Control of biochemical (e.g., integrin bioligands, growth factors, and drugs) and biophysical (e.g., stiffness, porosity, and geometry) properties of scaffolds to modulate dynamic signals influencing cell behavior is crucial for the fundamental understanding of mechanisms in cellular processes, such as regeneration, embryonic development, and tumor invasion. In this thesis, thermo-triggered reversible dynamic microenvironments were investigated mainly based on the thermoresponsive polymer PNIPAM to mimic intricate feedback loops between multi-responsive 3D ECM and cells that exist in vivo. Moreover, we also established a cyclic compressive stress of harnessing mechanical stimuli to study the mechanotransductive response, which provided a versatile approach to preclude the era of modern mechanobiology with computational tools.
In the first project, we successfully fabricated a biodegradable PCL scaffold via electrospinning, which can achieve the dynamic adhesion of endothelial cells. The cRGD-functionalized PNIPAM and antifouling linear polyglycerol (LPG) were anchored on the microfiber matrix based on polydopamine-triggered reactions. At low temperature (25 ºC), the human umbilical vein endothelial cells (HUVECs) were quickly attached to the surface via integrin αvβ3-cRGD interaction. However, the increase of the temperature to 37 ºC concealed the cRGD-PNIPAM to the LPG surface, which induced the cell release from the surface. (see Section 3.1). Compared with pure hydrophobic and hydrophilic interaction, using thermo-triggered PNIPAM only, cRGD-PNIPAM grafted surface exhibited higher cell attachment and release efficiency, especially under dynamic flow conditions. Moreover, the ratio of the adhesive factor on the surface was related to the attachment efficiency onto the scaffolds. The fiber matrices provided a suitable microenvironment for accelerating targeted endothelial cells spread and growth after primary screening. Therefore, this tunable dynamic system could dynamically modulate targeted cell attachment and detachment, which could be potentially applied for cell recruitment in vascular tissue engineering or cell isolation for downstream detection of diseases.
Furthermore, in order to understand how cells transduce material properties in the nonlinear dynamic microenvironment, we designed a dynamic nonlinear elastic of the fibrillar network to study the cell behavior under the reversible dynamic mechanical stimulation (RD-MS) with computational tools.
In the second project, we utilized a copolymer of acryloyl carbonated polycaprolactone P(CL-co-AC) with a responsive copolymer of N-isopropylacrylamide and 2-hydroxyethyl methacrylate P[(NIPAM-co-HEMA)] to introduce the fibrous character of native ECM by electrospinning. The synthetic thermally responsive hybrid microfibrous network shrunk at 37 °C and swelled at 25 °C, which allowed hMSCs to assimilate with their surroundings under the dynamic tuning of the local stiffness and geometric deformation by RD-MS. The nonlinear elastic fibrous networks increased focal adhesion ligand density, cell spreading, and polarization by recruiting fiber assembly under the RD-MS (see Section 3.2). In addition, compared to the hMSCs cultured under normal culture conditions, the tunable mechanics with multiple cycles from 37 to 25 °C highlighted the intimate control of cell-matrix interactions, which promoted nuclear translocation of YAP and osteogenic differentiation. The hMSCs could benefit more from integrating the combination of multiple stimuli under the RD-MS, such as stiffness, swelling behavior, and porosity, which may serve as a dynamic platform to closer mimic the complex natural system in vivo.
In the third project, we show that the commitment and differentiation of encapsulating hMSC spheroids in thermosensitive 3D hydrogels were simply altered by an interpenetrating poly (NIPAM-HEMA) nanogel to a gelatin methacryloyl (GelMA) network. This cell-laden hydrogel provided dynamic mechanics with a covalent crosslinking-coordinated with reversible physical network, which could regulate hMSCs in situ by dynamically stiffening soft niches via multicyclic changing temperatures from 25 to 37 ºC (see Section 3.3). Notably, the dynamic microenvironment gradually influenced the distribution from the basal to apical side and expression of nuclear lamin A/C and increased the YAP nuclear localization with cycles, which favors hMSCs undergoing osteogenesis (but not adipogenesis) in soft microniches. These findings highlight the central roles of the dynamic relationship between the biomechanical signals and mechanosensitive transcriptional regulators in the cellular mechanosensing.
To mimic natural ECM properties, this dynamic platform has provided a powerful tool to probe the effects of dynamic signals on cell behavior through mechanotransduction in many in vitro studies. We have provided a new insight into how cyclically reversible in situ changes in matrix mechanics affect cell fate based on thermo-triggered microenvironment in this thesis. However, there remain several challenges that need to be overcome in this field:
1. The temperature-triggered dynamic platform was very easy to operate and controlled cell adhesion, migration, and differentiation in vitro. However, it was difficult to integrate into the in vivo tissue microenvironment, where the temperature was relatively constant. Therefore, novel strategies for designing new materials can focus on providing in vivo spatiotemporal mechanics cues to make progress towards bridging the gap between in vitro and in vivo studies.
2. Although mechanotransduction has drawn much attention in the past five years, further studies can be focused on the specific mechanosensing mechanisms, such as embryonic development, vascular microenvironment, and cardiac fibrotic remodeling. Moreover, traction fore microscopy and computational tools can be employed to explain more complicated mechanisms.
Therefore, the development of the dynamic biomaterials for cell-ECM interactions will contribute to the understanding of the role of ECM in cell signaling, stem cell differentiation, and tissue repair.