Abstract
We present a non-destructive analytical calibration tool to allow quantitative assessment of individual calcium phosphates such as hydroxyapatite (HAP) from mixtures including brushite. Many experimental approaches are used to evaluate the mineralising capabilities of biomolecules including peptides. However, it is difficult to quantitatively compare the efficacy of peptides in the promotion of mineralisation when inseparable mixtures of different minerals are produced. To address this challenge, a series of hydroxyapatite and brushite mixtures were produced as a percent/weight (0–100%) from pure components and multiple (N = 10) XRD patterns were collected for each mixture. A linear relationship between the ratio of selected peak heights and the molar ratio was found. Using this method, the mineralising capabilities of three known hydroxyapatite binding peptides, CaP(S) STLPIPHEFSRE, CaP(V) VTKHLNQISQSY and CaP(H) SVSVGMKPSPRP, was compared. All three directed mineralisation towards hydroxyapatite in a peptide concentration dependent manner. CaP(V) was most effective at inducing hydroxyapatite formation at higher reagent levels (Ca2+ = 200 mM), as also seen with peptide-silk chimeric materials, whereas CaP(S) was most effective when lower concentrations of calcium (20 mM) and phosphate were used. The approach can be extended to investigate HAP mineralisation in the presence of any number of mineralisation promoters or inhibitors.
Highlights
In peptide driven hydroxyapatite (HAP) synthesis it is often the case that inseparable mixtures of various calcium phosphate crystalline materials are produced[3]
Patterns produced from highly crystalline materials require little processing so there is a minimal risk of loss of information[24]
infra-red spectroscopy (IR) and Raman spectroscopy were excluded as both require a higher level of processing on the spectra to gain quantifiable data
Summary
In peptide driven hydroxyapatite (HAP) synthesis it is often the case that inseparable mixtures of various calcium phosphate crystalline materials are produced[3]. In this study a series of HAP/brushite mixtures with increasing molar ratios of HAP to brushite (the most commonly formed calcium phosphate without mineralising directing influences present)[4,5,6,7] have been analysed with the intention of creating an analytical method applicable to the quantifiable estimation of, in this case, peptides that promote HAP mineralisation. Three HAP binding peptides, all of which were previously identified via phage display: CaP(S) STLPIPHEFSRE8–11, CaP(V) VTKHLNQISQSY10, 12–15 and CaP(H) SVSVGMKPSPRP8, 13, 16, 17, have been used in this study. An extension of the approach to quantify the effect on mineralisation of larger proteins such as silk chimeras built from mimics of spider silk MASP1 protein and the CaP(V) peptide is included[18]
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