Injectable Hydrogel with Magnetically Alignable PCL Fiber for Guiding Myogenic and Neurogenic Cell Alignment

다국어 초록 (Multilingual Abstract)

Injectable hydrogels offer a promising platform for treating irregular soft‐tissue defects; however, faithfully reconstructing the inherent anisotropic architecture of skeletal muscle and peripheral nerves following injection remains a significant challenge. In this study, we developed a magnetically alignable injectable hydrogel composed of gelatin methacryloyl (GelMA) incorporating fragmented polycaprolactone (PCL) microfibers embedded with magnetic nanoparticles (MNPs). This system enables rapid magnetic alignment of the fibers within seconds after injection, and subsequent UV crosslinking preserves anisotropy without compromising injectability or cytocompatibility.
The aligned hydrogels exhibited a compressive modulus of approximately 11 kPa even at a fiber content as low as 0.1%, a value comparable to randomly oriented scaffolds containing tenfold higher fiber density. This finding indicates that structural organization may have a greater influence on bulk mechanical stiffness than material quantity alone. In C2C12 myoblast cultures, fiber alignment promoted cell elongation and moderate orientation, while MyoD upregulation was observed only at fiber concentrations ≥0.5%. These results suggest that myogenic differentiation strongly depends on cell–fiber contact frequency rather than solely on bulk mechanical reinforcement.
Neural cell experiments further confirmed that the hydrogel platform maintains cytocompatibility and supports baseline RA/BDNF-induced differentiation. Although the aligned topography did not produce significant differences in neural marker expression under the current low-fiber and soft-matrix conditions, neurite outgrowth and differentiation capacity were preserved, indicating that chemical cues remain the primary driver of SH-SY5Y neurogenesis.
These findings highlight the importance of jointly optimizing structural, mechanical, and biochemical parameters to more effectively promote neural differentiation. Strategies such as increasing fiber content, modulating GelMA stiffness, incorporating additional neurotrophic factors, extending the differentiation period, and performing quantitative three-dimensional neurite reconstruction are expected to more clearly elucidate the effects of aligned topography on neural maturation and axonal guidance.
Overall, this magnetically alignable and injectable hydrogel provides a minimally invasive platform capable of generating spatially organized architectures while maintaining cytocompatibility for both myogenic and neurogenic cells. With further optimization, this system holds strong potential for engineering anisotropic tissues—such as skeletal muscle, tendons, and peripheral nerves—where structural alignment is essential for functional recovery.

목차 (Table of Contents)

• CHAPTER 1. Background 1
• 1.1 Principle of tissue engineering 2
• 1.2 Two-dimensional and three-di mensional culture systems 4
• 1.3 Hydrogel and three-dimensional scaffold systems 7
• 1.4 GelMA hydrogel 10
• 1.5 ECM-like biomaterials 13
• 1.6 Skeletal muscle and peripheral nerve 16
• 1.7 Existing alignment strategies 24
• 1.8 Mechanobiology of skeletal muscle and neuronal cells 26
• CHAPTER 2. Introduction 28
• CHAPTER 3. Experimental section 37
• 3.1 Chemicals and reagents 38
• 3.2 Fabrication of magnetically responsive PCL fiber 39
• 3.3 Preparation of MNP-PCL/GelMA hydrogel matrix 41
• 3.4 Characterization of fiber and hydrogel 43
• 3.5 Cell culture and encapsulation 44
• 3.6 Assessment of cell viability and morphology 46
• 3.7 Immunofluorescence staining 47
• 3.8 Statistical analysis 48
• CHAPTER 4. Results and Discussion 49
• 4.1. Fabrication and Characterization of MNP-PCL Microfibers 50
• 4.1.1 Fabrication of MNP-PCL Fibers through Electrospinning 52
• 4.1.2 Fiber Fragmentation through Alkaline Hydrolysis 53
• 4.1.3 Microfiber Dispersion Characteristics Within GelMA Hydrogels 55
• 4.2 Verification of Magnetic Field-Based Alignment Capability Following Injection 58
• 4.3. Mechanical Property Evaluation 63
• 4.3.1 Rheological Characterization: Oscillatory Rheology 64
• 4.3.2 Compression Test 66
• 4.3.3 Comparative Mechanical Property Analysis Based on Fiber Concentration and Alignment 69
• 4.4 Myoblast Viability and Morphological Responses 70
• 4.4.1 Cell Viability Assessment 71
• 4.4.2 Analysis of Cell Morphological Responses 73
• 4.4.3. Limiting Factors in Cellular Morphological Responses 77
• 4.5 Analysis of Muscle Differentiation Marker Expression in Aligned Structures 78
• 4.6 Assessment of Neural Cell Cytocompatibility 84
• 4.7 Analysis of Neural Differentiation Marker Expression 86
• 4.7.1 Time-Dependent Expression of Neural Differentiation Markers 87
• 4.7.2 Future Optimization and Enhancement Strategies 93
• CHAPTER 5. Conclusion 94
• References 97
• 국문초록 105