Introduction: The classic tissue engineering triad highlights the requirement of a porous scaffold, osteogenic cells and appropriate growth factor(s) to drive the healing stimulus, but in some instances all three elements are not required. This work focused on development of a tri-composite, biomimetic scaffold consisting of the marine derived biomaterial chitosan, hydroxyapatite (HA) particles and a marine derived growth factor (MDGF). The hypothesis was that a structure of chitosan/HA would mimic the natural bone architecture providing an appropriate scaffold for bone regeneration and MDGF would provide a biological stimulus. The aim of the current study was to develop methodology for producing such a tricomposite scaffold and provide an initial in vitro assessment of the components ability to support osteogenesis.
Methods: The proliferation and differentiation of human mesenchymal stem cells (MSCs) on chitosan, MDGF and HA individually and in combination, were investigated. For scaffold preparation, 1% chitosan solution was prepared in 0.05% acetic acid using purified chitosan and neutralized by adding 1M NaOH before freeze-drying. HA (nano-powder <200nm particle size) and MDGF were tested individually at concentrations of 200, 100, 50, 25, 12.5, 6.25 and 3.125μg/ml in complete growth medium. Tri-composite scaffolds were prepared using a poly-blend method adapted from Thein Han et al[1]. Scaffolds were characterised using SEM, FTIR and the porosity and density was measured by a liquid displacement method. Bone marrow derived human MSCs were isolated and expanded in culture. Cytotoxicity of single, bi and tri-composites were evaluated at 24, 48 and 72 hrs using reazurin assay; differentiation was assessed using alizarin red S assay or alkaline phosphatase activity assay at time points up to 25 days.
Results and Discussion: SEM studies showed that the tri-composite scaffold had a uniform porous structure - a key requirement for cell migration, proliferation and vascularisation. FTIR and XRF confirmed the presence of HA and MDGF in the scaffold which resulted in a slight decrease in porosity and increase in density. On its own, MDGF was found to inhibit cell proliferation at higher concentrations and at earlier time points: low concentrations and higher concentrations at 72 hours had no effect on proliferation. Similar results were seen with HA alone, however both HA and MDGF were found to increase MSC differentiation as measured by calcium deposition. Differentiation was significantly enhanced in MSCs cultured on the tri-composite, with increased alkaline phosphatase activity at days 17 and 25.
Figure 1. SEM of tricomposite scaffold
Conclusion: Cytotoxicity and differentiation studies of MSCs on bi and tri-composites showed that there was some evidence of a stimulatory effect on cell proliferation and very strong evidence that the tri-composite scaffold enhances osteogenic differentiation in excess of that seen by inclusion of the individual components. It can be concluded that the tri-composite is biocompatible, promotes osteogenesis and has the structural and compositional properties required of a scaffold for bone tissue engineering.
Funded by the Institute of Technology Tralee
References:
[1] Thein-Han WW, Misra RDK. Biomimetic chitosan-nanohydroxyapatite composite scaffolds for bone tissue engineering. Acta Biomaterialia 2009;5:1182-97.