Eleni Antoniadou

Background Critical-sized bone defects Do not heal spontaneously 500,000 bone repair procedures annually Trauma Resection Abnormal development Current clinical approaches Autograft Allograft Metallic implants Limitations 1. extended surgical time, 2. limited availability, 3. variable bone quality, 4. significant blood loss 5. donor-site morbidity

Background Osteoconductive materials Calcium phosphate, hydroxyapatite, Bioglass Osteoinductive materials Collagen, PLA, PLGA, Bioglass Materials usually conductive or inductive Bone is a collagen-hydroxyapatite composite Not both, so composites needed VEGF promotes angiogenesis May speed bone healing Osteoinductive stimulate the proliferation and differentiation of mesenchymal stem cells into bone-forming cells. Osteoconductive Scaffold for supporting the attachment of osteogenic precursor cells. Osteogenic Biological ability to directly create new bone. i.e. mesenchymal stem cells

Hypothesis Cell Source Mesenchymal Stem Cells Signals VEGF ECM PLGA + Bioglass coating Enhance bone regeneration 1. Improve vascularization 2. Better integration with native tissues Biomaterials approach

Reasoning PLGA Tailorable degradation properties Controlled growth factor release Bioglass Osteoconductive and inductive Mimics mineral composition in bone VEGF Promotes angiogenesis

Scaffold fabrication 3D, porous PLGA (85:15) VEGF incorporation Gas-foaming/particulate-leaching Bioglass coating Soak in slurry and dry overnight Scanning electron microscope In vitro release kinetics Radiolabeled VEGF In PBS, measure amount released over time

In vitro characterization Osteoconductive surfaceControlled growth factor release Bioglass (note crystal structure) Mimics bone hydroxyapatite PLGA Mimic bone collagen Good integration, + maintain surface Low error mg Matches PLGA degradation ~40% initial release diffusion outwards days ~60% 14 days

Endothelial Cell proliferation Endothelial cell culture Growth factor removal Insert 4 different groups of scaffolds bioglass-coated or uncoated scaffolds VEGF-releasing or blank Culture 72 hours Trypsinize and count cells Move scaffolds to new pre-seeded wells Repeat 72 hour cycle four times

Endothelial cell proliferation PLGA control + VEGF+bioglass+VEGF +bioglass Dissolution of bioglass? Additive effect? Comparable proliferation

MSC Differentiation Culture to passage 6 Statically seed onto sterilized scaffolds (4 groups) with Matrigel and α-MEM Add osteogenic supplements 10 mM β-glycerophosphate 50 ug/ml ascorbic acid 0.1 uM dexamethasone Culture on orbital shaker at 25 rpm Lyse cells and assay either after 1,2, or 4 weeks Alkaline phosphatase (spectrophotometer) Normalized by DNA (Hoechst dye + flourometer) Osteocalcin (ELISA)

Alkaline Phosphatase PLGA control + VEGF+bioglass+VEGF +bioglass In general, no major effects Bioglass trends lower ~20% variation

Osteocalcin PLGA control + VEGF+bioglass+VEGF +bioglass Again, in general, no major effects

In vivo critical defect model 9 mm diameter circular cranial defect in rats Full thickness (1.5-2 mm) Bioglass or bioglass + VEGF scaffolds implanted Euthanized after 2 or 12 weeks Fixation in formalin Scanned using micro-CT Bone volume fraction Bone mineral density Resolution 9 um

Analysis of blood vessel ingrowth Samples bisected, decalcified, parafin embedded Sectioned for histology 2 week samples immunostained with vWF (vessels) Light microscope, camera, and image analysis program Count blood vessels manually Normalize by tissue area Both treatments displayed significant increases in blood vessel density

Blood Vessel Density PLGA control +bioglass +VEGF +bioglass Density doubles compared to control! Most found near periphery

Micro-CT Analysis Side-view Initial callus has nearly bridged defect and is thickening Top-view Note healing bone doesn’t meet in center +bioglass +VEGF +bioglass

Bone Mineral Density +bioglass +VEGF +bioglass PLGA control ~25% increase Minor increase

Discussion Composite materials hybridize properties Local delivery of inductive factors from osteoconductive scaffolds Low concentrations of bioglass is angiogenic (500 ug) Mimic environment of natural healing (indirect) Upregulation of growth factors in surrounding cells? VEGF (3 ug) is much more potent (direct, focused) Relatively similar results in direct comparison

Discussion Localized, prolonged VEGF delivery Improved bone cell maturation over controls Increased bone mineral density Slight increase in bone volume Similar osteoid, but biomineralization is key Amount of bone unchanged, bone formation rate increases VEGF promotes establishment of vascular network Nutrient transport Supply progenitor cells to participate in healing

Discussion Lack of in vitro osteogenesis Low concentrations of bioglass -> angiogenic Higher concentrations of bioglass -> osteogenic Orders of magnitude greater Bioglass surface coating Limited by dissolution rate (ions) Inductive component Dissolution products upregulate important genes in osteoblasts

Important contributions Nutrient diffusion limitation Poor once tissue mineralizes Lacks vessels, blood supply Inner tissue becomes necrotic Scaffold eventually fails Inflammatory bone resorption Promoting angiogenesis is vital for long-term success Porosity Growth factors

Important Contributions Strengthened proposed link between bone remodeling and angiogenesis Bone remodeling process Could osteoporosis be a vascular disease?

Important Points Statistical significance vs. practical significance Is VEGF necessary? In vitro, no. In vivo, yes. Small animal models sometimes don’t scale up well Greater amounts of growth factors (expensive) Time of healing is a major consideration Just a snapshot, time depends on severity of defect Too long -> bone will resorb due to mechanical disuse

Criticisms No references for BMD of skull Too dense and bone becomes brittle Modulus mismatch -> stress concentrations -> fracture High BMD not necessarily a good thing! Passage 6 mesenchymal stem cells Slow phenotypic drift in vitro Earlier passage (~2-3) may show crisper effect Why no CT scan at week 2? Interesting to see early response

Main ideas Materials-based approach can lead to effective tissue engineering strategies (i.e. tissue engineering is more than just stem cell therapy) Reproducible Less risk than direct cellular therapy Strong, fundamental link between angiogenesis and bone formation Exploit through composite materials such as bioglass and growth factors like VEGF which promote both Goal: achieve a desired tissue response ECM degradation components Inductive factors released from the matrix

Thank you for your attention!!!