Hydrogel-based approaches for wound healing applications : temperature-responsive and photo-cross linkable hydrogel

다국어 초록 (Multilingual Abstract)

Skin cancer, particularly melanoma, stands as a significant global health threat due to its highly malignant and metastatic nature. Current treatment options, including surgery, chemotherapy, and immunotherapy, have inherent limitations, such as the risk of tumor recurrence post-surgery. This prompts the exploration of innovative approaches to enhance therapeutic efficacy, leading to the focus of our study on hydrogel-based solutions. Our investigation spans two interconnected chapters, each addressing distinct yet complementary aspects of skin-related challenges. The first chapter centers around the development of a hydrogel-based medical patch for melanoma treatment, aiming to overcome limitations associated with existing patches. Employing N-isopropylacrylamide (NIPAM), a temperature-sensitive polymer, our hydrogel responds to skin temperature, facilitating controlled drug delivery for effective skin regeneration. We navigate the challenges of slow temperature response associated with NIPAM by adopting a semi-IPN structure, where NIPAM intertwines with Poly(vinyl alcohol) (PVA), addressing hydrophobicity concerns and allowing precise control over substance release. The incorporation of doxorubicin (DOX), an anticancer drug, positions our hydrogel as a promising candidate for stable anticancer effects triggered by the skin’s temperature response. Simultaneously, the second chapter focuses on a hydrogel combining Collagen Methacryloyl (ColMA) and keratin, strategically chosen to capitalize on collagen's wound healing properties and keratin's ability to stimulate keratinocyte differentiation. This multifaceted approach synergizes lyophilized ColMA polymer with keratin, providing a unique solution for wound care beyond conventional dressings. The hydrogel actively promotes keratinocyte differentiation, resulting in augmented and expedited wound healing. The inclusion of ColMA, with its methacrylate modification, introduces tunable mechanical properties, enhancing adaptability to specific therapeutic requirements. Beyond wound care, the hydrogel holds promise in tissue regeneration and diverse biomedical applications. Both chapters collectively represent a pivotal step forward in advancing skin-related medical solutions. The dual-focus on melanoma treatment and wound healing, through innovative hydrogel formulations, underscores the potential to revolutionize current practices in regenerative medicine. The unique synergies harnessed in these approaches hold promises for enhanced therapeutic outcomes, fostering new frontiers in the field.

목차 (Table of Contents)

• Contents - i
• List of Figures v
• Abstract - ix
• Chapter 1. General Introduction 1
• 1.General Introduction 2
• 2. References - 3
• Chapter 2. Development of a Temperature-responsive Hydrogel
• Incorporating PVA into NIPAAm for Controllable Drug Release in
• Skin Regeneration 4
• 1. Introduction 5
• 2. Materials and methods 8
• 2.1. Materials - 8
• 2.2. Preparation of NIPAAm-PVA (N-P) hydrogels 8
• 2.3. Characterization of N-P hydrogels 10
• 2.4. Cell viability and proliferation 11
• 2.5. Release kinetics of DOX from N-P hydrogels 11
• 2.6. Evaluation of anti-cancer effect in vitro 12
• 2.7. Statistical analysis - 12
• 3. Results and discussion - 13
• 3.1. Fabrication and characterization of temperature responsive N-P hydrogel behavior-
• 13
• 3.2. Physical Characterization of N-P hydrogel according to temperature 16
• 3.3. In vitro biocompatibility and cell proliferation of N-P hydrogel 20
• 3.4. Correlation between degradation effect and drug release in vitro - 22
• 3.5. In vitro anti-cancer Effects of the DOX-loaded N-P Hydrogel - 25
• 4. Conclusions - 28
• 5. References 29
• Chapter 3. Keratin-infused ColMA-Based Photocrosslinked
• Hydrogels for Active Wound Healing 33
• 1. Introduction - 34
• 2. Materials and methods - 37
• 2.1. Materials 37
• 2.2. Preparation of Collagen methacryloyl (ColMA) 37
• 2.3. Preparation of keratin in-fused ColMA Hydrogel (ColMAK) 39
• 2.4. Characterization of ColMA hydrogels 39
• 2.5. Cell viability and proliferation 39
• 2.6. In vivo animal wound treatment of ColMAK hydrogels 40
• 2.7. Histological and IHC Evaluation 40
• 2.8. Statistical analysis - 41
• 3. Results and discussion - 42
• 3.1. Fabrication and characterization of ColMA hydrogel behavior 42
• 3.2. Physical Characterization of ColMA hydrogel 44
• 3.3. In vitro biocompatibility and cell proliferation of ColMA(K) hydrogel 46
• 3.4. In vivo animal wound healing assessment of ColMAK hydrogels 48
• 3.5. Histological evaluation in wound healing of ColMAK - 50
• 4. Conclusions - 52
• 5. References 53
• 국문요지 57
• Figure Contents
• Chapter 2. Development of a Temperature-responsive Hydrogel
• Incorporating PVA into NIPAAm for Controllable Drug Release in
• Skin Regeneration
• Figure 1.
• Schematic Illustration of temperature responsive N-P hydrogel. 7
• Table 1.
• Composition of hydrogel and manufacturing conditions. 9
• Figure 2.
• Characterization of N-P hydrogel: (a) DSC analysis, and (b) IR analysis. 15
• Figure 3.
• Evaluation of N-P hydrogel: (a) swelling ratio - representative fluorescence image (scale
• bar = 5 mm), (b) diameter measurement of N-P hydrogels (n = 3, ***p < 0.001), (c) SEM
• image (400 ⨉ and 2.0k ⨉, large scale bar = 100 um and small scale bar = 20 um), (d)
• storage and loss modulus (G’ and G”) of N-P hydrogels after swelling, shrinking, and
• reswelling, and (e) storage modulus comparison after swelling and reswelling (n = 3, *p <
• 0.05; **p < 0.01; ***p < 0.001). 19
• Figure 4.
• Evaluation of cell viability and proliferation of N-P hydrogel by transwell culture system:
• (a) representative LIVE/DEAD staining images of HaCat cells (scale bar = 275 um), (b)
• cell proliferation rate by CCK (n = 5, *p < 0.05; **p < 0.01). - 21
• Figure 5.
• Evaluation of N-P hydrogels according to amount of PVA: (a) degradation behavior of N-
• P hydrogels (n = 5), and (b) in vitro DOX release kinetics from N-P hydrogels (n = 5).
• 24
• Figure 6.
• In vitro anti-cancer effects of the DOX loaded N-P hydrogel by transwell system: (a) cell
• viability rate of B16-F10 melanoma cell by CCK (n = 5, **p < 0.01; ***p < 0.001), and
• (b) Fluorescence image of 2 h and 24 h (scale bar = 150 um, Endosome/lysosome and
• nuclei were stained with Lysotraker Green DND-26 and DAPI, respectively). 27
• Chapter 3. Keratin-infused ColMA-Based Photocrosslinked
• Hydrogels for Active Wound Healing
• Figure 1.
• Schematic Illustration of temperature responsive ColMAK hydrogel. 36
• Table 1.
• Composition of hydrogel and manufacturing conditions. 38
• Figure 2.
• Characterization of ColMA hydrogel: (a) IR analysis, and (b) NMR analysis. 43
• Figure 3.
• Rheological test of ColMA hydrogel (a) storage modulus and loss modulus after 4 ℃ and
• 37 ℃, and (b) storage modulus of comparison of 4 ℃ and 37 ℃, (c) storage modulus and
• loss modulus according to UV light time, (d) storage modulus of comparison according to
• UV light time. 45
• Figure 4.
• Evaluation of cell viability and proliferation of ColMA/k hydrogel by transwell culture
• system(a-c)/(d-f): (a) representative LIVE/DEAD staining images of HaCaT cells (scale
• bar = 650 um), (b) cell viability rate by LIVE/DEAD assay (n = 6, *p < 0.05), and (c) cell
• proliferation rate by CCK (n = 5, *p < 0.05; **p < 0.01), (d) representative LIVE/DEAD
• staining images of HaCaT cells (scale bar = 650 um), (e) cell viability rate by LIVE/DEAD
• assay (n = 6, *p < 0.05), and (f) cell proliferation rate by CCK (n = 5, *p < 0.05; **p <
• 0.01). 47
• Figure 5.
• In vivo wound healing assessment of multifunctional hydrogels. (a) Representative images
• of the wounds at different times. (b) Quantification of relative wound area on day 9 after
• treatment. (c) Quantification of wound contraction during the healing process. - 49
• viii
• Figure 6.
• Respectively images of skin wounds stained with hematoxylin and eosin (H&E),
• Masson’s Trichrome (MT) and ImmunoHistoChemistry (IHC) on days 12. - 51