BENG0011- Lecture 6.pdf

BENG0011- Manufacturing Regenerative Medicines: from Lab Bench to Industry Lecture 6: Bone Repair And Regeneration Dr Rana Khalife 1 Aims and objectives Understand the clinical problem Be able to explain Endochondral Ossification a...

BENG0011- Manufacturing Regenerative Medicines: from Lab Bench to Industry Lecture 6: Bone Repair And Regeneration Dr Rana Khalife 1 Aims and objectives Understand the clinical problem Be able to explain Endochondral Ossification and bone formation Understand the bone formation mechanism Be able to explain Bone tissue engineering and Bone fracture healing Be able to list Scaffold type and its formation Understand the Impact of bioreactors and perfusion in bone tissue engineering 2  The Clinical Problem - Causes Accidents  Falls from heights  Falls on ice or other unsafe surfaces  Overuse, particularly running or sports-related Diseases  Osteoporosis – weakening of living bone that has already been formed and is being remodeled due to calcium loss  Osteomalacia – weakening related to bone formation or to the bone building process due to vitamin D deficiency  Brittle bone disease – caused by a defect in the gene that produces type I collagen  Osteosarcoma – bone cancer 3 Bone Structure in the Human Body Two types of bone tissue: Compact Spongy Three types of cells: Osteoblasts (bone- forming cells) Osteoclasts (resorb or break down bone) Osteocytes (mature bone cells) 4 Figure 23-55 Molecular Biology of the Cell (© Garland Science 2008) 5 6 Figure 23-60 Molecular Biology of the Cell (C) Garland Science 2008) 7 Mesenchymal stem cells 8  Endochondral Ossification Gilbert, Scott F. 2000. Developmental Biology. 6th ed. Sunderland (MA): Sinauer Associates (A, B) MSCs condense and differentiate into cartilage cells (chondrocytes) to form a ‘bone template’ (C) Chondrocytes in the centre change in size, mineralise their extracellular matrix (e.g. with calcium carbonate), and their deaths allow blood vessels to enter (D, E) Blood vessels bring in osteoblasts, which bind to the cartilaginous matrix and deposit bone matrix 9 (F-H) Bone formation consist of ordered arrays of proliferating chondrocytes Key Points  Many bones are formed by endochondral ossification  MSCs can differentiate into several different cell types including chondrocytes and osteoblasts  Osteoblasts produce mineralized matrix, leading to bone formation  A fine balance between bone production (osteoblasts) and resorption (osteoclasts) takes place in healthy mature bone 10 Bone Injury And Intrinsic Regeneration  Bone regeneration and fracture healing occurs spontaneously when union of the fracture surfaces takes place – Periosteum progenitors – Osteoblast induction and anabolic shift – angiogenesis 11 12  Critical Sized Or Non-union Defects Do Not Heal Spontaneously  Open fractures – severe fracture – post-debridement – blast injury  Infection  Tumour resection  Osteonecrosis 13 Treatment Methods To Date  External fixation  Internal fixation (Ti screws and plates etc, ceramics, modified surfaces – e.g. HA- coated)  Immobilisation  Autologous bone graft treatments 14 Skeletal Stability: Current Treatment Options For Bone Defects Autogenous bone graft Distraction Salvage osteogenesis procedures Allogenic bone graft 15  Current Approaches in Bone Regeneration Autologous bone grafts – Surgical procedure (‘gold standard’). Bone graft harvesting from different donor sites  Histocompatibility  Non-immunogenic  Complicated and repeated surgery (infections!) Allografts – From human cadavers or living donors (devitalised) Bone-graft substitutes – Scaffolds of synthetic or natural biomaterials Growth factors – They induce mitogenesis of MSCs and their differentiation towards osteoblasts 16 Potential Functions of Bone Grafts  Osteogenesis - bone formation 1. Survival and proliferation of graft cells 2. Osteoinduction - host mesenchymal cells  Osteoconduction  Structural support 17 Graft Incorporation Within Host Tissue Five phases: 1. Hemorrhage 2. Inflammation 3. Vascular invasion 4. Osteoclastic resorbtion/ Osteoblastic apposition 5. Remodelling and reorientation 18 Tissue Engineered Bone – Considerations When Engineering 3D Tissues  Even distribution of cells throughout the entire construct  Even and timely distribution of oxygen to cells throughout the construct  Even and timely distribution of fresh nutrients to cells throughout the construct  Efficient removal of waste metabolites within the construct 19 Tissue Engineering Approach Bioactive 3D scaffold integrated with cells, proteins or genes to induce osteogenesis http://www.mech.kuleuven.be/en/bme/research/mechbio/ 20 21  Biomaterials used in Bone Regeneration A combination of inorganic and organic compounds is ideal to recreate the structure of native bone Bioactive ceramic materials (include Ca and P, present in native bone)  Hydroxyapatite (HA, Ca10(PO4)6(OH)2)  Tricalcium phosphate (TCP, Ca3(PO4)2) Collagen Glass ceramics 22  Calcium-phosphate The bioactive mechanism of these biomaterials consists of:  Formation of a layer of HA on the surface of the implant  This HA layer can increase the integration and incorporation of the biomaterial  Virtually all calcium phosphate materials exhibit this bioactivity Name Abbreviation Empirical Molar Formula Ca/P Ratio Tetra-calcium Phosphate TeCP Ca4(PO4)2O 2.00 Hydroxyapatite HA Ca10(PO4)6(OH)2 1.67 Tri-calcium Phosphate TCP Ca3(PO4)2 1.50 Octa-calcium Phosphate OCP Ca8H2(PO4)6 5H2O 1.33 Mono-calcium Phosphate MCPM Ca(H2PO4) 2H2O 1.00 Monohydrate 23 24 Tissue Engineered Bone – Considerations When Engineering 3D Tissues Also need to apply appropriate in vivo cues in the in vitro context of the bioreactor : – Biochemical : growth factors, cytokines.. – Nutritional : glucose and amino acids… – Biophysical: pH, oxygen tension, temperature… – Mechanical : scaffold topography, stiffness, mechanical stimulation 25 Tissue Engineered Bone - Considerations Types of bioreactor – Static versus perfusion Perfusion bioreactors extensively employed – Current gold standard – Perfusion seems to enhance bone formation  Mimic interstitial fluid flow that naturally invokes mechanical stimulation of bone formation – Flow patterns determine bone morphology 26 Bioreactor for bone TE 27 28 Perfusion Bioreactors For Enhanced Bone Formation  Improved distribution of cells throughout scaffold  Improved gaseous exchange – O2 delivery – CO2 removal  Improved nutrient transport – Delivery of fresh nutrients (e.g. glucose, amino acids, growth factors) – Removal of metabolites (e.g. lactate)  Induction of mechotransduction pathways 29 30 31 32 Perfusion Culture For Bone Tissue Engineering Grayson et al 2011 Biotech Bioeng 33 Perfusion And Cell Number Grayson et al 2011 Biotech Bioeng 34 Perfusion Culture For Bone Tissue Engineering 35 Enhancing cell retention and engraftment for improved tissue engineering of bone 36 37 38 39 40 41 42 43  Towards Scalable Tissue Engineered Bone Microcarrier-Based Approach:  Microcarriers can be manufactured to have dual function as a cell expansion substrate and an osteogenic biomaterial scaffold  Achieve scalable solutions (clinical requirement will vary between patients and over time)  Amenable to industrial biomanufacturing platforms  Plasticity to be clustered to meet unique shape and size requirements of patient 44 45 46 47 48 Summary  Advanced methods are now being employed to create new bone for grafting  Advanced technologies are required in order to realize the potential of tissue engineered bone for patient-specific needs  Tissue engineering community is developing novel scaffold fabrication methods, bioreactor design and culture methods to produce biomimetic tissues 49