Functional Replication of Tissue Microenvironment on Biomaterials


Student thesis: Doctoral Thesis

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Award date31 Jul 2019


In tissues, cells are in intimate contact with an extracellular microenvironment. In vitro cell culture substrates provide an important and convenient experimental platform for the study of cell-matrix interactions. A wide variety of cell culture substrates, from the coating of extracellular matrix (ECM) components on flat surfaces, engineered substrates with nanotopographies to decellularized tissue scaffolds, have been extensively studied and explored in the field of tissue engineering and regenerative medicine. Decades of research have shown that three major factors, topographical, biochemical and mechanical, in a tissue microenvironment are essential for cellular homeostasis and functions. However, current experimental models are too reductionist and simplistic to represent the complexity of the tissue microenvironment, hindering the detailed understanding of its functions and its exploration for therapeutic uses. In particular, the importance of the topographical, biochemical and mechanical factors in a tissue microenvironment have not been individually characterized, because it is impossible to alter one of these factors without simultaneously affecting the other two. The aim of my PhD study is to explore the innovative use of nanofabrication methods to functionally replicate tissue microenvironments on biomaterials.

Silica bioreplication (SBR) is a process that converts biological samples into silica replicas with high structural fidelity. In the first part of my PhD study, I explored the use of this technique to generate high-resolution replicas of intact mammalian tissues of different histological origins, including tendon, cartilage, skeletal muscle and spinal cord. SEM and AFM showed that the resulting replicas accurately preserved the three-dimensional ultrastructure of each tissue, while all biochemical components were eradicated by calcination. Such properties allow the uncoupling the topography of a tissue microenvironment from the biochemical and mechanical factors. Here, I showed that human mesenchymal stem cells (MSC) cultured on these replicas adopted vastly different morphology, suggesting that the topographical information of the tissue microenvironment captured by SBR could profoundly affect MSC biology. MSC cultured on tendon replica elongated and expressed tenocytes marker, while MSC cultured on flat silica surface and on the silica replica of skeletal muscle did not. Interestingly, MSC grown on the spinal cord replica developed into spheroids that resembled neurospheres morphologically and in terms of expression of neurosphere markers, and could be further differentiated into neuron-like cells, in the absence of any exogenous growth factors. Cell spreading and adhesion were investigated. MSC spreading is reduced when cultured on both spinal cord and skeletal muscle silica replicas. Interestingly MSC adhesion to the silica replicas has no significant different as compared to a normal flat glass surface. This part of my study reveals that tissue-specific topography was sufficient in initiating and directing differentiation of MSC, despite the absence of any organic, biochemical signals. SBR is a convenient and versatile method for capturing this topographical information on to a biologically inert material, allowing the functional characterization of the architecture of a tissue microenvironment that is previously impossible.

Besides SBR, I also explored the use of thermal scanning probe lithography (t-SPL) in replicating tissue topography. t-SPL is a nanofabrication technique in which an immobilized thermolabile resist, such as polyphthalaldehyde (PPA), is locally vaporized by a heated atomic force microscope (AFM) tip. Compared with other nanofabrication techniques, such as soft lithography and nanoimprinting lithography, t-SPL is more efficient and convenient as it does not involve time-consuming mask productions or complicated etching procedures, making it a promising candidate technique for the fast prototyping of nanoscale topographies for biological studies. Here, I established the direct use of PPA-coated surfaces as a cell culture substrate. I showed that PPA is biocompatible and that the deposition of allylamine by plasma polymerization on a silicon wafer before PPA coating can stabilize the immobilization of PPA in aqueous solutions. When seeded on PPA coated surfaces, human mesenchymal stem cells (MSCs) adhered, spread and proliferated in a manner indistinguishable from cells cultured on glass surfaces. This allowed us to subsequently use t-SPL to generate nanotopographies for cell culture experiments. As a proof of concept, we analyzed the surface topography of bovine tendon sections, previously shown to induce morphogenesis and differentiation of MSC, by means of AFM, and then “wrote” topographical data on PPA by means of t-SPL. The resulting substrate, matching the native tissue topography on the nanoscale, was directly used for MSC culture. The t-SPL substrate induced similar changes in cell morphology and focal adhesion formation in MSC compared to native tendon sections, suggesting that t-SPL can rapidly generate cell culture substrates with complex and spatially accurate topographical signals. This technique may greatly accelerate the prototyping of models for the study of cell-matrix interactions.

Taking together, this work reveals the previously unknown level of significant of tissue nanotopography in directing cell functions and MSC lineage commitment. My work also shows that the topographic cues can be functionally replicated onto synthetic materials. The developed protocol for modifying PPA-coated surfaces as cell culture substrate allows to the fast prototyping of cell culture substrate models. This protocol can be potentially benefit the development of tissue engineering.

    Research areas

  • Cell-matrix interaction, silica bioreplication, thermal scanning probe lithography, tissue engineering, biomaterial