Table of content
Abstract ... 1
Streszczenie ... 2
1. Introduction ... 4
2. Bone tissue – functions, properties and structure ... 7
2.1. Bone extracellular matrix (ECM) ... 9
2.2. Fracture healing and treatment of bone defects ... 11
3. Biomaterials for bone tissue engineering ... 13
3.1. Natural polymers ... 16
3.1.1. Proteins ... 16
3.1.2. Polysaccharides ... 16
3.1.3. Glycosaminoglycans (GAGs) ... 16
3.2. Synthetic biodegradable polymers ... 17
4. Processing polymers into scaffolds ... 21
4.1. Concept of three-dimensional (3D) structures ... 22
4.2. Scaffold fabrication techniques ... 24
5. Surface modification of the scaffold materials with ECM components ... 26
5.1. Collagen ... 27
5.2. Glycosaminoglycans (GAGs) ... 32
6. Aim and scope of the thesis ... 35
7. Materials and Methods ... 36
7.1.1 Poly(L-lactide-co-glycolide) ... 36
7.1.2. Collagen and GAGs as aECM coatings ... 36
7.1.3. Cells and media ... 37
7.2. Scaffold manufacturing ... 38
7.2.1. Scaffold preparation and microstructure modification ... 38
7.2.2. Coating scaffolds with collagen and GAGs – creation of artificial extracellular matrices (aECMs)………38
7.3. Scaffold characterization ... 39
7.3.1. Mechanical properties ... 39
7.3.2. Scanning electron microscopy (SEM) and optical microscopy ... 40
7.3.3. Microcomputer tomography (microCT) ... 40
7.3.4. Porosity and water uptake ... 40
7.3.6. Collagen amount (Sirius red) ... 41
7.3.7. GAG amount (toluidine blue) ... 42
7.4. Cell seeding and culture on scaffolds ... 42
7.4.1. Methods of cell culture and biological tests ... 42
7.4.2. Scanning electron microscopy (SEM) ... 43
7.4.3. MTT assay ... 43
7.4.4. Lactate dehydrogenase (LDH) assay ... 44
7.4.5. Alkaline phosphatase (ALP) assay ... 44
7.4.6. Mineralization – van Kossa staining and Calcium content ... 44
7.4.7. Gene expression ... 45
7.4.7.1. RNA isolation and cDNA synthesis ... 45
7.4.7.2. Semi-quantitative analysis ... 45
7.4.7.3. Quantitative analysis by real time polymerase chain reaction (qPCR) ... 46
7.4.8. Statistical analysis ... 47
8. Surface modified PLGA scaffolds with artificial extracellular matrices (aECMs) ... 48
8.1. Characterization of unmodified and surface modified scaffolds ... 48
8.2. Adhesion and proliferation of hMSCs ... 51
8.3. Osteogenic differentiation and gene expression of hMSCs ... 53
8.4. Discussion ... 58
9. PLGA scaffolds with mono- and bimodal pore distribution ... 65
9.1. Characterization of monomodal and bimodal scaffolds ... 65
9.2. Adhesion and proliferation of hMSCs ... 69
9.3. Osteogenic differentiation and gene expression of hMSCs ... 71
9.4. Discussion ... 75
10. Conclusion and Outlook ... 79
