An official website of the United States government
Abstract Mechanically tunable hydrogels are attractive platforms for 3D cell culture, as hydrogel stiffness plays an important role in cell behavior. Traditionally, hydrogel stiffness has been controlled through altering either the polymer concentration or the stoichiometry between crosslinker reactive groups. Here, an alternative strategy based upon tuning the hydrophilicity of an elastin‐like protein (ELP) is presented. ELPs undergo a phase transition that leads to protein aggregation at increasing temperatures. It is hypothesized that increasing this transition temperature through bioconjugation with azide‐containing molecules of increasing hydrophilicity will allow direct control of the resulting gel stiffness by making the crosslinking groups more accessible. These azide‐modified ELPs are crosslinked into hydrogels with bicyclononyne‐modified hyaluronic acid (HA‐BCN) using bioorthogonal, click chemistry, resulting in hydrogels with tunable storage moduli (100–1000 Pa). Human mesenchymal stromal cells (hMSCs), human umbilical vein endothelial cells (HUVECs), and human neural progenitor cells (hNPCs) are all observed to alter their cell morphology when encapsulated within hydrogels of varying stiffness. Taken together, the use of protein hydrophilicity as a lever to tune hydrogel mechanical properties is demonstrated. These hydrogels have tunable moduli over a stiffness range relevant to soft tissues, support the viability of encapsulated cells, and modify cell spreading as a consequence of gel stiffness.
more »
« less
Regulation of cell attachment, spreading, and migration by hydrogel substrates with independently tunable mesh size
Hydrogels are widely used as substrates to investigate interactions between cells and their microenvironment as they mimic many attributes of the extracellular matrix. The stiffness of hydrogels is an important property that is known to regulate cell behavior. Beside stiffness, cells also respond to structural cues such as mesh size. However, since the mesh size of hydrogel is intrinsically coupled to its stiffness, its role in regulating cell behavior has never been independently investigated. Here, we report a hydrogel system whose mesh size and stiffness can be independently controlled. Cell behavior, including spreading, migration, and formation of focal adhesions is significantly altered on hydrogels with different mesh sizes but with the same stiffness. At the transcriptional level, hydrogel mesh size affects cellular mechanotransduction by regulating nuclear translocation of yes-associated protein. These findings demonstrate that the mesh size of a hydrogel plays an important role in cell-substrate interactions.
more »
« less
- Award ID(s):
- 2011754
- PAR ID:
- 10500613
- Publisher / Repository:
- Elsevier
- Date Published:
- Journal Name:
- Acta Biomaterialia
- Volume:
- 141
- Issue:
- C
- ISSN:
- 1742-7061
- Page Range / eLocation ID:
- 178 to 189
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Abstract A majority of breast cancer deaths occur due to metastasis of cancer cells to distant organs. In particular, brain metastasis is very aggressive with an extremely low survival rate. Breast cancer cells that metastasize to the brain can enter a state of dormancy, which allows them to evade death. The brain microenvironment provides biophysical, biochemical, and cellular cues, and plays an important role in determining the fate of dormant cancer cells. However, how these cues influence dormancy remains poorly understood. Herein, we employed hyaluronic acid (HA) hydrogels with a stiffness of ~0.4 kPa as an in vitro biomimetic platform to investigate the impact of biochemical cues, specifically alterations in RGD concentration, on dormancy versus proliferation in MDA‐MB‐231Br brain metastatic breast cancer cells. We applied varying concentrations of RGD peptide (0, 1, 2, or 4 mg/mL) to HA hydrogel surfaces and confirmed varying degrees of surface functionalization using a fluorescently labeled RGD peptide. Post functionalization, ~10,000 MDA‐MB‐231Br cells were seeded on top of the hydrogels and cultured for 5 days. We found that an increase in RGD concentration led to changes in cell morphology, with cells transitioning from a rounded to spindle‐like morphology as well as an increase in cell spreading area. Also, an increase in RGD concentration resulted in an increase in cell proliferation. Cellular dormancy was assessed using the ratio of phosphorylated extracellular signal‐regulated kinase 1/2 (p‐ERK) to phosphorylated p38 (p‐p38) positivity, which was significantly lower in hydrogels without RGD and in hydrogels with lowest RGD concentration compared to hydrogels functionalized with higher RGD concentration. We also demonstrated that the HA hydrogel‐induced cellular dormancy was reversible. Finally, we demonstrated the involvement of β1 integrin in mediating cell phenotype in our hydrogel platform. Overall, our results provide insight into the role of biochemical cues in regulating dormancy versus proliferation in brain metastatic breast cancer cells.more » « less
-
Abstract Mechanical cues from the extracellular matrix (ECM) regulate vascular endothelial cell (EC) morphology and function. Since naturally derived ECMs are viscoelastic, cells respond to viscoelastic matrices that exhibit stress relaxation, in which a cell‐applied force results in matrix remodeling. To decouple the effects of stress relaxation rate from substrate stiffness on EC behavior, we engineered elastin‐like protein (ELP) hydrogels in which dynamic covalent chemistry (DCC) was used to crosslink hydrazine‐modified ELP (ELP‐HYD) and aldehyde/benzaldehyde‐modified polyethylene glycol (PEG‐ALD/PEG‐BZA). The reversible DCC crosslinks in ELP‐PEG hydrogels create a matrix with independently tunable stiffness and stress relaxation rate. By formulating fast‐relaxing or slow‐relaxing hydrogels with a range of stiffness (500–3300 Pa), we examined the effect of these mechanical properties on EC spreading, proliferation, vascular sprouting, and vascularization. The results show that both stress relaxation rate and stiffness modulate endothelial spreading on two‐dimensional substrates, on which ECs exhibited greater cell spreading on fast‐relaxing hydrogels up through 3 days, compared with slow‐relaxing hydrogels at the same stiffness. In three‐dimensional hydrogels encapsulating ECs and fibroblasts in coculture, the fast‐relaxing, low‐stiffness hydrogels produced the widest vascular sprouts, a measure of vessel maturity. This finding was validated in a murine subcutaneous implantation model, in which the fast‐relaxing, low‐stiffness hydrogel produced significantly more vascularization compared with the slow‐relaxing, low‐stiffness hydrogel. Together, these results suggest that both stress relaxation rate and stiffness modulate endothelial behavior, and that the fast‐relaxing, low‐stiffness hydrogels supported the highest capillary density in vivo.more » « less
-
Abstract There is a tremendous interest in developing hydrogels as tunable in vitro cell culture platforms to study cell response to mechanical cues in a controlled manner. However, little is known about how common cell culture techniques, such as serial expansion on tissue culture plastic, affect subsequent cell behavior when cultured on hydrogels. In this work, a methacrylated hyaluronic acid hydrogel platform is leveraged to study stromal cell mechanotransduction. Hydrogels are first formed through thiol‐Michael addition to model normal soft tissue (e.g., lung) stiffness (E ≈ 1 kPa). Secondary cross‐linking via radical photopolymerization of unconsumed methacrylates allows matching of early‐ (E ≈ 6 kPa) and late‐stage fibrotic tissue (E ≈ 50 kPa). Early passage (P1) human bone marrow mesenchymal stromal cells (hMSCs) display increased spreading, myocardin‐related transcription factor‐A (MRTF‐A) nuclear localization, and focal adhesion size with increasing hydrogel stiffness. However, late passage (P5) hMSCs show reduced sensitivity to substrate mechanics with lower MRTF‐A nuclear translocation and smaller focal adhesions on stiffer hydrogels compared to early passage hMSCs. Similar trends are observed in an immortalized human lung fibroblast line. Overall, this work highlights the implications of standard cell culture practices on investigating cell response to mechanical signals using in vitro hydrogel models.more » « less
-
Abstract Mesenchymal–epithelial transition (MET) is essential for tissue and organ development and is thought to contribute to cancer by enabling the establishment of metastatic lesions. Despite its importance in both health and disease, there is a lack of in vitro platforms to study MET and little is known about the regulation of MET by mechanical cues. Here, hyaluronic acid‐based hydrogels with dynamic and tunable stiffnesses mimicking that of normal and tumorigenic mammary tissue are synthesized. The platform is then utilized to examine the response of mammary epithelial cells and breast cancer cells to dynamic modulation of matrix stiffness. Gradual softening of the hydrogels reduces proliferation and increases apoptosis of breast cancer cells. Moreover, breast cancer cells exhibit temporal changes in cell morphology, cytoskeletal organization, and gene expression that are consistent with mesenchymal–epithelial plasticity as the stiffness of the matrix is reduced. A reduction in matrix stiffness attenuates the expression of integrin‐linked kinase, and inhibition of integrin‐linked kinase impacts proliferation, apoptosis, and gene expression in cells cultured on stiff and dynamic hydrogels. Overall, these findings reveal intermediate epithelial/mesenchymal states as cells move along a matrix stiffness‐mediated MET trajectory and suggest an important role for matrix mechanics in regulating mesenchymal–epithelial plasticity.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1016/j.actbio.2022.01.025
