Research Doctorate School in BIOmolecular Sciences PhD in BIOMATERIALS – XXIV Cycle (2009-2011)
A Novel Approach to the Fabrication of Polymeric Scaffolds for Tissue Engineering Applications
Carlos Miguel Domingues Mota
Supervisor: Prof. Emo Chiellini
Tutors: Dr. Federica Chiellini Dr. Dario Puppi
Laboratory of Bioactive Polymeric Materials for Biomedical and Environmental Applications (BIOLab) – Department of Chemistry and
Industrial Chemistry (University of Pisa)
I
Table of contents
1 INTRODUCTION ... 1
1.1 Tissue Engineering (TE) ...1
1.1.1 Scaffold-based TE approach...2
1.1.2 Biomaterials for scaffolds fabrication ...4
1.1.2.1 Natural polymers ... 4
1.1.2.2 Synthetic polymers ... 5
1.1.2.3 Bioactive ceramics ... 6
1.1.2.4 Composite materials ... 7
1.2 Techniques for Scaffolds Fabrication ...8
1.2.1 Solution electrospinning (ES) ...8
1.2.1.1 ES for production of multi-scale scaffolds ... 11
1.2.2 Melt-electrospinning (Melt-ES)... 12
1.2.3 Outlook over solution ES and melt-ES ... 15
1.2.4 Wet-spinning ... 16
1.2.5 Additive manufacturing (AM) techniques ... 23
1.2.5.1 Stereolithography (SLA) ... 23
1.2.5.2 Selective laser sintering (SLS) ... 27
1.2.5.3 Three-dimensional printing (3DP) ... 28
1.2.5.4 Fused deposition modeling (FDM) ... 30
1.2.5.5 Innovative AM equipments... 30
1.2.5.6 Organ Printing ... 32
1.2.6 Combined techniques and novel approaches ... 33
1.2.7 Rapid prototyping subtractive techniques ... 33
1.2.8 AM for surgical planning and medical devices ... 34
1.3 Conclusive Remarks and Future Perspectives ... 34
References ... 34
II – LIST OF ABBREVIATIONS ...49
III - GLOSSARY ...53
2 DEVELOPMENT OF ADVANCED TECHNOLOGIES FOR MANUFACTURING OF TISSUE ENGINEERING SCAFFOLDS ...56
Abstract ... 56
II
2.1 Introduction ... 56
2.2 Development of Hybrid Equipment Combining Additive Manufacturing (AM) and Electrospinning (ES) Technologies ... 57
2.2.1 Additive manufacturing hardware ... 58
2.2.2 Additive manufacturing software ... 59
2.2.3 Electrospinning (ES) ... 60
2.3 Development of Screw Extrusion-based Melt-electrospinning (Melt-ES) Apparatus .... 61
2.4 Modification of the AM Apparatus for Computer-controlled Wet-spinning ... 61
2.5 Development of a New Computer-controlled Wet-spinning Apparatus... 62
2.6 Conclusions... 63
References... 63
3 DUAL-SCALE POLYMERIC CONSTRUCTS AS SCAFFOLDS FOR TISSUE ENGINEERING ... 65
Abstract ... 65
3.1 Introduction ... 65
3.2 Experimental Section ... 68
3.2.1 Materials ... 68
3.2.2 Fabrication of dual-scale fibrous scaffolds ... 68
3.2.2.1 Production of 3D scaffolds by Bioextrusion ... 68
3.2.2.2 Electrospinning of polymer solution ... 69
3.2.3 Morphological characterization ... 69
3.2.4 Biological evaluation... 69
3.2.4.1 Cell culture onto PCL scaffolds ... 69
3.2.4.2 WST-1 cell proliferation assay ... 70
3.2.4.3 Confocal Laser Scanning Microscopy (CLSM) ... 70
3.2.5 Statistical evaluation ... 71
3.3 Results and Discussion ... 71
3.3.1 Development of dual-scale scaffolds... 71
3.3.1.1 Fabrication and morphological characterization of 3D PCL structures ... 72
3.3.1.2 Fabrication and morphological characterization of dual-scale scaffolds... 73
3.3.2 Biological results ... 75
III
3.4 Conclusions ... 79
References ... 79
4 DEVELOPMENT OF 3D WET-SPUN POLYMERIC SCAFFOLDS LOADED WITH ANTIMICROBIAL AGENTS FOR BONE ENGINEERING ...83
Abstract ... 83
4.1 Introduction ... 83
4.2 Materials and Methods ... 86
4.2.1 Materials ... 86
4.2.2 Fabrication of wet-spun meshes ... 86
4.2.3 Morphology analysis... 86
4.2.4 Drug loading... 86
4.2.5 In vitro drug release ... 87
4.2.6 In vitro biological evaluation... 87
4.2.6.1 WST-1 cell proliferation assay ... 87
4.2.6.2 Cytochemical stain... 87
4.2.6.3 Confocal laser scanning microscopy (CLSM) of the cell morphology... 88
4.2.6.4 Live/Dead assay... 88
4.2.7 Statistical analysis... 88
4.3 Results and Discussion ... 89
4.3.1 Meshes production and morphological analysis ... 89
4.3.2 Drug loading... 91
4.3.3 In vitro drug release ... 92
4.3.4 In vitro biocompatibility ... 93
4.4 Conclusions ... 96
References ... 97
5 ADDITIVE MANUFACTURING OF WET-SPUN POLYMERIC SCAFFOLDS FOR BONE TISSUE ENGINEERING ... 101
Abstract ... 101
5.1 Introduction ... 101
5.2 Materials and Methods ... 104
IV
5.2.1 Additive manufacturing of wet-spun scaffolds ... 104
5.2.1.1 Materials ... 104
5.2.1.2 Preparation of polymeric solutions ... 104
5.2.1.3 Fabrication of 3D polymeric scaffolds ... 104
5.2.2 Morphological characterization ... 105
5.2.2.1 Scanning electron microscopy (SEM) ... 105
5.2.2.2 Micro-computer tomography (µCT) ... 105
5.2.3 Mechanical characterization ... 106
5.2.4 Biological characterization ... 106
5.2.4.1 Cell seeding ... 106
5.2.4.2 Cell viability and proliferation ... 107
5.2.4.3 Alkaline phosphatase (ALP) activity ... 107
5.2.4.4 Mineralized matrix deposition analysis by alizarin red staining (ARS) ... 108
5.2.4.5 Cell morphology investigation by confocal laser scanning microscopy (CLSM)... 108
5.2.5 Statistical analysis ... 108
5.3 Results and Discussion ... 109
5.3.1 Additive manufacturing of 3D wet-spun structures... 109
5.3.2 Morphological analysis ... 111
5.3.3 Mechanical characterization ... 114
5.3.4 Biological characterization ... 116
5.4 Conclusions... 122
References... 122
6 ADDITIVE MANUFACTURING OF STAR POLY(Ε- CAPROLACTONE) WET-SPUN SCAFFOLDS FOR TISSUE ENGINEERING APPLICATIONS ... 126
Abstract ... 126
6.1 Introduction ... 127
6.2 Materials and Methods ... 129
6.2.1 Materials ... 129
6.2.2 Computer-aided wet-spinning apparatus ... 129
6.2.3 Solution preparation ... 130
6.2.4 Scaffolds production and design ... 130
6.2.5 Morphological analysis ... 131
6.2.6 Thermal properties ... 131
6.2.7 Biological evaluation... 132
6.2.7.1 Sterilization of the scaffolds ... 132
6.2.7.2 Cell culturing and cell seeding ... 132
6.2.7.3 Cell viability and cell proliferation ... 133
6.2.7.4 Quantification of collagen production ... 133
6.2.7.5 Alkaline phosphatase (ALP) activity ... 134
V
6.2.7.6 Mineralized matrix deposition... 134
6.2.7.7 Cell morphology investigation by confocal laser scanning microscopy (CLSM) ... 135
6.2.8 Statistical evaluation ... 135
6.3 Results and Discussion ... 135
6.3.1 Investigation of production parameters ... 136
6.3.1.1 Effect of polymer concentration (C*PCL) ... 137
6.3.1.2 Effect of deposition velocity (Vdep)... 139
6.3.1.3 Effect of solution feed rate (F)... 140
6.3.2 Fusion between filaments ... 142
6.3.3 Fibre morphology ... 142
6.3.4 HA loaded scaffolds... 143
6.3.5 Thermal properties ... 145
6.3.6 Biological evaluation ... 147
6.3.6.1 Cell viability and proliferation on scaffolds ... 147
6.3.6.2 Collagen production... 148
6.3.6.3 ALP activity measurement ... 149
6.3.6.4 Matrix mineralization analysis ... 150
6.3.6.5 Cell culture organization on scaffolds ... 151
6.4 Conclusions ... 153
References ... 153
7 ADDITIVE MANUFACTURING OF POLY(3-HYDROXYBUTYRATE- CO-3-HYDROXYHEXANOATE) SCAFFOLDS FOR ENGINEERED BONE DEVELOPMENT ... 158
Abstract ... 158
7.1 Introduction ... 159
7.2 Materials and Methods ... 161
7.2.1 Materials ... 161
7.2.2 Scaffold fabrication ... 161
7.2.3 Scanning electron microscope (SEM) analysis... 162
7.2.4 Crystalline degree ... 162
7.2.5 Scaffold porosity ... 162
7.2.6 Compressive mechanical characterization... 164
7.2.7 Biological evaluation ... 164
7.2.7.1 Scaffolds sterilization and conditioning ... 164
7.2.7.2 Cell culturing and cell seeding ... 165
7.2.7.3 Cell proliferation ... 165
7.2.7.4 Alkaline phosphatase (ALP) activity measurement ... 166
7.2.8 Statistical analysis... 167
7.3 Results and Discussion ... 167
VI
7.3.1 Investigation of processing parameters and morphological analysis ... 167
7.3.2 Scaffolds porosity ... 172
7.3.3 Compressive mechanical characterization ... 173
7.3.4 Biological evaluation... 174
7.3.4.1 Cell viability and proliferation ... 174
7.3.4.2 Alkaline phosphatase (ALP) activity measurement ... 176
7.4 Conclusion ... 177
References... 177
8 ADDITIVE MANUFACTURING OF 3D MELT-ELECTROSPUN STAR POLY(Ε-CAPROLACTONE) SCAFFOLDS ... 180
Abstract ... 180
8.1 Introduction ... 181
8.2 Materials and Methods ... 182
8.2.1 Materials ... 182
8.2.2 Melt-flow index (MFI) ... 182
8.2.3 Melt-electrospinning (Melt-ES) ... 182
8.2.4 Morphological analysis ... 183
8.2.5 Biological evaluation... 183
8.2.5.1 Preparation and sterilization of the scaffolds ... 183
8.2.5.2 Cell seeding and culturing ... 184
8.2.5.3 WST-1 tetrazolium salt cell proliferation assay ... 184
8.2.5.4 Cell viability by direct contact assay ... 184
8.2.5.5 Cell adhesion and proliferation assay ... 184
8.2.5.6 Quantification of collagen production ... 185
8.2.5.7 Cell morphology investigation by confocal laser scanning microscopy (CLSM)... 186
8.2.5.8 Morphological observation of cultured cells by scanning electron microscopy (SEM) 186 8.2.6 Statistical analysis ... 187
8.3 Results and Discussion ... 187
8.3.1 Melt-flow index (MFI) ... 187
8.3.2 Computer-aided melt-ES ... 187
8.3.2.1 Evaluation of the polymer extrusion flow rate (EFR) ... 189
8.3.2.2 Investigation of processing parameters... 190
8.3.2.3 Effect of the applied voltage (Vapp)... 195
8.3.2.4 Effect of processing temperature (Tproc)... 196
8.3.2.5 Effect of extrusion flow rate (EFR) ... 197
8.3.2.6 Effect of other processing parameters ... 197
8.3.2.7 Morphological analysis ... 198
8.3.2.8 Layer-by-layer fabrication of 3D melt-electrospun scaffolds ... 199
8.3.3 Biological characterization ... 200
VII
8.3.3.1 Cell viability and proliferation... 201
8.3.3.2 Collagen production... 202
8.3.3.3 Cell morphology investigation by confocal laser scanning microscopy (CLSM) ... 203
8.3.3.4 Morphological observation of cell-cultured scaffolds by SEM analysis ... 204
8.4 Conclusions ... 205
References ... 205
9 DEVELOPMENT OF NOVEL SCAFFOLDS FOR OTOLOGIC SURGERY APPLICATIONS ... 208
Abstract ... 208
9.1 Introduction ... 209
9.2 Materials and Methods ... 211
9.2.1 Materials ... 211
9.2.2 Scaffolds design ... 212
9.2.2.1 Partial ossicular replacement prosthesis (PORP) scaffold ... 212
9.2.2.2 Posterior canal wall (PCW) scaffold ... 213
9.2.2.3 Tympanic membrane (TM) scaffolds ... 214
9.2.3 Scaffold fabrication ... 215
9.2.3.1 Three-dimensional fibre deposition (3DF) technique ... 215
9.2.3.2 Electrospinning apparatus ... 216
9.2.4 Gas plasma treatment ... 217
9.2.5 Morphological analyses ... 217
9.2.5.1 Scanning electron microscopy (SEM) ... 217
9.2.5.2 Atomic force microscopy (AFM)... 218
9.2.6 Porosity ... 218
9.2.7 Biological evaluation of PORP scaffolds ... 218
9.2.7.1 Cell expansion of hMSCs ... 219
9.2.7.2 Cell culture of the PORP scaffolds ... 219
9.2.7.3 Methylene blue staining ... 220
9.2.7.4 DNA assay and ALP assay ... 220
9.2.7.5 Histology ... 221
9.2.8 Statistical evaluation ... 221
9.3 Results and Discussion ... 221
9.3.1 Scaffold manufacturing and characterization ... 221
9.3.1.1 PORP scaffold morphology ... 221
9.3.1.2 PCW scaffold morphology ... 225
9.3.1.3 TM scaffold morphology ... 227
9.3.2 Biological results of PORP scaffolds ... 232
9.3.2.1 Evaluation of cell distribution by means of methylene blue staining... 232
9.3.2.2 DNA assay ... 234
9.3.2.3 ALP assay... 235
VIII
9.3.2.4 Histology ... 236
9.3.2.5 SEM analysis of cell morphology ... 237
9.4 Conclusions... 239
References... 239
10 OVERALL CONCLUSIVE REMARKS ... 243
10.1 Development of Advanced Technologies for Manufacturing of Tissue Engineering Scaffolds ... 243
10.2 Dual-Scale Polymeric Constructs as Scaffolds for Tissue Engineering ... 243
10.3 Development of 3D Wet-spun Polymeric Scaffolds Loaded with Antimicrobial Agents for Bone Engineering ... 244
10.4 Additive Manufacturing of Wet-spun Polymeric Scaffolds for Bone Tissue Engineering ... 244
10.5 Additive Manufacturing of Star Poly(ε-caprolactone) Wet-spun Scaffolds for Tissue Engineering Applications ... 245
10.6 Additive Manufacturing of Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) Scaffolds for Engineered Bone Development ... 245
10.7 Additive Manufacturing of 3D Melt-electrospun Star Poly(ε-caprolactone) Scaffolds . ... 246
10.8 Development of Novel Scaffolds for Otologic Surgery Applications ... 246
