Eleni Antoniadou
Background
 Critical-sized bone defects
 Do not heal spontaneously
 500,000 bone repair procedures annually
 Trauma
 Resection
 Abnormal development
 Current clinical approaches
Limitations
1. extended surgical time,
 Autograft
2. limited availability,
 Allograft
3. variable bone quality,
 Metallic implants
4. significant blood loss
5. donor-site morbidity
Osteogenic
Background
Biological ability to
directly create new
bone. i.e.
mesenchymal stem
cells
 Osteoconductive materials
 Calcium phosphate, hydroxyapatite, Bioglass
Osteoconductive
 Osteoinductive materials
Scaffold for
supporting the
 Collagen, PLA, PLGA, Bioglass
attachment
of osteogenic
 Materials usually conductive or inductive
precursor cells.
 Bone is a collagen-hydroxyapatite composite
Osteoinductive
 Not both, so composites needed
stimulate
the proliferation and
 VEGF promotes angiogenesis
differentiation of
 May speed bone healing
mesenchymal stem
cells into
bone-forming cells.
Hypothesis
Cell Source
Mesenchymal Stem
Cells
Signals
VEGF
Biomaterials approach
ECM
PLGA +
Bioglass coating
Enhance bone regeneration
1. Improve vascularization
2. Better integration with native tissues
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 surface
Controlled growth factor release
~60% release @ 14 day
50% @7 days
Good
integration,
+ maintain
surface
Low error + 0.1 mg
Matches PLGA
degradation
PLGA
Mimic bone
collagen
Bioglass (note crystal structure)
Mimics bone hydroxyapatite
~40% initial release
diffusion outwards
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
Additive effect?
Comparable
proliferation
Dissolution of
bioglass?
PLGA
control
+ VEGF
+bioglass
+VEGF
+bioglass
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
In general, no major effects
PLGA
control
+ VEGF
+bioglass
+VEGF
+bioglass
~20% variation
Bioglass trends lower
Osteocalcin
Again, in general, no major effects
PLGA
control
+ VEGF
+bioglass
+VEGF
+bioglass
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
Density doubles
compared to
control! Most
found near
periphery
PLGA
control
+bioglass
+VEGF
+bioglass
Micro-CT Analysis
+bioglass
Top-view
Note healing
bone doesn’t
meet in center
+VEGF
+bioglass
Side-view
Initial callus has
nearly bridged
defect and is
thickening
Bone Mineral Density
Minor
increase
~25%
increase
PLGA
control
+bioglass
+VEGF
+bioglass
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!!!