A Biomaterial Based Approach to Evaluate the Chondrogenic Potential of Human Periosteum Derived Cells

The interdisciplinary field of tissue engineering sits at the intersection of cells, biomaterials, and biological stimulatory molecules. The interplay among these three components gives rise to promising strategies that, at their core, aim to design and create implants that can induce as well as support tissue regeneration and repair, even in a compromised environment. The versatility of biomaterials makes them a highly sought after avenue to pursue in order to advance scientific research. Biomaterials can be designed to act as a scaffold that mimics the local environment of the cells, they can be engineered to serve as a cellular delivery vehicle, and last but not least, they can be modified to sequester biological molecules, such as cytokines, and deliver them locally to the cells. These engineered biomaterials can be tailored to elicit a very specific cellular response, which in turn can have a profound impact on the success of the overall tissue engineering product. Thus, numerous scientific studies have explored the interaction between cells and biomaterials, such as microspheres or hydrogels, for tissue engineering purposes. More specifically, different hydrogels combined with relevant types of cells have been previously examined for the purpose of improving cartilage and bone regeneration. However, due to the complexity of the tissues themselves, researchers are constantly looking for ways to improve the outcomes for cartilage and bone tissue engineering.

Therefore, the general aim of this PhD dissertation was to evaluate different biomaterials, namely polyethylene glycol (PEG) hydrogels and gelatin microspheres, and their interaction with human periosteum-derived cells in culture in 3D. By understanding this aspect, we hoped to create an in vitro cartilaginous template that could subsequently lead to in vivo bone formation. To assess the potential of using these biomaterial based systems, one of the main objectives of this dissertation was to screen different formulations of PEG hydrogels and examine their capability to serve as a 3D system for expansion of progenitor cells as well as promotion of in vitro cellular chondrogenesis using different cell types. In the first part, hydrogel variables included the percentage of PEG macromer (2.5 %, 4 %, 6.5 %, 8 %) (w/v) used; the type of protease sensitive crosslinker utilized (VPMSMRGG or GPQGIWGQ), and the incorporation or lack of an adhesion binding molecule (RGD). From this initial screening, it was clear that the 6.5 % (w/v) constructs outperformed the other formulations. Furthermore, by using ATDC5 cells, human articular chondrocytes, and human periosteum-derived cells, in combination with proliferative culture conditions and chondrogenic differentiation culture conditions, we were able to show that the 6.5 % (w/v) PEG hydrogel composition crosslinked with the GPQGIWGQ protease sensitive peptide crosslinker was better.

These results prompted further investigation, in the second part, to determine a suitable composition of the PEG hydrogel system that could sustain the viability, promote the proliferation, and initiate the chondrogenic differentiation of encapsulated human periosteum-derived cells in combination with different culture media. Hydrogels in this part only varied in the presence or absence of RGD, and the second main variable was the stimulatory growth factor in their chondrogenic medium [transforming growth factor - β1 (TGF-β1) vs. bone morphogenetic protein - 2 (BMP-2)]. Through the analysis of cellular morphology, viability, proliferation, GAG production, and chondrogenic gene expression, it was determined that the 6.5 % (w/v) PEG hydrogel composed of the GPQGIWGQ crosslinker and the cell binding motif, RGD, was the most suitable construct within the scope of this dissertation. Moreover, these cell-laden hydrogels were ectopically implanted in vivo after chondrogenic priming in vitro to analyze their bone forming capacity. Although the explanted constructs displayed some osteogenic markers in gene expression analysis, there was no bone formation observed within these hydrogels.

In the last part of this work, growth factor loaded gelatin microspheres were incorporated within micromasses of human periosteum-derived cells in order to evaluate growth factor delivery via microspheres as an alternative to exogenous chondrogenic stimulation via the culture medium. Validated through microscopy images, histological analysis, and biochemical assays, the successful incorporation of these growth factor delivery systems displayed their potential to promote chondrogenesis of micromasses composed of human periosteum-derived cells in medium without exogenous growth factors.

In conclusion, this dissertation displayed that PEG hydrogels can be used as scaffolds to promote both the proliferation and chondrogenic differentiation of human periosteum-derived cells within 3D constructs with the same formulation. Moreover, gelatin microsphere based growth factor delivery systems can successfully be incorporated within micromasses of human periosteum-derived cells and promote their chondrogenic differentiation. Thus, this dissertation was able to open up new paths of research in the combination of biomaterials and progenitor cells that can be built upon for future bone and cartilage tissue engineering.