The Influence of Polymer Molecular Weight on Hyaluronic Acid Hydrogel Reinforcement of Articular Cartilage Open Access

Brackin, Riley (Spring 2023)

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Introduction: Osteoarthritis (OA) is a degenerative joint disease that often results from traumatic injury and causes significant disability. The disease is typically characterized by a gradual breakdown of articular cartilage, which is normally a dense extracellular matrix (ECM). As the degradation progresses, the ECM breaks apart, triggering a cascade of inflammatory signaling, including tumor necrosis factor–alpha (TNF-A) and interleukin-1-beta (IL-1b), which prompted the production of matrix-degrading enzymes. This perpetuates a cycle of further joint breakdown, ultimately contributing to the pathogenesis of osteoarthritis. Recent studies demonstrated that cartilage-infiltrating biomaterials can reinforce damaged cartilage and slow down the degradation of the ECM. This study specifically focuses on how the molecular weight of methacrylated hyaluronic acid (MeHA) impacts biomechanical fortification and preservation of articular cartilage in a pro-degradation environment.

Materials and Methods: MeHA (20 kDa, 75 kDa, and 100 kDa) was synthesized, and the degree of methacrylation of each was confirmed with NMR. The three hydrogel polymers were applied to juvenile bovine cartilage explants. Hydrogel mechanical testing was performed, and initial diffusion studies were conducted to confirm hydrogel infiltration into cartilage. Fortified cartilage explants were cultured for a two-week degradation period (10ng/mL IL-1b), during which media was sampled for proteoglycan loss. Explants were mechanically tested via Hertzian Indentation creep tests at the end of this two-week period.

Results: The degree of methacrylation was 30.28%, 35.5%, and 55.9% for the 20 kDa, 75 kDa, and 100 kDa polymers, respectively. All polymers produced significantly different compressive modulus values (p-value<0.0001). The 20 kDa MeHA applied to articular cartilage explants showed a significantly higher compressive modulus with the control condition, p-value<0.001, and IL-1b condition, p-value<0.01.

Discussion: The cartilage explants showed promising reinforcement with the 20 kDa MeHA polymer condition, particularly regarding compressive modulus. This suggests that a greater diffusion ability and distance are likely beneficial for mechanical reinforcement of cartilage, resulting in maintenance of these biophysical properties in degenerative conditions.

Clinical Relevance: Using MeHA with a smaller molecular weight may be beneficial in maintaining mechanical properties and proactively reducing articular cartilage degeneration.

Table of Contents

1. Introduction 2

1.1 Knee Joint Anatomy and Physiology 2

1.2 Motivation 4

1.3 Osteoarthritis 5

1.4 Hydrogels to Combat Chondrocyte Degeneration 10

1.5 Objectives and Significance 12

2. Materials and Methods 14

Aim1: Methacrylation of Hyaluronic Acid and Gel Mechanics 14

2.1 Methacrylate Hyaluronic Acid Reaction 14

2.2 Gel Mechanics 17

Aim 2: MeHA Hydrogel Diffusion 17

2.3 Explant Dissection and Processing 17

2.4 Diffusion Studies (integration and fortification) 18

Aim 3: Application of MeHA to Living Explants 19

2.5 Living Explant Degenerative Culture 19

2.6 Mechanical Testing of Degenerative Culture 21

2.7 DMMB Assay 23

2.8 Safranin-O Fast Green Staining 23

2.9 Statistical analysis 24

3. Results 25

Aim 1: Methacrylation of Hyaluronic Acid and Gel Mechanics 25

3.1 NMR of MeHA Polymers with Interpretations 25

3.2 Gel Mechanics 28

Aim 2: MeHA Hydrogel Diffusion 30

3.3 Tissue Penetration of Degenerated Cartilage Images of Diffusion 30

Aim 3: Application of MeHA to Living Explants 31

3.4 Living Cartilage Explant Mechanics 31

3.5 Proteoglycan Loss 33

3.6 Imaging 35

4. Discussion 37

5. Conclusion 43

6. Supplementary Information 44

7. References 46

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