Development and biological evaluation of fluorophosphonate-modified hydroxyapatite for orthopaedic applications

Abstract

There is an incentive to functionalise hydroxyapatite (HA) for orthopaedic implant use with bioactive agents to encourage superior integration of the implants into host bone. One such agent is (3S) 1-fluoro-3-hydroxy-4-(oleoyloxy) butyl-1-phosphonate (FHBP), a phosphatase-resistant lysophosphatidic acid (LPA) analogue. We investigated the effect of an FHBP-HA coating on the maturation of human (MG63) osteoblast-like cells. Optimal coating conditions were identified and cell maturation on modified and unmodified, control HA surfaces was assessed. Stress tests were performed to evaluate coating survivorship after exposure to mechanical and thermal insults that are routinely encountered in the clinical environment. MG63 maturation was found to be three times greater on FHBP-modified HA compared to controls (p < 0.0001). There was no significant loss of coating bioactivity after autoclaving (P = 0.9813) although functionality declined by 67% after mechanical cleaning and reuse (p < 0.0001). The bioactivity of modified disks was significantly greater than that of controls following storage for up to six months (p < 0.001). Herein we demonstrate that HA can be functionalised with FHBP in a facile, scalable manner and that this novel surface has the capacity to enhance osteoblast maturation. Improving the biological performance of HA in a bone regenerative setting could be realised through the simple conjugation of bioactive LPA species in the future. Depicted is a stylised summary of hydroxyapatite (HA) surface modification using an analogue of lysophosphatidic acid, FHBP. a HA surfaces are simply steeped in an aqueous solution of 2 μM FHBP. b The polar head group of some FHBP molecules react with available hydroxyl residues at the mineral surfaces forming robust HA-O-P bonds leaving acyl chain extensions perpendicular to the HA surface. These fatty acyl chains provide points of integration for other FHBP molecules to facilitate their self-assembly. This final surface finish enhanced the human osteoblast maturation response to calcitriol, the active vitamin D3 metabolite.

Polydopamine-Lysophosphatidate-Functionalised Titanium: A Novel Hybrid Surface Finish for Bone Regenerative Applications

“…Evidence of osteoblast maturation was via total alkaline phosphatase (ALP) activity-specifically, the quantification of p-NP formation from p-NPP. In support of our previous work using FHBP [25], the direct functionalisation of Ti with this LPA analogue generated a surface that was superior to blank Ti at supporting 1,25D-induced MG63 maturation (Figure 4). The increase in p-NP generation for FHBP-Ti compared to blank Ti was significant by approximately 2.2-fold (p < 0.0001).…”

“…As a phosphonic acid, FHBP would be predicted to bond robustly to Ti [34,35]. When we steeped Ti in a 2-µM solution of FHBP, the resultant surface supported 1,25D-induced osteoblast maturation akin to what we have observed previously for hydroxyapatite [25]. However, the extent of cellular maturation was further enhanced when FHBP was applied to Ti in a mildly alkaline solution of DHC.…”

“…Using 10mM Tris (pH 8.8) as the diluent, FHBP was prepared at a final concentration of 2 µM, and a portion was immediately dispensed onto samples (1 mL/Ti disc). The FHBP concentration was informed from our previous work with this compound for Ti and hydroxyapatite (HA) functionalisations [7,25]. The remaining solution was subsequently combined to solid dopamine hydrochloride (DHC) to achieve a final mass concentration of 2 mg/mL, and the resultant solution was dispensed (1 mL/Ti disc) within a minute of gentle mixing.…”

Development of a facile fluorophosphonate-functionalised titanium surface for potential orthopaedic applications

“…Secondary ion mass spectrometry (SIMS) analysis concentrated on control (blank) and 2 ​μM FHBP-coated specimens. The FHBP steeping concentration was informed from our previous work pertaining to the FHBP-functionalisation of HAp [ 27 ]. Two different initial surface finishes of the Ti6Al4V discs were used, as-received (presenting a relatively rough surface) and polished (smooth at the micro-scale).…”

“…The original, as-received surface finish proved to be too rough for the HS-AFM technique. Therefore, analysis concentrated on imaging control (blank) and 2 ​μM FHBP-coated polished specimens, as informed from our previous work pertaining to the functionalisation of HAp with FHBP [ 27 ].…”