The Design, Synthesis, and Evaluation of Polyphosphazenes for Hard Tissue Engineering
Open Access
- Author:
- Morozowich, Nicole Lynn
- Graduate Program:
- Chemistry
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- April 19, 2013
- Committee Members:
- Harry R Allcock, Dissertation Advisor/Co-Advisor
James Patrick Runt, Committee Member
Benjamin James Lear, Committee Member
Raymond Edward Schaak, Committee Member - Keywords:
- Polyphosphazne
Bioerodible
Biomimetic
Hard Tissue Engineering
Vitamin
Antioxidant
Hydroxyapatite
Hydrogel
ToF-SIMS - Abstract:
- Synthetic bone grafts that promote the natural mineralization process would be excellent candidates for the repair or replacement of bone defects. This dissertation describes the development of a new class of biomimetic polyphosphazenes for use as implantable scaffolds, which were designed to better mimic the chemical and physical properties of bone. Chapter 1 outlines the development, evaluation, and current status of bioerodible polyphosphazenes specifically optimized for biomedical applications. The polymers were developed because of their ability to sensitize the polymers to hydrolysis to benign small molecules that can be metabolized or excreted from the body. The largest class of these polymers consists of macromolecules with amino acid ester side groups and these are the main focus of the chapter. However, a variety of polymers with other side groups also show promise as bioerodible species, and these are mentioned later in the chapter. Chapter 2 discusses the synthesis of polyphosphazenes containing various vitamin substituents, and their subsequent characterization and hydrolytic instability. The incorporation of biomolecules that stimulate bone growth into a biodegradable polymer scaffold would provide a long term beneficial effect upon hydrolysis. Specifically, vitamins L1, E, and B6 were used because of their biocompatibility, their importance in a variety of biological functions, and their potential to increase the mechanical properties of the resulting polymers, thus making these materials promising candidates for hard tissue engineering scaffolds. Chlorine replacement reactions were carried out initially with the small molecule, hexachlorocyclotriphosphazene, as a model for high polymeric, poly(dichlorophosphazene). Due to the steric hindrance generated by vitamin E as a substituent, co-substituted polymers were synthesized with both glycine ethyl ester or sodium ethoxide as the second substituent. Similarly, vitamin B6 was co-substituted with glycine ethyl ester or phenylalanine ethyl ester to favor biodegradability. To prevent crosslinking via multifunctional reagents, the hydroxyl groups in vitamin B6 were protected and subsequently deprotected under acidic conditions after side group linkage to the polymer backbone. The glass transition temperatures of the polymers ranged from -24.0 to 44.0 °C. Hydrolysis of the polymers in de-ionized water at 37 °C was used as an initial estimate of their hydrolytic sensitivity. Different solid polymers underwent 10 % to 100 % weight loss in six weeks with the generation of a broad pH range of ~2.5 to 9. The weight loss during preliminary hydrolysis experiments was attributed to cleavage of the polymer backbone and/or the polymers becoming soluble in the aqueous media during hydrolytic reactions. These polymers were not further explored for use as hard tissue grafts because of their fast hydrolysis profiles. To address this issue, chapter 3 discusses the synthesis of polyphosphazenes containing the antioxidant, ferulic acid, and amino acid esters as co-substituents. The synthesis protocol utilized the replacement of chlorine atoms in poly(dichlorophosphazene) by ferulic acid with either glycine, alanine, valine, or phenylalanine ethyl ester. Ferulic acid protects cells from free radical damage, and its steric characteristics have the potential to generate polymeric materials with high mechanical strength for hard tissue engineering scaffolds. Incorporation of the amino acid esters allows for control over the polymer hydrolysis while releasing the antioxidant at different rates. Macromolecular substitution reactions were carried out utilizing the allyl ester of ferulic acid to prevent side reactions during halogen replacement. The allyl protecting group was then removed using mild conditions. The polymers were characterized by 1H, 31P, GPC, and DSC techniques. Static water contact angles were measured to monitor the change in hydrophobicity/hydrophilicity. Before deprotection, the water contact angles were 82 – 88 ° and after deprotection the water contact angles were 56 – 71 °. A pH-dependent hydrolysis study revealed that the polymers are hydrolytically sensitive and decomposed ~ 5 – 25 % over 8 weeks yielding a final pH between 6.0 – 6.5. Polymer hydrolysis resulted in the release of ferulic acid. The polymers were photocrosslinked by a [2+2] cycloaddition of the ferulic acid moieties induced by exposure to long-wave UV light. The crosslinking was monitored by UV-spectroscopy via the apparent decrease in the 320 nm absorbance, attributed to the cyclization of the ferulic acid moieties. Increased UV exposure time led to an increase in the level of crosslinking. After 60 seconds, the polymers were crosslinked ~ 41 – 62 %. Hydrolysis experiments of the crosslinked materials showed only a 0 – 5 % weight loss after 8 weeks with a pH between 6.6 – 7.0. In order for the polymers to be used as a synthetic bone graft they should promote the natural mineralization process of hydroxyapatite (HAp). Chapter 4 describes the mineralization behavior of the antioxidant containing polyphosphazenes when exposed to a solution of simulated body fluid (SBF). All polymers contained ferulic acid (antioxidant), co – substituted with different amino acid esters linked to the polyphosphazene backbone. Differences in the side groups determined the hydrophobicity or hydrophilicity of the resulting polymers. All the polymers mineralized monocalcium phosphate monohydrate (MCPM), a type of biological apatite. However, the mineralization process (amount of deposition and length of time) was dependent on the hydrophilicity or hydrophobicity of the polymers. The polymer-apatite composites were examined by electron scanning microscopy (ESEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), and thermogravametric analysis (TGA). Weight gain data were also obtained. To verify that the nucleation process was due to the presence of calcium and phosphate, two standard solutions were prepared: one solution (NaCl Solution) contained only sodium chloride and the second solution (mSBF) was similar to SBF except without known crystal – growth inhibitors such as Mg2+ and HCO3-. No mineralization occurred when the polymers were exposed to the NaCl Solution, but mineralization took place when exposed to mSBF. The apatite phase produced was hydroxyapatite (HAp). The mineralization process in mSBF was much more extensive, with all samples gaining more weight than when exposed to SBF. A similar trend was also found (as in the case of SBF), with the amount of deposition and length of deposition time depending on the hydrophilicity/hydrophobicity of the polymer. These results suggest that the nucleation process is due to calcium and phosphate, and the absence of crystal growth inhibitors allows for the rapid nucleation of HAp. In both cases, the mineralization process was favored on hydrophilic surfaces (static water contact angle of 56–65°) versus hydrophobic surfaces (71–86°). These polymers are currently being tested for their osteocompatibility. Chapter 5 discusses the synthesis of phosphonate [PO(OEt2)] and phosphonic acid [PO(OH)2] containing polyphosphazenes because of their ability to bind hydroxyapatite [Ca10(PO4)6(OH)2], which comprises 70 wt% of bone. These polymers could mimic the natural bone healing mechanism, making them excellent candidates for implantable bone grafts. Two synthetic protocols have been developed to obtain the polymers, herein referred to as prior- and post-side group assembly. Prior-assembly required the synthesis of a phosphonate-containing side group before attachment to the polyphosphazene backbone through nucleophilic substitution, whereas post-assembly required the synthesis of a polyphosphazene containing free amino groups to which the phosphonate can be coupled by Michael addition after polymer synthesis. The final step for both routes required the deprotection of the phosphoester to the corresponding phosphonic acid. The polymers were characterized by 1H and 31P NMR, GPC, and DSC techniques. A six week hydrolysis study using phosphate buffered saline (PBS) determined their hydrolytic sensitivity. All the polymers were hydrolytically sensitive, as required for this purpose, and decomposed ~ 2 – 50 % by week six. The hydrolysis products were analyzed by UV-Vis techniques and their release was monitored over the course of the experiment. These results are in agreement with percent solid mass loss data. In general, all the phosphonic acid polymers hydrolyzed at a faster rate than their corresponding phosphoester derivatives. Chapter 6 discusses the mineralization potential of phosphoester and phosphonic acid containing polyphosphazenes during exposure to a solution of simulated body fluid (SBF) for a period of four weeks. Although all the polymers showed an initial mineralization response, the amount of deposition and the time scale, were dependent on the side group chemistry of the polymers. After exposure to SBF for one week, all the polymers mineralized HAp. After three weeks in SBF, polymers containing phosphoester substituents showed no significant change, with a weight gain of < 1 %, while polymers containing phosphonic acid substituents underwent a significant increase in the amount of mineralized HAp, with weight gains between 5 - 10 %. The morphology of mineralized features was observed with Environmental Scanning Electron Microscopy (ESEM). However, due to the structural complexity of the mineralized polymers, the identity of the mineralized phase could not be definitively identified using traditional characterization techniques such as energy dispersive spectroscopy (EDS), X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), or X-ray photoelectron spectroscopy (XPS). Time-of-flight secondary ion mass spectrometry (ToF-SIMS), a technique not previously explored for this type of application, successfully reveals details of the chemistry associated with the mineralized phase. The remaining chapters discuss the synthesis and fabrication of injectable scaffolds for hard tissue engineering. Specifically, chapter 7 discusses the bioerodible behavior of polyphosphazenes containing serine and threonine amino acids, which contain two different functional points of attachment to a phosphazene backbone (N- and O-terminus), were synthesized using an improved technique and their hydrolysis behavior was investigated. In the aqueous media the solid polymers yield hydrolysis products phosphate, ammonia, and the amino acid, and ethanol, and have the potential to be used in several different biomedical applications, ultimately determined by their hydrolysis behavior. A pH- dependent hydrolysis study revealed that all the polymers are hydrolytically sensitive, regardless of the type of linkage to the polyphosphazene backbone, although the hydrolysis rates are different. Polymers with amino acid esters linked through the N-terminus hydrolyzed between 16 and 60 % within a six week period. This is the fastest reported hydrolysis of any amino acid ester substituted polyphosphazene. The mechanism of hydrolysis is by bulk erosion as monitored by environmental scanning electron microscopy (ESEM). Polymers with the amino acid units linked through the O-terminus are soluble in water; thus their percent weight loss in aqueous media could not be determined. However, 31P NMR spectroscopy confirmed their hydrolytic sensitivity and the formation of phosphates and oligomeric species, which increased during the six week hydrolysis period. Complete hydrolysis did not occur within six weeks. The O-linked species are under investigation to use as injectable hydrogel scaffolds. Chapter 8 discusses the synthesis and characterization of amino-containing polyphosphazenes and the development of novel injectable hydrogel systems based on the Schiff base reaction between the amino-polyphosphazenes and aldehyded-dextran polymers. The amino-polyphosphaznes were characterized by several methods including 1H NMR, 31P NMR, solid state 13C NMR, GPC, and DSC techniques. The aldehyded-dextran polymers were characterized by solid state 13C NMR, FTIR, and their aldehyde content was determined by titration. After physical mixing of the two components at room temperature and physiological pH, different gelation times were observed. The gelation time was found to be dependent on the percent of oxidized dextran, the higher the oxidation percentage, the faster the gelation time. The hydrogel properties were evaluated using rheology, themal characterization, and microscopy. Hydrogels with low- and high-crosslink density were observed.