Microenvironmental Guidance of Early Cell Mechanoresponse and Precise Matrix Deposition for Meniscus Tissue Engineering Public

Abstract

The meniscus is a fibrocartilaginous structure in the knee joint that plays a crucial role in load distribution, shock absorption, and joint stability. Meniscus injuries are a significant orthopaedic challenge, often leading to joint degeneration and osteoarthritis. While tissue-engineered scaffolds show promise for meniscal regeneration, understanding early cellular responses to biomaterial environments remains crucial for optimizing scaffold design. Additionally, the spatial heterogeneity of meniscal extracellular matrix (ECM) is crucial to recapitulating the native function of the tissue. This study investigates how 3D hydrogel microenvironments influence early cell mechanoresponse and ECM deposition, focusing on marrow stromal cells (MSCs) and meniscal fibrochondrocytes (MFCs), for the eventual goal of optimizing the design of tissue-engineered meniscus scaffolds.

Using fibrin-based hydrogels, we fabricated fiber-reinforced microenvironments that emulate the mechanical anisotropy of native meniscus tissue. MSCs encapsulated within fiber-reinforced constructs exhibited heterogeneous morphological and mechanosensitive responses, which were analyzed using principal component analysis (PCA) and agglomerative hierarchical clustering (AHC). This approach identified three distinct cell subpopulations based on cell morphology and nuclear translocation of YAP, a key mechanotransduction marker. Notably, high-response MSCs preferentially localized near stiff polymer fibers, suggesting a distance-dependent mechanosensitive response.

To further explore early cell-matrix interactions, we used methacrylated gelatin and hyaluronic acid hydrogels (GelMA/MeHA) to investigate microenvironmental influence of nascent ECM production by MFCs. It was found that MFCs possess an inherent ability to deposit aligned matrix within 14 days, and increasing MeHA content in GelMA hydrogels suppressed matrix anisotropy, demonstrating that biomaterial composition directly influences meniscal ECM organization. Various GelMA/MeHA formulations promoted differential distributions of protein and glycosaminoglycan (GAG) deposition by MFCs within 3 days, providing a promising avenue by which to precisely guide ECM deposition in tissue engineered meniscus.

Overall, this study provides novel insights into early cell-matrix interactions in meniscal tissue engineering. By identifying spatially responsive cell populations and their mechanosensitive behaviors, we establish a framework for optimizing fiber-reinforced scaffolds. These findings contribute to the design of next-generation meniscus replacements that control cellular guidance and ECM deposition.  Future studies will focus on integrating these biomaterial strategies into preclinical models to assess long-term tissue maturation and biomechanical performance.

Table of Contents

Table of Contents

Introduction                                                                                                                       1-6

Materials and Methods                                                                                                   7-18

Cell Preparation, Culture, and Native Meniscus Sectioning                                           7

Fibrin Gel Fabrication and Mechanical Testing                                                           7-8

Cell Encapsulation and Hydrogel Culture – Fibrin Gels                                                  8

Immunofluroescent Staining – Fibrin Gels                                                                   8-9

Image Acquisition and Cell Parameter Extraction – Fibrin Gels                               9-10

Analysis Pipeline, PCA, AHC – Fibrin Gels                                                             10-11

Methacrylated Gelatin/Hyaluronic Acid Gel Fabrication                                               11

Nascent Matrix Labeling and Culture of GelMA/MeHA Hydrogels                          11-12

Immunofluorescent Staining – GelMA/MeHA Gels                                                  12-13

Imaging and Cell Parameter Extraction – GelMA/MeHA Gels                                     13

Pentanoate-Functionalized Hyaluronic Acid and Peptide Functionalization           13-14

Statistics                                                                                                                   17-18

Results                                                                                                                         19-35

Discussion                                                                                                                  36-40

Conclusions                                                                                                                       41

References                                                                                                                   42-51

About this Honors Thesis

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School	Emory College
Department	Biology
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Biology, Cell Engineering, Biomedical
Mot-clé	Biomaterials Mechanoresponse Hydrogels Tissue Engineering Meniscus
Committee Chair / Thesis Advisor	Jay Patel, Emory University
Committee Members	Megan Cole, Emory University Miguel Reyes, Emory University

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