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Functionalized and Structurally Enhanced ECM Scaffolds: Advancing Regenerative Medicine : 기능화와 구조특성 강화를 통한 세포외 기질 지지체 기반의 재생 치료제 개발

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dc.contributor.advisor황석연-
dc.contributor.author김범석-
dc.date.accessioned2023-11-20T04:26:20Z-
dc.date.available2023-11-20T04:26:20Z-
dc.date.issued2023-
dc.identifier.other000000178262-
dc.identifier.urihttps://hdl.handle.net/10371/196560-
dc.identifier.urihttps://dcollection.snu.ac.kr/common/orgView/000000178262ko_KR
dc.description학위논문(박사) -- 서울대학교대학원 : 공과대학 협동과정 바이오엔지니어링전공, 2023. 8. 황석연.-
dc.description.abstractDecellularized ECM (dECM) play a crucial role in the development of advanced biomaterials for regenerative medicine. This thesis presents an in-depth investigation of decellularization techniques, specifically incorporating dECM to regenerative application using additive


engineering skills and more conservative process to fabricate dECM scaffold. The current decellularization methods and dECM applications presented partial loss of extracellular matrix and unstable structural support in tissue environments, that possibly corelated to regeneration ability of dECM in vivo. To amend this problem, in the further chapters, the protein loading cryogel fabrication and supercritical fluid-based technology were applied to develop ideal dECM derived biomaterial.
The brain dECM cryogels were fabricated by cross-linking method utilizing heparin sulfate which have negative charge features. This approach aimed to mimic the complex composition of native brain tissues and growth factor loading affinity for support of neural tissue formation. The cryogel sustained their 3D structure in various stress conditions and controlled of growth factor releasing kinetics. The brain dECM cryogels exhibited excellent biocompatibility, allowing cell adhesion, proliferation, and tissue-like growth within the scaffold.
The comparative study of decellularization methods revealed the superiority of the supercritical fluid technique in preserving the native tissue architecture and extracellular matrix (ECM) components. By eliminating cellular components while retaining crucial ECM proteins,


this technique offers a promising approach to create biomimetic scaffolds. Quantitative analysis of tissue protein contents demonstrated the higher preservation of ECM (collagen, GAG), and other key proteins when using the supercritical fluid technique. Moreover, supercritical flow-based decellularization method showed higher neuronal cell differentiation efficiency and formal nerve tissue regeneration also.
Overall, this thesis highlights the potential of cryogels as three- dimensional scaffolds and additive loading of specific growth factor improves regeneration ability of own dECM and shows application versatility in tissue engineering applications. Additionally, the utilization of the supercritical fluid technique for efficient decellularization, preserving the native ECM components, and maintaining the structural integrity of tissues.
These techniques provide a platform for developing advanced biomaterials that closely mimic the native tissue environment, offering potential for regenerative medicine and tissue engineering approaches.		-
dc.description.abstractDecellularized ECM (dECM)은 뛰어난 생체적합성과 원재료 내 의 조직 재생 성분 덕분에 조직공학용 재료로서 널리 이용되어 왔습니 다. 이 논문에서는 탈세포화 재료의 응용방법에 대해 중점적으로 다루 고 있으며, 특히 dECM 스캐폴드를 제조하기 위해 다른 기능성 고분자 를 도입하거나 새로운 탈세포 방법을 사용하여 현재 사용하고 있는 dECM의 활용기술의 한계점을 극복하고자 하였습니다. 현재의 탈세포 화 방법 및 dECM 응용기법들은 dECM의 재생 능력과 깊게 연관되어 있는 원조직 유래고분자, 단백성분이 다량 손실 된다는 문제가 있기에 이를 보완하고자 조직 유래 성분을 최대한 보존하는 새로운 탈세포 방


법과 추후에 조직 특이적 단백 성분을 보충해줄 수 있는 생체 재료 제작 기법을 개발하였습니다.
먼저 음전하 특성을 갖는 헤파린 설페이트기를 뇌 조직 유래 탈세 포와 가교 하여 양전하 특성의 성장인자를 담지 및 서 방출하는 cryogel 을 제작하였습니다. 이 접근법은 원 조직의 복잡한 세포외 기질 구조와 뇌 조직 재생을 위한 성장 인자를 동시에 제공할 수 있다는 장점을 가지 고 있습니다. 제작된 cryogel은 다양한 스트레스 조건에서 3D 구조를 유지하였고 헤파린 설페이트 양에 따른 성장 인자 방출양을 조절할 수 있었습니다. 뇌 탚세포 조직 기반 cryogel을 이용한 외상성 뇌 손상 (traumatic brain injury) 모델 재생 실험에서 cryogel 은 뛰어난 생체 적합성을 나타내는 동시에 스캐폴드 내에서 신경세포의 부착, 증식 을 촉진하는 것을 확인 하였습니다.
초임계 유체를 이용한 탈세포화 방법을 이용한 신경 조직 지지체 제 작 실험에서 초임계 유체 기반 탈세포 기법은 기본 조직 구조 및 세포외 기질(ECM) 구성 요소를 보존하는데 있어 기존의 계면활성제를 이용한 기법에 비해 우수함을 증명하였습니다. 초임계 유체 기술은 탈 세포된 신경조직 내의 콜라겐 및 기타 주요 단백질이 기존의 계면활성제를 이 용한 기법에 비해 더 많이 보존되었으며 또한 인장강도, 탄성도 등의 기


계적 물성변화 또한 초임계 유체 기반 탈세포 기법으로 제조된 신경 조 직이 원조직과 더 가까운 특성을 보였습니다. 이러한 높은 단백 보존율, 원조직 유사 물성을 가지는 탈세포 조직 지지체는 높은 신경 세포의 분 화 효율과 동물조직에서 3차원 지지체로서 조직내의 세포 부착 지점을 제공함으로 기능성 조직의 형성을 유도함을 확인하였다.
전반적으로, 이 논문은 기본 ECM 구성 요소 보존 및 조직의 구조적 안전성 유지를 위한 초임계 유체 기술과 조직 특이적인 성장인자 전달 을 위한 음전하 기반의 고분자를 화학적 가교로 탈세포 조직에 도입하 는 연구를 정리하였습니다. 2개의 방법모두 기존의 탈세포화기법의 문 제점인 원조직 유래 조직 재생 인자가 탈세포 과정 중 소실되는 현상을 보완할 방법으로 개발된 실험 기법들이며 이러한 기술의 조합은 재생 의학 및 조직 공학 분야에 탈세포 조직을 도입하기 위한 새로운 솔루션 을 제공하여 원조직을 이상적으로 모방하는 생체 재료를 개발하기 위한 플랫폼을 제공할 수 있을 것으로 예상됩니다.		-
dc.description.tableofcontentsTable of content
Abstract i
Table of content v
List of Figures xiv
List of Tables xvi
CHAPTER ONE: INTRODUCTION １
1.1 Overview １
1.2 Objective of the thesis ２
1.3 Organization of thesis ４
CHAPTER TWO: ５
THE SCIENTIFIC BACKGROUND AND RESEARCH PROGRESS ５
2.1 Biomaterials for tissue engineering ５
2.1.1 Polymeric material for tissue engineering and their applications ５

2.1.2 Decellularized ECM for tissue engineering １３
2.1.3 Application of decellularization technique to tissue engineering １８
2.2 Decellularization principle and methods ２５
2.2.1 Principle of decellularization process and traditional decellularization methods ２５
2.2.2 Supercritical fluid-based decellularization ３０
CHAPTER THREE: ３８
FABRICATION OF NGF RELEASING CRYOGEL WITH BRAIN DECELLULARIZED ECM FOR TBI (TRAUMATIC BRAIN INJURY) TREATMENT ３８
3.1 Introduction ３８
3.2 Materials and method ４２
3.2.1 Decellularization of brain tissue ４２
3.2.2 Paraffin sectioning of brain tissues ４３
3.2.2.1 H&E staining of brain tissues ４３

3.2.2.2 Trichrome staining of brain tissues ４４
3.2.2.3 Safranin-O staining ４５
3.2.2.4 DNA quantification in brain tissues ４５
3.2.2.5 Hydroxy proline quantification in brain tissues ４６
3.2.2.6 DMMB assays for quantifications of sGAG ４７

3.2.3 Characterization of brain dECM and heparin derived cryogels ４８

3.2.3.1 Preparation of brain dECM and heparin derived cryogel ４８

3.2.3.2 Reaction efficiency calculation ４８
3.2.3.3 FTIR analysis of cryogel ４９
3.2.3.4 Microstructure analysis of cryogel ４９
3.2.3.5 Swelling ratio calculation of cryogel ４９
3.2.4 Mechanical property characterization of cryogel ５０
3.2.4.1 Compressive modulus measurements of cryogels ５０
3.2.4.2 Rheological property characterization of crygoels ５０
3.2.4.3 Cryogel injectability test ５１
3.2.5 Heparin mediated protein sustain release kinetics measurement. ５１
3.2.6 PC12 cells culture and differentiation ５１
PC12 differentiation with croygel encapsulated NGF ５１
3.2.6.1 PC12 culture on the cryogel ５２
3.2.6.2 ß-3tubluin staining of PC12 cells ５２
3.2.7 Regeneration of TBI animal model with cryogel ５３
3.2.7.1 Brain defect model design and cryogel application in vivo ..５
３
3.2.7.2 Fluorescence staining of brain tissues ５３
3.2.8 Statistics analysis ５４
3.3 Results ５６
3.3.1 Decellularization of the brain tissue and component characterization native tissue and brain dECM ５７
3.3.2 Fabrication and characterization of brain dECM based cryogel. ６０
3.3.3 Rheological property characterization of fabricated brain dECM derived cryogel ６３
3.3.4 Characterization of injectability of fabricated cyrogels. ６４
3.3.5 Heparin dependent protein loading efficiency of cyrogels and NGF correlated PC12 cell differentiation ratio characterization….６６
3.3.6 PC12 culture and differentiation on the NGF loaded cryogels７０
3.3.7 In vivo application of cryogel (De1.0HS0.3) and characterization of vessel formation ７１
3.3.8 In vivo application of cryogel (De1.0HS0.3) and characterization of neuronal cell migration in host tissues ７５
3.3.9 In vivo application of cryogel (De1.0HS0.3) and characterization of neuronal cell recruitment in to the cryogel network .７ ８
3.4 Discussion ７９
3.5 Conclusion ９３
3.6 Supplementary figure ９６
4.1 Introduction １００
4.2 Material & methods １０４
4.2.1 Isolation and decellularization of sciatic nerve tissues １０５
4.2.2 Histological assessment of sciatic nerve tissues. １０６
4.2.2.1 Paraffin sectioning of nerve tissues １０６
4.2.2.2 H&E staining of nerve tissues １０７
4.2.2.3 Trichrome staining of nerve tissues １０７
4.2.2.4 Safranin-O staining １０８
4.2.3 ECM characterization in nerve tissues １０９
4.2.3.1 DMMB assay for quantifications of sGAG １０９
4.2.3.2 DNA quantification in nerve tissues １０９
4.2.3.3 Hydroxyproline quantification in nerve tissues １１０
4.2.3.4 Proteomic evaluation of tissue samples １１１
4.2.4 Mechanical property characterization of nerve tissues １１２
4.2.4.1 Extension force test of nerve tissues １１２
4.2.4.2 Compression force test １１２
4.2.5 Neuronal cell culture １１２
4.2.5.1 PC12 cell cultures for proliferation and differentiation.１１２
4.2.5.2 PC12 cell differentiation on nerve tissues １１３
4.2.5.3 PC12 cell imaging with confocal microscopy １１３
4.2.6 Surface image of decellularized ECM with SEM １１４
4.2.7 In vivo experiment and characterization of regeneration of tissues １１４
4.2.7.1 Nerve tissue implants to subcutaneous area for immune reaction characterization １１４
4.2.7.2 dECM implantation in sciatic nerve defect model １１５
4.2.7.3 Gait analysis of sciatic nerve defect model １１５
4.2.7.4 Evaluation of muscular tissue regeneration １１６
4.2.7.5 Histological assessment of sciatic nerve tissues １１６
4.2.7.6 Luxol fast blue staining of regenerated nerve tissues １１７
4.2.8 Statistics analysis １１７
4.3 Results １１９
4.3.1. Decellularized ECM scaffold fabrication and characterization
of ECM １２０
4.3.2 Mechanical characterization of decellularized tissue samples .１ ２３
4.3.3 PC12 differentiation on native and decellularized tissue slice samples １２６
4.3.4 Immune cell infiltration of implanted tissue samples １３０
4.3.5 Motor function and muscular tissue regeneration after implantation in vivo １３２
4.3.6 Nerve tissue regeneration assessment after tissue implantation
in vivo １３５
4.4 Discussion １３７
4.5 Conclusion １４７
4.6 Supplementary figure １４９
CHAPTER FIVE: CONCLUDING REMARKS １５４
BIBLIOGRAPHY １５６
국문초록 １５７


List of Figures

Figure 2.1 Commonly applied synthetic polymers to tissue engineering. Copy right (2009), Royal Society of Chemicals. 8
Figure 2.2 Natural derived polymers for tissue engineering and cell responsive abilities. Copy right (2021), springer nature 11
Figure 2.3 Scheme of 3D scaffold application to in vivo, cell environments by providing of chemical and physical cues 17
Figure 2.4 Description about host immune cell and ECM interaction in tissue environments.. 17
Figure 2.5 dECM applications in various tissue engineering strategies 24
Figure 2.6 Basic information of super critical flow process 34
Figure 3. 1 ECM structure analysis and quantification in native and decellularized brain tissues (dECM). 56
Figure 3. 2 ECM structure analysis and quantification in native and decellularized brain tissues (dECM). 59
Figure 3.3 Rheological characteristics measurements of the cryogels 62
Figure 3.4 Injectability characterization of the cryogels 65
Figure 3.5 Sustain release profile of the proteins and characterization of neuronal cell differentiation ability followed by releasing of NGF from cryogels 67
Figure 3.6 3D culture of PC12 cells on NGF encapsulated cryogels for 21 days. 70
Figure 3.7 In vivo brain defect model developement and assessments of vascularization of brain tissues, 72
Figure 3.8 Neuronal tissue regeneration assessment post implantation of cryogels in infarct area of host tisseus. 74
Figure 3.3 Neuronal tissue regeneration assessment post implantation in implanted crygel. areas. 77
Figure S3.1 Zeta potential of heparin sulfate and decellularized brain ECM 96
Figure. S3.2 Immune cell population of infarct area after surgery A) Representative


images of Iba-1 images in infarct area of host tissues 97
Figure S3.3 Immune cell population in implanted cryogel area 98
Figure 4.1 Composition analysis of native tissue and decellularized tissues processed with different decellularization methods 119
Figure 4.2 Mechanical characterization of native and decellularized tissues. (A) Representative image of extension test method of wet tissue 123
Figure 4. 3 PC12 cell culture and differentiation on nerve tissue slices. (A-B) Class III β-tubulin and actin staining of PC12 cell cultured on native and decellularized tissues. 125
Figure 4.4 PC12 cell culture and differentiation on nerve tissue slices for a long- term period. (A) PC12 cell differentiation on nerve tissues for 21 days 128
Figure 4.5 Immune cell infiltration ratio characterization of native tissues and dECM after implantation to skin area 129
Figure 4. 6 Muscular regeneration and motor function recovery in vivo. (A) Nerve tissue implantation at nerve gap defects 131
Figure 4. 7 Gap-43 and S-100 fluorescence staining of sciatic nerves post- implantation at 4,8 weeks. 134
Figure 4. 8 Luxol fast blue staining of cross sectioned regenerated nerves after 8weeks. 136
Figure S4. 1 Polymeric composition analysis and contents measurements of decellularized tissues processed with various decellularization methods 149
Figure S4. 2 SEM image of native tissues and decellularized tissues 150
Figure S4. 3 PC12 differentiation cultured on TCP plates with/without NGF 151
Figure S4.4 Fluorescence data of PC12 cells cultured for 3,7 days on nerve tissue slcies. -152
Figure S4. 5 Quantitative data of native tissues. (A) Gap-43, S-100 staining of native tissues. 153



List of Tables

Table 2.1 Description about traditional decellularization methods 29
Table 1.2 Supercritical fluid based decellularization with various parameter conditions and tissue sources. 37
Table 3.1 Decellularized ECM and heparin sulfate concentrations in each cryogel group. 59		-
dc.format.extentxv, 185-
dc.language.isokor-
dc.publisher서울대학교 대학원-
dc.subjectDecellularization-
dc.subjectHeparin sulfate-
dc.subjectSupercritical fluid-
dc.subjectExtracellular matrix-
dc.subjectBiomaterials-
dc.subject.ddc660.6-
dc.titleFunctionalized and Structurally Enhanced ECM Scaffolds: Advancing Regenerative Medicine-
dc.title.alternative기능화와 구조특성 강화를 통한 세포외 기질 지지체 기반의 재생 치료제 개발-
dc.typeThesis-
dc.typeDissertation-
dc.contributor.AlternativeAuthorBeok seok Kim-
dc.contributor.department공과대학 협동과정 바이오엔지니어링전공-
dc.description.degree박사-
dc.date.awarded2023-08-
dc.identifier.uciI804:11032-000000178262-
dc.identifier.holdings000000000050▲000000000058▲000000178262▲-
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