Development of a prevascularized bone implant *** Entwicklung eines prävaskularisierten Knochenimplantats Doctoral thesis for a doctoral degree Julius-Maximilians-Universität Würzburg submitted by Christoph Rücker born in Mellrichstadt Würzburg 2018 Submitted on: Members of the committee Chairperson: Primary supervisor: Prof. Dr. Heike Walles Supervisor (second): PD Dr. Alois Palmetshofer Date of public defense ………………………………………………………………… Date of receipt of certificates …………………………………………………………. Table of contents List of Figures ...................................................................................................... I List of Tables ...................................................................................................... II Abbreviations ..................................................................................................... III Abstract .............................................................................................................. V Zusammenfassung ............................................................................................ VI Introduction ............................................................................................... 1 1.1 Bone ...................................................................................................... 1 1.1.1 Bone structure ................................................................................ 1 1.1.2 Bone healing and blood supply ....................................................... 3 1.1.3 Bone defects ................................................................................... 5 1.2 Tissue engineering and bioreactor technology ...................................... 5 1.3 Relevant cell types for bone tissue engineering .................................... 7 1.3.1 Mesenchymal stem cells ................................................................. 7 1.3.2 Microvascular endothelial cells ....................................................... 8 1.3.3 Endothelial progenitor cells ............................................................. 9 1.3.4 Synergies in MSC and EPC .......................................................... 11 1.4 Strategies for bone regeneration ......................................................... 12 1.4.1 Synthetic biomaterials ................................................................... 12 1.4.2 Bone graft prefabrication following the in vivo bioreactor principle 14 1.4.3 Naturally derived decellularized scaffolds ..................................... 16 1.5 Growth factors and the special role of BMP ........................................ 18 1.6 Animal models ..................................................................................... 20 1.7 Translation of research into clinical application ................................... 20 1.8 Aim of the thesis .................................................................................. 22 Material ..................................................................................................... 23 Methods ................................................................................................... 37 3.1 Cell culture methods............................................................................ 37 3.1.1 Isolation of primary human cells ................................................... 37 3.1.2 Isolation of primary ovine cells ...................................................... 39 3.1.3 Cell culture conditions ................................................................... 40 3.1.4 Passaging of cells ......................................................................... 40 3.1.5 Cell counting and cell viability determination ................................ 40 3.1.6 Freezing of cells ............................................................................ 41 3.1.7 Thawing of cells ............................................................................ 41 3.1.8 Differentiation of human and ovine mesenchymal stem cells ....... 41 3.2 Generation of the cell-free biological vascularized scaffold BioVaSc- TERM ® ................................................................................................ 44 3.3 Generation of prevacularized bone implants ....................................... 45 3.3.1 Seeding of BioVaSc-TERM ® with microvascular endothelial cells 45 3.3.2 Seeding of MSCs onto ß-TCP granules ........................................ 45 3.3.3 Combination of reseeded BioVaSc-TERM ® and seeded ß-TCP ... 46 3.3.4 Prevascularized bone implantation in the ovine tibia .................... 46 3.3.5 Prevascularized bone implantation in the mandibular angle ......... 46 3.4 Characterization .................................................................................. 48 3.4.1 Histology ....................................................................................... 48 3.4.2 Immunohistochemistry .................................................................. 56 3.4.3 MTT assay .................................................................................... 57 3.4.4 Fluorescent staining ...................................................................... 58 3.4.5 Acetylated low-density lipoprotein assay ...................................... 58 Results ..................................................................................................... 60 4.1 Mobile Incubator .................................................................................. 60 4.2 Endothelial progenitor cells ................................................................. 62 4.3 Differentiation of human and ovine mesenchymal stem cells .............. 63 4.4 BioVaSc-TERM ® decellularization ....................................................... 65 4.5 GMP conform documentation system ................................................. 68 4.6 Seeding of TCP granules .................................................................... 74 4.7 BioVaSc-TERM ® reseeding ................................................................. 74 4.8 Animal experiment: Tibia defect in sheep ............................................ 76 4.9 Animal experiment: Defect in the mandibular angle in sheep .............. 81 Discussion ............................................................................................... 87 5.1 Mobile incubator .................................................................................. 87 5.2 Animal models ..................................................................................... 89 5.3 Bone graft implantation into critical size defects in sheep tibia and mandibale ............................................................................................ 93 5.4 Consideration of the GMP principles ................................................... 99 Outlook ................................................................................................... 102 References ............................................................................................. 103 Appendix ................................................................................................ 118 8.1 Affidavit ............................................................................................. 118 8.2 Acknowledgement ............................................................................. 119 List of Figures Figure 19: CAD model of the mobile incubator. ................................................ 60 Figure 20: Schematic illustration of the bioreactor setup. ................................. 61 Figure 21: Heating of the incubation room of mobile incubator. ....................... 62 Figure 1: EPC isolated from peripheral blood. .................................................. 63 Figure 2: Histological analysis of human and ovine MSC differentiation. ......... 64 Figure 3: Histological and immunohistochemical analysis of human and ovine MSC differentiation. .......................................................................................... 65 Figure 4: Histological analysis of human and ovine MSC differentiation. ......... 65 Figure 5: Staining of ECM of deceullularized porcine small intestine segments (BioVaSc-TERM ®). ........................................................................................... 66 Figure 6: Quality assessment of decellularized porcine jejunum (BioVaSc- TERM ®). ........................................................................................................... 67 Figure 7: ß-TCP granules seeded with MSC. ................................................... 74 Figure 8: BioVaSc-TERM ® after resseding with EPCs. .................................... 74 Figure 9: Immunohistological staining of section of reseeded BioVaSc-TERM ®. ......................................................................................................................... 76 Figure 10: Tibial defect creation and bone graft implantation. .......................... 77 Figure 11: Histological staining of tibial samples of different donors. ............... 78 Figure 12: Immunohistochemical staining of tibial implant sample D. .............. 79 Figure 13: Bone regeneration after 6 months. .................................................. 80 Figure 14: Mandible defect creation and bone graft implantation. .................... 82 Figure 15: X-ray follow up after 6 months. ........................................................ 83 Figure 16: Transverse section of the mandibular angle defect treated with