Worldwide 500,000 cases of maxillofacial cancer are diagnosed each year. After surgery, the reconstruction of large bone defect is often required. The induced membrane approach (Masquelet, 2000) is one of the strategies, but exhibits limitations in an oncological context (use of autografts with or without autologous cells and Bone Morphogenetic Proteins). The objectives of this work are to develop an injectable osteoinductive and osteoconductive composite matrix composed of doped strontium (Sr) hydroxyapatite (HA) particles dispersed within a polysaccharide scaffold, to evaluate in vitro their ability to stimulate osteoblastic differentiation of human mesenchymal stem cells (hMSC) and to stimulate in vivo bone tissue regeneration.

HA particles were synthesized with different ratios of Sr. X-ray diffraction (XRD), Inductively Coupled Plasma (ICP), and particle size analysis (Nanosizer™) were used to characterize these particles. HA and Sr-doped HA were dispersed at different ratios within a pullulan-dextran based matrices (Autissier, 2010), Electronic scanning microscopy Back Scattering Electron microscopy (ESEM-BSE) and ICP were used to characterize the composite scaffolds. In vitro assays were performed using hMSC (cell viability using Live/Dead assay, expression of osteoblastic markers by quantitative Polymerase Chain Reaction). Matrices containing these different particles were implanted subcutaneously in mice and analyzed by Micro-Computed Tomography (micro-CT) and histologically (Masson's trichrome staining) after 2 and 4 weeks of implantation.

XRD analysis was compatible with a carbonated hydroxyapatite and patterns of Sr-doped HA are consistent of Sr substitution on HA particles. Morphological evaluation (TEM and Nanosizer™) showed that HA and Sr-doped HA particles form agglomerates (150 nm to 4 µm). Matrices composed with different ratios of HA or Sr-doped-HA, exhibit a homogenous distribution of the particles (ESEM-BSE), whatever the conditions of substitution. In vitro studies revealed that Sr-doped HA particles within the matrix stimulates the expression of osteoblastic markers, compared to non-doped HA matrices. Subcutaneous implantation of the matrices demonstrated the formation of a mineralized tissue. Quantitative analyses show that the mineralization of the implants is dependent of the amount of HA particles dispersed, with an optimal ratio of 5% of particles. Histological analysis revealed osteoid tissue in contact to the matrix.

In conclusion, the ability of this injectable composite scaffold to promote ectopically tissue mineralization is promising for bone tissue engineering. Osseous implantation in a femoral bone defect in rats is now in progress. 5% of doped HA particles were implanted within the induced membranes in a context of radiotherapy procedure. Micro-CT analyses are ongoing. This new matrix could represent an alternative to the autografts for the regeneration of large bone defects in an oncological context.

First works focuses on the characterization (physical and biological) of this biomaterial. Current work had studied osteoinductive and osteoconductive capacity of these hydrogels. In vivoresults highlight a significant bone reconstruction two months after implantations on bone lesions in mice.

Bone is a dynamic and vascularized tissue that has the ability of naturally healing upon damage. Nevertheless, in the case of critical size defects this potential is impaired. Present approaches mainly consider autografts and allografts, which presents several limitations. Bone Tissue Engineering (BTE) is based on the use of 3D matrices to guide both cellular growth, differentiation to promote bone regeneration. Hence, matrices can contain biological materials such as cells and growth factors. Our project aims to design a hydrogel for BTE, particularly for bone lesion filling. We previously showed that a porous 3D hydrogel, Glycosyl-Nucleoside-Fluorinated (GNF) is: 1) non-cytotoxic to clustered human Adipose Mesenchymal Stem Cells (hASCs), 2) bioinjectable and 3) biodegradable. Therefore, this novel class of hydrogels show promise for the development of therapeutic solutions for BTE [1]. The hypothesis of this research was that improving the capacity to promote the adhesion of cells by adding collagen gel matrices and bone morphogenic protein 2 (BMP-2) to improve the bone regenerative potential of this gel. Collagen is a protein matrix well known for its cytocompatibility [2]. BMP-2, have been shown ability to induce bone formation in combination with an adequate matrix [3]. Thereby, the overall aim of this work was to design, develop and validate a new composite hydrogel for BTE.

GNF was prepared as previously described in detail[1], at a concentration of 3% (w/v). Type I-collagen gel was prepared from rat-tail tendons at a concentration of 4 g/L [2]. hASCs were isolated from human adipose tissue in our laboratory. To establish a suitable microenvironment for cell proliferation and differentiation cells were seeded in collagen and then GNF gel was added and the resulting mixture was blended, BMP-2 (InductOs ® Kit) is added to this preparation (5µm BMP-2/ml). Fluorometry was used to follow BMP2 release in vitro andin vivo(NOG mices;n=6), orthotopic calvariumbone critical defect (3.3 mm) has been selected to challenge the bone repair.

Adding collagen hydrogel improve cell adhesion, survivals and proliferation rather than simple GNF hydrogel. This novel gel composite has the ability to sustain hASCs adhesion and differentiation towards the osteoblastic lineage (positive ALP cells). Fluorometry showed the ability of our hydrogel to prolong the residence of BMP-2 (in vitro and in vivo) compared to collagen hydrogel sponges. Implantation of hydrogel containing hASC and BMP-2 has shown encouraging results in bone reconstruction: 2 months after implantation of biomaterials a significant bone reconstruction can be observed using X-Ray imaging.

Adding collagen to GNF allowed to obtain gels showing satisfactory cell-behaviour. In parallel, the presence of GNF hydrogel helps to improve mechanical properties of the biomaterial (hydrogel stability and controlled release of BMP-2). The first in vivostudies have shown encouraging bone regeneration capacity of these hydrogels. The implantation performed on a larger number of animals and quantitative microCT analysis will enable us to judge the effectiveness of this hydrogel as a new injectable biomaterial for BTE.

This work was partially supported by NSERC-Canada, FRQ-NT-Quebec, FRQ-S- Quebec, and CFI-Canada. Mathieu Maisani was awarded of a NSERC CREATE Program in Regenerative Medicine (www.ncprm.ulaval.ca).