Poster abstract: Discovering Bioactive Compounds For Bone Growth: The Osteogenicity Of Marine Organism-derived Extracts

Introduction
Novel medicines from natural sources, or bioactives, hold potential for the treatment of a wide variety of clinical conditions. This project focuses on screening marine extracts for their osteogenic potential, with a view to developing novel medical treatments for conditions such as osteoporosis and the stimulation of fracture repair. The need for such novel treatment options is evidenced by current costs to the healthcare system, with estimates that the economic cost of osteoporosis for the UK alone was €5.4 billion in 2010, whilst that value was €37.4 billion for Europe as a whole(1).

As the project commenced in October 2014 work is still at the development stage, with initial blind screening currently taking place for a total 43 marine extracts. These extracts, from organisms such as algae, sponges and ascidians, were produced by Dr Margaret Rae of NUIG from organisms collected during the first two years of the Beaufort Marine Biodiscovery Research Programme.

It is hypothesized that some of these extracts may contain osteogenic factors capable of stimulating osteoblast activity and/or enhancing bone mineralisation. As a result, our leading experimental focus will be on outcome measures of osteoblast proliferation, differentiation and mineralisation in vitro. If an extract is shown to have a positive effect it may be suitable for application to areas of bone trauma, or for use in conjunction with synthetic bone substitutes.

Materials and methods
Marine extracts will first be reconstituted in an appropriate solvent. Our preliminary screen of the extracts will involve the use of PicoGreen, alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) assays on human foetal osteoblasts (hFOBs). Subsequent work will be carried out on a subset of these extracts using mesenchymal stem cells (MSCs) sourced from human bone marrow. Further outcome measures will include ELISA deduced osteocalcin expression and q-RT PCR for osteogenic markers such as osteopontin, bone sialoprotein and Cbfa-1/Runx2 expression. In addition, any enhancement in mineralisation will be detected via von Kossa and quantitative Alizarin Red S staining. The xCELLigence system, which allows frequent short term measures of both cell attachment and number, will be used to support PicoGreen results.

Discussion
The preliminary hFOB screen will allow future experiments to be focused on those extracts showing the greatest osteogenic potential. Subsequent experiments will investigate the ability of these extracts to stimulate MSCs, sourced from human bone marrow, to differentiate down the osteogenic lineage.

Discovery of unique bioactives in marine invertebrates is an active field, with the majority of previous work focusing on establishing the in vitro potential of marine extracts to promote and regulate osteoblast differentiation, leading to enhanced levels of mineralization(2,3). However, considering the vast number of potential marine extracts, work in this area is still in its infancy, whilst the mechanisms of many extract effects are poorly understood4. Mineralising organisms present the most likely source of bioactive compounds for bone growth, with a long evolutionary history of producing molecules to support calcified skeletal tissue formation.

Post screening there are numerous avenues for further research, including osteoclast related studies, work to elucidate the molecular mechanisms leading to the extracts effect and in vivo model testing. Overall, it is hoped that the current project will identify novel therapeutic compounds for multiple orthopaedic applications.

References
1. Hernlund E. et al. Arch. Osteoporos. 8: 136, 2013 2.O’Gorman D. M. et al. Phytother. Res. 26: 375-380, 2012
3. Nguyen M. et al. J. Aquat. Food. Prod. T. 23: 436-446, 2014 4. Green D. et al. Mar. Drugs 11:1203-20, 2013

Acknowledgements
The Beaufort Marine Research Award.