Nicholas R. Krogman

Polyphosphazene/Nano-Hydroxyapatite Composite Microsphere Scaffolds for Bone Tissue Engineering

The nontoxic, neutral degradation products of amino acid ester polyphosphazenes make them ideal candidates for in vivo orthopedic applications. The quest for new osteocompatible materials for load bearing tissue engineering applications has led us to investigate mechanically competent amino acid ester substituted polyphosphazenes. In this study, we have synthesized three biodegradable polyphosphazenes substituted with side groups, namely, leucine, valine, and phenylalanine ethyl esters. Of these polymers, the phenylalanine ethyl ester substituted polyphosphazene showed the highest glass transition temperature (41.6 degrees C) and, hence, was chosen as a candidate material for forming composite microspheres with 100 nm sized hydroxyapatite (nHAp). The fabricated composite microspheres were sintered into a three-dimensional (3-D) porous scaffold by adopting a dynamic solvent sintering approach. The composite microsphere scaffolds showed compressive moduli of 46-81 MPa with mean pore diameters in the range of 86-145 microm. The 3-D polyphosphazene-nHAp composite microsphere scaffolds showed good osteoblast cell adhesion, proliferation, and alkaline phosphatase expression and are potential suitors for bone tissue engineering applications.

Dipeptide-based polyphosphazene and polyester blends for bone tissue engineering.

Polyphosphazene-polyester blends are attractive materials for bone tissue engineering applications due to their controllable degradation pattern with non-toxic and neutral pH degradation products. In our ongoing quest for an ideal completely miscible polyphosphazene-polyester blend system, we report synthesis and characterization of a mixed-substituent biodegradable polyphosphazene poly[(glycine ethyl glycinato)(1)(phenyl phenoxy)(1)phosphazene] (PNGEG/PhPh) and its blends with a polyester. Two dipeptide-based blends namely 25:75 (Matrix1) and 50:50 (Matrix2) were produced at two different weight ratios of PNGEG/PhPh to poly(lactic acid-glycolic acid) (PLAGA). Blend miscibility was confirmed by differential scanning calorimetry, Fourier transform infrared spectroscopy, and scanning electron microscopy. Both blends resulted in higher tensile modulus and strength than the polyester. The blends showed a degradation rate in the order of Matrix2<Matrix1<PLAGA in phosphate buffered saline at 37 degrees C over 12 weeks. Significantly higher pH values of degradation media were observed for blends compared to PLAGA confirming the neutralization of PLAGA acidic degradation by polyphosphazene hydrolysis products. The blend components PLAGA and polyphosphazene exhibited a similar degradation pattern as characterized by the molecular weight loss. Furthermore, blends demonstrated significantly higher osteoblast growth rates compared to PLAGA while maintaining osteoblast phenotype over a 21-day culture. Both blends demonstrated improved biocompatibility in a rat subcutaneous implantation model compared to PLAGA over 12 weeks.

The influence of side group modification in polyphosphazenes on hydrolysis and cell adhesion of blends with PLGA.

Polyphosphazenes have been synthesized with tris(hydroxymethyl)amino methane (THAM) side groups and with co-substituents glycine ethyl ester and alanine ethyl ester. The THAM side group was linked to the polyphosphazene backbone via the amino function. The three pendent hydroxyl functions on each THAM side group were utilized for hydrogen bonding association with poly(glycolic-lactic acid) (PLGA). Co-substitution of the polyphosphazene with both THAM and glycine or alanine ethyl esters was employed to avoid the insolubility of the single-substituent THAM-substituted polyphosphazenes. Both poly[(tris(hydroxymethyl)aminomethane)(ethyl glycinato)phosphazene] and poly[(tris(hydroxymethyl)aminomethane)(ethyl alanato)phosphazene] (1:1 ratio of side groups) were blended with PLGA (50:50) or PLGA (85:15). DSC analysis indicated miscible blend formation, irrespective of the detailed molecular structure of the polyphosphazene or the composition of PLGA in the blend. Hydrolysis studies of the polyphosphazene:PLGA (50:50) blends indicated that the PLGA component hydrolyzed more rapidly than the polyphosphazene. Primary osteoblast cell studies showed good cell adhesion to the polymer blends during 14 days, but subsequent limited cell spreading due to increased surface roughness as the two polymers eroded at different rates.

Biomimetic, bioactive etheric polyphosphazene-poly(lactide-co-glycolide) blends for bone tissue engineering.

The long-term goal of this work is to develop biomimetic polymer-based systems for bone regeneration that both allow for neutral pH degradation products and have the ability to nucleate bonelike apatite. In this study, the etheric biodegradable polyphosphazene, poly[(50%ethyl glycinato)(50%methoxyethoxyethoxy)phosphazene] (PNEG(50)MEEP(50)) was blended with poly(lactide-co-glycolide) PLAGA and studied their ability to produce high-strength degradable biomaterials with bioactivity. Accordingly, two blends with weight ratios of PNEG(50)MEEP(50) to PLAGA 25:75 (BLEND25) and 50:50 (BLEND50) were fabricated using a mutual solvent approach. Increases in PNEG(50)MEEP(50) content in the blend system resulted in decreased elastic modulus of 779 MPa when compared with 1684 MPa (PLAGA) as well as tensile strength 7.9 MPa when compared with 25.7 MPa (PLAGA). However, the higher PNEG(50)MEEP(50) content in the blend system resulted in higher Ca/P atomic ratio of the apatite layer 1.35 (BLEND50) when compared with 0.69 (BLEND25) indicating improved biomimicry. Furthermore, these blends supported primary rat osteoblast adhesion and proliferation with an enhanced phenotypic expression when compared with PLAGA. These findings establish the suitability of PNEG(50)MEEP(50)-PLAGA biodegradable blends as promising bioactive materials for orthopedic applications.

Novel factor-loaded polyphosphazene matrices: Potential for driving angiogenesis

Currently employed bone tissue engineered scaffolds often lack the potential for vascularization, which may be enhanced through the incorporation of and regulated release of angiogenic factors. For this reason, the objective here was to fabricate and characterize protein-loaded amino acid ester polyphosphazene (Pphos)-based scaffolds and evaluate the novel sintering method used for protein incorporation, a method which will ultimately allow for the incorporation of proangiogenic agents. To test the hypothesis, Pphos and their composite microspheres with nanocrystalline hydroxyapatite (Pphos-HAp) were fabricated via the emulsion solvent evaporation method. Next, bovine serum albumin (BSA)-containing microsphere matrices were created using a novel solvent–non-solvent approach for protein loading. The resulting protein (BSA) loaded circular porous microsphere based scaffolds were characterized for morphology, porosity, protein structure, protein distribution and subsequent protein release pattern. Scanning electron microscopy revealed porous microsphere scaffolds with a smooth surface and sufficient level of sintering, illustrated by fusion of adjacent microspheres. The porosity measured for the poly(ethyl phenylalanato:glycinato)phosphazene (PNPhGly) and poly(ethyl phenylalanato:glycinato)phosphazene-hydroxyapatite (PNPhGly-HAp) scaffolds were 23 ± 0.11% and 18 ± 4.02%, respectively, and within the range of trabecular bone. Circular dichroism confirmed an intact secondary protein structure for BSA following the solvent sintering method used for loading and confocal microscopy verified that FITC-BSA was successfully entrapped both between adjacent microspheres and within the surface of the microspheres while sintering. For both Pphos and their composite microsphere scaffolds, BSA was released at a steady rate over a 21 day time period, following a zero order release profile. HAp particles in the composite scaffolds served to improve the release profile pattern, underscoring the potential of HAp for growth factor delivery. Moreover, the results of this work suggest that the solvent–non-solvent technique for protein loading is an optimal one that will allow for future development of angiogenic factor-loaded Pphos matrices with the capacity to invoke neovascularization.