Gelatin Nanoparticle-Assisted Stem Cell Spheroid Therapeutics for Enhanced Tissue Regeneration = 극미세 젤라틴 나노파티클이 융합된 생체 이식형 줄기세포 스페로이드 개발

다국어 초록 (Multilingual Abstract)

Nanoengineered cues integrated with three-dimensional (3D) cell systems offer a powerful route to amplify therapeutic functions by regulating cell-material interactions across molecular to tissue scales. By embedding bio-responsive nanomaterials within cell-assembled architectures, such hybrid constructs create tunable interfaces that bridge engineered design and dynamic biological environments, addressing long-standing challenges in regenerative medicine.
In this thesis, we developed ultra-tiny gelatin nanoparticles (GNPs) as intrinsically biocompatible, degradable nano-cues and integrated them with mesenchymal stem cell (MSC) spheroids to build hybrid therapeutics. The GNP-MSC spheroids were engineered to maximize interactions within complex biological milieus by enhancing cell-cell cohesion, adhesion, proliferation, and lineage commitment while preserving paracrine potency. GNPs within the spheroid microenvironment established ECM-mimetic signaling niches that concentrated morphogens and supported efficient transduction, thereby guiding cellular behaviors essential for tissue repair. Furthermore, the bioimplantable GNP-MSC spheroids provided spatial guidance, enabling synergistic regulation of cytoskeletal organization and of osteogenic and cutaneous regenerative programming.
We demonstrated that GNP-assisted spheroids modulated angiogenesis, tempered inflammatory responses, and accelerated tissue regeneration through controlled material properties including biopolymer chemistry and degradability tailored to the target tissue. In vivo, the platform promoted functional repair in two representative models: a 5 mm critical size calvarial defect in C57BL/6 mice and an 8 mm full-thickness dorsal skin wound in Sprague Dawley rats, yielding enhanced vascularization, matrix remodeling, and defect closure compared with conventional spheroids. Collectively, these results positioned GNP-integrated spheroids as a versatile, scalable platform for activating and sustaining regenerative programs, from drug-augmented cell delivery to advanced tissue- engineering scaffolds. The design principles established here advance highly adaptive, functional biomaterials for next-generation therapies in musculoskeletal and soft-tissue repair and provide a generalizable blueprint for microenvironmental bioactive systems.

국문 초록 (Abstract)

(국문초록)
3차원 세포 시스템에 나노공학적 단서를 통합하는 전략은 분자 수준부터 조직 수준에 이르는 세포–소재 상호작용을 정밀하게 조절함으로써 치료 기능을 증폭시키는 강력한 접근법이다. 세포가 자가 조립된 구조체 내부에 생체반응성 나노소재를 통합하면, 공학적으로 설계된 요소와 동적 생체 환경을 연결하는 조절 가능한 인터페이스가 형성되며, 이는 재생의학 분야의 고질적인 한계를 해결하는 데 기여할 수 있다.
본 학위논문에서는 생체적합성이 높고 생분해가 가능한 극미세 젤라틴 나노입자(gelatin nanoparticles, GNPs)를 나노공학적 단서로서 개발하고, 이를 중간엽 줄기세포(mesenchymal stem cells, MSC) 스페로이드와 결합하여 하이브리드 세포 치료 플랫폼을 구축하였다. GNP–MSC 스페로이드는 복잡한 생체 미세환경에서의 상호작용을 극대화하도록 설계되었으며, 세포–세포 응집과 접착, 증식 및 계통(lineage) 관련 기능을 강화하면서도 파라크린(paracrine) 효능을 유지하도록 공학적으로 최적화되었다. 스페로이드 내부 미세환경에 분포한 GNP는 세포외기질(ECM) 모사 신호 niche를 형성하여 형태형성인자(morphogen)의 국소 농축과 효율적 신호전달을 유도하고, 조직 재생에 필요한 세포 거동을 상향 조절하였다. 또한 생체 이식이 가능한 GNP–MSC 스페로이드는 결손부 유지 및 정착 효과를 제공하여 세포골격 조직화와 골형성 및 피부 재생 프로그램을 시너지 있게 조절하였다.
나아가 본 연구는 GNP-MSC 스페로이드가 생체고분자 화학과 분해성 등 표적 조직에 맞게 조절된 소재 특성을 통해 혈관신생을 조절하고, 염증 반응을 완화하며, 조직 재생을 가속화함을 입증하였다. 동물 실험에서는 C57BL/6 마우스의 5 mm 임계 크기 두개골 결손 모델과 Sprague–Dawley 랫드의 8 mm 전층 등 피부 결손 모델에서 GNP–MSC 스페로이드가 기존 스페로이드 대비 혈관화, 기질 재형성 및 결손 폐쇄를 유의하게 향상시켜 기능적 조직 회복을 촉진하였다. 종합하면, 본 연구 결과는 GNP-MSC 통합 스페로이드가 약물 보강 세포 전달부터 고도화된 조직공학 스캐폴드까지 확장 가능한, 범용적이고 스케일업이 가능한 재생 활성화 플랫폼임을 제시한다. 본 논문에서 확립한 설계 원리는 근골격계 및 연조직 재생 치료를 위한 차세대 기능성 생체재료 개발에 기여하며, 미세환경 기반 생체활성 시스템의 일반화 가능한 설계의 청사진을 제공한다.

목차 (Table of Contents)

• Table of Contents
• Abstract
• 1. Introduction 1
• 1.1. General introdcution 1
• 1.2. Objectives 4
• 1.3. References 5
• 2. Chapter 2. Gelatin Nanoparticle-Assisted Stem Cell Spheroid Therapeutics for Enhanced Tissue Regeneration 7
• 2.1. Summary 8
• 2.2. Introduction 9
• 2.3. Materials and Methods 13
• 2.3.1. Fabrication and Physicochemical Characterization of GNPs 13
• 2.3.2. Evaluation of Drug Release from GNPs 14
• 2.3.3. Synthesis of GNPs using Gelatin from Various Manufacturers 14
• 2.3.4. Buffer- and Surfactant-Assisted Enhancement of GNP Colloidal Stability 15
• 2.3.5. Maintenance of Mesenchymal (MSC) Stem Cells 15
• 2.3.6. Synthesis of Gelatin Microparticles (GMPs) 16
• 2.3.7. Cell Viability Comparison with GMPs and GNPs 16
• 2.3.8. Generation of GMP-MSC Spheroids and GNP-MSC Spheroids 17
• 2.3.9. Scanning Electron Microscopy 18
• 2.3.10. Co-culture GNP-MSC spheroids with HUVECs 18
• 2.3.11. Live/Dead Assay 19
• 2.3.12. EdU Proliferation Assay 19
• 2.3.13. Immunofluorescence Staining 20
• 2.3.14. Alizarin Red S Staining 21
• 2.3.15 Human Angiogenesis Array 21
• 2.3.16. Western Blotting 21
• 2.3.17. In vivo Analysis 22
• 2.3.18. Statistical Analysis 24
• 2.4. Results and Discussion 25
• 2.4.1. Fabrication and Physicochemical Characterization of GNPs 25
• 2.4.2. Characteristics and properties of GNP-incorporated with MSC spheroids 27
• 2.4.3. Formation of GNP-incorporated with MSC Spheroids with Enhanced Function and Structural Integrity 28
• 2.4.4. GNP-incorporated with MSC Spheroid Platform: Mechanism of Action 31
• 2.4.5. GNP-incorporated with MSC Spheroids Assisted Enhancement in Hard and Soft Tissue Regeneration in vivo 34
• 2.5. Conclusion 49
• 2.6. References 72
• 3. Concluding Remarks 81
• 국문초록 83
• List of Figures
• Figure 2.1. Ultra-tiny GNP-incorporated with MSC Spheroids as a Therapeutic Platform for Enhanced Tissue Regeneration 50
• Figure 2.2. Evaluation of GNP-incorporated with MSC Spheroid Platform for their Functional Ability in vitro 52
• Figure 2.3. Proposed Mechanisms of GNP-MSC Spheroid Platform for Enhanced MSC Proliferation 54
• Figure 2.4 Evaluation of the Pro-angiogenic Effects of GNP-MSC Spheroids in a HUVEC Model 56
• Figure 2.5. Synergistic Enhancement of Bone Tissue Regeneration through in vivo Incorporation of GNP-MSC Spheroids 58
• Figure 2.6. Synergistic Enhancement of Skin Tissue Regeneration through in vivo Incorporation of GNP-MSC Spheroids 60
• Figure S2.1. Schematic of the Fabrication Process of GNPs using Two-step Desolvation Method 62
• Figure S2.2. Time-dependent Drug Release Profile from GNPs 63
• Figure S2.3. Synthesis and Analysis of GNPs by Manufacturer and Grade 64
• Figure S2.4. Enhancement of GNP Dispersibility and Characterization 65
• Figure S2.5. Optimization of GNP-MSC Spheroids 66
• Figure S2.6. SEM Images indicating GNP-MSC Spheroid Groups Cultured for 7 Days Under Varying Cell Densities (0.5 × 10³ , 1 × 10³ , and 1.5 × 10³ cells) and GNP Concentrations (0.35, 1, and 1.75 mg/mL GNPs) 67
• Figure S2.7. Analysis of Spheroid Size and Diameter Based on Varying GNP Concentrations After Optimizing the Cell Density to 1 × 10³ Cells per well 68
• Figure S2.8. Fabrication, Purification, and Biocompatibility Evaluation of GMPs in MSC Co-culture 69
• Figure S2.9. SEM Images Showing Cell-cell and Cell-ECM Interactions in MSC-only Spheroid 70
• Figure S2.10. Evaluation of the Bone-differentiation Effects of GNPs in a MSC Model 71