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Tissue engineered bone formation, substitution and regeneration: application of porous calcium phosphate scaffold materials and growth factors.

Deliverables

Sintered porous materials have a very low specific surface area and the release of active proteins is essentially determined by physical processes (diffusion of molecules), which are difficult to control. The coating applied to the inner pores of the ceramic is a nanocrystalline apatite with a very high specific surface area and a high surface reactivity. It can bind active molecules and release them either as a consequence of crystal maturation, surface displacement (by ions or other molecules) and/or cell activity. The nanocrystalline apatite may have their chemical composition and surface characteristics adapted for a specific use. Multilayer coatings can be realized with sequential release of active molecules. The coating process has been registered as an "enveloppe Soleau" (number 145689, 22/10/2002) at the French National Institute of Industrial Property
OBL have listed all the current defects cases of craniomaxillofacial surgery and dentistry area. It has also been concluded that there is no need to make FEA studies in the case of non-load bearing devices, which are the most common in craniomaxillofacial or dentistry surgery.
An appropriate method for loading and incorporating the growth factor has been achieved and this is a vacuum technique. Release kinetic studies have shown that an incorporated growth factor (growth hormone) can be released; this can be applied to other GF. The loading dose required for sustained release over a specific time period has been determined. The released GF has retained biological activity, as indicated by tests using primary human osteoblast-like cells
Typically, information on the pore size distribution in a sample is derived from such methods as image analysis. This can be time consuming and requires access to specialised equipment. In a structure containing more than one level of porosity these methods can lead to misleading results. Where the analyser fails to differentiate between macropores and the interconnections between them the data will be skewed to the smaller end of the spectrum. An alternative method was therefore developed based on techniques commonly used by porous sintered metal filter manufacturers (ASTM F902). This gas permeability technique produces a dimension for the diameter of an equivalent cylindrical capillary based on an idealised approximation of the porous structure. By analysing SEM micrographs of a range of the samples produced by our foaming method, we were able to establish a relationship that accurately related the results of the gas permeability test to mean macropore size of our porous microstructures. The advantages of this technique are that it is simple to set up and quick to perform. It is envisaged that the data produced by this test could be the basis of a quality control procedure allowing rapid verification and validation of the production process
A proprietary method for producing 3-Dimensional porous scaffolds with controlled highly interconnected porosity (open), <85%> as well as substrates with gradient porosity has been developed. The method has been optimised for biocompatible calcium phosphate materials such as Hydroxyapatite (HA) and Tricalcium phosphate (TCP). Laboratory samples and prototypes have been produced. In vitro and in vivo performance of the materials have been documented and show very good results. This method provides an alternative production technique for the production of scaffolds for bone replacement materials and for tissue engineering. The scaffolds are indicated for bone tissue regeneration with and without activation using cells, Platelet rich plasma and growth factors. FIN-Ceramica Faenza developed an innovative method for the production of Ca/P 3-D scaffold. The scaffold is characterised with high porosity degree (70-90 vol.%) and controlled porosity (micro, macro and interconnection porosity). The Ca/P3-D scaffold are indicated for bone defect regeneration, to be use alone or in combination with cells, PRP or growth factors. A European Patent Application has been filed.
A unique foaming technology produces a ceramic foam microstructure comprising of cells, windows and struts. The cells are formed from a series of struts, which are fired to near theoretical density. By varying the processing parameters, the material can be produced with porosities between 50 and 90%. The size of the macropores and the interconnections between can be readily adjusted. The gel casting method employed allows the production of near net shape forms with either simple or complex geometry. The product can also be easily machined using conventional equipment. This technique has now been developed and optimised for use with biocompatible materials (calcium phosphates) such as hydroxyapatite (HA), tri-calcium phosphate (TCP) and biphasic HA/TCP. From the batches produced, discs for in vitro studies and blocks and granules for in vivo studies and physical and mechanical characterisation have been inputted into the PORELEASE project. Produced under controlled conditions and specifications using the ISO EN 13485 standard, HI-POR's scaffolds are approved for CE marking as Class III medical devices.
New scaffolds for bone regeneration present characteristics have to undergo a series of controls before being "passed". A reliable way of verifying the ability of the scaffolds to allow bone formation and vascularisation is the use of ectopic bone formation assays in a small animal model. Bone formation has been therefore assessed by implanting "in vivo" human Osteoprogenitor Cells (Bone Marrow Stromal Cells) combined to the new bioceramic scaffolds subcutaneously in immunodeficient mice. In this system human bone formation occurs within few weeks. This model system also allows the study of bone scaffolds interaction. Preliminary results have indicated excellent bone formation and in general superior properties of new scaffold for bone regeneration
FTIR spectroscopy and X-rays diffraction (XRD) techniques have been used to control the purity of the synthesised calcium phosphate powders and of the produced ceramics as well as to evaluate the modifications of explanted scaffold from small animals. Improvements of existing methods have been achieved to identify and quantify the main impurities, to evaluate the ceramic HA/TCP ratio, the organic matrix deposition and bone neoformation on explanted calcium phosphate scaffolds. Surface composition and physical properties changes can interfere with the sintering process as well as with the biological behaviour of the bioceramics. Mg and carbon have been identified by X-ray photoelectron spectroscopy (XPS) and surface energy determined by the contact angle method (Owens-Wendt theory). All these surface and bulk improved characterisation procedures will be published as they can be applied to other samples in other programs. The proceedings of the Bioceramics 16th conference held in Porto in 2003 were published (Processing of Ca-P ceramics, surface characteristics and biological performance by S. Cazalbou et al, Key Engineering Materials, vols. 254-256 (2004), 833-836). The writing of another manuscript on the improvement of the analysis procedures of explanted Ca-P ceramic samples is in progress