Investigating Cellular Mechanotransduction of Wound Healing within a Macroporous Biomaterial

Mechanical cues, such as extracellular matrix (ECM) stiffness, regulate fibroblast [1] and macrophage phenotype [2] and coordinate macrophage–fibroblast interactions [3]. Furthermore, the time scales over which matrix mechanics change are relevant, as cells are exposed to dynamic changes rather than static conditions during wound healing [4–9]. In this context, biomaterials capable of stiffening in situ and in the presence of cells have been used to identify new disease-relevant mechanotransduction mechanisms [4–9]. However, these studies often employ 2D culture and lack the dimensionality of native tissue. While 3D culture methods are improving, 3D mechanobiology studies are often unable to reproduce 2D findings. One challenge when studying the effects of matrix stiffness in 3D is that stiffness and cell confinement are often coupled [10]. Therefore, we aim to design a biomaterial suitable for 3D, in situ stiffening to mimic changes to the dermis during fibrosis and wound healing. Importantly, our design allows for stiffness tuning independent of confinement and macrophage incorporation for co-culture to study inflammation. We adapted Methacrylated Hyaluronic Acid (MeHA) a material compatible with 2D in situ stiffening studies via photo-initiated polymerization [5,6], to create a 3D macroporous gel, comprised of fibrous microgels. We hypothesize the macroporous nature of the gel will allow fibroblast spreading, matrix deposition, and macrophage migration. We hypothesize that our platform will allow us to dynamically increase matrix stiffness over different time scales, by tuning photopolymerization parameters, without increasing cell confinement, and therefore allow us to identify new mechanotransduction pathways contributing to dermal inflammation, macrophage polarization, myofibroblast activation, and macrophage–fibroblast crosstalk in 3D. References [1] Vallée, A. & Lecarpentier, Y., Cell & Bioscience (2019) DOI 10.1186/s13578-019-0362-3. [2] Atcha, H. et al., Nature Communications (2021) DOI 10.1038/s41467-021-23482-5. [3] Pakshir, P. et al, Nature Communications (2019) DOI 10.1038/s41467-019-09709-6. [4] Chen, Z. & Lv, Y., Composites Part B: Engineering (2022). DOI 10.1016/j.compositesb.2022.110162. [5] Caliari, S. et al., Scientific Reports (2016). DOI 10.1038/srep21387 (2016). [6] Guvendiren, M. & Burdick, J. A., Nature Communications (2012). DOI 10.1038/ncomms1792/. [7] Mabry, K. M, et al., Biomaterials (2015). DOI 10.1016/j.biomaterials.2015.01.047. [8] Ondeck, M. G. et al., Proceedings of the National Academy of Sciences of the United States of America (2019). 10.1073/pnas.1814204116. [9] Kumar, A. et al., Nature Biomedical Engineering (2019). DOI 10.1073/pnas.1814204116. [10] Pathak, A. et al Proceedings of the National Academy of Sciences of the United States of America (2012). DOI 10.1073/pnas.1118073109.

Mechanical cues, such as extracellular matrix (ECM) stiffness, regulate fibroblast [1] and macrophage phenotype [2] and coordinate macrophage–fibroblast interactions [3]. Furthermore, the time scales over which matrix mechanics change are relevant, as cells are exposed to dynamic changes rather than static conditions during wound healing [4–9]. In this context, biomaterials capable of stiffening in situ and in the presence of cells have been used to identify new disease-relevant mechanotransduction mechanisms [4–9]. However, these studies often employ 2D culture and lack the dimensionality of native tissue.

While 3D culture methods are improving, 3D mechanobiology studies are often unable to reproduce 2D findings. One challenge when studying the effects of matrix stiffness in 3D is that stiffness and cell confinement are often coupled [10]. Therefore, we aim to design a biomaterial suitable for 3D, in situ stiffening to mimic changes to the dermis during fibrosis and wound healing. Importantly, our design allows for stiffness tuning independent of confinement and macrophage incorporation for co-culture to study inflammation. We adapted Methacrylated Hyaluronic Acid (MeHA) a material compatible with 2D in situ stiffening studies via photo-initiated polymerization [5,6], to create a 3D macroporous gel, comprised of fibrous microgels.

We hypothesize the macroporous nature of the gel will allow fibroblast spreading, matrix deposition, and macrophage migration. We hypothesize that our platform will allow us to dynamically increase matrix stiffness over different time scales, by tuning photopolymerization parameters, without increasing cell confinement, and therefore allow us to identify new mechanotransduction pathways contributing to dermal inflammation, macrophage polarization, myofibroblast activation, and macrophage–fibroblast crosstalk in 3D.

References
[1] Vallée, A. & Lecarpentier, Y., Cell & Bioscience (2019) DOI 10.1186/s13578-019-0362-3.
[2] Atcha, H. et al., Nature Communications (2021) DOI 10.1038/s41467-021-23482-5.
[3] Pakshir, P. et al, Nature Communications (2019) DOI 10.1038/s41467-019-09709-6.
[4] Chen, Z. & Lv, Y., Composites Part B: Engineering (2022). DOI 10.1016/j.compositesb.2022.110162.
[5] Caliari, S. et al., Scientific Reports (2016). DOI 10.1038/srep21387 (2016).
[6] Guvendiren, M. & Burdick, J. A., Nature Communications (2012). DOI 10.1038/ncomms1792/.
[7] Mabry, K. M, et al., Biomaterials (2015). DOI 10.1016/j.biomaterials.2015.01.047.
[8] Ondeck, M. G. et al., Proceedings of the National Academy of Sciences of the United States of America (2019). 10.1073/pnas.1814204116.
[9] Kumar, A. et al., Nature Biomedical Engineering (2019). DOI 10.1073/pnas.1814204116.
[10] Pathak, A. et al Proceedings of the National Academy of Sciences of the United States of America (2012). DOI 10.1073/pnas.1118073109.

Masters Thesis focused on developing the protocol for characterizing phenotype of cells during in situ stiffening while also helping quantify material properties of granular MeHA gels for 3D culture. Skills required from the student side (all ideal but not necessary): -Cell culture experience -Immunofluorescent Staining and/or Microscopy -Experience with either hydrogels or microfluidic devices Skills to be learnt during the project: -3D cell culture -MeHA gel synthesis -Mechanical Sieving -Loading Gels and Cells into a microfluidic device -In Situ Photopolymerization method -Confocal Imaging -Rheometry -MeHA synthesis (if interested)

Masters Thesis focused on developing the protocol for characterizing phenotype of cells during in situ stiffening while also helping quantify material properties of granular MeHA gels for 3D culture.

Skills required from the student side (all ideal but not necessary):
-Cell culture experience
-Immunofluorescent Staining and/or Microscopy
-Experience with either hydrogels or microfluidic devices

Skills to be learnt during the project:
-3D cell culture
-MeHA gel synthesis
-Mechanical Sieving
-Loading Gels and Cells into a microfluidic device
-In Situ Photopolymerization method
-Confocal Imaging
-Rheometry
-MeHA synthesis (if interested)

Dr Jaimie Mayner, Macromolecular Engineering Lab, DMAVT Email: jmayner@ethz.ch Please email me, sharing your CV and transcript, if you are interested in the position so we can schedule a meeting. Don't hesitate to reach out if you have any questions!

Dr Jaimie Mayner,
Macromolecular Engineering Lab, DMAVT
Email: jmayner@ethz.ch
Please email me, sharing your CV and transcript, if you are interested in the position so we can schedule a meeting. Don't hesitate to reach out if you have any questions!