Stem Cells and Tissue Engineering

Eleni Antoniadou

BackgroundCritical-sized bone defects

Do not heal spontaneously500,000 bone repair procedures annually

TraumaResectionAbnormal development

Current clinical approachesAutograftAllograftMetallic implants

Limitations1. extended surgical time, 2. limited availability, 3. variable bone quality, 4. significant blood loss 5. donor-site morbidity

BackgroundOsteoconductive materials

Calcium phosphate, hydroxyapatite, BioglassOsteoinductive materials

Collagen, PLA, PLGA, BioglassMaterials usually conductive or inductive

Bone is a collagen-hydroxyapatite compositeNot both, so composites needed

VEGF promotes angiogenesisMay speed bone healing

Osteoinductive

stimulatethe proliferation

anddifferentiation of

mesenchymal stem cells into

bone-forming cells.

Osteoconductive

Scaffold for supporting the

attachmentof osteogenic

precursor cells.

OsteogenicBiological ability to directly create

new bone. i.e.mesenchymal

stem cells

HypothesisCell Source

Mesenchymal Stem Cells

SignalsVEGF

ECMPLGA +

Bioglass coating

Enhance bone regeneration1. Improve vascularization

2. Better integration with native tissues

Biomaterials approach

ReasoningPLGA

Tailorable degradation propertiesControlled growth factor release

BioglassOsteoconductive and inductiveMimics mineral composition in bone

VEGFPromotes angiogenesis

Scaffold fabrication3D, porous PLGA (85:15)

VEGF incorporation Gas-foaming/particulate-leaching

Bioglass coating Soak in slurry and dry overnight

Scanning electron microscopeIn vitro release kinetics

Radiolabeled VEGF In PBS, measure amount released over time

In vitro characterizationOsteoconductive surface Controlled growth factor release

Bioglass (note crystal structure)Mimics bone hydroxyapatite

PLGAMimic bone collagen

Good integration,+ maintain surface

Low error + 0.1 mgMatches PLGA

degradation

~40% initial releasediffusion outwards

50% @7 days~60% release @ 14 days

Endothelial Cell proliferationEndothelial cell culture

Growth factor removalInsert 4 different groups of scaffolds

bioglass-coated or uncoated scaffoldsVEGF-releasing or blank

Culture 72 hoursTrypsinize and count cellsMove 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 DifferentiationCulture 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 rpmLyse cells and assay either after 1,2, or 4

weeksAlkaline 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 model9 mm diameter circular cranial defect in rats

Full thickness (1.5-2 mm)Bioglass or bioglass + VEGF scaffolds

implantedEuthanized after 2 or 12 weeks

Fixation in formalinScanned using micro-CT

Bone volume fraction Bone mineral density Resolution 9 um

Analysis of blood vessel ingrowthSamples bisected, decalcified, parafin

embeddedSectioned for histology

2 week samples immunostained with vWF (vessels)Light microscope, camera, and image analysis

programCount blood vessels manually

Normalize by tissue areaBoth 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-viewInitial callus has nearly bridged defect and is thickening

Top-viewNote healing bone doesn’t meet in center

+bioglass +VEGF +bioglass

Bone Mineral Density

+bioglass +VEGF +bioglass

PLGA control

~25% increaseMinor

increase

DiscussionComposite 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

DiscussionLocalized, prolonged VEGF delivery

Improved bone cell maturation over controls Increased bone mineral density Slight increase in bone volume

Similar osteoid, but biomineralization is keyAmount of bone unchanged, bone formation rate

increasesVEGF promotes establishment of vascular

networkNutrient transportSupply progenitor cells to participate in healing

DiscussionLack of in vitro osteogenesis

Low concentrations of bioglass -> angiogenicHigher concentrations of bioglass ->

osteogenic Orders of magnitude greater

Bioglass surface coatingLimited by dissolution rate (ions)Inductive component

Dissolution products upregulate important genes in osteoblasts

Important contributionsNutrient diffusion limitation

Poor once tissue mineralizesLacks vessels, blood supply

Inner tissue becomes necroticScaffold eventually failsInflammatory bone resorption

Promoting angiogenesis is vital for long-term success

PorosityGrowth factors

Important ContributionsStrengthened proposed link between bone

remodeling and angiogenesis Bone remodeling process

Could osteoporosis be a vascular disease?

Important PointsStatistical significance vs. practical

significanceIs VEGF necessary? In vitro, no. In vivo, yes.

Small animal models sometimes don’t scale up wellGreater amounts of growth factors (expensive)

Time of healing is a major considerationJust a snapshot, time depends on severity of

defectToo long -> bone will resorb due to mechanical

disuse

CriticismsNo references for BMD of skull

Too dense and bone becomes brittleModulus mismatch -> stress concentrations ->

fracture High BMD not necessarily a good thing!

Passage 6 mesenchymal stem cellsSlow phenotypic drift in vitroEarlier passage (~2-3) may show crisper effect

Why no CT scan at week 2?Interesting to see early response

Main ideasMaterials-based approach can lead to

effective tissue engineering strategies (i.e. tissue engineering is more than just stem cell therapy) ReproducibleLess risk than direct cellular therapy

Strong, fundamental link between angiogenesis and bone formationExploit through composite materials such as

bioglass and growth factors like VEGF which promote both

Goal: achieve a desired tissue responseECM degradation componentsInductive factors released from the matrix

Thank you for your attention!!!