Evaluation of Bone Regeneration using Exosomes derived from induced-Mesenchymal Stem cells combined with Small Intestinal Submucosa in Calvarial Defect Model : 유도 중간엽 줄기세포 유래 엑소좀과 소장 점막하층 복합체를 이용한 두개골 결손 모델에서의 골 재생 평가

다국어 초록 (Multilingual Abstract)

This study aimed to evaluate the efficacy of exosomes derived from human-induced pluripotent stem cell-derived mesenchymal stem cell combined with a small intestinal submucosa (SIS) scaffold for bone regeneration in rat calvarial defect model. Thirty six rats were divided into six groups based on the exosome treatment concentration: Sham, Control (defect-only group), SIS/N (only SIS treated group), SIS/Exo-L (SIS combined with low doses of exosomes), SIS/Exo-M (SIS combined with medium doses of exosomes), and SIS/Exo-H (SIS combined with high doses of exosomes). Histologic, histomorphometric and immunohistochemical analyses were conducted at 4 and 8 weeks postoperatively. Histological analysis revealed that osteoid tissue was confirmed in the SIS/Exo-M and SIS/Exo-H at 4 weeks postoperatively. By 8 weeks, osteoid tissue was confirmed across all treatment groups: SIS/N, SIS/Exo-L, SIS/Exo-M, and SIS/Exo-H. At histomorphometric and immunohistochemical analyses, the defects of control were filled predominantly with fibrous tissue. The groups treated with SIS and exosomes showed regenerative processes quantified by a significant increase in the tissue thickness of calvarial defects area, and the expression of CD31, CD34, CD105, and PCNA, at both time points. Exosome-treated groups exhibited significantly increased numbers of osteoblasts and osteoclasts, suggesting active bone regeneration, alongside elevated bALP expression. Osseous tissue within the defect area increased while the fibrous tissue decreased, in a dose- and time- dependent manner with exosome treatment. These results suggest that exosomes combined with SIS scaffolds synergistically facilitate bone regeneration and indicate exosome-enhanced SIS holds promise as a potent, cell-free therapeutic strategy for critical bone defects.

국문 초록 (Abstract)

본 연구의 목표는 인간 유도만능줄기세포 유도 중간엽 줄기세포 유래 엑소좀과 소장 점막하층 복합체를 이용하여 랫트 두개골 결손 모델에서 골 재생에 미치는 효능을 평가하는 것이다. 랫트 36마리를 처치에 따라 6개의 군으로 나누었다: Sham, Control (골결손만 일으킨 군), SIS/N (골결손부에 SIS만 처리한 군), SIS/Exo-L (골결손부에 SIS와 저농도의 엑소좀을 처리한 군), SIS/Exo-M (골결손부에 SIS와 중간농도의 엑소좀을 처리한 군), 그리고 SIS/Exo-H (골결손부에 SIS와 고농도의 엑소좀을 처리한 군). 수술 후 4주 및 8주에 조직학적, 조직형태계측적, 및 면역조직화학적 평가를 수행하였다. 수술 후 4주차 조직검사 결과, SIS/Exo-M과 SIS/Exo-H에서만 osteoid tissue가 확인되었다. 8주차 조직검사 결과에서는 SIS/N, SIS/Exo-L, SIS/Exo-M, 그리고 SIS/Exo-H를 포함한 모든 처치 그룹에서 osteoid tissue가 확인되었다. 조직형태계측 및 면역조직학적 분석에서, control의 결손 부위는 주로 섬유조직으로 채워져 있었다. SIS 및 엑소좀 처치 그룹들은 두 시점 모두에서 두개골 결손 부위의 thickness와 CD31, CD34, CD105, PCNA 발현 세포 수의 유의미한 증가를 보였다. 엑소좀 처치 그룹들에서 osteoblast와 osteoclast 수의 유의미한 증가와 더불어 bALP 발현 증가를 나타냈는데, 이는 활발한 골 재생이 진행되고 있음을 의미한다. 결손 부위 내의 osteoid tissue 증가 및 섬유조직의 감소는 엑소좀 처치 농도 및 시간에 비례하는 양상을 보였다. 이러한 결과는 엑소좀과 SIS scaffold의 결합이 골 재생을 상조적으로 촉진함을 시사하며, critical bone defect에 대한 cell free therapy로서의 가능성을 가지고 있음을 나타낸다.

목차 (Table of Contents)

• 1. INTRODUCTION 1
• 2. MATERIALS AND METHODS 5
• 2.1. iMSC-Exos preparation 5
• 2.2. SIS scaffold preparation 5
• 2.3. Animals 6
• 2.4. Surgical procedure for calvarial defect model 7
• 2.5. Evaluation 8
• 2.5.1. Tissue sampling and histological analysis 8
• 2.5.2. Immunohistochemistry 9
• 2.5.3. Histomorphometric analysis 10
• 2.6 Statistical analysis 11
• 3. RESULTS 15
• 3.1. Histopathologic analysis 15
• 3.1.1. HE stained general histopathological analysis 15
• 3.1.2. MT-stained histopathological analysis 16
• 3.1.3. SO-stained histopathlogical analysis 17
• 3.2. Histomorphometric analysis 18
• 3.2.1. Total calvaria thickness 18
• 3.2.2. Inflammatory cell numbers 19
• 3.2.3. Osteoclast cell numbers 19
• 3.2.4. Osteoblast cell numbers 20
• 3.2.5. SO-stained cell numbers 20
• 3.2.6. Osteoid tissues 21
• 3.2.7. MT+ fibers 22
• 3.3. Immunohistochemistry 22
• 3.3.1. Immunohistochemical analysis of Hematopoietic stem cell marker (CD34 and 45) immunoreactive cells 23
• 3.3.2. Immunohistochemical analysis of mesenchymal stem cell marker (CD105) immunoreactive cells 24
• 3.3.3. Immunohistochemical analysis of angiogenesis marker (CD31) immunoreactive cells 25
• 3.3.4. Immunohistochemical analysis of osteoblast marker (bALP) immunoreactive cells 25
• 3.3.5. Immunohistochemical analysis of cell proliferation marker (PCNA) immunoreactive cells 26
• 4. DISCUSSION 54
• 5. CONCLUSION 62
• 6. REFERENCES 63
• (Abstract) 82
• (초 록) 84