Tissue Engineering Example:Tissue Engineering Example:Combinatorial Effects of Osteoconductive Combinatorial Effects of Osteoconductive

and Osteoinductive Elements in Bone and Osteoinductive Elements in Bone RegenerationRegeneration

Stephanie PasquesiStephanie Pasquesi

BIOE 506BIOE 506

April 27, 2009April 27, 2009

Coating of VEGF-releasing Coating of VEGF-releasing scaffolds with bioactive glass for scaffolds with bioactive glass for angiogenesis and bone angiogenesis and bone regenerationregeneration

J. Kent Leach J. Kent Leach a,ba,b, Darnell Kaigler , Darnell Kaigler bb, Zhuo Wang , Zhuo Wang bb, Paul H. , Paul H. Krebsbach Krebsbach bb, David J. Mooney , David J. Mooney a,ba,b

aa Division of Engineering and Applied Sciences, Harvard University, Cambridge, MA 02138, USA Division of Engineering and Applied Sciences, Harvard University, Cambridge, MA 02138, USAbb School of Dentistry, Biologic and Materials Sciences, University of Michigan, Ann Arbor, MI 48109, School of Dentistry, Biologic and Materials Sciences, University of Michigan, Ann Arbor, MI 48109,

USAUSA

Biomaterials (2006)Biomaterials (2006)

DefinitionsDefinitionsOsteoconductionOsteoconduction – the ability of some materials to – the ability of some materials to serve as a scaffold to which bone cells can attach, serve as a scaffold to which bone cells can attach, migrate, grow, and dividemigrate, grow, and divideOsteoinductionOsteoinduction – the capacity of normal – the capacity of normal chemicals in the body to stimulate primitive stem chemicals in the body to stimulate primitive stem cells or immature bone cells to grow and mature, cells or immature bone cells to grow and mature, forming healthy bone tissueforming healthy bone tissueNeovascularizationNeovascularization – – the formation of functional the formation of functional microvascular networks with red blood cell microvascular networks with red blood cell perfusion (i.e. formation of new blood vessels)perfusion (i.e. formation of new blood vessels)– Different from angiogenesis: protrusion and outgrowth of Different from angiogenesis: protrusion and outgrowth of

capillary buds from capillary buds from pre-existing pre-existing blood vesselsblood vessels

MitogenMitogen – chemical substance, usually a protein, – chemical substance, usually a protein, which promotes cell division and mitosiswhich promotes cell division and mitosis

Current Bone Graft MaterialsCurrent Bone Graft Materials

AutograftsAutografts– Graft tissue (bone) from the Graft tissue (bone) from the

patientpatientProblems: chronic pain at Problems: chronic pain at site of bone harvestation, site of bone harvestation, limited supplylimited supply

AllograftsAllografts– Graft tissue (bone) from Graft tissue (bone) from

someone other than the patientsomeone other than the patientProblems: immune rejection, Problems: immune rejection, risk of disease transmissionrisk of disease transmission

Metallic ImplantsMetallic ImplantsProblems: immune rejection, Problems: immune rejection, different mechanical different mechanical properties, stress risers in properties, stress risers in existing bone, risk of poor existing bone, risk of poor positioning by surgeon, etc.positioning by surgeon, etc.

Synthetic MatricesSynthetic Matrices

Scaffolds of synthetic material (effectively synthetic Scaffolds of synthetic material (effectively synthetic ECM) to provide a support for osteoblast ECM) to provide a support for osteoblast proliferation in critically-sized bone defectsproliferation in critically-sized bone defects– Once osteoblasts attach to scaffold and osteogenesis is Once osteoblasts attach to scaffold and osteogenesis is

induced, scaffold dissolves away, leaving new healed induced, scaffold dissolves away, leaving new healed bone in its placebone in its place

Currently can present osteoinductive growth factors Currently can present osteoinductive growth factors from within matrices, but lack the osteoconductivity from within matrices, but lack the osteoconductivity of conventional graft materialsof conventional graft materialsInterest in developing a composite material allowing Interest in developing a composite material allowing for delivery of osteoinductive macromolecules and for delivery of osteoinductive macromolecules and possessing osteoconductive propertiespossessing osteoconductive properties

VEGFVEGF

VEGF = vascular endothelial VEGF = vascular endothelial growth factorgrowth factor

Protein, endothelial cell Protein, endothelial cell mitogenmitogen– Well known for angiogenesis, Well known for angiogenesis,

also important in osteogenesisalso important in osteogenesisPromotes neovascularization, Promotes neovascularization, bone turnover, osteoblast bone turnover, osteoblast migration and mineralizationmigration and mineralization

OsteoinductiveOsteoinductive

Bioactive Glass (BG)Bioactive Glass (BG)

Osteoconductive, surface active, glass-ceramic Osteoconductive, surface active, glass-ceramic material composed of several oxidized mineralsmaterial composed of several oxidized minerals– Good adhesive bonding capacity with bone and some Good adhesive bonding capacity with bone and some

connective tissuesconnective tissues

Some studies have shown it may exhibit both Some studies have shown it may exhibit both osteoconductive and osteoinductive propertiesosteoconductive and osteoinductive properties– Hattar et al (2005), Bosetti et al (2005)Hattar et al (2005), Bosetti et al (2005)

Others show stronger matrices and accelerated Others show stronger matrices and accelerated deposition of hydroxyapatite layer in vitrodeposition of hydroxyapatite layer in vitro– Suggests improved integration upon placement in Suggests improved integration upon placement in

vivovivoMaquet et al (2003), Verrier et al (2004), Lu et al (2005), Yao Maquet et al (2003), Verrier et al (2004), Lu et al (2005), Yao et al (2005)et al (2005)

HypothesisHypothesis

Adding an osteoconductive (BG) surface Adding an osteoconductive (BG) surface to VEGF (osteoinductive) releasing to VEGF (osteoinductive) releasing scaffolds serving as synthetic ECM will scaffolds serving as synthetic ECM will enhance bone regeneration through enhance bone regeneration through improved vascularization and integration improved vascularization and integration with native tissueswith native tissues

Procedure OverviewProcedure Overview

VEGF incorporated into 3D porous scaffolds VEGF incorporated into 3D porous scaffolds made from poly(lactide-co-glycolide) for made from poly(lactide-co-glycolide) for localized protein deliverylocalized protein deliveryScaffold surface coated with bioactive glass to Scaffold surface coated with bioactive glass to enhance osteoconductivityenhance osteoconductivityInvestigated in vitro modelsInvestigated in vitro models– HMVEC (human microvascular endothelial cell) HMVEC (human microvascular endothelial cell)

proliferationproliferation– Progenitor cell differentiationProgenitor cell differentiation

Investigated in vivo modelsInvestigated in vivo models– NeovascularizationNeovascularization– Bone regenerationBone regeneration

Scaffold FabricationScaffold Fabrication

3 3 μμg VEGF incorporated in g VEGF incorporated in polymeric scaffolds by gas polymeric scaffolds by gas foaming/particulate foaming/particulate leaching processleaching processScaffold coated with BG by Scaffold coated with BG by soaking in ethanol to soaking in ethanol to reduce hydrophobicity and reduce hydrophobicity and then submerging in a BG then submerging in a BG slurry in distilled waterslurry in distilled water– BG deposited was 0.5 +/- BG deposited was 0.5 +/-

0.2 mg 0.2 mg BG particulate on scaffold.

Scaffold VEGF ReleaseScaffold VEGF Release

VEGF released in a VEGF released in a sustained fashion sustained fashion over 18 daysover 18 days

Radio-labeled VEGF Radio-labeled VEGF was used as a tracerwas used as a tracer

In Vitro TestingIn Vitro Testing

HMVECs grown in wells containing four HMVECs grown in wells containing four different scaffold typesdifferent scaffold types– Uncoated Blank Scaffolds (BL)Uncoated Blank Scaffolds (BL)– Uncoated VEGF-releasing Scaffolds (V)Uncoated VEGF-releasing Scaffolds (V)– BG Coated Blank Scaffolds (BGBL)BG Coated Blank Scaffolds (BGBL)– BG Coated VEGF-releasing Scaffolds (BGV)BG Coated VEGF-releasing Scaffolds (BGV)

In Vitro Testing – HMVEC ProliferationIn Vitro Testing – HMVEC ProliferationAll groups compared to All groups compared to control demonstrated control demonstrated increased HMVEC increased HMVEC proliferation through day 6proliferation through day 6

Enhanced proliferation in Enhanced proliferation in BGBL samples was not BGBL samples was not detectable by day 9detectable by day 9

BG coating has an additive BG coating has an additive proliferation affect when proliferation affect when comparing V to BGV comparing V to BGV samplessamples– By days 10-12 proliferation By days 10-12 proliferation

rate of BGV decreased with rate of BGV decreased with respect to that of Vrespect to that of V

Filled – BL (control)Open – V (VEGF, no coating)Horizontal Striped – BGBL (BG, no VEGF)Vertical Striped – BGV (BG and VEGF)

In Vitro Testing – Progenitor Cell In Vitro Testing – Progenitor Cell DifferentiationDifferentiation

Scaffolds were seeded Scaffolds were seeded with hMSCs (human with hMSCs (human mesenchymal stem cells)mesenchymal stem cells)Alkaline phosphatase Alkaline phosphatase expressionexpression– Indicator of progenitor cell Indicator of progenitor cell

differentiationdifferentiationNo significant differences No significant differences between different scaffoldsbetween different scaffolds

Osteocalcin secretionOsteocalcin secretion– Secreted differentiation Secreted differentiation

markermarkerNo significant differences No significant differences between different scaffoldsbetween different scaffolds

Filled – BL, Open – V, Horizontal Striped – BGBL, Vertical Striped – BGV

In Vivo TestingIn Vivo Testing

9mm diameter hole made in 9mm diameter hole made in

Lewis rat craniaLewis rat crania

2 types of implant2 types of implant– BG coated scaffold with VEGF (BGV)BG coated scaffold with VEGF (BGV)– BG coated control scaffold (BGC)BG coated control scaffold (BGC)

At 2 weeks, some rats euthanized and scaffolds At 2 weeks, some rats euthanized and scaffolds scanned for neovascularizationscanned for neovascularization

At 12 weeks, other rats euthanized and scaffolds At 12 weeks, other rats euthanized and scaffolds inspected for bone regenerationinspected for bone regeneration

In Vivo Testing - NeovascularizationIn Vivo Testing - Neovascularization

2 week samples were 2 week samples were tested for the presence of tested for the presence of blood vessels by blood vessels by immunostaining for vWF immunostaining for vWF (von Willebrand Factor)(von Willebrand Factor)– vWF: glycoprotein present in vWF: glycoprotein present in

large quantities in large quantities in subendothelial matrices subendothelial matrices

Vessels = circular, dark Vessels = circular, dark brown (arrows)brown (arrows)

BGC – top, BGV - bottom

In Vivo Testing - NeovascularizationIn Vivo Testing - Neovascularization

Significantly more (p<0.001) vessels in BGV than BGC Significantly more (p<0.001) vessels in BGV than BGC scaffoldsscaffolds– BGV displayed 117 BGV displayed 117 ±± 20 vessels/cm 20 vessels/cm22

– BGC displayed 66 BGC displayed 66 ±± 8 vessels/cm 8 vessels/cm22

Area between dashed lines: scaffold aloneArea between dashed lines: scaffold alone– 36 36 ±± 9 vessels/cm 9 vessels/cm2 2 (unpublished)(unpublished)

In Vivo Testing – Bone RegenerationIn Vivo Testing – Bone Regeneration

12 week samples were scanned for bone 12 week samples were scanned for bone regeneration by microCT imagingregeneration by microCT imaging– Left: Distribution of new mineralized tissueLeft: Distribution of new mineralized tissue– Right: Nearly complete bridging of defect by new Right: Nearly complete bridging of defect by new

mineralized tissuemineralized tissue

In Vivo Testing – Bone RegenerationIn Vivo Testing – Bone Regeneration

Bone volume fractionBone volume fraction– BGV slightly higher, no BGV slightly higher, no

significant differencesignificant differenceBGV: 20 BGV: 20 ± ± 4% 4% BGC: 14 BGC: 14 ±± 6% 6%

Bone Mineral DensityBone Mineral Density– BGV shows significant BGV shows significant

increase (p=0.02) vs. BGCincrease (p=0.02) vs. BGCBGV: 177 BGV: 177 ±± 17 mg/cm 17 mg/cm33

BGC: 135 BGC: 135 ±± 27 mg/cm 27 mg/cm33

– Area between dashed lines: Area between dashed lines: scaffold alonescaffold alone

120 ±120 ± 20 mg/cm 20 mg/cm33 (unpublished)(unpublished)

Conclusions - BGConclusions - BG

BG coating induces significant increase in BG coating induces significant increase in proliferation of endothelial cells in vitro and in proliferation of endothelial cells in vitro and in vivovivo– Angiogenesis further increased with the delivery Angiogenesis further increased with the delivery

of VEGF from BG coated scaffoldsof VEGF from BG coated scaffolds

Large difference in masses of BG (500 Large difference in masses of BG (500 μμg) g) and VEGF (3 and VEGF (3 μμg) needed for similar g) needed for similar response response – Suggests that angiogenic effects of BG may be Suggests that angiogenic effects of BG may be

indirectindirect

Conclusions - BGConclusions - BG

Did not show osteogenic response of BG unlike Did not show osteogenic response of BG unlike prior studiesprior studies– Relatively low concentrations of BG used in this Relatively low concentrations of BG used in this

model were enough to elicit angiogenic response, model were enough to elicit angiogenic response, higher concentrations may yield a more robust higher concentrations may yield a more robust osteogenic reactionosteogenic reaction

Previous studies used larger concentrations, packing the Previous studies used larger concentrations, packing the defect area with BGdefect area with BG

Osteoconductivity of BG was limited by Osteoconductivity of BG was limited by dissolution rate of coatingdissolution rate of coating

BG coating offers inductive component not BG coating offers inductive component not available through other osteoconductive available through other osteoconductive materialsmaterials

Conclusions - VEGFConclusions - VEGF

Prolonged delivery of VEGF improves Prolonged delivery of VEGF improves maturation of newly formed bone maturation of newly formed bone – Significant increase in bone mineral densitySignificant increase in bone mineral density– Slight increase in bone volume fractionSlight increase in bone volume fraction

Expected from prior studiesExpected from prior studies

Defect regeneration may benefit from Defect regeneration may benefit from localized VEGF presentationlocalized VEGF presentation– Establishes a vascular network for nutrient Establishes a vascular network for nutrient

transport, potentially supplying progenitor transport, potentially supplying progenitor cells for healingcells for healing

Overall ConclusionsOverall Conclusions

Strong linkage between angiogenesis and Strong linkage between angiogenesis and bone regenerationbone regenerationCombinatorial approaches of delivering Combinatorial approaches of delivering osteoinductive factors from osteoinductive factors from osteoconductive scaffolds provide osteoconductive scaffolds provide therapeutic benefittherapeutic benefit– May achieve desired tissue response by May achieve desired tissue response by

capitalizing on degradation components of capitalizing on degradation components of synthetic ECM and inductive factors released synthetic ECM and inductive factors released from the matrixfrom the matrix

Questions?Questions?