Bone grafts are crucial for the treatment of bone defects caused by tumor excision. The gold standard is autograft but their availability is limited. Allografts are an alternative, but there is a risk of rejection by the immune system. The tissue engineering field is trying to develop vascularized bone grafts, using innovative biomaterials for surgery applications. While the gold standard in bone graft in dentistry is the use of decellularized bovine bone particles (Bio-Oss®), our work has produced a polysaccharide-based composite matrix (composed of PUllulan, DextraNand particles of HydroxyApatite (PUDNHA), as a new scaffold for promoting bone formation and vascularization of the tissue. In the context of bone tissue regeneration, the function of osteoblast and endothelial cells has been extensively studied, while the impact of osteocytes has been regarded as secondary. Nonetheless, the osteocytes represent 90–95% of bone cells and are responsible for orchestration of bone remodeling.

Here, we propose an original method to analyze the interaction between bone and biomaterials, after in vivo implantation of the matrix PUDNHA in an experimental sheep model. Our objectives are to analyze the network established by osteocytes in the newly formed tissue induced by the matrix, as well as their interactions with the blood vessels.

Sheep have been implanted with the Bio-Oss® or the PUDNHA using the sinus lift technique. After 3 (3M) and 6 months (6M), the animals were euthanazied and the explants were fixed, analyzed by X-ray, embedded in Methylmetacrylate/Buthylmetacrylate and analyzed histologically by Trichrome staining. Thereafter, the samples (n=3/group) were polished using different sand papers. A final polish was realized using a 1µm Diamond polishing compound. The blocks were incubated 10 or 30s with 37% phosphoric acid to remove the mineral on the surface, then dipped in 2.6% sodium hypochlorite to remove the collagen. The samples were air dried overnight, metallized with Gold palladium the following day, before being imaged with a SEM.

As expected, PUDNHA activates bone regeneration in this sinus lift model after 3M and 6M. X-ray analysis and histological data revealed more bone regeneration at 6M versus 3M in both groups. With this acid eching technique, we were able to visualize the interface of bone with the biomaterials. This treatment coupled with SEM analysis, confirmed the increase of bone formation with time of implantation in both groups. In addition, SEM images revealed that osteocyte alignment and their network were different in the new regenerated bone compared to the host bone. Moreover, images showed the direct contact of the osteocytes with the blood vessels formed in the new regenerated bone.

This acid eching technique can be useful in the field of biomaterials to see the relationship between cells, blood vessels and the material implanted and understand how the new bone is forming around the different biomaterials.

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.