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Matrix mechanics controls cell fate by modulating the bonds between integrins and extracellular matrix (ECM) proteins. However, it remains unclear how fibronectin (FN), type 1 collagen, and their receptor integrin subtypes distinctly control force transmission to regulate focal adhesion kinase (FAK) activity, a crucial molecular signal governing cell adhesion/migration. Here we showed, using a genetically encoded FAK biosensor based on fluorescence resonance energy transfer, that FN-mediated FAK activation is dependent on the mechanical tension, which may expose its otherwise hidden FN synergy site to integrin α5. In sharp contrast, the ligation between the constitutively exposed binding motif of type 1 collagen and its receptor integrin α2 was surprisingly tension-independent to induce sufficient FAK activation. Although integrin α subunit determines mechanosensitivity, the ligation between α subunit and the ECM proteins converges at the integrin β1 activation to induce FAK activation. We further discovered that the interaction of the N-terminal protein 4.1/ezrin/redixin/moesin basic patch with phosphatidylinositol 4,5-biphosphate is crucial during cell adhesion to maintain the FAK activation from the inhibitory effect of nearby protein 4.1/ezrin/redixin/moesin acidic sites. Therefore, different ECM proteins either can transmit or can shield from mechanical forces to regulate cellular functions, with the accessibility of ECM binding motifs by their specific integrin α subunits determining the biophysical mechanisms of FAK activation during mechanotransduction.

Thixotropic materials, which become less viscous under stress and return to their original state when stress is removed 1 , have been used to deliver gel–cell constructs 2 and therapeutic agents 3 . Here we show that a polymer–silica nanocomposite thixotropic gel can be used as a three-dimensional cell culture material. The gel liquefies when vortexed—allowing cells and biological components to be added—and resolidifies to trap the components when the shear force from spinning is removed. Good permeability of nutrients and gases through the gel allows various cell types to proliferate and be viable for up to three weeks. Human mesenchymal stem cells cultured in stiffer gels developed bone-like behaviour, showing that the rheological properties of the gel can control cell differentiation. No enzymatic 4 , chemical 5 , 6 , or photo-crosslinking 7 , 8 , 9 , changes in ionic strength 10 , 11 , 12 , 13 , 14 or temperature 15 , 16 are required to form or liquefy the gel, offering a way to sub-culture cells without using trypsin—a protease commonly used in traditional cell culture techniques. Polymer–silica nanocomposite gels can be used to culture cells in a three-dimensional environment, offering a way to propagate cells without using enzymes to dissociate them from the surface of conventional cell culture flasks. This approach relies on the dependence of the viscosity of the gel on stress

Materials engineered to elicit targeted cellular responses in regenerative medicine must display bioligands with precise spatial and temporal control. Although materials with temporally regulated presentation of bioadhesive ligands using external triggers, such as light and electric fields, have recently been realized for cells in culture, the impact of in vivo temporal ligand presentation on cell–material responses is unknown. Here, we present a general strategy to temporally and spatially control the in vivo presentation of bioligands using cell-adhesive peptides with a protecting group that can be easily removed via transdermal light exposure to render the peptide fully active. We demonstrate that non-invasive, transdermal time-regulated activation of cell-adhesive RGD peptide on implanted biomaterials regulates in vivo cell adhesion, inflammation, fibrous encapsulation, and vascularization of the material. This work shows that triggered in vivo presentation of bioligands can be harnessed to direct tissue reparative responses associated with implanted biomaterials. Transdermal light-triggered activation of cell-adhesive peptides on the surface of implanted hydrogels alters cell–material interactions, such as cell adhesion and spatial patterning, and fibrous encapsulation and vascularization of the material.

Background Mesenchymal stem cells (MSCs) are of great interest in bone regenerative medicine due to their osteogenic potential and trophic effects. However, challenges to large-scale production of MSCs can hinder the translation of MSC therapies. 3D Microcarrier (MC)-based MSC culture presents a scalable and cost-effective alternative to conventional methods of expansion in 2D monolayers. Furthermore, biodegradable MCs may allow for MC-bound MSC delivery without enzymatic harvest for selected applications such as bone healing. However, the effects of cell expansion on microcarriers and enzymatic cell harvest on MSC phenotype and osteogenic differential potential are not well understood. In this study, we characterized human fetal MSCs (hfMSCs) after expansion in 3D microcarrier spinner or 2D monolayer cultures. Following expansion, we compared osteogenic differentiation of cultures seeded with 3D MC-harvested, 3D MC-bound and conventional 2D monolayer (MNL)-harvested cells when cultured in osteogenic induction media on collagen-coated plates. Results Fetal MSCs expanded on both 3D agitated Microcarriers (MC) and 2D Plastic static monolayer (MNL) cultures express high levels of MSC surface markers. MC-harvested hfMSCs displayed higher expression of early osteogenic genes but slower mineralization kinetics compared to MNL-harvested MSCs during osteogenic induction. However, in the comparison between MC-bound and MC-harvested hfMSCs, osteogenic genes were upregulated and mineralization kinetics was accelerated in the former condition. Importantly, 3D MC-bound hfMSCs expressed higher levels of osteogenic genes and displayed either higher or equivalent levels of mineralization, depending on the cell line, compared to the classical monolayer cultures use in the literature (MNL-harvested hfMSCs). Conclusion Beyond the processing and scalability advantages of the microcarrier culture, hfMSCs attached to MCs undergo robust osteogenic differentiation and mineralization compared to enzymatically harvested cells. Thus biodegradable/biocompatible MCs which can potentially be used for cell expansion as well as a scaffold for direct in vivo delivery of cells may have advantages over the current methods of monolayer-expansion and delivery post-harvest for bone regeneration applications.

Bone formation is a complex physiological process which is orchestrated by multiple microenvironmental cues such as soluble factors, cell-cell interactions and the extracellular matrix. Integrins are heterodimeric transmembrane receptors consisting of α and β subunits which mediate cell interactions with the extracellular matrix. Beta 1 integrins encompass the majority of integrins and represent the main integrin binding partners of collagen I, the most abundant extracellular matrix component of bone. The central goals of this dissertation project were to elucidate the role of β1 integrins on bone development and healing in vivo, and to design biomimetic α2β1 integrin-specific polyethylene glycol hydrogels to enhance bone healing within segmental bone defectsBecause global β1 knockout mice are embryonically lethal, in order to study the role of β1 integrins in vivo, we used the Cre-Lox system to generate mice with conditional beta 1 integrin deletions in osteolineage cells at three stages: (1) mesodermal cells [under Twist 2/Dermo 1], (2) osteoprogenitors [under the osterix promoter] and (3) mature osteoblasts and osteocytes [under the osteocalcin promoter]. We found that β1 integrin deletion in mesodermal cells severely impaired pre-natal skeletal mineralization, particularly in the calvarium, and also resulted in late-stage embryonic lethality. In contrast, β1 integrin deletion in pre-osteoblasts resulted in viable but runted mice with decreased cranial mineralization, tooth defects, impaired femur development and some perinatal mortality. Finally, mice with β1 integrin null osteoblasts and osteocytes displayed very mild bone phenotypes with no change in femur fracture healing capacity. Taken together, these results suggest that β1 integrins play an important role inthe early bone formation process but are not essential for the function of mature osteoblasts and osteocytes.We also sought to engineer a biomimetic bone graft substitute by incorporating the following two bioactive components into a matrix metalloproteinase (MMP)-sensitive synthetic polyethylene glycol (PEG) hydrogel: (1) the collagen I-mimetic triple-helical synthetic ligand GFOGER, which specifically binds to the pro-osteogenic α2β1 integrin, and (2) recombinant human bone morphogenetic protein 2 (rhBMP-2). We synthesized PEG hydrogels incorporating GFOGER or the commonly used non-integrin selective adhesive peptide, RGD, in equimolar densities and studied hMSC differentiation responses to each of these surfaces. We then examined the effects of treating murine radial segmental defects with either GFOGER functionalized PEG-MAL hydrogels or GFOGER gels which also incorporated a low dose of rhBMP-2. Our data indicated that GFOGER hydrogels enhanced bone healing compared to empty defects and that incorporating low dose rhBMP-2 in GFOGER gels further improved bone formation. We evaluated the roles of the GFOGER ligand and the MMP-sensitive crosslinker, GCRDVPMSMRGGDRCG (VPM), in this response by comparing bone formation in defects treated with non-degradable hydrogels, degradable hydrogels lacking the GFOGER ligand, and in defects treated with degradable GFOGER hydrogels. Minimal bone formation occurred in response to PEG hydrogels which were not functionalized with any adhesive ligand and there was no bone formation in non-degradable PEG hydrogels, indicating that adhesive function and degradability are essential to bone regeneration in response to GFOGER hydrogels. Our examination of rhBMP-2 dose response within GFOGER hydrogels suggested that low 0.02mg/ml (0.03 μg) dose was sufficient for robust healing, but that the medium 0.04 mg/ml (0.06 μg) dose increased bone volume and mineral density within the defect compared to the low dose. The high 0.2 mg/ml (0.3 μg) BMP-2 dose induced less bone formation within the defect than the medium dose and altered the structure of the ulna so that it encircled the radius and fused with the radius. FMT analysis and in vitro BMP release assays revealed that GFOGER hydrogels provided sustained release of rhBMP-2. Finally, we evaluated the bone regeneration capacity of low dose rhBMP-2 delivery from GFOGER functionalized PEG hydrogels in comparison with collagen sponges, the clinical standard for BMP-2 delivery. We observed superior bone healing in response to GFOGER hydrogel treatment. In conclusion, our bioengineered integrin-specific hydrogel may be a promising bone graft substitute for the treatment of large bone defects.