Hypoxia-mimicking bioactive glass/collagen glycosaminoglycan composite scaffolds to enhance angiogenesis and bone repair.

One of the biggest challenges in regenerative medicine is promoting sufficient vascularisation of tissue-engineered constructs. One approach to overcome this challenge is to target the cellular hypoxia inducible factor (HIF-1α) pathway, which responds to low oxygen concentration (hypoxia) and results in the activation of numerous pro-angiogenic genes including vascular endothelial growth factor (VEGF). Cobalt ions are known to mimic hypoxia by artificially stabilising the HIF-1α transcription factor. Here, resorbable bioactive glass particles (38 μm and 100 μm) with cobalt ions incorporated into the glass network were used to create bioactive glass/collagen-glycosaminoglycan scaffolds optimised for bone tissue engineering. Inclusion of the bioactive glass improved the compressive modulus of the resulting composite scaffolds while maintaining high degrees of porosity (>97%). Moreover, in vitro analysis demonstrated that the incorporation of cobalt bioactive glass with a mean particle size of 100 μm significantly enhanced the production and expression of VEGF in endothelial cells, and cobalt bioactive glass/collagen-glycosaminoglycan scaffold conditioned media also promoted enhanced tubule formation. Furthermore, our results prove the ability of these scaffolds to support osteoblast cell proliferation and osteogenesis in all bioactive glass/collagen-glycosaminoglycan scaffolds irrespective of the particle size. In summary, we have developed a hypoxia-mimicking tissue-engineered scaffold with pro-angiogenic and pro-osteogenic capabilities that may encourage bone tissue regeneration and overcome the problem of inadequate vascularisation of grafts commonly seen in the field of tissue engineering.

[1]  Gavin Jell,et al.  The effects of strontium-substituted bioactive glasses on osteoblasts and osteoclasts in vitro. , 2010, Biomaterials.

[2]  J. Vacanti,et al.  Tissue engineering : Frontiers in biotechnology , 1993 .

[3]  Aldo R Boccaccini,et al.  Effect of bioactive glasses on angiogenesis: a review of in vitro and in vivo evidences. , 2010, Tissue engineering. Part B, Reviews.

[4]  Hong-Hee Kim,et al.  Stabilization of hypoxia-inducible factor-1alpha is involved in the hypoxic stimuli-induced expression of vascular endothelial growth factor in osteoblastic cells. , 2002, Cytokine.

[5]  F. O'Brien,et al.  Addition of hydroxyapatite improves stiffness, interconnectivity and osteogenic potential of a highly porous collagen-based scaffold for bone tissue regeneration. , 2010, European cells & materials.

[6]  N. Faucheux,et al.  Differentiation of preosteoblasts using a delivery system with BMPs and bioactive glass microspheres , 2007, Journal of materials science. Materials in medicine.

[7]  Jay R Lieberman,et al.  The role of growth factors in the repair of bone. Biology and clinical applications. , 2002, The Journal of bone and joint surgery. American volume.

[8]  M. Longaker,et al.  VEGF expression in an osteoblast-like cell line is regulated by a hypoxia response mechanism. , 2000, American journal of physiology. Cell physiology.

[9]  Esther Novosel,et al.  Vascularization is the key challenge in tissue engineering. , 2011, Advanced drug delivery reviews.

[10]  Delbert E Day,et al.  Bioactive glass in tissue engineering. , 2011, Acta biomaterialia.

[11]  Eileen Gentleman,et al.  Bioactive Glass Scaffolds for Bone Regeneration , 2007 .

[12]  Aldo R Boccaccini,et al.  A review of the biological response to ionic dissolution products from bioactive glasses and glass-ceramics. , 2011, Biomaterials.

[13]  H. Redmond,et al.  Is human fracture hematoma inherently angiogenic , 2000 .

[14]  Jos Malda,et al.  The roles of hypoxia in the in vitro engineering of tissues. , 2007, Tissue engineering.

[15]  T. H.,et al.  The Journal of Bone and Joint Surgery , 2006 .

[16]  Gunasekaran Kumar,et al.  Morbidity at Bone Graft Donor Sites , 2014 .

[17]  R. Buttyan,et al.  Acute intravesical infusion of a cobalt solution stimulates a hypoxia response, growth and angiogenesis in the rat bladder. , 2003, The Journal of urology.

[18]  Ling Wei,et al.  Transplantation of hypoxia-preconditioned mesenchymal stem cells improves infarcted heart function via enhanced survival of implanted cells and angiogenesis. , 2008, The Journal of thoracic and cardiovascular surgery.

[19]  David J Mooney,et al.  Coating of VEGF-releasing scaffolds with bioactive glass for angiogenesis and bone regeneration. , 2006, Biomaterials.

[20]  Amir A. Al-Munajjed,et al.  The healing of bony defects by cell-free collagen-based scaffolds compared to stem cell-seeded tissue engineered constructs. , 2010, Biomaterials.

[21]  Jeffrey A Hubbell,et al.  Peptide-matrix-mediated gene transfer of an oxygen-insensitive hypoxia-inducible factor-1alpha variant for local induction of angiogenesis. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[22]  Julian R. Jones,et al.  Extracellular matrix formation and mineralization on a phosphate-free porous bioactive glass scaffold using primary human osteoblast (HOB) cells. , 2007, Biomaterials.

[23]  J. K. Leach,et al.  Proangiogenic Potential of a Collagen/Bioactive Glass Substrate , 2008, Pharmaceutical Research.

[24]  E. Keshet,et al.  Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis , 1992, Nature.

[25]  F. O'Brien,et al.  The effect of dehydrothermal treatment on the mechanical and structural properties of collagen-GAG scaffolds. , 2009, Journal of biomedical materials research. Part A.

[26]  A. Berdal,et al.  Potential of biomimetic surfaces to promote in vitro osteoblast-like cell differentiation. , 2004, Biomaterials.

[27]  Larry L Hench,et al.  Third-Generation Biomedical Materials , 2002, Science.

[28]  C. Rudd,et al.  Cytocompatibility and Effect of Increasing MgO Content in a Range of Quaternary Invert Phosphate-based Glasses , 2010, Journal of biomaterials applications.

[29]  F. O'Brien,et al.  The effects of collagen concentration and crosslink density on the biological, structural and mechanical properties of collagen-GAG scaffolds for bone tissue engineering. , 2009, Journal of the mechanical behavior of biomedical materials.

[30]  L. Gibson,et al.  The effect of pore size on cell adhesion in collagen-GAG scaffolds. , 2005, Biomaterials.

[31]  M. Ricci,et al.  Development of a scalable procedure for fine calcium alginate particle preparation , 2010 .

[32]  S. Stacker,et al.  The vascular endothelial growth factor family: signalling for vascular development. , 1999, Growth factors.

[33]  Fergal J O'Brien,et al.  Addition of hyaluronic acid improves cellular infiltration and promotes early-stage chondrogenesis in a collagen-based scaffold for cartilage tissue engineering. , 2012, Journal of the mechanical behavior of biomedical materials.

[34]  H. Redmond,et al.  Vascular endothelial growth factor stimulates bone repair by promoting angiogenesis and bone turnover , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[35]  G. Semenza,et al.  Life with Oxygen , 2007, Science.

[36]  Julian R. Jones,et al.  Hypoxia inducible factor-stabilizing bioactive glasses for directing mesenchymal stem cell behavior. , 2015, Tissue engineering. Part A.

[37]  L L Hench,et al.  Gene-expression profiling of human osteoblasts following treatment with the ionic products of Bioglass 45S5 dissolution. , 2001, Journal of biomedical materials research.

[38]  N. Epstein,et al.  Pros, cons, and costs of INFUSE in spinal surgery , 2011, Surgical neurology international.

[39]  G. Jell,et al.  Synthesis and characterization of hypoxia-mimicking bioactive glasses for skeletal regeneration , 2010 .

[40]  T. Clemens,et al.  Activation of the hypoxia-inducible factor-1α pathway accelerates bone regeneration , 2008, Proceedings of the National Academy of Sciences.

[41]  F. O'Brien,et al.  Understanding the effect of mean pore size on cell activity in collagen-glycosaminoglycan scaffolds , 2010, Cell adhesion & migration.

[42]  F. O'Brien,et al.  Evaluation of early healing events around mesenchymal stem cell-seeded collagen–glycosaminoglycan scaffold. An experimental study in Wistar rats , 2011, Oral and Maxillofacial Surgery.

[43]  Johnny Huard,et al.  VEGF Improves, Whereas sFlt1 Inhibits, BMP2‐Induced Bone Formation and Bone Healing Through Modulation of Angiogenesis , 2005, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[44]  Antonios G Mikos,et al.  Dual delivery of an angiogenic and an osteogenic growth factor for bone regeneration in a critical size defect model. , 2008, Bone.

[45]  Larry L. Hench,et al.  Genetic design of bioactive glass , 2009 .

[46]  M. Vallet‐Regí,et al.  Interaction of an ordered mesoporous bioactive glass with osteoblasts, fibroblasts and lymphocytes, demonstrating its biocompatibility as a potential bone graft material. , 2010, Acta biomaterialia.

[47]  F. O'Brien,et al.  Development and characterisation of a collagen nano-hydroxyapatite composite scaffold for bone tissue engineering , 2010, Journal of materials science. Materials in medicine.

[48]  Astrid Hamm,et al.  Efficient transfection method for primary cells. , 2002, Tissue engineering.

[49]  Wei Fan,et al.  Hypoxia-mimicking mesoporous bioactive glass scaffolds with controllable cobalt ion release for bone tissue engineering. , 2012, Biomaterials.

[50]  F. O'Brien,et al.  Evaluation of the ability of collagen–glycosaminoglycan scaffolds with or without mesenchymal stem cells to heal bone defects in Wistar rats , 2012, Oral and Maxillofacial Surgery.

[51]  Thomas D. Schmittgen,et al.  Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. , 2001, Methods.

[52]  C. Kirkpatrick,et al.  Induction of apoptosis in human microvascular endothelial cells by divalent cobalt ions. Evidence for integrin-mediated signaling via the cytoskeleton , 2001, Journal of materials science. Materials in medicine.

[53]  F. O'Brien,et al.  Osteoblast activity on collagen-GAG scaffolds is affected by collagen and GAG concentrations. , 2009, Journal of biomedical materials research. Part A.

[54]  Junzo Tanaka,et al.  The effect of calcium ion concentration on osteoblast viability, proliferation and differentiation in monolayer and 3D culture. , 2005, Biomaterials.

[55]  Atsushi Namiki,et al.  Hypoxia Induces Vascular Endothelial Growth Factor in Cultured Human Endothelial Cells (*) , 1995, The Journal of Biological Chemistry.

[56]  F. O'Brien.  Biomaterials & scaffolds for tissue engineering , 2011 .

[57]  Fergal J O'Brien,et al.  Influence of freezing rate on pore structure in freeze-dried collagen-GAG scaffolds. , 2004, Biomaterials.