A central focus of research within the tissue engineering field is aimed at developing biomaterials that will instruct cells to produce a desired tissue. Such strategies are seen as critical for the repair of complex tissue interfaces that are inherently difficult to fix using strictly a materials- or cell-based approach. One such tissue is the enthesis, which connects soft (i.e. cartilage-type) to hard (i.e. bone) tissue. Previous studies have demonstrated that a material’s surface chemistry can be utilized to induce mesenchymal stem cells (MSCs) to differentiate into enthesis-type chondrocytes (cartilage cells) and osteoblasts (bone cells) in-vitro. However, the development of an industrially-relevant surface functionalization technique that can be applied to FDA-approved TE materials remains a major challenge.
This thesis focuses on the characterization and optimization of plasma polymer surfaces to enhance the in-vitro differentiation of MSCs to chondrocytes (cartilage cells) and osteoblasts (bone cells). In the first part of this study, heptylamine (HApp) and propionaldehyde (PApp) plasma polymers were deposited onto silicon wafers and FDA-approved poly (ε-caprolactone) (PCL) polymers using different deposition times and discharge powers. It was demonstrated that heptylamine and propionaldehyde could be utilized to generate amine and carbonyl-type pp, respectively. All plasma parameters investigated produced an even surface coating that was sufficiently thick (i.e > 7.9 nm) to mask the chemical properties of the underlying substrate, without significantly changing the physical properties of it. Interestingly, pp chemistry varied more depending on the discharge power, and not the time.
To demonstrate the feasibility of applying pp to 3D surfaces, scaffolds consisting of either a single PCL phase or bi-phasic PCL and PCL with hydroxyapatite (PCL/nHA) phases were fabricated utilizing the thermally induced phase separation (TIPS) process. HApp and PApp were then deposited onto the 3D scaffolds, and HApp was detected up to a distance of 2 mm into the scaffolds under most conditions. Whilst PApp penetration could not be analyzed with XPS, staining showed that carboxylic acid functionality was introduced to the scaffolds as a result of the PApp process.
To mimic cell culture conditions, which typically require the use of protein-rich fetal bovine serum (FBS), the pp surfaces were incubated with FBS and characterized to determine the amount and types of proteins adsorbed. Despite the different surface chemistries presented, the amount and type of proteins adsorbed to the surfaces varied minimally between samples. Although the presence of vitronectin (VN) could not be definitively confirmed, this protein was further investigated for its potential interaction with surfaces. Specifically, VN was adsorbed to PApp surfaces and its conformation was examined using ToF-SIMS. Interestingly, amino acid fragments corresponding to the RGD cell adhesion peptide sequence were more prevalent on VN adsorbed to PApp than PCL samples. To examine the effect of the adsorbed proteins (i.e. FBS or VN) on cell attachment, MSCs were seeded in serum-free media onto either FBS or VN-adsorbed, or bare plasma polymer surfaces. In general, more cells attached to bare pp than FBS-adsorbed surfaces, and VN-adsorbed surfaces further enhanced cell over bare pp surfaces. Interestingly, MSC attachment was slightly higher on PApp samples than PCL with VN-adsorbed, and this result is consistent with the finding that RGD cell adhesion peptides are more prevalent on Papp-treated surfaces.
Plasma polymer surfaces (2D and 3D) were then evaluated for their effect on MSC differentiation to chondrocytes and osteoblasts. On 2D surfaces, chondrogenic differentiation was best supported by PApp, as evidenced by the aggregation of cells and up-regulation of chondrogenic markers on those surfaces. Consistent with 2D results, 3D PCL scaffolds with PApp appeared to best support chondrogenesis, as these surfaces gave rise to glycosaminoglycan (GAG) and cartilage rich ECM, characteristic of chondrocytes. Osteogenic differentiation did not vary as much on 2D plasma polymer surfaces as compared to PCL, but VN-adsorbed to PApp surfaces gave rise to cells that morphologically resembled bone, and showed protein expression and up-regulated gene-level expression for osteogenic markers. 3D scaffolds consisting of PCL/nHA best supported osteogenesis, as evidenced by the up-regulation of osteogenic markers and the presence of mineralized matrix in the SEM images of these scaffolds. Scaffolds cultured in mixed media (i.e. containing osteogenic and chondrogenic growth factors) displayed similar results to the scaffolds in individual growth factors, that is, PApp appeared to best for chondrogenesis whereas PCL/nHA scaffolds appeared to support osteogenesis.
From these results, it is apparent that various surface chemistries can promote MSC differentiation to chondrocytes and osteoblasts. Furthermore, these effects may be enhanced through the preferential display of certain peptides (e.g. RGD) upon adsorption of proteins to the pp surfaces. The findings presented in this thesis provide crucial insight into the impact of pp on MSC differentiation outcomes in two and three dimensions, and further, these outcomes collectively represent a major step toward the greater challenge of developing a suitable construct for engineering the osteochondral-linkage present at the enthesis.