Biological functionalisation of silicon substituted hydroxyapatite porous ceramics for bone tissue engineering – Céram-Os

The Céram-Os project is a multidisciplinary basic research. It involves four complementary academic partners in the fields of ceramic processing, bioorganic chemistry, cell biology and restorative surgery.
The chronological participation of the partners is intended to set up a complete new method for bone tissue regeneration. The project includes inorganic-organic materials processing, physico-chemical characterization of materials, in vitro and in vivo biological evaluations.
The project is divided into five thematic main tasks. Two additional tasks are specifically devoted to the project coordination and to the valorization of scientific results, which includes industrial prospective for further development of scaffolds for applications in bone tissue engineering.

Carbonate and silicate co-substituted hydroxyapatite (CSiHA) and silicon substituted hydroxyapatite (SiHA) powders were synthesized by precipitation in aqueous medium. Dense and flat ceramic pellets were produced by cold isostatic pressing and sintering. They have been used in order to establish and characterize the protocol of surface functionalization. These flat substrates will also be used to assess the biological properties of functionalized ceramics. This part of the program includes the assessment of sterilization for further animal experimentation.

The work done over the first 18 months of the project focused on the synthesis of carbonate and silicate co-substituted apatites (CsiHA) and silicon substituted apatite (SIHA) powders, on the fabrication of dense ceramic substrates and on the development of an immobilization protocol of the (73-92) peptide of the rh-BMP-2 protein onto the surface of these substrates. The specific strategy of grafting by silanization, specific coupling reactions and click-chemistry has allowed to obtain a covalent grafting of a low density of peptides (absence of polymer film on the surface of the ceramic) on the SiHA ceramics. The results obtained on the functionalization and characterization of functionalized dense ceramics have been the subject of an oral communication in an international conference.
Parallel to this work, the study of the bioactivity of the peptide sequence (73-92) of the rh-BMP2 compared to that of the complete protein was conducted. The results obtained using several protocols with two cell lines show that in contrast to the complete protein, the peptide did not induce any of the two signaling pathways (activity of alkaline phosphatase via the route of p38 / MAPK and induction of «BMP responsive elements« via the route of Smads) conventionally induced by rh-BMP2 protein.

Contracted work for the forthcoming period of the project are identical to those initially planned. The use of peptide (73-92) is abandoned because it did not give a positive result of bioactivity, Only rh-BMP2 protein will be implemented in further work in accordance with the replacement solution provided initially. The main actions will focus on the adaptation of grafting protocol to the complete protein. Protocols for the study of in vitro release kinetics of protein are currently being investigated.
The in vivo study of bioceramic functionalized rh-BMP2 protein will be programmed in a second time. It will be performed by subcutaneous implantation in rats. The animals will be handled according to the European guidelines for care and use of laboratory animals. Detailed animal testing protocols will be submitted for prior authorization from the Ministry of Research through an Ethics Committee.
In this light, ceramic scaffolds, shaped by a model replica process and then sintered, will be functionalized by the protein and sterilized before implantation.

Oral communication :
Elaboration of osteoinductive phosphocalcic bioceramics for bone tissue engineering
E. Poli, C. Damia, V. Chaleix, V. Sol, D. Marchat, N. Douard, D. Logeart, J. Brie, E. Champion
14th International Conference and Exhibition of the European Ceramic Society, EcerS 2015, Toledo (Spain), 23-25 Juin 2015

The reconstruction of large bone defects resulting from trauma, cancer or malformations remains a significant clinical issue. The current approaches include autografts, allografts or synthetic biomaterials but none of them are really satisfactory.
Calcium phosphate hydroxyapatite ceramics (HAC) have been used as synthetic bone graft substitutes for more than 30 years. Their biological properties (i.e. osteoconductivity) have led to the implantation of highly macroporous parts that conduct bone ingrowth within their interconnected framework of pores. However, the natural formation of new bone tissues is limited to small dimensions (up to about 1 cm from bone apposition). Thus, it should be biologically stimulated in order to extend applications of porous HAC to the repair of large osseous defects.
In this context our project relates to bone tissue engineering. It aims to develop a new generation of osteoinductive ceramic scaffolds able to maintain tissue volume and shape, control osteogenesis, guide and activate bone ingrowth and promote the vascular integration. The Céram-Os project is a multidisciplinary basic research. It involves four complementary academic partners in the fields of ceramic processing, bioorganic chemistry, cell biology and restorative surgery. The strategy is to functionalize the HAC surface with a peptide sequence of recombinant human bone morphogenetic protein (rh-BMP2) that exhibits osteoinductive properties, stimulates periosteal cells proliferation, and induces osteogenesis. The peptide will be immobilized onto the substrate surface via a biodegradable linker, which allows a controlled release of the osteoinductive molecule over time.
The project includes four main objectives:
(i) Manufacturing dense pellets and macroporous scaffolds made of silicon substituted hydroxyapatite Ca10(PO4)5.6(SiO4)0.4(OH)1.6 (SiHAC).
The powder will be synthesized by wet precipitation. The ceramic scaffolds will be shaped through a template casting process followed by sintering.
(ii) Biofunctionalisation of SiHAC
The functionalisation will be based on the use of a degradable linker covalently grafted to SiHAC surface and supporting rh-BMP2 peptide.
(iii) In vitro evaluation of the bioactivity of biofunctionalized SiHAC and release characteristics of peptide.
The biological activity of the immobilized peptide will be investigated using two complementary BMP responsive cell lines, (C3H10-BRE-Luc and C2C12 cells). The release kinetics of immobilized peptide will be characterized in culture medium and the bioactivity of the released peptide will be checked. Such evaluations will include the validation of the sterilization strategy.
(iv) In vivo biological evaluation of biofunctionalized scaffolds.
Osteogenic potential of biofunctionalized scaffolds and BMP peptide dose-response relationship will be evaluated in a subcutaneous rat model. Bone promotion and ingrowth in scaffolds prepared in various conditions (e.g. bare scaffolds, scaffolds with adsorded rh-BMP2 or scaffolds biofunctionalized with BMP-2 peptide) will be quantified using micro-CT and histomorphometry analyses.

Our strategy is based on the osteoinductive and vasculogenic potential of rh-BMP2 associated with the osteoconductive properties of calcium phosphate macroporous scaffold. The ceramic scaffold will supply continuous structural support (size and shape) and provide mechanical stability and integrity to the construct with high stiffness like bone. Its biofunctionalization will bring the ability to release active rh-BMP2 peptides at a desired rate for new bone formation and ingrowth.
The development of this new generation of functionalized bioceramics will represent a major shift towards "smart" materials able of promoting the bone formation. It will provide an adapted surgical solution for the filling and repair of large bone defects as an alternative to cancellous bone autograft or metal implants.