. This highly invasive surgery involves complete removal of the painful disc and replacement with a bone graft. Posterior fusion is another method used in fusing a spinal

21 unit. It involves the fixation of the posterior facets and lamina which are located

posterior to the disc while leaving the degenerated disc in place. Synthetic internal fixation devices are sometimes used to stabilize the spinal level. Pain relief as a result of lumbar fusion comes from the stabilization achieved through elimination of motion. Studies show a wide range in success rates of spinal fusion, from 32% to 98% [35]. This is in part due to a poorly defined definition of success after back surgery. Spinal fusion often leads to newer problems such as bone graft donor site pain, pseudoarthosis, and spinal stenosis [45]. Fusing two vertebral bodies alters the normal biomechanical properties of the spinal unit by increasing motion and stresses on the adjacent intervertebral joints [6, 35, 43]. The degeneration rate may be accelerated in adjacent discs due to these increased stresses, creating more instability and pain [46]. Highly rigid internal fixation devices have been suspected of causing disuse osteoporosis and stress shielding, which results in muscle atrophy and decrease in bone density [47, 48]. Ray [32] made projections for the number and cost of discectomy and fusion surgeries for the year 2000 by combining census data with statistics from the National Center for Health Statistics. These projections are found in Table 1.

22
Table 1. Projections for discectomy and fusion surgeries in the United States for the year 2000 [32] Surgery Discectomy Fusion

Number % of Population Cost / Surgery Failures Total Cost

254,000 0.094 % $40,000 20,000 (8%) $8.8 billion

65,000 0.024 % $80,000 9,800 (15%) $6 billion

These data suggest that 1 out of 1064 members of the general U.S. population had discectomy surgery while 1 out of 4167 underwent fusion surgery during the year 2000. The total cost associated with each surgery includes the cost of procedure and post operative care. The cost to repeat a failed procedure was included as well. Rays cost projections do not include work compensation and other procedures. Bao and Yuan [6] state that 700,000 spine procedures are performed in the United States each year. Based on these figures the total cost caused by back pain and treatment is approximately $50 billion per year.
2.4 Intervertebral Disc Prostheses

Problems with the currently available surgical techniques have led researchers to investigate the idea of a prosthetic intervertebral disc. Other joints susceptible to

degeneration, like the hip, knee, and knuckle, can be successfully replaced with prostheses [43]. Artificial joint designs replaced the popular fusion procedures that were once commonly performed for diseased knee and hip joints.

23 For any prosthetic intervertebral disc design, there are general requirements. The design must involve biocompatible materials that will not cause local tissue reactions, carcinogenesis, or organ toxicity [43, 48]. The wear resistance of the materials used in the design need to be taken into consideration because wear particles can lead to a foreign body response [48]. The fatigue properties of the material need to be sufficient to withstand the number of loading cycles to which the intervertebral disc is subjected. The mechanical properties of a prosthetic intervertebral disc, including stiffness, yield and fatigue strength, and viscoelastic properties, should match or exceed that of a normal healthy disc [43]. Kinematically, the joint should be stable, that is, not susceptible to allowing movement that exceeds that allowed by a healthy functional spinal unit [2, 43, 48]. Finally, the prosthetic disc design should allow for a safe surgical implantation.
2.4.1 Total Disc Replacements

Total disc replacement has been considered for discs that have undergone extensive degeneration. The prosthesis would serve as a preferable treatment to fusion because it allows for biomechanical movement between two adjacent vertebral bodies. The benefit of replacing both the nucleus and annulus is that the effectiveness of surgery is not dependent on the integrity of the annulus or degree of degeneration [6]. However, total intervertebral discs replacements, which are mostly multicomponent designs, are susceptible to problems such as interfacial bonding and wear [6]. Total intervertebral disc implants must also have adequate fixation to the vertebral endplates. For comparative purposes, Figure 8a shows an image of a healthy functional intervertebral disc between two vertebral bodies. Figure 8b shows the position of a native intervertebral disc between outlines of the vertebral bodies. One of the earliest

24 prosthetic disc designs, shown in Figure 8c, was developed by Fernstrom [49]. His design involved inserting a stainless steel ball bearing into the interbody space in human patients. X-rays of the Fernstrom disc taken 4-7 years after the implantation showed disc space narrowing in 74% of the patients due to the migration of the balls into the vertebral bodies [49]. Knowles developed an all-metal disc spacer to be placed posteriorly to act as a wedge, taking up the compressive load on the posterior elements [50]. Spacer devices, such as these, do not restore any flexibility to the vertebral unit. The advantage of using an all-metal intervertebral device is the fatigue strength of metals. Hedman and Kostuik [2, 48] claim that an effective design would have to be able to withstands 100 million cycles, equivalent to 40 years of use. This goal for the fatigue life of the implant is appropriate for disc replacements because the average patient suffering lower back pain is around 40 years old, much younger than the average age of patients requiring hip and knee implants [6]. Hedman and Kostuik [2, 48] have designed an all-metal disc, shown in Figure 8d, that is composed of two Ti-6Al-4V (Ti alloy with 6wt% Al and 4wt% V) springs placed between either hot isotactically pressed or forged CoCrMo endplates with CoCr beads sintered to the endplates to ensure bony ingrowth fixation [2, 48]. The endplates are also connected with a hinge on the posterior side of the device. This device was fatigue tested up to 100 million cycles in vitro which is 6.5 times greater than the longest wear and fatigue tests performed on orthopedic implants [48]. Studies by Schmiedberg et al. [51] showed that the device generates wear particles from both the spring/endplate interface and hinge/pin interfaces. These particles can elicit a cellular response resulting in inflammation or bone resorbtion [51]. Patil designed a disc system, shown in Figure 8e, composed of cup-shaped metal endplates connected

25 by multiple stainless steel springs [52]. The design was to direct stresses away from the intervertebral disc with cup-shaped endplates. Lee et al. [43] suggested that this design may present many problems such as wear debris and fatigue failure of the springs. Bao and Yuan report that short term testing of an artificial disc made with two Ti-6Al-4V springs in a sheep model showed no tissue ingrowth into the springs or hinges [6]. Lee et al. [43] state that spring system designs such as those proposed by Kostuik and Patil grossly oversimplify spinal motion and are subject to tissue interpenetration that can potentially disrupt motion. The SB Charite disc, developed by German orthopedic surgeons, Buttner-Janz and Schellnack in conjunction with Link has been the design that has undergone the most clinical trials [53-55]. The latest generation of this disc is shown in Figure 8f. Radiographic images of this device are shown in Figure 9. The multicomponent device consists of an ultra-high molecular weight polyethylene (UHMWPE) sliding core positioned between two Co-Cr-Mo endplates. The device does not attempt to mimic the natural discs mechanical stiffness. Kostuik [48] claims that the SB Charite disc design

is unconstrained with a center of rotation that is too anterior and projects that the polyethylene core will undergo cold flow in about 4 years. Lee et al. [43] suggests that dislocation in vivo is probable due to the unconstrained nature of the sliding polyethylene core of the SB Charite disc. Early clinical tests on the first SB Charite discs showed high occurrence of problems such as core or plate dislodgement and core fracturing [32]. The tests were halted and the device was redesigned. Over 2000 SB Charite prosthesis have been implanted in patients since 1984 [6]. Griffith et al. [56] performed the most extensive clinical trial on the third generation SB Charite disc and found significant

26 decreases in patient back and leg pain following implantation of the disc on an analog scale. European clinical trials over 2 to 5 years show satisfactory results in 63% of the patients with the prosthesis [57]. In one clinical study, problems with the implant seemed to be centered around the endplate/implant interface [58]. David [58] claims that the SB Charite disc does not match the anatomical nature of the vertebral endplates. It appears that no studies have been performed to characterize the wear that potentially can occur between the surfaces of the metal endplates and polyethylene core. However,

polyethylene wear particles generated in UHMWPE artificial hips have been shown to cause inflammation and osteolysis [59]. Researchers began to evaluate soft polymeric materials due to their mechanical similarity to the natural disc material. It is generally believed that better tissue tolerance will exist with implant materials that are closer in mechanical properties to the natural disc [43]. A design by Stubstad and Urbaniak [60, 61] consisted of three layers, a silicone core sandwiched between two layers of Dacron mesh embedded silicone. The purpose of the Dacron covering on the superior and inferior caps was to promote tissue ingrowth and implant fixation [60, 61]. The disc was tested in the chimpanzee model, where infection and resorbtion of the adjacent bone was reported [61]. Furthermore, dislodgement of the prosthesis occurred if there was not an exact fit [61]. Edeland [62] proposed a silicon design that was capped with polyethylene base plates. Another design by Edeland [63] involved a silicone nucleus design constrained by silicone or polyethylene annulus replacements on the bases and outer circumference. The Acroflex disc, developed by Steffee [64, 65] in collaboration with Acromed Corporation, has been implanted in humans in the United States. The implant, shown in

27 Figure 8g, is composed of a hexane-based rubber bonded to two Ti-6Al-4V endplates that are sintered with Ti beads [64, 65]. Out of 6 patients who underwent replacement surgery with the Acroflex disc, 4 had satisfactory results [66, 67]. The two discs that failed suffered fractures of the rubber core [66, 67]. The Acroflex disc was the first intervertebral disc prosthesis approved by the FDA to undergo clinical trials under the Investigational Device Exemption [35]. However, in 1990, 2-mercaptobenzothiazole, a chemical used in the vulcanization process of the rubber core, was found to be carcinogenic in rats and clinical use was stopped [66, 67]. Another polymeric disc, designed by Lee and Parsons [43, 45, 68, 69], is shown in Figure 8h. They designed a device that would replace all of the disc except for the outermost annular layers. Their composite design was composed of a polysiloxane

modified styrene-ethylene/butylenes copolymer (C-Flex) core to mimic the softness of the nucleus [68, 70, 71]. They surrounded the core with a stiffer polyurethane reinforced with Dacron fiber lamina to mimic the annulus [68, 70, 71]. They used stiffer

polyurethane endplates coated with a woven jacket to frame the core [68, 70, 71]. The jacket has also been coated with hydroxyapatite to promote bone ingrowth from the vertebral endplates [68, 70, 71]. Bao and Huan state that a lack of fixation between the implant and the vertebral bodies is what has prevented the Lee device from being tested clinically [6]. Ambrosio et al. / investigated the potential (PHEMA/PCL) use of poly(2with

hydroxyethylmethacrylate)

polycaprolactone

embedded

poly(ethylene therephthalate) (PET) fibers [72].

When studied under compression,

28 increases in PCL and PET content increased the implants mechanical strength and stiffness, which they found to be close to that of a canine intervertebral disc [72].
2.4.2 Nucleus Pulposus Replacement

Researchers began to realize the potential benefits of only replacing the dehydrated nucleus. The approach entails a less invasive posterior surgery as well as a return of the annulus to its healthy natural tension [5]. This approach would not be suitable for cases in which the disc is in the later stages of degeneration. Because these devices are not intended to be fixed to the vertebrae, no problems with endplate fixation exist for a nucleus replacement. Also, the surgical time required for a nucleus

replacement procedure should approach the time required for a discectomy in contrast to the longer time required for more invasive complete disc replacements using the prostheses described previously [6]. Garcia showed that a polyurethane elastomer can be polymerized in situ within a nucleus cavity at physiological temperatures rather than at the traditional processing temperature of 100C [73]. Since polymerization is an exothermic process, localized heating of surrounding tissue needs to be considered. In addition, injection pressure needs to be closely monitored to make sure the nuclear cavity is filled and that the monomer and oligomer solution doesnt herniate through the annulus prior to curing. Schulmann [74] saw this problem with his work with in situ polyurethane polymerization. Hou [75] tested a silicone rubber as a nucleus replacement in vitro and in monkeys. The results from the monkey model showed no adverse reactions from

surrounding tissue [75]. Froning worked on designs involving fluid filled bladders that

29 more closely mimicked the nucleuss fluid properties [76]. One of his designs is shown in Figure 8i. However, fluid filled bladders pose the problem of critical rupture, as is often seen with Silicone and Saline breast implants, and therefore would not be suitable nucleus prostheses. In the late 1980s, Ray and Corbin [32, 77-79] designed cylindrical implants to be inserted after removal of the nucleus. The side by side implants are shown in Figure 8j. They consisted of fiber-weaved shells of biodegradable poly(glycolic acid) (PGA) that is filled with a thixotropic gel such as hyaluronic acid once it is positioned during surgery [32, 77-79]. With swelling, the shell degrades as tissue begins to penetrate and grow. The implants have been investigated for controlled release of therapeutic agents such as anti-inflammatory drugs [79]. It was believed that difficulty with sealing the fluid into the capsules caused them to abandon this design [6]. In the mid 1990s, Ray et al. [80, 81] embarked on another design that involved side by side implants positioned in the mediallateral position rather than anterior-posterior like the earlier design. A polyacrylonitrile (PAN) hydrogel surrounded by flexible but inelastic extended polyethylene fiber was proposed [80, 81]. The size of the implant requires a hole to be cut in the annulus larger than incisions commonly used for discectomy [6]. It has been reported that out of 101 patients who received the implant, 17 suffered implant extrusion [6]. They reported that for the patients that did not suffer implant extrusion, pain relief was common [6]. One potential problem with implants consisting of multiple cylindrical components is that they are free to move around within the area left by the removal of the native nucleus until tissue ingrowth occurs. Improper positioning of these two swollen cylinders can change the biomechanical behavior. Their shape characteristics do not ensure the transfer

30 of compressive stresses into tensile stresses in the annulus fibrosus, as it occurs in a healthy intervertebral disc. Bao and Higham developed a poly(vinyl alcohol) (PVA) nucleus pulposus replacement that restores function to the intervertebral disc by mimicking both the mechanical and physiological properties of the disc [6, 35, 82-87]. The hydrogel

material, containing 70% water, acts similarly to the nucleus in that it absorbs and releases water depending on the applied load [6, 35, 83]. Bao [87] has proposed methods for dehydrating the hydrogel nucleus substitutes to facilitate a less invasive insertion into the spine. Bao and Higham have proposed forming a nucleus replacement from two cylindrical segments similar to Rays design to ease insertion and reduce the size of the hole needed in the annulus [86]. A baboon test model of the PVA nucleus showed no adverse local or systematic tissue reaction [6]. They report the PVA nucleus substitute to have a compressive modulus greater than 4 MPa and a compressive strength greater than 1 MPa [83]. Prosthetic implants using PVA should not be considered stable within the physiological environment due to the fact that PVA is a semicrystalline, hydrophilic polymer that can undergo dissolution. The dissolution process involves an unfolding of PVA crystal chains that join the amorphous region of the polymer, disentangle, and eventually dissolve [88, 89]. Polymer chain dissolution results in a network with a decreased mechanical stiffness resulting from a larger network mesh size. Larger

crystals, which undergo a slower dissolution process, are found in semicrystalline PVA hydrogels that have higher PVA molecular weights [88]. Felt et al. [90, 91] have investigating a disc design involving the injection of curing polyureathane in situ in conjunction with a balloon catheter and delivery balloon.

31 The design was tested with a human cadaver model and showed that the device is able to restore disc height and disc modulus [6]. Bao and Yuan have also proposed the use of an aperture sealing device in conjunction with an implantable PVA nucleus replacement [92].

32

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

(j)

Figure 8. (a) Vertebral spinal unit (b) with detail of intervertebral disc. Prosthetic disc design by: (c) Fernstrom, (d) Hedman and Kostuik, (e) Patil, (f) Buttner-Janz, (g) Steffee et al., (h) Lee and Parsons, (i) Froning, (j) Ray [32].

33

Figure 9. Radiographic images of SB Charite disc designed by Buttner-Janz show the position of the polyethylene core between 2 Co-Cr-Mo endplates. Core position can be determined by the radiographically opaque metal ring that surrounds the core [15].

34
2.4.3 Tissue Engineering Approach

Stone [93] has attempted to regenerate the intervertebral disc by constructing a scaffold of biocompatible and bioresorbable glycosaminoglycan fibers. Cells penetrate and grow into the scaffold while the scaffold dissolves within the body. A tissue

engineering approach to repairing the intervertebral disc has also been developed by Gan and Ducheyne [94, 95]. They implanted nucleus pulposus cells onto PLGA (polylactideco-glycolide) and bioactive glass substrates [94]. Their results suggest that both the PLGA and bioactive glass substrates had cell adhesion and proliferation. The bioactive glass with a calcium phosphate rich layer was found to induce cellular activity much better than the PLGA [94]. Contrary to their promising results, the avascular

characteristics of the nucleus region of the intervertebral disc make the tissue-engineering approach impractical. Cell migration, adhesion and growth onto a scaffold are not likely to occur in a nuclear cavity that is devoid of vascularized tissue.
2.5 PVA/PVP Hydrogels

PVA is a polymer that has been studied extensively for potential biomedical applications. The swelling, chemical, and mechanical properties of PVA were

determined in the late 1970s by Peppas and Merrill [96, 97]. Oka et al. [98] found no inflammatory or degenerative changes in the articular cartilage or synovial membrane surrounding their artificial PVA cartilage after 8-52 weeks. PVA hydrogels can be produced from solution via repeated freezing and thawing cycles that increase the order of the crystals, changing the dissolution properties, mesh size, and diffusion properties of the polymer [99-101]. Mondino et al. [102] recently showed that gamma irradiation treatments increase the tensile strength of PVA hydrogels. The ability of PVA to release

35 therapeutic drugs from its polymer network has been studied extensively [89, 103-105]. PVA has recently been investigated as a potential keratoprostheses [106] and as a promising material for a bioartificial pancreas design [107]. Stammen et al. [108] has proposed using a freeze-thawed PVA hydrogel in a number of applications including cartilage replacement and spine disc replacement. They characterized the mechanical properties of these hydrogels crosslinked by the freeze-thaw process. They saw an increase in tangent compressive modulus between 1-18 MPa from 10-60% strain [108]. They also found shear tangent modulus in the range of 0.1-0.4

MPa, depending on strain magnitude [108]. PVP is a hydrogel that has been used for a number of biomedical applications. It was used as a colloidal plasma substitute in World War II [109] and has been used in soft contact lenses [59]. It has recently been investigated by Kao et al. for applications such as single-layer hydrogel wound dressings and tissue adhesives [110]. Risbud and Bhat [111] evaluated the biocompatibility of PVP / -chitosan hydrogel membranes. They found the membranes to be biocompatible. Risbud and Bhat [111] also found that the additions of PVP gave the hydrogel polymer network increased strength due to the viscoelastic properties of polymer [111]. Characterization of PVA/PVP copolymer blends has shown that a higher PVP molecular weight within the blend will lead to more interactions between PVA and PVP and higher blend crystallinity [112]. The single glass transition temperature (Tg) value observed for PVA/PVP blends verifies the blends miscibility [113]. Interactions

between PVA and PVP occur through interchain hydrogen bonding between the carbonyl group of PVP and the hydroxyl group on PVA as shown in Figure 10. These interactions

36 have been studied with FTIR and NMR [114, 115]. Hydrogen bonding interactions involving carbonyl and hydroxyl groups have also been seen in ethyl isobutyrate (EIB) / 4-ethylphenol (EPh) mixtures, poly(4-vinyl phenol) (PVPh) / poly(methyl methacryalate) PMMA blends, and poly(4-vinyl phenol) (PVPh) / poly(vinyl pyrrolidone) PVP blends [116-119].

37

O H O N N O

O H

O H O

O H

O H

PVA
Interchain Hydrogen Bonding

N N O

O N

PVP
O

Figure 10. Interchain Hydrogen bonding within a PVA/PVP blend occurs between carbonyl groups on PVP and hydroxyl groups on PVA.

38
3 3.1 Experimental - Materials Synthesis and Testing Methods Hydrogel Synthesis

Polymer blends were prepared with poly(vinyl alcohol) (PVA) and poly(vinyl pyrrolidone) (PVP). PVA (99.0-99.8% hydrolyzed) was obtained from Dupont

(Wilmington, DE) in two grades: 71-30 ( M n =48,400-52,800, M w =138,400-146,500) and 90-50 ( M n =74,800-79,200, M w =89,500-97,770). PVP was obtained from Aldrich (Milwaukee, WI) ( M w =10,000) and Sigma (St. Louis, MO) ( M w =40,000). molecular weights and ratios of the polymers used in the blends are shown in Table 2.
Table 2. PVA/PVP polymer blends that were prepared PVP / PVA (Powder Mass Basis) 0 / 100 0.5 / 99.5 0.75 / 99.25 1 / 99 5 / 95 10 / 90 17.5 / 82.5 25 / 75 50 / 50 75 / 25 PVA M w 143,000 PVP M w 10,000 PVP M w 40,000 PVA M w 95,000 PVP M w 10,000 PVP M w 40,000

The

A0 A0.5 A0.75 A1 A5 A10 A17.5 A25 A50 A75

B0 B0.5 B0.75 B1 B5 B10 B17.5 B25 B50 B75

C0 C0.5 C0.75 C1 C5 C10 C17.5 C25 C50 C75

D0 D0.5 D0.75 D1 D5 D10 D17.5 D25 D50 D75

39 Dried polymers were weighed using a Denver Instruments M-120 balance with a sensitivity of 0.0001g. Polymer solutions (10% w/w) were prepared by dissolving the polymers in DI water. The beakers were sealed with DuraSeal and placed in an oven at 90C for 12-24 hours. The polymer solutions were removed from the oven and stirred for 10 minutes. This was followed by 10 minutes of sonication to remove air bubbles. Each of the solutions was caste into polyethylene petri dishes and 8in x 12in rectangular PMMA molds. The solutions were placed back in the oven set at 37C to evaporate the DI water. Evaporation took between 36-48 hours to complete. Upon drying, residual water was removed from the polymer films in a vacuum oven at 35C with an absolute pressure of 5 in Hg.
3.2 Equilibrium Swelling Analysis

The polymer blends were swollen in DI water for 1-2 hours to form gels. Circular discs were cut from the gels with a 8.35 mm ID brass hole punch and then dried in an oven at 37C to evaporate the DI water that was introduced in swelling. The circular discs were further dried in a vacuum oven overnight at 35C with an absolute pressure of 5 in Hg to further remove water from the network. The dry mass of the circular discs (n=3) for each polymer was measured. The discs were then swelled with 30 ml of DI water in glass jars. The discs were kept at 37C in a shaker bath shaking at 60 RPM for the first 4 days. The discs were then kept at 37C in a waterbath. The masses of each swollen gel were measured after 1 hr, 2 hr, 4 hr, 8 hr, 1 day, 2 days, 4 days, and 1 week swelling and weekly thereafter up to 120 days. Solutions were changed after each measurement. After 120 days of swelling in DI water, the circular thin film discs were

40 dried in an oven at 37C and then in a vacuum oven at 35C around an absolute pressure of 5 in Hg. Dry masses of the discs were then measured. The density of each of the polymers was calculated prior to swelling by measuring the weight of the three discs in heptane. The mass of the disc measured in heptane was compared to the mass of the dry polymer discs in air prior to swelling. The following equation which is based on the buoyancy of the polymer in heptane was used to calculated the density of the polymer blends:

! polymer =
3.3 Chemical Analysis

( mdry mheptane )

! heptane mdry

(3.1)

Attenuated Total Reflectance - Fourier Transform Infrared Spectroscopy (ATRFTIR) tests were conducted to examine the chemistry of the blends using a Nicolet (Madison, WI) model 560 with ATR attachment. Evaporated polymer blends were placed on a ZnSe crystal for analysis. Samples were examined as prepared (n=3) as well as following 8 weeks of swelling in Phosphate buffered saline solution (n=1). The spectra of the as-prepared films were obtained with 4 cm-1 resolution (1.928 cm-1 data spacing) and 1024 scans. The spectra of the samples that were swollen for 8 weeks and then dried were obtained with 1 cm-1 resolution (0.482 cm-1 data spacing) and 1024 scans. Spectra analysis was performed with the Nicolet OMNIC software package.
3.4 Network Characterization with Rubber Elasticity Theory

Six 60 mm x 90 mm sheets were cut out of the larger thin films that were caste for each copolymer blend. The sheets were swollen in PBS (pH=7.4) at 37C in 500 ml glass jars. The jars were shaken at 60 RPM in a 37C shaker water bath for 2 days. After

41 2 days, the jars were moved to a 37C water bath. After 2 days, 1 week, 2 weeks, 4

weeks, and 8 weeks of immersion in vitro, thin strips were cut from the swollen hydrogels on a natural rubber surface with a cutting tool shown in Figure 11. The tool was prepared by gluing four Feather Disposable Scalpels together with glue. The width of the sample, determined by the distance between the scalpel blades, varied from 4.25 mm to 4.65 mm. The lengths of the tensile samples were all greater than 75 mm. Widths of the tensile samples were dependent on the film thickness that remained after the caste polymer blend was dried. The dimensions of the swollen strips adhered to the ASTM Standard Test Method for Tensile Properties of Thin Plastic Sheeting [120]. Sample width and thickness were measured with digital veneer calipers having a resolution of 0.01 mm. Tensile tests were performed on the thin film strips for each of the blends after 2 days, 1 week, 2 weeks, 4 weeks, and 8 weeks of swelling in vitro. Tensile tests were run on each blend (n=5) on an Instron Series Materials Testing System Series 4442 (Canton, MA) with a 500 N load cell. The distance between the tensile grips at the start of each test was 50 mm. The thin film samples were extended 12.5 mm (25% extension) at a rate of 5 mm/min as proposed by the ASTM standard [120]. Axial load and displacement values were recorded at a rate of 20 pts/sec with a computer interface. The tensile data was used to calculate the tensile modulus of the gels. Also, the rubber elasticity theory was used to relate the tensile modulus and degree of swelling of the gels with the characteristics of the hydrogel network structure, namely, average molecular weight between crosslinks, M c , and crosslink density, X.

42
3.5 Thermal Analysis

The PVA/PVP blends were studied using a differential scanning calorimeter (DSC) (TA Instruments (Newark, DE)). DSC tests were performed on samples that were cut from the polymer film that was caste after the water had evaporated. The experiments involved heating the polymer from 25C to 250C at a rate of 5C/min. The mass of each sample (usually between 4.0 mg and 6.0 mg) was measured, encapsulated within an aluminum pan and lid, and sealed with a standard die press. The melting point and enthalpy change with melting were determined with Universal Analysis software.
3.6 Statistical Analysis

Selected results from the previously described tests were compared statistically using a two sided analysis of variance and Students t test. The sample sizes for each of the characterization tests were stated in the previous sections. Compared results that were found to be statistically different at the 95% confidence level are annotated with a * signifying that p<0.05. Compared results that were found to be statistically different at the 99.5% confidence level are annotated with a ** signifying that p<0.005.

43

Figure 11. The device that was used to cut PVA/PVP films into tensile strips was formed from four Feather Disposable Scalpels that were glued together. A natural rubber cutting surface was used.

44
4 4.1 Results Swelling Characterization

Swelling experiments involve measuring the mass and/or volume of a hydrogel over an immersion time. following equation: A mass swelling coefficient, q, can be calculated by the

q=

m m dry

(4.1)

mdry is the initial dry mass of the polymer disc and m is the mass as a function of time. A plot of q vs. time for the blends having a PVA M w of 143,000 and a PVP M w of 10,000 is shown in Figure 12. The pure PVA hydrogel and blends prepared with up to 25% PVP reached a maximum in swelling after 1-2 days of immersion. Equilibrium swelling, which was reached shortly thereafter, was sustained for 120 days in vitro (q=2.3 2.6). The range seen in mass swelling coefficients corresponds to 57 62% water w/w. Addition of PVP resulted in an increase in swelling coefficient. Blends prepared with 50% and 75% PVP had a significantly higher maximum swelling; q=3.3 and q=4.6 respectively. However, these 2 blends failed to reach a stable equilibrium over the immersion time. Blends prepared with these higher percentages of PVP proved to be more brittle in the dry state and more prone to tear when being handled during weighing after immersion. The densities of the gels ranged from 1.25 1.29 g/ml. Density values of the dry polymers were used to convert mass swelling coefficients to volume swelling coefficients with the following equation:

Q = 1 + (q 1) ! 2,r

(4.2)

45 The reciprocal of the volume swelling coefficient, Q, is the polymer volume fraction, 2,s. Polymer volume fractions are required in the calculation of network characteristics in Section 4.3.2.

46

5 4.5 4 3.5 3 2.5 2 1.5 1 0 20 40 60 80 100 120

PVP %

75 % H20

0 0.5 0.75 1 5 10 25 50

50 % H20

75

Time (days)
Figure 12. Mass swelling coefficient as a function of time of immersion. Blends prepared with higher portions of PVP swelled to a greater extent however blends prepared with excess PVP suffered dissolution and failed to reach a stable equilibrium swelling.

47
4.2 4.2.1 Dissolution of Hydrogels Mass Loss Analysis

The dry masses of the circular discs that were immersed in vitro for 120 days, m120, were compared to the initial dry mass of the discs, mdry, to determine the extents of dissolution within the polymer networks. calculated with the following equation: Percentage of polymer mass loss was

% Polymer Mass Loss =

mdry m120 m dry

100%

(4.3)

Figure 13 shows a plot of polymer mass loss for the 143K PVA / 10K PVP blend. Discs made from pure PVA (%PVP=0) had 6.9% mass loss over 120 days in
vitro. Small additions of PVP to the polymer network resulted in a decrease in mass loss

with the minimum percentage mass loss suffered by the blend prepared with 99% PVA and 1% PVP (2.8%). Blends with 0.75% PVP and 1% PVP showed statistically

significant less dissolution than pure PVA (p < 0.05).

48

% Polymer Mass Loss

70 60 50 40 30 20 10 0 0 0.5 0.75 1 5 10 25 50 75

* *

% PVP
Figure 13. Polymer mass loss after 120 days swelling as a function of PVP composition. Polymer mass loss reached a minimum at 0.75% and 1% PVP.

49
4.2.2 Chemical Analysis

Three dry thin film samples of each polymer blend were tested with ATR-FTIR to determine the content of the films after being crosslinked by evaporation. A typical absorbance spectrum obtained for the PVA/PVP blends is shown in Figure 14. The notable absorbance peaks of the blend, with respect to this study, are identified in Figure 14. There is a proportional relationship between the height of the absorbance peak around 1142 cm-1 and the degree of crystallinity of the PVA network [88, 96, 97]. The peak results from a C-O stretching mode that is influenced by hydrogen bonding. The 1142 cm-1 band is a shoulder on the larger peak centered around 1090 cm-1. This peak in PVA is the characteristic alcohol peak due to C-O stretching. Carbonyl bonds, which are attached to the pyrrolidone rings, result in stretching modes seen with the absorbance peaks at 1657 cm-1 and 1567 cm-1. These two peaks represent the free and hydrogen bonded carbonyl stretching modes for the PVA / PVP blend respectively. It is assumed that a hydrogen bonded carbonyl is an indication of an intermolecular secondary interaction between the carbonyl oxygen on a PVP chain and a hydroxyl group along a PVA chain. Li and Brisson [116, 117] analyzed similar twin carbonyl peaks for PVPh / PMMA blends, which have a very similar functional group pairing to the PVA / PVP blend. They found carbonyl peaks for the PVPh / PMMA blend to be at 1733 cm-1 and 1707 cm-1. The addition of PVP to a pure PVA polymer network causes many changes to the spectra, the most notable of which are due to the carbonyl double bond as shown in Figure 15. Other smaller changes were seen in the 1200-1500 cm-1 range but these peaks were not specifically identified. A large increase in the height of the 1657 cm-1 peak was

50 seen when the proportion of PVP in the blend was increased. However, the hydrogen bonded carbonyl peak at 1567 cm-1 showed small increases in relative intensity up to the blend containing 95% PVA and 5% PVP. For blends that were prepared with higher fractions of PVP (10%, 17.5%, and 25%), the hydrogen bonded carbonyl peak at 1567 cm-1 became shadowed and enveloped behind the larger free carbonyl peak at 1657 cm-1. Distinguishing between the two peaks above 5% PVP was impossible as seen in Figure 15. The heights of the free carbonyl peak at 1657 cm-1 and the alcohol peak at 1090 cm-1 were determined from spectra from three different films of each of the blends. The baseline chosen for each spectra was used to determine the heights of both peaks. The ratio of the peaks on each of the spectra was calculated and an average peak height ratio (1657 cm-1 / 1090 cm-1) was calculated for each blend composition. For the samples that had just been crosslinked via evaporation, an increase in peak height ratio was seen with increasing content of PVP in the blend. After 56 days of immersion in vitro and

subsequent drying, one film from each blend was analyzed again with ATR-FTIR and the peak height ratio was calculated. Figure 16 shows the results from this dissolution study. A percentage decrease in peak height ratio over the 56 days immersion can be calculated that roughly estimates the fractional loss of free carbonyl groups from the polymer network. Figure 17 shows that blends prepared with PVP percentages 5% and greater have a fractional loss of free carbonyl groups between 70-90%, indicating a significant dissolution of PVP out of the polymer network.

51

1090 1142

1657 1567

Figure 14. Typical ATR-FTIR spectra for PVA/PVP blend. The spectra shown was prepared with 95% PVA / 5% PVP. The four noted absorbance peaks are labeled in cm-1.

52

% PVP

1657

1567

Figure 15. ATR-FTIR Spectra of PVA/PVP blends (143K PVA / 10K PVP) prior to swelling. Intensity of the free C=O peak increases with PVP%. The hydrogen bonded C=O peak is not detectable above 5% PVP due to its relative intensity to the larger and broader free C=O peak.

53

Peak Height Ratio (1657/1090)

1.2 1.0 0.8 0.6 0.4 0.2 0.0 0.5 1 5 PVP% 10 17.5 25 Prior to immersion (n=3) After 56 days immersion (n=1)

Figure 16. Change in peak height ratio of blend spectra over 56 days immersion in vitro. PVP not incorporated within the polymer network is free to dissolve into solution. An equilibrium value of free C=O in each gel is reached after 56 days immersion.

54

1 Fractional loss of free C=O 0.8 0.6 0.4 0.2 0 0 5 10 15 PVP% 20 25 30

Figure 17. Fractional loss of free carbonyl bonds as a function of PVP composition. The fraction loss was determined by the change in free C=O peak height ratio with ATRFTIR spectra analysis.

55
4.3 4.3.1 Mechanical Analysis Youngs Modulus

Youngs Modulus values were calculated by measuring the slopes of plots of tensile stress versus tensile strain between 0 and 8.3% strain. The plots remained

relatively linear within this strain range. A sample plot showing how Youngs Modulus values were measured is shown in Figure 18. Values were calculated for each of the PVA/PVP blends after 2, 7, 14, 28, and 56 days immersion and are plotted in Figure 19. For each of the blends, only slight changes were seen over the immersion time. After 2 days of immersion, the films had reached maximum swelling and were approaching equilibrium swelling. The film prepared with pure PVA showed a decrease in modulus between 2 days and 7 days immersion that remained the same throughout the duration of the study. Films that were prepared with small additions of PVP to the polymer network showed some deviations in modulus over time. Tests of blends containing 0.5% and 1% PVP showed that maximum stiffness was reached within 7-14 days, after which a decrease in modulus occurred with continued immersion. Blends prepared with 17.5% and 25% PVP showed stable Youngs Moduli values throughout the immersion study. There was a general decrease in Youngs Modulus with increasing PVP composition after each of the immersion times. Between 2 and 56 days of swelling there was a significant decrease in the Youngs modulus for the pure PVA sample. For films prepared with 5 and 10% PVP, Youngs moduli increased significantly between 2 and 56 days of immersion.

56

0.25 Tensile Stress (MPa) 0.20 0.15 0.10 0.05 0.00 0 0.02 0.04 0.06 0.08 0.1 Strain (mm/mm)
E

Figure 18. Youngs Modulus (E) was calculated by determining the slope for the plot of tensile stress as a function of tensile strain.

57

3.0 2.5 E (MPa) 2.0 1.5 1.0 0.5 0.0 0 0.5 1 5 % PVP 10 17.5 25

2 days

7 days

14 days

28 days

56 days

Figure 19. Youngs Modulus of PVA/PVP polymer blend thin films as a function of PVP composition after various times of immersion in vitro.

58
4.3.2 Network Characterization with Rubber Elasticity Theory

The rubber elasticity theory can be used to calculate a tensile modulus, G that is related to the polymer volume fraction, 2,s, by the following equation:

" $ 1 $
2

= G#2,s 1 3

(4.4)

where is tensile stress and is polymer elongation [121, 122]. Figure 20 shows a sample plot of how the tensile modulus, G, was calculated from tensile and swelling data. Tensile stress and modulus can be used with the rubber elasticity theory, which is traditionally written for shear stress and modulus, because of the anisotropy of the hydrogel samples being tested. The slope was determined from the initial 8.3% of strain. The samples tested generally exhibited a linear response in this initial region. The slope of the plot in Figure 20 was calculated for each of the films after 2, 7, 14, 28, and 56 days of swelling in vitro. However, the films may have not reached equilibrium swelling at the 2 and 7 day time periods, thereby making the polymer volume fraction values, 2,s, at those time periods inaccurate. Therefore, G values were only calculated for the gels after 14, 28, and 56 days of swelling in vitro. Using the rubber elasticity theory for small strains (<20%), it is possible to calculate the effective average molecular weight between crosslinks, M c , for networks crosslinked without a solvent being present [123, 124]. The following equation was used to calculate M c from the tensile modulus, G, equilibrium polymer volume fraction, 2,s, and polymer density, 2,r:

" $ 1 $
2

= ! 2,r RT

1 Mc

2 Mn

# 2,s1 3

(4.5)

59 Here, M n is the average polymer network molecular weight prior to crosslinking. For the PVA/PVP blends, M n was calculated on a weight fraction basis using the M n values of the PVA and PVP polymers. M c values for the 143K PVA / 10K PVP series of blends were calculated after 14, 28, and 56 days of swelling and are shown in Figure 21. The M c values for the gels in this molecular weight series remained relatively stable from 14 to 56 days of swelling. One hydrogel blend containing 0.5% PVP showed an increase in M c between 14 and 56 days (p<0.05). The films prepared with up to 10% PVP had similar M c values to the pure PVA sample. A crosslinking ratio or crosslinking density, X, can be calculated from the M c values with the following equation [125]:

X=

Mo 2Mc

(4.6)

where Mo is the molecular weight of one repeat unit in the network structure. For the PVA/PVP hydrogels, the ATR-FTIR spectral analysis (Section 4.2.2) has shown that PVP chains not hydrogen bonded to the network structure, suffer significant dissolution. Therefore, it was estimated that Mo for the hydrogel blends is close to 44 g/mol which is the molecular weight of one vinyl alcohol repeat unit. Figure 22 shows the crosslinking densities for the 143K PVA / 10K PVP series after 14, 28, and 56 days of swelling. The crosslinking density remained stable for each blend except for the 0.5% PVP which showed a decrease between 14 and 56 days of swelling (p<0.05). A statistical decrease in crosslinking density with increasing PVP content was only seen for blends prepared with greater than 10% PVP content.

60

0.3 0.25

(MPa)

0.2
G

0.15 0.1

0.05 0 0 0.05 0.1 0.15 0.2 0.25 (-1/3) 2 (-(1/ )) 2,s 0.3 0.35

Figure 20. Sample plot showing how tensile modulus (G) was determined. The slope was measured between 0 and 8.3% strain.

61

7000 6000 5000

Mc
14 days 28 days 56 days

4000 3000 2000 1000 0 0 0.5 1 % PVP 5 10 25

Figure 21. Effective molecular weight between crosslinks ( M c ) after 14, 28, and 56 days of immersion for 143K PVA / 10K PVP blend series.

62

0.012 0.01 0.008 0.006 0.004 0.002 0 0 X

*
14 days 28 days 56 days

0.5

1 % PVP

10

25

Figure 22. Crosslinking density (X) after 14, 28, and 56 days of immersion for 143K PVA / 10K PVP blend series.

63
4.4 Thermal Analysis

The DSC scans for the PVA (143K) / PVP (10K) blend are shown in Figure 23. The melting temperature and heat of melting (the area created by the melting endotherm) were determined for each scan using the Universal Analysis software provided by TA Instruments. By using selected limits, the software performs an integration on the

melting peak. Using a 5C/min heating rate led to clear melting endotherms; however, step transitions such as Tg were not easily identifiable at such a slow rate. Figure 24 shows the typical baseline that was chosen with the integration software. Scans were integrated with a changing baseline to better characterize the melting enthalpy of the blends. The melting points of the blends, as determined by DSC, are shown in Figure 25. The 99.5% PVA / 0.5% PVP blend had a higher melting point than the pure PVA hydrogel (227.5C vs. 225.1C) (p<0.05). Further additions of PVP to the blend caused the melting point to decrease. The change in enthalpy due to melting of the PVA (143K) / PVP (10K) blend is shown in Figure 26. Blends containing 1% PVP and 5% PVP had significantly higher changes in enthalpy due to melting than the pure PVA blend. Additions of PVP above 5% (10%, 17.5%, and 25% PVP) resulted in decreases in the change in enthalpy due to melting.

64

%PVA / %PVP

75 / 25 82.5 / 17.5 90 / 10 95 / 5 99 / 1 99.5 / 0.5

100 / 0

Figure 23. DSC scans of PVA/PVP blends (143K PVA / 10K PVP) showing changes in Tm with PVP composition.

65

Figure 24. Typical analysis of melting in a DSC scan with integration performed with a changing baseline using Universal Analysis software provided by TA Instruments.

66

232 T melt (oC) 228 224 220 216 0

0.5

5 10 % PVP

17.5

25

Figure 25. Melting points (Tm) of PVA/PVP blends (143K PVA / 10K PVP) prior to immersion as a function of PVP composition.

67

120 100

** *

Hmelt (J/g)

80 60 40 20 0 0 0.5 1 5 %PVP 10 17.5 25

Figure 26. Changes of enthalpy at melting (Hmelt) of PVA/PVP blends (143K PVA / 10K PVP) prior to immersion as a function of PVP composition.

68
4.5 Effects of M w on Hydrogel Properties

Along with the series of compositions using 143K M w PVA and 10K M w PVA (Series A) that has previously been reported, 3 other series of compositions (B, C, and D) were processed and analyzed. These 3 series were investigated to determine the The series that will now be

importance that M w has on a hydrogels properties. presented alongside the A series are found in Table 3.

Table 3. Molecular weight series Series PVA M w PVP M w

A B C D

143,000 143,000 95,000 95,000

10,000 40,000 10,000 40,000

The following tests were performed on these 3 additional series of blends:

= Equilibrium swelling characterization with determination of polymer density (120 days immersion in vitro) = Mass loss analysis after 120 days immersion in vitro = Tensile Testing after 2, 7, 14, 28, and 56 days immersion in vitro to determine Youngs Modulus, M c , and X = Thermal analysis with DSC prior to immersion to determine Hmelt and Tm
4.5.1 Equilibrium Swelling Characterization

When the equilibrium swelling data is presented for a common blend composition but with varying M w of PVA and PVP, a swelling / M w relationship can be investigated. For the swelling of the pure PVA samples shown in Figure 27, the two PVA M w s that were tested yielded very similar swelling results.

69 However when PVP was added to PVA in the 1% PVP, 5% PVP, and 10% PVP blends (Figures 28, 29, and 30), the hydrogel swelling showed dependence on the M w of the PVA. The higher molecular weight PVA attained a higher equilibrium swelling coefficient. The molecular weight of the PVP did not seem to affect the swelling

characteristics at these blend compositions containing relatively small amounts of PVP. For blends that were prepared with larger percentages of PVP, shown in Figures 31 and 32 for 25% and 50% PVP, equilibrium swelling was dependent on the molecular weight of the PVP that was used.
4.5.2 Mass Loss Analysis

Dissolution analysis over 120 days immersion in vitro, presented as a mass loss percentage in Figure 33, showed that the percentage of mass loss over 120 days immersion was not dependent on the M w of the PVP used. Also, the blends prepared with the 95,000 M w PVA did not show a minimum in mass loss for small additions of PVP as was shown with the 143,000 M w PVA.
4.5.3 Youngs Modulus

To compare the blends prepared with different PVA and PVP M w s, the Youngs Moduli for blends after 56 days of immersion in vitro were plotted. Figures 34 and 35 show that Youngs Modulus was only marginally dependent on M w of the PVP. The 90% PVA / 10% PVP blend with a 95K M w PVA and a 40K M w PVP showed an exceptionally high modulus following each immersion time. It did not follow the trend seen earlier in which Youngs Modulus decreased when the blends PVP content was greater than 10%. Figures 36 and 37 show that for PVA rich blends (1% PVP), the

70 lower M w PVA had a higher tensile modulus. Once again, in Figure 37, the 10% PVP blend had showed an uncharacteristically high tensile modulus.
4.5.4 Network Characterization with Rubber Elasticity Theory

Comparing the characteristics of hydrogel networks by examining molecular weight between crosslinks and crosslinking density offers a way to investigate the influence that M w has on the properties of the hydrogel blend. As previously shown in Figures 21 and 22, M c and as a result X changed only slightly between 14 and 56 days of immersion. Therefore, comparisons of these network characteristics can be made after 56 days of swelling only. Figure 38 shows the effective molecular weight between

crosslinks after 56 days of swelling. The M c values for the pure PVA samples show the role that M w plays in formation of the polymer network. The 95K PVA resulted in a tighter network having almost half the effective molecular weight separating crosslinks than the 143K PVA after 56 days of immersion. Small additions of 10K PVP to 143K PVP (B Series) resulted in a decrease in M c . The 1% PVP sample in the B series had a M c of 2375 g/mol while the

pure PVA sample in that series had a M c of 3472 g/mol. However, the pure PVA sample prepared with the 95K M w PVA had the lowest M c after 56 days of immersion, 1814 g/mol, making it the tightest network of all the gels tested. Additions of PVP to the 95K PVA did not result in any decrease in the M c . The 10% PVP gel with 95K PVA and 40K PVP had a M c that was uncharacteristically low when compared to the 10% PVP gels prepared with in the A, B, and C series. This corresponds to the observation that

71 Youngs Modulus for this blend was high compared to the other blends prepared with 10% PVP. Figure 39 shows the crosslinking density for the gels after 56 day of immersion. The crosslinking density of the 95K PVA was almost double that shown by the gel made from pure 143K PVA. The density increased with small additions of PVP (>5%) for the B series containing 143K PVA and 40K PVP. highest for the pure 95K PVA.
4.5.5 Thermal Analysis

However, crosslinking density was

Thermal properties of pre-swollen hydrogel blends were also shown to be dependent on M w as seen in Figures 40 and 41. The melting point for the 95K M w PVA was larger than the melting point for the 143K M w PVA. The melting points for all the M w combinations for 1%, 5%, and 10% PVP were similar. For the 17.5% and 25% PVP blends, the melting point was solely dependent on the PVP M w , with the higher M w PVP exhibiting a higher melting point. The change in enthalpy due to melting was not as dependent on PVP M w . However, for the pure PVA samples, the lower M w sample showed a higher change in enthalpy due to melting. The 99% PVA / 1% PVP blend with the higher M w PVA showed a higher Hmelt than blend processed with the lower M w PVA. The 95% PVA / 5% PVP blend had the highest Hmelt of all the blends tested (109.7 J/g).

72

2.6 2.2 1.8 1.4 1 0 20 40 60 80 time (days) 100 120

143K PVA 95K PVA

Figure 27. Mass swelling coefficient (q) for pure PVA hydrogels as a function of immersion time.

73

2.6 2.2 1.8 q 1.4 1 0 20 40 60 80 time (days) 100 120

143K PVA / 10K PVP 95K PVA / 10K PVP 143K PVA / 40K PVP 95K PVA / 40K PVP

Figure 28. Mass swelling coefficient (q) for 99% PVA / 1% PVP hydrogel blends as a function of immersion time.

74

2.6 2.2 1.8

143K PVA / 10K PVP 143K PVA / 40K PVP 95K PVA / 40K PVP

1.4 1
0

95K PVA / 10K PVP

20

40

60 80 time (days)

100

120

Figure 29. Mass swelling coefficient (q) for 95% PVA / 5% PVP hydrogel blends as a function of immersion time.

75

3 2.6 2.2
q

1.8
143K PVA / 10K PVP 143K PVA / 40K PVP 95K PVA / 40K PVP

1.4 1 0

95K PVA / 10K PVP

20

40

60 80 time (days)

100

120

Figure 30. Mass swelling coefficient (q) for 90% PVA / 10% PVP hydrogel blends as a function of immersion time.

76

3 2.6 2.2
q

1.8
143K PVA / 10K PVP 143K PVA / 40K PVP 95K PVA / 40K PVP

1.4 1 0

95K PVA / 10K PVP

20

40

60 80 time (days)

100

120

Figure 31. Mass swelling coefficient (q) for 75% PVA / 25% PVP hydrogel blends as a function of immersion time.

77

4 3.5 3 2.5 2 1.5 1 0 20 40 60 80 time (days) 100 120

143K PVA / 10K PVP 95K PVA / 10K PVP 143K PVA / 40K PVP 95K PVA / 40K PVP

Figure 32. Mass swelling coefficient (q) for 50% PVA / 50% PVP hydrogel blends as a function of immersion time.

78

40 35 % Polymer Mass Loss 30 25 20 15 10 5 0 0 0.5 0.75 1 5 % PVP 10 25 50

143K PVA / 10K PVP 95K PVA / 10K PVP 143K PVA / 40K PVP 95K PVA / 40K PVP

Figure 33. Polymer mass loss after 120 days swelling as a function of PVP composition. Polymer mass loss was not dependent on PVP M w .

79

3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 0

143K PVA / 10K PVP

143K PVA / 40K PVP

E (MPa)

0.5

5 PVP %

10

17.5

25

Figure 34. Influence of PVP M w on Youngs Modulus for 143,000 M w PVA after 56 days immersion.

80

4
95K PVA / 10K PVP 95K PVA / 40K PVP

3 E (MPa) 2 1 0 0 0.5 1 5 PVP % 10 17.5 25

Figure 35. Influence of PVP M w on Youngs Modulus for 95,000 M w PVA after 56 days immersion.

81

4
143K PVA / 10K PVP 95K PVA / 10K PVP

3 E (MPa) 2 1 0 0 0.5 1 5 PVP % 10 17.5 25

Figure 36. Influence of PVA M w on Youngs Modulus for 10,000 M w PVP after 56 days immersion.

82

4
143K PVA / 40K PVP 95K PVA / 40K PVP

3 E (MPa) 2 1 0 0 0.5 1 5 PVP % 10 17.5 25

Figure 37. Influence of PVA M w on Youngs Modulus for 40,000 M w PVP after 56 days immersion.

83

7000
143K PVA / 10K PVP 143K PVA / 40K PVP 95K PVA / 40K PVP

6000 5000 4000

95K PVA / 10K PVP

* *

Mc
3000 2000 1000 0

0.5

1 % PVP

10

25

Figure 38. Effective molecular weight between crosslinks ( M c ) after 56 days of immersion as a function of PVP composition.

84

0.016 0.012 X 0.008 0.004 0

* *

143K PVA / 10K PVP 143K PVA / 40K PVP 95K PVA / 10K PVP 95K PVA / 40K PVP

0.5

1 % PVP

10

25

Figure 39. Crosslinking density (X) after 56 days of immersion as a function of PVP composition.

85

236
143K PVA / 10K PVP 143K PVA / 40K PVP 95K PVA / 40K PVP

232 T melt (oC)

95K PVA / 10K PVP

*
228

*
224 220 216 0 0.5 1 5 % PVP 10 17.5 25

Figure 40. Melting Point (Tm) for PVA/PVP blends as a function of PVP composition.

86

140 120 Hmelt ( J/g) 100 80 60 40 0

143K PVA / 10K PVP 95K PVA / 10K PVP

143K PVA / 40K PVP 95K PVA / 40K PVP

0.5

5 %PVP

10

17.5

25

Figure 41. Enthalpy change due to melting (Hmelt) for PVA/PVP blends as a function of PVP composition.

87
5 5.1 Discussion Equilibrium Swelling

The swelling characterization of blends prepared with up to 25% PVP did not differ significantly from the pure PVA hydrogel. For these blends, equilibrium swelling was reached quickly and stability was shown over 120 days. However, blends prepared with larger portions of PVP showed instability in swelling, especially the blend prepared with 75% PVP. Hydrogen bonding between hydroxyl groups on PVA chains allowed sections of the PVA chain to crystallize. The steric hindrance of the pyrrolidone groups in PVP restricted the chains to being amorphous. PVP is more hydrophilic than PVA, an observation that was noted when solvating the polymer powders. The amorphous nature of the PVP and its higher affinity for water made PVP dominant blends swell to a greater extent and show less stability under physiological conditions.
5.2 5.2.1 Dissolution of Hydrogels Mass Loss Analysis

The pure PVA hydrogel lost 6.9% of its dry mass over 120 days of immersion. Additions of 0.75% PVP and 1% PVP to the PVA network resulted in over 50% reductions in percentage of polymer mass that was lost into solution. Interchain

hydrogen bonding between the hydroxyl group on the PVA chains and carbonyl groups on PVP chains resulted in an increased stability in the polymer network. These

secondary interactions between the PVA and PVP served as physical crosslinks and limited the dissolution of polymer out of the polymer network and into solution. Larger additions of PVP to the network led to higher percentages of polymer mass loss. Instability at larger PVP percentages was due to the interruption of PVA

88 crystal segments with bulky pyrrolidone rings. Crystalline segments of PVA chain have less chain solvation and greater stability than amorphous regions in a PVA network. The minimum in mass loss at 1% PVP, seen in Figure 13, was the result of a balance between stability gained through the introduction of interchain PVA/PVP hydrogen bonding and the instability introduced when PVP sterically interrupts PVA crystal segments.
5.2.2 Chemical Analysis

The purpose of analyzing the gels with ATR-FTIR was to determine changes in the chemistry of the PVA/PVP blends with immersion. The reduction in peak height ratio for each blend to a common value after 56 days immersion is evidence that PVP chains not incorporated into the polymer network through hydrogen bonding selectively leach out of the blend. The fractional loss of free carbonyl bonds that is plotted in Figure 17 shows that addition of excess PVP into the blend only resulted in a higher percentage of unincorporated PVP that left the network.
5.3 5.3.1 Mechanical Analysis Youngs Modulus

The Youngs Modulus of the pure PVA sample decreased 17% from 2 days to 56 days of immersion while Youngs Moduli of the 0.5 and 1% PVP blends only decreased 5% and 7% respectively over that same time period. The Youngs Modulus for the 5% and 10% PVP blends increased by 21% and 40% between 2 and 56 days. These

increases could be due to the peak that is seen in swelling around 2 days of immersion. The swelling coefficients partially decreased after this peak in swelling, resulting in a tightening of their polymer networks. This correlates with an increase in Youngs

Modulus that was seen over that immersion time.

89
5.3.2 Network Characterization with Rubber Elasticity Theory

The calculation of molecular weight between crosslinks ( M c ) and crosslinking density (X) is preferable to characterizing the gels by their Youngs Modulus values. Youngs Modulus values of two gels having different network structures can be similar because the gels reach different degrees of equilibrium swelling. Calculating the

network characteristics involves combining the swelling and mechanical data in the rubber elasticity theory to determine the size of the polymer network and its degree of crosslinking. The hydrogel networks molecular weight between crosslinks and degree of crosslinking did not change significantly between 14 to 56 days of immersion, except for the blend prepared with 0.5% PVP. This blend showed a decrease in crosslinking density between 14 and 56 days of immersion. Within the 143K PVA / 10K PVP molecular weight series, additions of up to 10% PVP to the network did not result in changes to the M c values. The blend prepared with 25% PVP had a more open network having a higher M c and lower X than the other blends in the A molecular weight series. The PVP in this blend that did not hydrogen bond to PVA chains dissolved into solution, leaving a network with larger pores.
5.4 Thermal Analysis

The change in enthalpy due to melting (Hmelt) can be correlated to the energy to melt PVA crystallites and disrupt interchain hydrogen bonding interactions. Hmelt was higher for the blends prepared with 1% and 5% PVP than the pure PVA hydrogel. Enthalpy change due to melting can be used to determine the degree of crystallinity of a semi-crystalline polymer by comparing it to literature values for a theoretically perfect

90 PVA crystal [105]. For this analysis, Hmelt was also a measure in part of the degree of physical crosslinking. For the 1% and 5% PVP blends, interchain hydrogen bonding caused a slight increase in the enthalpy change during melting. Moderate amounts of PVP in a blend served to stabilize the crystal structures, thus increasing Hmelt. The melting point increase shown in Figure 40 that was seen with small additions of PVP indicates that physical crosslinking made the crystalline segments in the polymer more stable. The decrease in crystallinity and melting point for blends with greater than 10% PVP is an indication that the PVP chains are interrupting PVA crystallites due to steric hindrances of the bulky pyrrolidone group.
5.5 Effects of M w on Hydrogel Properties

The properties of the PVA/PVP blends depend on the M w of both the PVA and PVP. With pure PVA, the 95K and 143K M w polymers showed similar swelling

profiles. The PVA hydrogel with a 95K M w exhibited greater mechanical stiffness than 143K M w PVA under physiological conditions. The M c values of PVA dominant blend were lower for the 95K M w PVA blends. Using a lower M w PVA in the blend resulted in a tighter network that had a higher crosslinking density. This finding was supported by the thermal analysis that showed the 95K M w PVA had a greater degree of physical crosslinking than the 143K M w PVA. For PVA/PVP blends prepared with moderate fractions of PVP (1%, 5%, and 10%), the influence of the PVP M w on swelling was negligible. The higher M w PVA had higher mass swelling coefficients at these compositions because it had a greater molecular weight between crosslinks ( M c ). Hydrogels with looser networks were able to

91 imbibe more solution into their network structures and thus have a higher equilibrium swelling coefficient due to this greater M c . However, this logic was not seen with the pure PVA hydrogels in which swelling did not seem to be dependent on the M w that was used to prepare the gels. Blends prepared with larger fractions of PVP (25% and 50% PVP) showed a greater dependence of swelling on the M w of the PVP that was used. When the longer chain PVP was used, the network was able to take in more PBS. However, for blends with greater than 10% PVP, the tensile properties were similar regardless of PVP M w . Polymer dissolution was not statistically dependent on the M w of the PVA or PVP used. However, Figure 33 shows that blends prepared with 143K PVA generally suffered less polymer dissolution than blends prepared with 95K PVA. This is expected because longer polymer chains would take longer to leave the polymer network through reptation than shorter polymer chains. However for PVP, no significant difference in dissolution was found for the two M w s that were tested. Table 4 compares the properties of the 4 blends prepared with 1% PVP. The blend that shows the greatest combination of ideal properties is the B series gel prepared with 99% 143K PVA and 1% 40K PVP. Comparing this blend to the pure PVA sample containing 143K PVA shows that PVP stabilized the polymer network. The 1% addition of 40K PVP was more successful in stabilizing the polymer network than the 1% addition of 10K PVP as shown in Figure 38. Both of these gels suffered a similar mass loss percentage over 120 days of immersion as shown in Figure 33, but the decrease in M c that was found for the blend using 40K PVP was much greater than that using the 10K PVP. The longer chain length in the higher M w PVP resulted in better crosslinking of

92 the PVA crystallites through interchain hydrogen bonding. This is confirmed by the DSC data which showed the B series polymer with 1% PVP to have a higher degree of initial crosslinking than the A series 1% PVP polymer before the polymer were swelled. The pure PVA gel prepared with 95K PVA and many blends prepared with the 95K M w PVA showed an even higher degree of crosslinking than the optimum blend prepared with 99% 143K PVA and 1% 40K PVP. However, blends prepared with 95K PVA underwent

greater amounts of polymer dissolution than the optimum blend.

Table 4. Comparison of the properties of blends prepared with 1% PVP

Series M w PVA used M w PVP used Mass Swelling Coefficient (56 days) % Mass Loss (120 days) Youngs Modulus (MPa) (56 days) M c (g/mol) (56 days) X (56 days) Tm (C) (as prepared) Hmelt (J/g) (as prepared)

A 143K 10K 2.35 2.8 1.51 3788 0.0058 226.3 84.8

B 143K 40K 2.39 2.9 2.50 2375 0.0093 226.7 98.4

C 95K 10K 2.20 5.7 3.08 1949 0.0113 226.4 92.9

D 95K 40K 2.18 8.3 2.29 2529 0.0087 226.9 99.3

93
5.6 Summary

Table 5 shows a design chart summarizing how blends can be prepared to achieve various hydrogel properties.

94
Table 5. Design chart for PVA/PVP hydrogel blends Property Effect of PVP concentration Effect of PVA M w
0% PVP not dependent on PVA M w Mass Swelling Coefficient, q q with PVP% 1, 5, and 10% PVP higher M w PVA had higher q values 25 and 50% PVP not dependent on PVA M w % Mass Loss Minimum at 1% PVP for series A and B (0.5-5% PVP) For A,C, and D series, M c increases with PVP conc. For B series, M c is minimized at 1% PVP Generally remains unchanged up to 10% PVP, however for B series showed a maximum at 1% PVP Decreases for blends containing greater than 10% PVP For higher M w PVA, Tm was maximum between 0.5-5% PVP. For lower M w PVA, Tm decreased with increasing PVP% Hmelt (J/g) (as prepared) Generally, maximum between 1-5% PVP for each M w series Independent of PVA
M w for pure PVA/PVP

Effect of PVP M w

1, 5, and 10% PVP not dependent on PVP M w 25 and 50% PVP higher M w PVP had higher q values

Lower for higher M w of PVA Higher for the higher PVP M w for gels less than 1% PVP Independent of PVA M w for gels greater than 1% PVA

Independent of M w of PVP

Effective Molecular Weight between Crosslinks, M c ,(g/mol)

Only important for A and B series blends with 1% PVP which showed the higher M w PVP to result in a lower M c

Crosslinking Density, X

Independent of PVA M w above 1% PVP

Independent of PVP M w above 1% PVP

Tm (C) (as prepared)

blends Tm Higher for lower M w PVA Generally, lower M w PVA results in higher Hmelt

Higher M w PVP resulted in higher Tm

Independent of PVP M w

95
6 Conclusions

PVA/PVP blends have potential to serve as materials for a nucleus pulposus replacement. The stability of gels containing moderate amounts of PVP has been shown with equilibrium swelling studies, dissolution experiments, and mechanical analysis to determine characteristics of polymer network structure. Blends gain their stability and superior network properties through the mechanism of interchain hydrogen bonding between hydroxyl groups on PVA chains and carbonyl groups on PVP chains. These secondary interactions have been seen with other polymer blends with similar side group chemistry. Blends prepared with larger portions of PVP suffered significant polymer dissolution due to the interruption of PVA crystallites with large bulky pyrrolidone rings. ATR-FTIR analysis verified that PVP chains not incorporated into the PVA network structure dissolved out of the gel. This dissolution explains why these gels prepared with higher amounts of PVP swell more and exhibit weaker mechanical properties. The M w of the PVA and PVP that was used to prepare these blends plays an important role in determining the hydrogels properties. Lower M w PVA was shown to result in a polymer network that was tighter and less swellable, possessing a higher density of crosslinks than the higher M w PVA that was tested. However, the higher

M w PVA showed far superior network stability in mass loss experiments, due to longer chains taking a longer time to dissolve out of the polymer network. PVP M w was only shown to be significant for blends prepared with larger portions of PVP. Higher M w PVP resulted in greater swelling. Also, for blends containing 143K M w PVA, a higher M w PVP seemed to better stabilize the network than the lower M w PVP. The blends that possessed the best combination of superior polymer mass retention along with a

96 significant mechanical stability when compared to the pure PVA samples was prepared with 99% PVA (143K) and 1% PVP (40K).

97
7 7.1 Recommendations for Further Research Processing of Hydrogels

Crosslinking hydrogels by evaporation is a method that cannot be used to process cylinder shaped replacements for the nucleus pulposus. Other processing methods that allow for more variations in shape such as repeating freezing and thawing cycles on a polymer solution need to be used to make suitable nucleus replacements from PVA/PVP hydrogel blends. The influence that the processing method has on material properties can be investigated. Also, the freeze/thaw blends should be tested to determine whether the trends that were seen with M w and PVP concentration for evaporated thin films still apply. Evaporation crosslinking leads to possible inconsistencies with processing. Crosslinking by evaporation creates polymer films that have material properties inherently dependent on the thickness of the film. Thicker films, that take longer to dry, essentially are processed with a higher degree of crystallinity than thinner films of the same blend composition. More time is provided for the PVA crystals to grow. The thickness variation in the films that were tensile tested ranged from 0.35 to 0.75 mm. For a majority of the samples, the thickness ranged from 0.45 to 0.55 mm. The process of freezing and thawing a hydrogel is much more easily controlled than the evaporation crosslinking process.
7.2 Quantitative Analysis of Hydrogen Bonding

Analysis of the hydrogen bonded carbonyl stretching band at 1567 cm-1 is limited with the ATR-FTIR method. At larger PVP concentrations, the free carbonyl stretching band is too intense compared to the hydrogen bonded band and obscures the hydrogen

98 bonded band, making it undetectable. Studying the blends with NMR would offer better detail of hydrogen bonding within the blend than ATR-FTIR spectral analysis. Direct evidence of the existence of interchain hydrogen bonding is possible with NMR [126].
7.3 New Criteria Needed for Nucleus Pulposus Replacements

The tensile tests performed in this study were intended to measure the mechanical stiffness of the hydrogels and calculate network structural characteristics.

Physiologically, a tensile test does not replicate the stress states under which the nucleus is subjected. However, a tensile test is appropriate in measuring the mechanical

properties of polymer thin films. A new set of mechanical performance criteria is needed for hydrogel nucleus implants that are designed to expand under compression, restoring tension to the annulus fibrosus layers. The function of these types of implants is closer to that of the native nucleus than the traditional total intervertebral disc replacements. Measurements of a bulk modulus from radially constrained compression tests would provide a better measurement tool in deciding whether a nucleus replacement can mimic the mechanics of a native nucleus. Furthermore, the fatigue performance of these hydrogel blends needs to be studied to predict the effect that cyclic loading will have on the stability and material properties of the gel. Creep analysis will determine the extent of flow that this polymer hydrogel will undergo while being subjected to constant prolonged loads that are seen with the native nucleus.

99
7.4 Drug Delivery Capabilities of PVA/PVP Hydrogel Blends

The use of hydrogel blends offers the potential of designing the nucleus replacement with drug delivery capabilities. The interaction of therapeutics with the PVA / PVP hydrogel blend should be investigated.

100
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