Novel treatment combining plasma–activated water for skin wound regeneration by regulation of redox metabolism
플라즈마수를 결합한 새로운 치료법의 산화 환원 대사 조절을 통한 피부창상 재생 촉진 효과 분석
- 주제(키워드) Plasma-activated water , Silk-based biomaterials , Redox species , Cell migration , Wound regeneration , Selective cytotoxicity
- 주제(DDC) 610
- 발행기관 아주대학교
- 지도교수 김철호
- 발행년도 2023
- 학위수여년월 2023. 8
- 학위명 박사
- 학과 및 전공 일반대학원 의학과
- 실제URI http://www.dcollection.net/handler/ajou/000000032932
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
The impaired healing of irradiated skin wounds is a noteworthy clinical issue that poses a challenge to the widespread use of radiation therapy. Despite numerous attempts to treat this condition, it is still deemed an intractable wound. Innovative treatment approaches are therefore necessary to tackle the wound complications in irradiated areas. To promote rapid regeneration of skin wounds, activation of re-epithelialization by keratinocytes migration as leading cells is crucial in the early stages of injury. Plasma medicine has been known to have beneficial effect in wound healing. We conducted in vitro and in vivo experiments to elucidate the action and underlying molecular mechanism of novel microwave plasma-activated water (PAW) on promoting skin cell migration. The study also demonstrated that increased intracellular reactive oxygen species (ROS) level played a role as driving force in activating cell migration, which was controlled by oxidases and mitochondria. To explore the additional advantageous effect of PAW on wound healing, the study assessed the antibacterial properties of PAW against major pathogens. PAW showed high competence in sterilizing bacteria and posed no harm to healthy skin cells. The mediators of sterilization in PAW were investigated by loss of function test. Finally, the study utilized PAW to treat a composite gel made of silk fibroin and fibrin (S-F) to examine whether the controlled release of PAW from the S-F hydrogel could trigger beneficial cellular responses in a radiotherapy-related skin wound while also modifying the microstructure of the S-F hydrogel, thereby functioning as biomaterials establishing regenerative microenvironment. The scanning electron microscopy and Fourier-transform infrared spectroscopy analyses demonstrated that the application of PAW modified both the microstructure and chemical bonds of the S-F gel, which indicated that PAW enhanced the mechanochemical properties of the S-F gel. Wound healing assays and immunofluorescence staining revealed that treatment of the PAW treated S-F hydrogel led to favorable improvements in cell viability, morphology, and extracellular matrix depositions, which are crucial for the regenerative process of the wound. Using six-week-old C57/BL6 mice, we developed an irradiated skin-flap murine model with S-F hydrogel placing underneath the raised skin flap. In postoperative analysis of the flap tissue suggested that compared to the groups treated with S-F hydrogel only or control, the PAW-treated S-F hydrogel significantly enhanced wound regeneration and reduced the inflammatory response. Taken together, our study demonstrated that PAW-treated S-F hydrogel as a novel material that can considerably improve irradiated wound restoration by effectively promoting re-epithelialization without damaging epithelial cells by selective cytotoxicity. The additional antimicrobial effect and improvement of the regenerated tissue quality are expected to alleviate chronic radiated tissue distortion and subsequent clinical complications.
more목차
Introduction 1
Materials and Methods 4
A. PAW generation by microwave plasma system 4
B. Measurement of PAW conductivity 5
C. Cell culture 5
D. Cell viability of normal skin cells 6
E. Cell migration assay 6
F. Gelatin zymogram assay 7
G. Quantitative RT-PCR 7
H. Western blot 7
I. Immunocytochemistry 8
J. Measurement of intracellular ROS 9
K. Mitochondrial membrane potential assay (∆Ψm assay) 9
L. Transmission electron microscopy 9
M. In vivo skin wound regeneration study with murine model 10
N. Hematoxylin and eosin staining 11
O. Immunohistochemistry staining 11
P. Solid agar plate assay 11
Q. Extracellular RONS analysis 12
R. Ion chromatography and Griess assay 13
S. Production of PAW-treated silk–fibrin (S-F) composite hydrogel 13
T. Cell viability and morphology analysis on PAW-treated S-F hydrogel 14
U. Microstructure analysis of PAW-treated S-F composite gel 15
V. Tensile property and elasticity assessment of PAW-treated S-F hydrogel 15
W. Analysis of nitric oxide release and degradation of PAW-treated S-F hydrogel 16
X. Assessment of in vitro ECM synthesis on S-F hydrogel 16
Y. Irradiated wound regeneration study by S-F hydrogel treated murine model 17
Z. Immunofluorescent staining analysis of restored radiated wound tissue 18
Results 19
A. Physicochemical properties of PAW by generation of reactive species 19
B. The mortality of normal skin cells was not induced by PAW treatment 20
C. The enhanced cellular migration by PAW was confirmed with increased MMP-2 expressions 21
D. PAW promotes cell migration by modulating focal adhesion molecules 23
E. PAW promotes intracellular ROS signaling-dependent cell migration with mitochondrial activity 25
F. ROS scavenger undermines the PAW-induced promotion of keratinocyte migration 27
G. PAW improves acute murine wound regeneration by promoting skin cell migration 29
H. Sterilization effects of PAW 32
I. The major extracellular effector of PAW was the NO3− isomer 32
J. Treatment of PAW-applied silk-fibrin hydrogel resulted in selective cytotoxicity on both normal and cancer cells 35
K. Surface modification of silk-fibrin hydrogel by PAW treatment improved material properties and biocompatibility 37
L. PAW treatment promoted extracellular matrix accumulation of cells on silk–fibrin hydrogel 39
M. PAW-treated silk-fibrin composite gel improved the regeneration of radiated skin wounds in vivo 41
N. Neovascularization and the anti-inflammatory effect of the PAW-treated silk-fibrin composite gel in radiation wounds of murine skin 43
Discussion 46
References 57
