Polyphosphazene/nano-hydroxyapatite composite microsphere scaffolds for bone tissue engineering

Abstract

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.

Figure 1

Structure of (a) general polyphosphazene (PPH) and (b) poly[bis(ethyl phenylalaninato)phosphazene] (PNEPhA). PNEPhA is a biodegradable polymer with a glass transition temperature close to popular polyester PLAGA 85/15.

Figure 2

Scanning electron micrographs of polymer (a) PNEPhA, and composite (b) 90PNEPhA-10nHAp, (c) 80PNEPhA-20nHAp and (d) 70PNEPhA-30nHAp microspheres. The presence of nHAp on the microsphere surface resulted in surface roughness, and the roughness increased with nHAp loading. However, composite microspheres did not form beyond 30% of nHAp loading

Figure 3

Schematics of composite microsphere sintering: a solvent/non-solvent approach. In the presence of a dynamic solvent, microsphere surfaces swell and open up the polymer chains. The chain interaction between the adjacent microspheres leads to microsphere bonding. With continued solution evaporation (solvent more than non-solvent), the dynamic solvent transforms from a poor solvent to a non-solvent state, resulting permanent bonding between the adjacent microspheres.

Figure 4

Scanning electron micrographs showing the morphology of the scaffolds sintered using solvent/non-solvent compositions of (a) 15T-85H, (b) 17.5T-87.5H, (c) 20T-80H and (d) 22.5T-77.5H. Three-dimensional scaffolds showed an interconnected pore structure and the inset shown for every scaffold confirms the bonding between the adjacent microspheres. Where T is for THF and H is for hexane.

Figure 5

Effect of solvent/non-solvent composition on scaffold mechanical properties, where (a) is compressive modulus, and (b) is compressive strength. Both compressive modulus and the compressive strength increase with increase with THF content in the dynamic solvent. (*)Denotes significant difference with p<0.05.

Figure 6

Effect of solvent/non-solvent composition on scaffold porosity properties, where (a) is median pore diameter, and (b) is pore volume (%). Both pore size and percent of porosity, evaluated using mercury porosimetry, show a decreasing trend with increasing THF content in the dynamic solvent. (*) denotes significant difference with p<0.05.

Figure 7

Scanning electron micrograph showing primary rat osteoblast cell proliferation recorded at day 7. Cell presence is clearly seen in the microsphere adjoining areas. Well spread cells on the microsphere surface (other than the microsphere junctions) are shown in the inset.

Figure 8

Cytoskeletal actin distribution of primary rat osteoblast cells grown on composite microsphere matrix for (a) 2, (b) 6 and (c) 12 days. The circled region shows higher initial cell proliferation at the microsphere adjoining areas. DAPI (nuclei stain) emission is not included because of its interference with polymer PNEPhA blue emission.

Figure 9

Primary rat osteoblast cell proliferation (MTS assay) on PNEPhA-20nHAp, PLAGA-20nHAp composite scaffolds and planar TCPS. Progressive growth on TCPS surface is a sign of a healthy PRO culture. (*) Denotes significant difference with p<0.05.

Figure 10

Alkaline phosphatase activity expressed by primary rat osteoblast cells on PNEPhA-20nHAp, PLAGA-20nHAp composite scaffolds and planar TCPS. (*) Denotes significant difference with p<0.05.

Figure 11

Macro, micro and nano structure of PNEPhA-20 nHAp composite microsphere scaffolds. (a) Optical image showing cylindrical (10 mm length & 4.5 mm diameter) and disk (2 mm thick & 8 mm diameter) shaped scaffolds fabricated using the dynamic solvent sintering method. Cylindrical scaffolds were used for mechanical testing, and disk shaped scaffolds for porosity and in vitro cell studies. (b) SEM showing the microstructure of the scaffolds where the adjacent microspheres are fused via the dynamic solvent sintering method. (c) High magnification scanning electron micrograph showing nano HAp particle dispersion on a microsphere surface. The circled regions show nHAp mono (solid line) and poly (dotted line) dispersion.