||Development of Three Dimensional Polyethylene Glycol Based Hydrogel for Tissue Engineering Scaffold Application
||Department of BioMedical Engineering
Two-photon laser scanning microscopy
Poly (ethylene glycol) diacrylate
Adipose-derived stem cell
Articular cartilage degeneration which is hard to repair by itself because of no nerves and blood vessels has become a public health issue since average of lifespan is increasing. Many repairing approaches did not restore the normal hyaline cartilage but fibrocartilage instead. Previous researches were limited by barrier of mass transfer, improper induction sequence, cell anchorage, or few amount of available cells. Buildup of the monitoring systems that simulate physiological conditions enables us to inspect the events of electrical and chemical communications between cells. In this study, we fabricated poly (ethylene glycol) diacrylate (PEGDA) into three kinds of devices by customized mold. PEGDA is hydrophilic, biocompatible and photo-crosslink-able. In addition, adipose-derived stem cells (ASC) were harvested as the standard cells to evaluate whether the systems were functional.
First product was thin film of PEGDA featuring micro-pattern created by two-photon laser scanning microscopy. Surface of the film was grafted RGD peptide so that cells could partially adhere to the surface of polymer. Second product was micro-well which hosted high quantity but small population of cells. The bonding between silane and PEGDA was competed by protein; thus, we were able to control the delamination speed of micro-well from culture stage. The last product was scaffold for cartilage tissue engineering with novel design to reduce barrier of mass transfer. ASC were encapsulated in PEGDA and induced by co-culture strategy to stimulate the protein express which are the characteristic of chondrogenesis. Compared with treatment of exogenous supplement of growth factors, co-culture strategy achieved the same results.
In general, different manufacturing process granted three devices which shared the same material with the abilities to monitor, house, and induce chondrogenesis. ASC is an ideal autologous cell source because it is abundant in quantity and has outstanding differential potential. Future work is to fully utilize these devices to acquire information of regulating chondrogenesis. Ultimate goal is to integrate three devices into tissue engineering scaffold with real-time surveillance on proliferation and differentiation status.
Table of contents v
List of tables viii
List of figures ix
Chapter 1 Introduction 1
1.1 Background 1
1.1.1 Compositions and properties of cartilage 1
1.1.2 Cartilage degeneration and approaches of repair 2
1.2 Scaffolds 4
1.2.1 Design of scaffold 4
1.2.2 Natural biomaterials 5
1.2.3 Artificial biomaterials 6
1.3 Two-photon laser scanning microscopy 8
1.4 Micro-well array 10
1.5 Cell Source 10
1.5.1 Differentiated cells of normal tissue 10
1.5.2 Stem cells 11
1.5.3 Bone marrow stem cells 12
1.5.4 Adipose-derived stem cells 12
1.6 Environment 14
1.6.1 Mechanical stimulation 14
1.6.2 Biochemical stimulation 14
1.6.3 Co-culture system 15
1.7 Purpose and specific aims 16
1.7.1 Purpose 16
1.7.2 Aims 17
Chapter 2 Materials and methods 21
2.1 Materials and instruments 21
2.2 Cell culture 24
2.2.1 Harvest of ASC 24
2.2.2 Harvest of chondrocytes 24
2.2.3 Cell maintenance and passage 25
2.3 Gelation of PEGDA 25
2.3.1 Customized mold to load PEGDA solution 25
22.214.171.124 PEGDA film with micro-pattern created by TPLSM 25
126.96.36.199 Micro-well array with controllable delamination hosted cells 25
188.8.131.52 PEGDA encapsulated ASC to induce chondrogenesis by co-culturing with xenogeneic chondrocytes 26
2.3.2 Parameters to crosslink PEGDA 26
2.4 Mechanical property of PEGDA gel 26
2.5 Cell membrane fluorescent stain-Cell linker 27
2.6 Biochemical evaluation 27
2.6.1 Cell viability assay 27
2.6.2 Western blot 28
2.7 Histological section 29
Chapter 3 Results 30
3.1 PEGDA film with micro-pattern created by TPLSM 31
3.2 Micro-well array with controllable delamination hosted cells 33
3.3 PEGDA encapsulated ASC to induce chondrogenesis by co-culturing with xenogeneic chondrocytes 35
3.3.1 Geometric structure of scaffold 35
3.3.2 Young’s modulus of PEGDA hydrogel 35
3.3.3 Cell morphology within scaffold 38
3.3.4 Cell viability in hydrogel 39
3.3.5 Subcutaneous implant of hydrogel loaded with cell 41
184.108.40.206 Observation at the surface of PEGDA after implantation 41
220.127.116.11 Fluorescence image of cell within hydrogel 42
3.3.6 Sections of PEGDA hydrogel scaffold 43
3.3.7 Change of protein secretion of ASC upon treatment 47
Chapter 4 Discussion 48
4.1 PEGDA film with micro-pattern created by TPLSM 48
4.2 Micro-well array with controllable delamination hosted cells 48
4.3 PEGDA encapsulate ASC to induce chondrogenesis by co-culturing with xenogeneic chondrocytes 50
4.4 Integration of three tools 57
Chapter 5 Conclusion 59
Curriculum vitae 66
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