AbstractThe broad aim of this work was to investigate the in vitro osteoclast (OC) resorption of calcium phosphate (CaP) biomaterials. Resorption of CaP biomaterials is traditionally assessed using an OC resorption assay where resorption pits formed on the CaP surface are analysed by microscopy techniques and quantified on the basis of pit number, pit area or pit volume. Apart from the time consuming nature of the analysis techniques, there are many limitations associated with the current methods. Pit area measurements (2D) have become common practice when assessing CaP biomaterial resorption in vitro but it is not a precise indicator of resorption; variations in pit depth are not taken into consideration and it is unsuitable for use on porous materials where visualisation of internal structures is difficult or for use on materials with rough surfaces where determination of individual pits would be difficult. A 3D quantification of bioresorption is available but requires specialised, expensive equipment.
An appropriate measure of resorption was required to be more efficient and more cost-effective than the current available in vitro methods, but most importantly, to directly correlate with pit area measurements and have the ability to be used in a broad range of in vitro experiments. Thus, the aim of this work was to establish the suitability of several outcome measures as indicators of OC resorption in vitro that directly correlate with the current pit area measurements.
An OC resorption assay was developed for the generation of actively resorbing OC in vitro. Several experiments were conducted in order to optimise the resorbability of the generated OC. Cell culture variables included: cell type, OC differentiation factor concentration, media type, cell seeding density, pH and cell culture period.
Once the OC assay was established several outcome measures were assessed as potential indicators of resorption in vitro, namely; the correlation of percentage area resorbed in vitro with Ca and P mineral release into cell culture medium, OC number and OC activity. In order to accurately correlate pit area with the alternative outcome measures the assay was carried out using dense beta-Tricalcium Phosphate (P-TCP). The rationale for using a dense substrate was two-fold. Firstly, it allowed pit formation and area to be analysed devoid of material anomalies or pores, which are frequently observed with less dense substrates and can give rise to discrepancies within results. Secondly, the current 2D methods for measuring resorption perform best on a substrate free from imperfections.
This body of work has established two main outcome measures of bioresorption in vitro that correlate with pit area measurements; Ca and P mineral release into the culture medium and OC specific enzyme activity. Both outcome measures assess bioresorption using biochemical techniques. These outcome measures have shown potential as indicators of bioresorption in vitro and will prove invaluable for improving the fundamental understanding of OC resorption of CaP biomaterials.
|Date of Award||2015|
|Supervisor||Fraser Buchanan (Supervisor), Susan Clarke (Supervisor), Nicholas Dunne (Supervisor) & John Nelson (Supervisor)|