Développement par génie tissulaire d’un substitut osseux humain prévascularisé

Authors: Kawecki, Fabien
Advisor: Auger, François A.Fradette, Julie
Abstract: Bone tissue engineering is a field of regenerative medicine that allows the production of substitutes from the patient's cells in association with either biomaterials and/or growth factors, such as bone morphogenetic proteins (BMPs). The chemical composition or the xenogeneic origin of these biomaterials can lead to healing failures that would result in the non-integration of the graft to the surrounding native tissue or the rejection by the patient's body. Moreover, in the absence of vascularization, the center of thick tissues produced will end up with reduced or insufficient intake of nutrients and oxygen. This can significantly reduce graft survival and post-implantation healing. We hypothesized that the prevascularization of these osseous substitutes will provide a capillary network that can promote in vitro osteogenesis and in vivo bone healing. The overall goal of this thesis is to develop a new model of a prevascularized human osseous cell sheet produced by the self-assembly method using human adipose-derived stem cells (hASCs). The aims are: 1) Determine the pro-angiogenic potential of these osseous cell sheets, and study the development of laser-assisted bioprinted microvascular structures. 2) Characterize the formation of a capillary network produced by random seeding of endothelial cells and evaluate its impact on osteogenesis, biomineralization, and the healing of calvarial bone defects. 3) Improve osteogenesis of the tissues using bone morphogenetic protein (BMP) treatment and determine the potential of osseous substitutes, co-cultured or not with endothelial cells, to heal alveolar bone defects. Thus, hASCs have been induced towards an osteogenic differentiation pathway to form manipulable osseous cell sheets. These cell sheets showed a pro-angiogenic profile with the secretion of molecules, such as vascular endothelium growth factor (as high as non-induced cell sheets) and angiopoietin-1 (2.3-fold higher than non-induced cell sheets), that can promote the formation and the maintenance of a capillary network. Large endothelial structures were formed by laser-assisted bioprinting of human umbilical vein endothelial cells (HUVECs). The alignment of these structures depends on the cellular orientation of the stacked cell sheets. Prevascularized osseous tissues, which were vascularized by the random seeding method, allowed the formation of a capillary network 2.1-fold denser and 3.7-fold more connected compared to non-induced tissues. However, prevascularization delayed osteogenesis and biomineralization in vitro, with decreases in osteocalcin secretion (1.7-fold) and hydroxyapatite formation (1.6-fold) in the matrix. Prevascularization of the osseous grafts revealed an improvement of their survival (5.7-fold) after 12 weeks of implantation in calvarial bone defects created in immunodeficient rats. In addition, these results suggest that the prevascularization of osseous tissue does not interfere with the healing of cranial bone defects. To improve the potential of the model, the osteogenic effect of BMP-9 treatment on hASCs, as well as the impact of their co-culture with HUVECs for 21 days, were investigated. BMP-9 treatment of osseous tissues significantly increased the activity of alkaline phosphatase (3-fold), while prevascularization significantly increased the thickness (2-fold) and the mechanical properties (percent deformation: 1.6-fold, Young modulus: 3.6-fold and tensile strength: 3.7-fold) of the osseous tissues after 21 days of co-culture. Untreated, prevascularized or BMP-9-treated osseous and stromal tissues were grafted for 10 weeks into alveolar bone defects created in immunodeficient rats following tooth extractions. A surgical bone filler biomaterial was used as a positive control. Micro-computed tomography scans and histologic observations revealed elevated bone healing when the defects are grafted with BMP-9-treated or non-BMP-treated osseous tissues. In addition, these defects exhibited a similar bone volume fraction at the implantation site after 10 weeks compared to those filled with the biomaterial. Finally, this new model of prevascularized human osseous tissue could ultimately offer clinicians an advantageous solution for the treatment of small bone defects and represent, for fundamental researchers, a powerful in vitro research tool.
Document Type: Thèse de doctorat
Issue Date: 2019
Open Access Date: 7 June 2021
Permalink: http://hdl.handle.net/20.500.11794/69302
Grantor: Université Laval
Collection:Thèses et mémoires

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