The physiological effects of 1,25 vitamin D3 (1,25D) are well known and the previously held dogma was that this was the only active vitamin D metabolite. A number of methods have been employed to demonstrate the effects of 24,25-dihydroxyvitamin D3 (24,25D) on osteoblast maturation responses, in the presence of FHBP, ((3S) 1-Fluoro-3-hydroxy-4-(oleoyloxy)butyl-1-phosphonate), an agonist of lysophosphatidic acid (LPA). These include alkaline phosphatase (ALP) expression and investigation of the role of CYP27B1, which is the enzyme responsible for converting 24,25D to 1,24,25D. Ketoconazole, which inhibits the actions of CYP27B1, as well as an enzyme-linked immunosorbant assay (ELISA) for CYP27B1 were used. The results clearly demonstrate that 24,25D stimulates maturation of MG63 cells when combined with FHBP. It has also been shown that the metabolite is not converted to another active form (for example, 1,24,25D) within osteoblasts, due to the absence of CYP27B1. 24,25D is an active vitamin D metabolite and exerts its effects in a bone fide manner, rather than following conversion to another active metabolite in osteoblasts. Given it is non-calcaemic, this metabolite has the exciting potential of being used in a bone regenerative setting in orthopaedic applications.
Successful osseointegration requires the production of a mechanically competent collagenous matrix, by osteoblasts, at the implant site. Lysophosphatidic acid (LPA) is a bioactive lipid which we discovered interacts with vitamin D3 (D3) to secure human osteoblast (hOB) maturation on both titanium (Ti) and hydroxyapatite. We therefore covalently attached LPA and a related compound, (3S) 1-fluoro-3-hydroxy-4-butyl-1-phosphate (FHBP), to both solid and porous Ti discs and seeded them with hOBs to assess their ability to support D3-induced cell maturation. Solid functionalised discs were washed and reused a further two times, whilst other discs were stored for 6 months. Increased alkaline phosphatase (ALP) activity indicated that both LPA and FHBP-modified Ti serve as superior substrates for securing D3-induced hOB maturation compared to unmodified metal (p < 0.001). Although total ALP activity was less for cells on recycled discs and after storage, enzyme levels were still significantly greater compared to hOBs grown on control Ti. LPA and D3 co-treatment also resulted in an increase in osteocalcin (∼17ng/ml versus 6ng/ml for D3 alone, P < 0.001) and collagen synthesis (∼310pg/ml versus <10pg/ml for D3 alone, P < 0.001). Research is ongoing to evaluate the efficacy of our modified Ti surfaces to secure hOB formation from their stem cell progenitors.
