Phosphate metabolism is central to the bone formation pathway. Phosphate is shuttled through the cell membrane to the mitochondria, where it is polymerised to form adenosine triphosphate. Once exocytosed the ATP may then be cleaved to form pyro and orthophosphates, the balance of which can determine whether mineralisation occurs or not. We are developing a range of materials at the University of Birmingham that have been formulated so that they can influence this balance, with the potential either to drive or prevent mineralisation from occurring. This talk will describe how we have used this process to develop materials that can be used to stimulate bone formation around an implant or to prevent the formation of pathological bone. It will also talk about the steps that we have taken to move these therapies towards clinical trial.
Bone is a hierarchically structured hard tissue that consists of approximately 70 wt% low-crystallinity hydroxyapatite. Intricate tubular channels, such as Haversian canals, Volkman's canals, and canaliculi are a preserved feature of bone microstructure. These structures provide pathways for vasculature and facilitate cell-to-cell communication processes, together supporting viability of cellular components and aiding in remodeling processes. Unfortunately, many commercial bone augmentation materials consist of highly crystalline phases that are absent of the structuring present within the native tissue they are replacing. This work reports on a the development of a novel bone augmentation material that is able to generate biologically analogous tubular calcium phosphate mineral structures from hydrogel-based spheres that can be packed into defects similar to those encountered in vivo. Calcium loaded spheres were made by adding 5 wt% agar powder to 1 M calcium nitrate solutions, before heating the mixture to 80–90 oC and feeding droplets of gel into a reservoir of liquid nitrogen. Deposition of tubular mineral was initiated by exposure to ammonium phosphate solutions at concentrations between 500 mM and 1 M, and was characterized by micro-XRF mapping, XRD and SEM techniques. For an ex vivo model, human bone tissue was collected from patients undergoing elective knee replacement surgery. The United Kingdom National Research Ethics Service (East of Scotland Research Ethics Service) provided ethical approval (11/ES/1044). The augmented defect of the model was characterised by micro-XRF mapping and micro-CT techniques.Background
Experimental
Investigations into the response to implant debris tend to concentrate on how a population of cells proliferate in the presence of implant material, and how the regulation of cytokines change. For the problem of cobalt-chromium (CoCr) implants this has been done for osteoblasts and osteoclasts to understand how bone resorption, leading to aseptic loosening, is occurring. However, investigating the formation of the extracellular-matrix (ECM) may give a better indication of the mechanisms occurring. ECM is excreted from cells and is important for adhesion, structure, signaling and growth. Type I collagen is the most abundant protein in the ECM and is known to direct tissue development and is therefore a key part of understanding the mechanism behind aseptic loosening. 3T3-fibroblasts were seeded in Dulbecco's Modified Eagle Medium (DMEM) and supplemented with 100mM ascorbic acid. Every 48hours cells were fed with DMEM and doped with Co and Cr ions until fixation. Sirius Red dye was used to bind to the type I collagen, then removed using NaOH and analysed using UV absorption to show relative amounts of collagen. Type I collagen gel was formed in the presence of Co and Cr ions with and without DMEM and the fibers were imaged using AFM.Background
Methods
Calcium orthophosphates, such as hydroxyapatite (Ca5(PO4)3OH) (HA), have long been employed as bone graft materials. Recent work has suggested that calcium pyrophosphate (Ca2P2O7) (CaPy) may strongly stimulate bone deposition. In this study we compare calcium orthophosphate and pyrophosphate precipitates as suitable bone regeneration materials. As well as HA, two forms of pyrophosphate precipitate were compared in this work: amorphous calcium pyrophosphate (amCaPy) and star particle calcium pyrophosphate (stCaPy). Briefly, 0.15M Na4P2O7·10H2O and 0.3M Ca2Cl·2H2O solutions of equivalent volume were combined and left to age before performing a series of filtration and re-suspension steps upon the precipitate. Drying yielded amCaPy powder. stAmPy was produced by the same procedure however the pH of the starting solutions were altered to pH7 before combination.Background
Methods
Calcium phosphate (CaP) particles have attracted great interest as transfection reagents, yet little is known about their mechanism of internalisation. We report live cell time-course tracking of CaP particles during internalisation and the influence of Ca:P ratio on transfection efficiency. Relatively recent work has seen calcium phosphate (CaP) salts used for the delivery of biological materials into cells in the form of peptides, polymers and DNA sequences. Calcium phosphate salts have a critical safety advantage over other vectors such as viruses in that they pose no risk of pathogenicity due to mutation and show no apparent cytotoxicity. Previous work within the group showed that Ca:P ratio influenced the transfection efficiency, but the fate of the particles on internalisation is yet unknown. The difficulty in tracking the particles can be related to the visual similarity to granulation within the cells. Using a surface modification method that enables the fluorescent labeling of silicon-substituted hydroxyapatite (SiHA) particles, we have tracked the internalisation of the particles to understand their mechanism of entry and how particle composition may influence transfection efficiency.Summary Statement
Introduction
