Tissue Engineering Example: Combinatorial Effects of Osteoconductive and Osteoinductive Elements in Bone Regeneration Stephanie Pasquesi BIOE 506 April 27, 2009

Coating of VEGF-releasing scaffolds with bioactive glass for angiogenesis and bone regeneration J. Kent Leach a,b, Darnell Kaigler b, Zhuo Wang b, Paul H. Krebsbach b, David J. Mooney a,b a Division of Engineering and Applied Sciences, Harvard University, Cambridge, MA 02138, USA b School of Dentistry, Biologic and Materials Sciences, University of Michigan, Ann Arbor, MI 48109, USA Biomaterials (2006) Biomaterials (2006)

Definitions Osteoconduction – the ability of some materials to serve as a scaffold to which bone cells can attach, migrate, grow, and divide Osteoinduction – the capacity of normal chemicals in the body to stimulate primitive stem cells or immature bone cells to grow and mature, forming healthy bone tissue Neovascularization – the formation of functional microvascular networks with red blood cell perfusion (i.e. formation of new blood vessels) –Different from angiogenesis: protrusion and outgrowth of capillary buds from pre-existing blood vessels Mitogen – chemical substance, usually a protein, which promotes cell division and mitosis

Current Bone Graft Materials Autografts –Graft tissue (bone) from the patient Problems: chronic pain at site of bone harvestation, limited supply Allografts –Graft tissue (bone) from someone other than the patient Problems: immune rejection, risk of disease transmission Metallic Implants Problems: immune rejection, different mechanical properties, stress risers in existing bone, risk of poor positioning by surgeon, etc.

Synthetic Matrices Scaffolds of synthetic material (effectively synthetic ECM) to provide a support for osteoblast proliferation in critically-sized bone defects –Once osteoblasts attach to scaffold and osteogenesis is induced, scaffold dissolves away, leaving new healed bone in its place Currently can present osteoinductive growth factors from within matrices, but lack the osteoconductivity of conventional graft materials Interest in developing a composite material allowing for delivery of osteoinductive macromolecules and possessing osteoconductive properties

VEGF VEGF = vascular endothelial growth factor Protein, endothelial cell mitogen –Well known for angiogenesis, also important in osteogenesis Promotes neovascularization, bone turnover, osteoblast migration and mineralization Osteoinductive

Bioactive Glass (BG) Osteoconductive, surface active, glass-ceramic material composed of several oxidized minerals –Good adhesive bonding capacity with bone and some connective tissues Some studies have shown it may exhibit both osteoconductive and osteoinductive properties –Hattar et al (2005), Bosetti et al (2005) Others show stronger matrices and accelerated deposition of hydroxyapatite layer in vitro –Suggests improved integration upon placement in vivo Maquet et al (2003), Verrier et al (2004), Lu et al (2005), Yao et al (2005)

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

Procedure Overview VEGF incorporated into 3D porous scaffolds made from poly(lactide-co-glycolide) for localized protein delivery Scaffold surface coated with bioactive glass to enhance osteoconductivity Investigated in vitro models –HMVEC (human microvascular endothelial cell) proliferation –Progenitor cell differentiation Investigated in vivo models –Neovascularization –Bone regeneration

Scaffold Fabrication 3 μg VEGF incorporated in polymeric scaffolds by gas foaming/particulate leaching process Scaffold coated with BG by soaking in ethanol to reduce hydrophobicity and then submerging in a BG slurry in distilled water –BG deposited was 0.5 +/- 0.2 mg BG particulate on scaffold.

Scaffold VEGF Release VEGF released in a sustained fashion over 18 days Radio-labeled VEGF was used as a tracer

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

In Vitro Testing – HMVEC Proliferation All groups compared to control demonstrated increased HMVEC proliferation through day 6 Enhanced proliferation in BGBL samples was not detectable by day 9 BG coating has an additive proliferation affect when comparing V to BGV samples –By days proliferation rate of BGV decreased with respect 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 Differentiation Scaffolds were seeded with hMSCs (human mesenchymal stem cells) Alkaline phosphatase expression –Indicator of progenitor cell differentiation No significant differences between different scaffolds Osteocalcin secretion –Secreted differentiation marker No significant differences between different scaffolds Filled – BL, Open – V, Horizontal Striped – BGBL, Vertical Striped – BGV

In Vivo Testing 9mm diameter hole made in Lewis rat crania 2 types of implant –BG coated scaffold with VEGF (BGV) –BG coated control scaffold (BGC) At 2 weeks, some rats euthanized and scaffolds scanned for neovascularization At 12 weeks, other rats euthanized and scaffolds inspected for bone regeneration

In Vivo Testing - Neovascularization 2 week samples were tested for the presence of blood vessels by immunostaining for vWF (von Willebrand Factor) –vWF: glycoprotein present in large quantities in subendothelial matrices Vessels = circular, dark brown (arrows) BGC – top, BGV - bottom

In Vivo Testing - Neovascularization Significantly more (p<0.001) vessels in BGV than BGC scaffolds –BGV displayed 117 ± 20 vessels/cm 2 –BGC displayed 66 ± 8 vessels/cm 2 Area between dashed lines: scaffold alone –36 ± 9 vessels/cm 2 (unpublished)

In Vivo Testing – Bone Regeneration 12 week samples were scanned for bone regeneration by microCT imaging –Left: Distribution of new mineralized tissue –Right: Nearly complete bridging of defect by new mineralized tissue

In Vivo Testing – Bone Regeneration Bone volume fraction –BGV slightly higher, no significant difference BGV: 20 ± 4% BGC: 14 ± 6% Bone Mineral Density –BGV shows significant increase (p=0.02) vs. BGC BGV: 177 ± 17 mg/cm 3 BGC: 135 ± 27 mg/cm 3 –Area between dashed lines: scaffold alone 120 ± 20 mg/cm 3 (unpublished)

Conclusions - BG BG coating induces significant increase in proliferation of endothelial cells in vitro and in vivo –Angiogenesis further increased with the delivery of VEGF from BG coated scaffolds Large difference in masses of BG (500 μg) and VEGF (3 μg) needed for similar response –Suggests that angiogenic effects of BG may be indirect

Conclusions - BG Did not show osteogenic response of BG unlike prior studies –Relatively low concentrations of BG used in this model were enough to elicit angiogenic response, higher concentrations may yield a more robust osteogenic reaction Previous studies used larger concentrations, packing the defect area with BG Osteoconductivity of BG was limited by dissolution rate of coating BG coating offers inductive component not available through other osteoconductive materials

Conclusions - VEGF Prolonged delivery of VEGF improves maturation of newly formed bone –Significant increase in bone mineral density –Slight increase in bone volume fraction Expected from prior studies Defect regeneration may benefit from localized VEGF presentation –Establishes a vascular network for nutrient transport, potentially supplying progenitor cells for healing

Overall Conclusions Strong linkage between angiogenesis and bone regeneration Combinatorial approaches of delivering osteoinductive factors from osteoconductive scaffolds provide therapeutic benefit –May achieve desired tissue response by capitalizing on degradation components of synthetic ECM and inductive factors released from the matrix

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