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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session E

Poster I13 - Understanding 3D Cell-Matrix Interactions Using Microfiber-Hydrogel Composite Materials

Saturday, October 26, 2024

10:00 AM – 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Sabriye Yilmaz

Undergraduate Researcher
Rowan University
Cherry Hill, New Jersey, United States

Co-Author(s)

Umu S. Jalloh (she/her/hers)

Graduate Student at Rowan University
Rowan University
Glassboro, New Jersey, United States
JP

Jay Patel, PhD

Assistant Professor
Emory University, United States

Primary Investigator(s)

Sebastian Vega, PhD

Associate Professor
Department of Biomedical Engineering, Rowan University, Glassboro, NJ and Department of Orthopedic Surgery, Cooper Medical School of Rowan University, Camden, NJ, New Jersey, United States

Introduction: Cells have the remarkable ability to sense and respond to physical cues from their local ECM, and there are major differences in cell-matrix interactions between cells on 2D and in 3D engineered environments. For instance, adherent cells on 2D matrices experience increased cell spreading and matrix mechanosensing with increasing substrate stiffness, whereas 3D cell spreading and matrix mechanosensing is regulated by cell-mediated material remodeling/degradation, which does not necessarily depend on stiffness. Although hydrogels with enzymatically degradable moieties can induce 3D cell spreading and matrix mechanosensing, there is poor control over cellular orientation, which is critical for engineering aligned tissues such as the meniscus and skeletal muscle. We hypothesize that cells can sense stiffness while embedded in a soft-stiff 3D biomaterial composite, and that this phenomenon can be leveraged to control 3D cellular alignment. To test our hypothesis, we encapsulated MSCs (mesenchymal stem cells) in enzymatically degradable hydrogels in the presence of a stiff, linear microfiber. Using this platform, we studied the effects of microfiber diameter and hydrogel network interconnectivity on MSC alignment.

Materials and

Methods: Microfiber formation: PCL (polycaprolactone) microfibers were formed by melting PCL (80,000 Mn) pellets (80 °C) that were pulled with forceps. Norbornene-PCL (Nor-PCL) microfibers were formed by mixing PCL with 8-arm PEG-Nor (20 kDa, JenKem) at 10 wt% (Figure 1A). By varying forceps pull-rate, small (PCL 215 µm; Nor-PCL 313 µm) and large (PCL 458 µm; Nor-PCL 588 µm) diameter microfibers were formed.

Formation of microfiber-hydrogel composites: Hydrogels were created using a thiol-ene click chemistry reaction between HANor (norbornene-modified hyaluronic acid) and di-thiolated enzymatically degradable crosslinker. Hydrogel solutions consisted of HANor (3 wt%), di-thiolated crosslinker (2.5 mM), photoinitiator (Irgacure 2959, 0.05 wt%), and cell adhesion RGD peptide (2 mM) in MSC culture medium (αMEM, 10% fetal bovine serum, 1% penicillin/streptomycin). MSC pellets were resuspended in hydrogel solution (1E6 cells/mL) and added to a cylindrical mold (8 mm diameter, 1 mm height) attached to glass (12 mm diameter). Four groups were evaluated for morphology: (1) small PCL fiber, (2) small Nor-PCL fiber, (3) large PCL fiber, and (4) large Nor-PCL fiber. Gels in each group were then photopolymerized using UV light (10 mW/cm², 5 minutes) and cultured in MSC culture medium at 37 °C (Figure 1B).

Assessing MSC viability and alignment: to examine viability, Live/Dead staining was performed on microfiber-gel composite groups after 5 days in culture. To examine alignment, microfiber-gel groups were stained with phalloidin (1:100) and Hoechst (1:1000), and confocal microscopy images were used to quantify single-cell conformity indices (Figure 1, Equation 1) using ImageJ software.

Results, Conclusions, and Discussions:

Results: Encapsulated MSCs showed high viability in hydrogels regardless of microfiber presence, size, or inclusion of norbornene, with over 92% viable cells across all groups (Figure 1C). 5 days post-encapsulation, there was noticeable variation in angle deviation from fiber, aspect ratio, and conformity index (Eq. 1) among cells (Figure 1D-E). The conformity index shows the amount of alignment of the cell to the stiff fiber by accounting for the cell angle and amount of cell spreading, as reported by Pucha et al. [1]. The conformity index showed a negative correlation based on distance from the fiber for each microfiber group (Figure 1F). However, the R-squared values were small, indicating there was weak correlation.

Cells were then binned based on distance in 75 µm increments from the fiber to examine average conformity index (Figure 1G). Nor-PCL fibers had a slightly higher average conformity index than PCL fibers of equivalent size. For both PCL and Nor-PCL, the larger fibers had higher average conformity values. However, these trends were not statistically significant (p-values > 0.05).

Discussion: These findings suggest that 3D cell alignment can be controlled in a soft-stiff hydrogel environment and increased microfiber diameter and connectivity (via inclusion of Nor) increases MSC alignment. However, the correlations were poor and not statistically significant, warranting a deeper level of analysis. In a recent study evaluating the effects of 3D cell alignment in soft-stiff microenvironments, a set of cellular and nuclear descriptors were analyzed using Agglomerative Hierarchical Clustering (AHC) [1]. This approach yielded three distinct clusters that showed statistically significant trends. Additionally, YAP (yes-associated protein) is a mechanosensitive transcriptional coactivator that can be included in the conformity index equation to attain single-cell morpho-mechanical responses [1].

Conclusions: In this study, we found that fiber diameter and connectivity can regulate 3D cell alignment in soft-stiff material composites. We are currently investigating how microfiber tension may augment these effects. Tension has a proportional relationship with stiffness and could potentially increase the amount of conformity. These findings have the potential for guiding the design of fiber-reinforced scaffolds for engineering aligned tissues including peripheral nerve, the meniscus, and skeletal muscle.

Acknowledgements (Optional): References

[1] Pucha, S. A., Hasson, M., Solomon, H., McColgan, G. E., Robinson, J. L., Vega, S. L., Patel, M. J. Revealing early spatial patterns of cellular responsivity in fiber-reinforced microenvironments. Mary Ann Liebert, Inc., 2024.