Tissue engineering for evaluation and treatment of craniofacial abnormalities

The overall objective of this thesis work is to apply tissue engineering strategies for better understanding and treatment of craniofacial abnormalities. Craniofacial abnormalities can be caused by traumatic events or various diseases processes and are associated with either bone loss or excessive bone formation. 3D hydrogel culture model, derived from tissue engineering strategies, was used to study the pathogenesis of Apert syndrome, an autosomal dominant disorder that is caused by one of two specific missense substitution mutations (S252W and P253R) in fibroblast growth factor receptor 2 (FGFR2). Osteoblasts from Apert Fgfr2+/S252W and Fgfr2+/P253R mice were expanded and characterized in 2D, and then respectively encapsulated in 3D hydrogels and their phenotype in 3D in vitro was compared to that of in vivo tissue specimens. Complementary to the in vivo findings, both mutations were found to be associated with increased osteoblastic association after in vitro 3D culture. Data presented in this thesis suggested several mechanisms that may underlie the Apert syndrome pathogenesis including altered bone matrix remodeling and changes in the FGFR2 response to FGF ligands. Some differences between Fgfr2+/P253R cells and Fgfr2+/252W cells were also noted. These findings suggested the feasibility of applying tissue engineering as a novel approach to study disease processes and expanded our ability to evaluate mutant mice. Furthermore, for both S252W and P253R mutant cells, co-culturing with wild-type cells using a 3D hydrogel model resulted in changes in expression levels of mutant osteoblasts differentiation markers and matrix turn-over genes to levels that were comparable to the wild type controls. These results suggest a normalization effect on the mutant cells during the co-culture with wild type cells and provide the rationale for further investigation of the potential of cell therapy in treating Apert syndrome.; The second part of the thesis focused on improving scaffold design and exploring several stem cell lines as potential cell sources for craniofacial defects repair. RGD conjugated PEODA hydrogel was found to promote the osteogenesis of mesenchymal stem cells (MSCs) in a dosage-dependent manner, with 2.5 mM being the optimal concentration. An optimal cell density (20 million/ml) was then determined for human mesenchymal stem cell based bone tissue engineering using hydrogel as a scaffold. In addition to the adult MSCs, several embryonic stem cell lines were also explored as potential cell sources for bone tissue engineering, using the cell-adhesive hydrogel as well as a solid PLGA/PLLA/Hydroxyapatite composite scaffold. Osteogenesis was examined both in vitro and in vivo in a mouse cranial defect model. It has been shown that both human embryonic stem cell-derived cell line and embryonic germ-derived cell line can be induced to form early osteoid-like structures when seeded on the PLGA/PLLA/Hydroxyapatite composite scaffold and implanted in vivo without causing tumors. This work represents an initial attempt in exploring the potential of using embryonic cells for bone tissue engineering applications.