The development of bio-mimetic materials for tissue reconstruction through the systematic study of cell-matrix interactions

Abstract

The mission of tissue engineering is to recapitulate the natural process of tissue formation by assembling cells into synthetic scaffold. This relies on the understanding of the functions and properties of the tissue microenvironment (TME), the specific extracellular environment within endogenous tissues. Although existing studies demonstrated the effect of each of the topographical, mechanical and biochemical properties on cell behaviors in isolation, the effect of these properties within the native TME are complicated and ill defined. This thesis aims to investigate how topographical, mechanical and biochemical features of natural TME contribute to the modulation of the biochemistry, morphology and functions of cells, and to translate this knowledge into the fabrication of biomaterials.

Tissue cryosections as a cell culture model system was established. It allowed robust assessment of cell phenotypes in a near-natural TME. Mesenchymal stem cells (MSC) cultured on bone, cartilage and tendon cryosections adopted different morphology, supporting the idea that tissue cryosections forms a robust platform for cell-TME studies. Then, Achilles tendon TME was chosen for proof of concept. This tendon cryosection induced different cell types to adopt different morphologies, indicating that the effect of TME is cell type specific. The proliferation of MSC cultured on cryosection was suppressed, however it was instructed to commit tenogenic differentiation. Then, the necessity of TME topographical properties in forming this instruction was delineated by seeding MSC onto cross-sectional tendon cryosection. Although this surface contained native biomechanical and biochemical cues, it could not promote differentiation. This highlighted the necessity of topographical cues within the TME.

Next, nano-grooved titanium surface that resembles the topographical cues of tendon TME was used to replicate the function of TME. This surface successfully promoted morphogenesis of MSC but not differentiation. This implicated that biomechanical and biochemical cues are both necessary for instructing desired cell phenotypes. The proteomes of MSC cultured on nanogrooved and planar surfaces were then studied using quantitative proteomics. This revealed some expected changes such as up regulation of cytoskeleton and cell-adhesion proteins, suggesting mechanotransduction events might have been induced by nano-grooved surface. However, expressions of RNA-binding proteins were also regulated, representing novel findings. These proteins were also found in the proteome of cellmicroenvironment interface identified through the use of subcellularfractionation and proteomics. This consolidated their involvement in cellmatrix interactions.

The topographical and mechanical properties of cryosection were replicated by using bioimprinting. This imprint induced the morphogenesis of MSC, but tenocytic differentiation was induced only when collagen 1 was coated. However incorrect mechanical properties would abolish such phenotypic guidance. This suggests that topographical, mechanical and biochemical information in a TME are individually indispensable, and it is possible to functionally reconstruct a TME by bioimprinting and ECM protein coating.

In summary, this study investigated the topographical, mechanical and biochemical properties in tendon TME and their combined effect on controlling cell phenotypes. It illustrates that biomimetic approach that mimics these three properties of a tissue can effectively control cell phenotypes. Further investigation on better biomimetic methods and its molecular mechanisms will help establishing strategies for constructing functional tissues.