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.

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).