Quantifying the efficiency of hydroxyapatite mineralising peptides

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+ = 200mM), as also seen with peptide-silk chimeric materials, whereas CaP(S) was most effective when lower concentrations of calcium (20mM) and phosphate were used. The approach can be extended to investigate HAP mineralisation in the presence of any number of mineralisation promoters or inhibitors

Identification of a Cardiac Specific Protein Transduction Domain by In Vivo Biopanning Using a M13 Phage Peptide Display Library in Mice

Background: A peptide able to transduce cardiac tissue specifically, delivering cargoes to the heart, would be of significant therapeutic potential for delivery of small molecules, proteins and nucleic acids. In order to identify peptide(s) able to transduce heart tissue, biopanning was performed in cell culture and in vivo with a M13 phage peptide display library. Methods and Results: A cardiomyoblast cell line, H9C2, was incubated with a M13 phage 12 amino acid peptide display library. Internalized phage was recovered, amplified and then subjected to a total of three rounds of in vivo biopanning where infectious phage was isolated from cardiac tissue following intravenous injection. After the third round, 60% of sequenced plaques carried the peptide sequence APWHLSSQYSRT, termed cardiac targeting peptide (CTP). We demonstrate that CTP was able to transduce cardiomyocytes functionally in culture in a concentration and cell-type dependent manner. Mice injected with CTP showed significant transduction of heart tissue with minimal uptake by lung and kidney capillaries, and no uptake in liver, skeletal muscle, spleen or brain. The level of heart transduction by CTP also was greater than with a cationic transduction domain. Conclusions: Biopanning using a peptide phage display library identified a peptide able to transduce heart tissue in vivo efficiently and specifically. CTP could be used to deliver therapeutic peptides, proteins and nucleic acid specifically to the heart. © 2010 Zahid et al

Porous polymer/hydroxyapatite scaffolds: characterization and biocompatibility investigations.

Contains fulltext : 79508.pdf (Publisher’s version ) (Closed access)Poly-lactic-glycolic acid (PLGA) has been widely used as a scaffold material for bone tissue engineering applications. 3D sponge-like porous scaffolds have previously been generated through a solvent casting and salt leaching technique. In this study, polymer-ceramic composite scaffolds were created by immersing PLGA scaffolds in simulated body fluid, leading to the formation of a hydroxyapatite (HAP) coating. The presence of a HAP layer was confirmed using scanning electron microscopy, energy dispersive X-ray spectroscopy and Fourier transform infrared spectroscopy in attenuated total reflection mode. HAP-coated PLGA scaffolds were tested for their biocompatibility in vitro using human osteoblast cell cultures. Biocompatibility was assessed by standard tests for cell proliferation (MTT, WST), as well as fluorescence microscopy after standard cell vitality staining procedures. It was shown that PLGA-HAP composites support osteoblast growth and vitality, paving the way for applications as bone tissue engineering scaffolds