Z29 - Laminin-332 Conjugated Electrospun Tissue Scaffolds for Enhanced Cell Adhesion

Saturday, October 11, 2025

10:00 AM - 11:00 AM PDT

Location: Exhibit Hall F, G & H

Presenting Author(s)

JF

Jack Flaggert

Undergraduate Student
Dartmouth College, United States

Co-Author(s)

Adelaide Cagle, B.S.

Ph.D. Candidate
Dartmouth College
Hampstead, New Hampshire, United States

Last Author(s)

HN

Hung V.-T. Nguyen, Ph.D.

Assistant Professor of Engineering
Dartmouth College
Hanover, New Hampshire, United States

Primary Investigator(s)

Katherine Hixon

Principle Investigator
Dartmouth College, Thayer School of Engineering
Hanover, New Hampshire, United States

Introduction: : The field of biomaterials continues to seek new strategies to enhance the performance and compatibility of materials used in the body. Synthetic materials such as electrospun fibers (ESFs) are commonly used in biomaterials and tissue engineering due to their superior mechanical and tunable properties [1]. ESFs are particularly valued for their high surface area and porosity, which facilitate cell infiltration. However, synthetic scaffolds often lack the biological markers and binding sites present in natural materials [1]. When a permanent implant is required—especially one requiring resistance to degradation, for implant support—synthetic polymers are ideal; however, implementation can be challenging if the surrounding tissue does not adhere spontaneously to the scaffold surface. In transcutaneous or soft tissue applications, high tissue turnover and fibrotic encapsulation further complicate long-term adhesion. This is commonly addressed by incorporating bioactive molecules into the synthetic material [2]. However, previous studies have typically incorporated these biomolecules into the ESF solution, resulting in functional groups embedded within the fibers and not accessible on the surface [3]. To address this, we propose post-electrospinning surface conjugation of bioactive proteins to the ESFs surface, ensuring that functional molecules are presented only on the accessible fiber surfaces. This approach could yield robust, porous scaffolds with durable bioactivity for tissue support and repair. In this study, we focus on the conjugation of laminin, a basement membrane protein known to promote integrin-mediated cell adhesion, to ESF meshes made with FDA-approved polymethyl methacrylate (PMMA) and polyethylene terephthalate glycol (PETG) [4,5,6].

Materials and

Methods: : A polymer blend was prepared by dissolving 1g of PMMA and 1g of PETG in 20 mL of Hexafluoro-2-propanol using a rotary tube mixer. The solution was electrospun using a Spinbox (Bionicia Fluidnatek) onto a rotating mandrel (~1000 rpm) to produce aligned fibers and a flat collector for non-aligned fibers. Meshes were imaged via scanning electron microscopy (SEM), then functionalized through surface hydrolysis followed by carbodiimide crosslinking. Individual ESF squares were submerged in 1M sodium hydroxide for 45 minutes, then protonated with a 5-minute 0.1M hydrochloric acid wash. For carbodiimide-mediated conjugation, 479.25mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 287.73 mg of N-hydroxysuccinimide (NHS) were added to 50 mL of 1M 2-(N-morpholino)ethanesulfonic acid buffer (Fig. 1). ESFs were incubated in this solution to generate reactive NHS esters, then transferred to phosphate-buffered saline (PBS) containing the target protein and incubated for 24 hours at room temperature on a shaker. Samples were thoroughly washed with PBS to remove unbound protein. Two proteins were evaluated: rat-derived collagen and recombinant human laminin-332, the latter known for promoting integrated-mediated cell adhesion [4]. Conjugation was confirmed via fluorescence microscopy. Collagen binding was assessed using anti-collagen primary and fluorescent secondary antibodies; laminin was detected similarly. SEM was used to evaluate scaffold morphology post-electrospinning, post-hydrolysis, post-carbodiimide attachment, and post-protein-conjugation. Fluorescence imaging was used to quantify protein retention and assess specificity by comparing non-functionalized ESFs incubated with protein.

Results, Conclusions, and Discussions:: SEM of the PMMA/PETG hybrid ESFs revealed morphologies consistent with prior studies. ESFs collected on a rotating mandrel exhibited high alignment, while those collected on a stationary collector displayed random orientation (Fig. 2A, B). Neither surface hydrolysis nor protein conjugation resulted in visible changes to ESF morphology (Fig. 2C-F).
Fluorescence microscopy confirmed successful conjugation of rat collagen and human laminin-332 to the ESFs. Rat collagen-conjugated scaffolds exhibited increased fluorescence under 488 nm excitation compared to non-functionalized controls (Fig. 3D–F). Laminin-332-conjugated scaffolds showed even stronger fluorescence (Fig. 3G), with negligible signal in unconjugated samples, suggesting a more efficient or robust conjugation for laminin-332.
These results support the feasibility of creating bioactive, protein-functionalized ESFs with preserved morphological integrity. The enhanced fluorescence observed for laminin-332 suggests it may provide greater surface availability or stronger binding, making it particularly promising for applications involving cell adhesion and integration. The aligned architecture of the ESFs may further support cell ingrowth and directional migration. Ongoing studies are evaluating cytotoxicity and cell compatibility. A 14-day human dermal fibroblast (hDFB) viability assay is underway to assess short-term cellular response to the modified scaffolds. In parallel, cell adhesion studies are quantifying hDFB attachment to ESFs with and without laminin-332 conjugation. Given laminin’s widespread use as a culture substrate to promote adhesion and proliferation, increased cell attachment is anticipated [7]. Together, these results highlight the potential of post-electrospinning protein conjugation as a modular strategy for enhancing synthetic scaffold bioactivity. Future work will focus on long-term cell viability and integration in physiologically relevant tissue models.

Acknowledgements and/or References (Optional): : [1] Jenkins, Thomas Lee, and Dianne Little. “Synthetic Scaffolds for Musculoskeletal Tissue Engineering: Cellular Responses to Fiber Parameters.” Npj Regenerative Medicine, vol. 4, no. 1, Jun. 2019, p. 15. www.nature.com, https://doi.org/10.1038/s41536-019-0076-5.

[2] Gordon, David J., et al. “Modification of Titanium Alloy Surfaces for Percutaneous Implants by Covalently Attaching Laminin.” Journal of Biomedical Materials Research Part A, vol. 94A, no. 2, Aug. 2010, pp. 586–93. DOI.org (Crossref), https://doi.org/10.1002/jbm.a.32735.

[3] Cruz-Maya, Iriczalli, et al. “Comparative Study on Protein-Rich Electrospun Fibers for In Vitro Applications.” Polymers, vol. 12, no. 8, Jul. 2020, p. 1671. PubMed Central, https://doi.org/10.3390/polym12081671.

[4] Hohenester, Erhard. “Structural Biology of Laminins.” Essays in Biochemistry, vol. 63, no. 3, May 2019, pp. 285–95. PubMed Central, https://doi.org/10.1042/EBC20180075.

[5] Dondani, Jay R., et al. “Surface Treatments of PEEK for Osseointegration to Bone.” Biomolecules, vol. 13, no. 3, 3, Mar. 2023, p. 464. www.mdpi.com, https://doi.org/10.3390/biom13030464.

[6] Scott Lucas. Medical Device Material Performance Study Polyethylene Terephthalate (PET) Safety Profile. 24 Nov. 2020, https://www.fda.gov/media/155399/download.

[7] Rodin, Sergey, et al. “Long-Term Self-Renewal of Human Pluripotent Stem Cells on Human Recombinant Laminin-511.” Nature Biotechnology, vol. 28, no. 6, Jun. 2010, pp. 611–15. www.nature.com, https://doi.org/10.1038/nbt.1620.