Abstract
There is continued focus on the development of new biomaterials and associated biological testing methods needed to reduce the time taken for their entry to clinical use. The application of Raman spectroscopy to the study of individual cells that have been in contact with biomaterials offers enhanced in vitro information in a potentially non-destructive testing regime. The work presented here reports the Raman spectral analysis of discreet U-2 OS bone cells after exposure to hydroxyapatite (HA) coated titanium (Ti) substrates in both the as-deposited and thermally annealed states. These data show that cells that were in contact with the bioactive HA surface for 7 days had spectral markers similar to those cultured on the Ti substrate control for the same period. However, the spectral features for those cells that were in contact with the annealed HA surface had indicators of significant differentiation at day 21 while cells on the as-deposited surface did not show these Raman changes until day 28. The cells adhered to pristine Ti control surface showed no spectral changes at any of the timepoints studied. The validity of these spectroscopic results has been confirmed using data from standard in vitro cell viability, adhesion, and proliferation assays over the same 28-day culture period. In this case, cell maturation was evidenced by the formation of natural bone apatite, which precipitated intracellularly for cells exposed to both types of HA-coated Ti at 21 and 28 days, respectively. The properties of the intracellular apatite were markedly different from that of the synthetic HA used to coat the Ti substrate with an average particle size of 230 nm, a crystalline-like shape and Ca/P ratio of 1.63 ± 0.5 as determined by SEM-EDX analysis. By comparison, the synthetic HA particles used as a control had an average size of 372 nm and were more-rounded in shape with a Ca/P ratio of 0.8 by XPS analysis and 1.28 by SEM-EDX analysis. This study shows that Raman spectroscopy can be employed to monitor single U-2 OS cell response to biomaterials that promote cell maturation towards de novo bone thereby offering a label-free in vitro testing method that allows for non-destructive analyses.
Highlights
Methods to accurately test the biological response of cells to biomaterials, and the devices fabricated from them, are continually being developed
Hydroxyapatite (HA) (Ca10(PO4)6OH2) has been widely used as a coating on Ti to promote bone cell adhesion, proliferation and maturation and, to support the formation of functional bone tissue [4, 5]. This osteoconductive bioactive material is similar in chemical structure to bone apatite, it has a stoichiometric Ca/P ratio of 1.67 whereas, natural apatite is typically non-stoichiometric and either deficient in Ca or P, and it stimulates bone cells to attach, migrate and proliferate along its surface [6]
The presence of X-ray photoelectron spectroscopy (XPS) peaks namely Ca 2p, O 1s and P 2p confirmed that CaP, in this case HA, was successfully coated onto Ti substrates with Ca 2p and P 2p peaks at 347.43 and 133.43 eV with atomic concentrations of 13.25% and 16.37%, respectively (Fig. 1a)
Summary
Methods to accurately test the biological response of cells to biomaterials, and the devices fabricated from them, are continually being developed. In the case of in vitro testing techniques, it is common to investigate the response of specific cells to a biomaterial in the context of a proposed clinical application for the attendant device such as, for tissue repair, restoration, or replacement purposes In this context, the appropriate measure of biocompatibility must extend from bioinert to bioactive systems wherein cells adhere, proliferate and differentiate on the biomaterial or device depending on specific properties [1]. Surface modification and/or coating of Ti with other materials is commonly used to minimise or eliminate these issues In this regard, hydroxyapatite (HA) (Ca10(PO4)6OH2) has been widely used as a coating on Ti to promote bone cell adhesion, proliferation and maturation and, to support the formation of functional bone tissue [4, 5]. This osteoconductive bioactive material is similar in chemical structure to bone apatite, it has a stoichiometric Ca/P ratio of 1.67 whereas, natural apatite is typically non-stoichiometric and either deficient in Ca or P, and it stimulates bone cells to attach, migrate and proliferate along its surface [6]
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