(B-80) Mechanical Characterization and Cellular Responses of Biomimetic dECM-MeHA Scaffolds for Fibrous Tissue Engineering

Friday, October 13, 2023

9:30 AM – 10:30 AM PDT

Location: Exhibit Hall - Row B - Poster # 80

Presenting Author(s)

LH

Lucy Hederick

Undergraduate Student
University of Pennsylvania, Northwestern University
Wayne, Pennsylvania, United States

Co-Author(s)

ZL

Zizhao Li

Graduate Student
University of Pennsylvania, United States
YZ

Yujia Zhang

Graduate Student
University of Pennsylvania
Philadelphia, Pennsylvania, United States

Primary Investigator(s)

SH

Su-Jin Heo

Professor
University of Pennsylvania, United States

Introduction:: Musculoskeletal injuries are challenging to fully repair due to limited vascularity and poor endogenous healing abilities in tissues like tendons, knee meniscus, and the rotator cuff. Tissue-engineered scaffolds offer a promising solution by stimulating tissue repair through increased cell proliferation, differentiation, and extracellular matrix deposition [1]. Among the various scaffold designs, biomimetic scaffolds have garnered attention, combining essential extracellular matrix proteins, such as collagen and proteoglycans, with polymeric hydrogels to mimic the native tissue microenvironment. The success of a biomimetic scaffold hinges on its ability to synergistically replicate the mechanical properties of native tissue while facilitating optimal cellular growth and differentiation in the target cells [2]. However, the optimal parameters regarding mechanical properties for constructing a tissue-engineered scaffold that fulfills both roles have yet to be fully elucidated [3]. Therefore, to address this crucial knowledge gap, we present a study involving the construction of biomimetic scaffolds using fixed decellularized extracellular matrix (dECM) and varying methacrylated hyaluronic acid (MeHA) polymer stiffness. The mechanical modulus and bioactivity of each scaffold construct will be thoroughly evaluated. By optimizing the polymeric and mechanical characteristics of these biocompatible scaffolds, we aim to pave the way for their clinical incorporation and their potential to significantly enhance the healing of musculoskeletal injuries.

Materials and Methods:: MeHA of varying stiffness was produced according to the previous protocol [4] by varying amounts of methacrylic anhydride (Sigma) added to sodium hyaluronate (Lifecore) solution. To produce dECM-MeHA hydrogels for electrospinning, 2% w/v dECM in dECM acidic solution (synthesized according to the previous protocol [4]) was combined with 4% w/v MeHA (of either 35%, 50%, 70%, or 100% methacrylic anhydride concentration), 2% w/v PEO, 0.5% w/v Irgacure 2959, and distilled water. Scaffolds were electrospun at a rate of 1 mL dECM-MeHA gel per hour and then crosslinked under UV light for 30 minutes after collection. Scaffold fiber diameter was measured in dry and hydrated environments using an environmental scanning electron microscope (ESEM). Scaffolds electrospun onto circular glass slides were chemically crosslinked using Genipin solution for three days before seeding bovine mesenchymal stem cells (bMSCs). Cell attachment was assessed using Cell Counting Kit-8 (CCK8) on Day 1. On Day 3, we extracted histone proteins from these scaffolds for Western Blot analysis to evaluate epigenetic modifications.

Results, Conclusions, and Discussions:: In this study, we successfully fabricated four dECM-MeHA scaffolds with varying MeHA stiffness, ranging from 35% (softest) to 100% (stiffest). Upon immersion in PBS, both the softest (35% dECM-MeHA) and stiffest (100% dECM-MeHA) scaffolds exhibited a significant increase in fiber diameter with a more significant increase in the softest group (Fig. 1). Successful cell attachment to each scaffold was indicated by CCK8 assays. Absorbance significantly increased with 100% dECM-MeHA scaffolds, indicating that increased MeHA stiffness within the scaffold likely enhances cell attachment (Fig. 2). The Western Blot analysis revealed increased levels of acetylation of Histone H3K9 (H3K9ac, a marker for gene activation), and trimethylation of H3K27 (H3K27me3, a marker for gene suppression), in the 35% dECM-MeHA scaffold relative to other groups. This result suggests that the scaffold stiffness regulates cellular gene expression and differentiation (Fig. 3). In conclusion, stiffness-tunable scaffolds were successfully created as evidenced by fiber diameters in dry and hydrated environments. While the stiffest scaffold promoted the highest level of cell attachment, attachment was successful in each scaffold. Additionally, epigenetic modulations occurred in cells of each scaffold type. Taken together, these results provide valuable insights for optimizing the design of tissue-engineered scaffolds that can effectively promote fibrous tissue repair and regeneration.

Acknowledgements (Optional): :

References (Optional): :

Sujeeun LY, Goonoo N, Ramphul H, et al. Correlating in vitro performance with physico-chemical characteristics of nanofibrous scaffolds for skin tissue engineering using supervised machine learning algorithms. Royal Society Open Science. 2020;7(12):201293. doi:10.1098/rsos.201293

Gilpin A, Yang Y. Decellularization Strategies for Regenerative Medicine: From Processing Techniques to Applications. Biomed Res Int. 2017; 2017. doi:10.1155/2017/9831534

Zhang J, Wehrle E, Adamek P, et al. Optimization of mechanical stiffness and cell density of 3D bioprinted cell-laden scaffolds improves extracellular matrix mineralization and cellular organization for bone tissue engineering. Acta Biomaterialia. 2020;114:307-322. doi:10.1016/j.actbio.2020.07.016

Zizhao Li, Se-Hwan Lee, et al. Tunable Extracellular Matrix-Based Multiphasic Nanofibrous Scaffold Systems for Rotator Cuff Enthesis Repair, Annual Meeting of Orthopaedic Research Society 2023.