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Competitive Protein Binding Influences Heparin-Based Modulation of Spatial Growth Factor Delivery for Bone Regeneration.

ABSTRACT: Tissue engineering strategies involving the in vivo delivery of recombinant growth factors are often limited by the inability of biomaterials to spatially control diffusion of the delivered protein within the site of interest. The poor spatiotemporal control provided by porous collagen sponges, which are used for the clinical delivery of bone morphogenetic protein-2 (BMP-2) for bone regeneration, has necessitated the use of supraphysiological protein doses, leading to inflammation and heterotopic ossification. This study describes a novel tissue engineering strategy to spatially control rapid BMP-2 diffusion from collagen sponges in vivo by creating a high-affinity BMP-2 sink around the collagen sponge. We designed an electrospun poly-?-caprolactone nanofiber mesh containing physically entrapped heparin microparticles, which have been previously demonstrated to bind and retain large amounts of BMP-2. Nanofiber meshes containing 0.05 and 0.10?mg of microparticles/cm2 demonstrated increased BMP-2 binding and decreased BMP-2 release in vitro compared with meshes without microparticles. However, when microparticle-containing meshes were used in vivo to limit the diffusion of BMP-2 delivered by using collagen sponges in a rat femoral defect, no differences in heterotopic ossification or biomechanical properties were observed. Further investigation revealed that, although BMP-2 binding to heparin microparticles was rapid, the presence of serum components attenuated microparticle-BMP-2 binding and increased BMP-2 release in vitro. These observations provide a plausible explanation for the results observed in vivo and suggest that competitive protein binding in vivo may hinder the ability of affinity-based biomaterials to modulate growth factor delivery.

SUBMITTER: Hettiaratchi MH

PROVIDER: S-EPMC5549832 | biostudies-other | 2017 Jul

REPOSITORIES: biostudies-other

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Competitive Protein Binding Influences Heparin-Based Modulation of Spatial Growth Factor Delivery for Bone Regeneration.

Hettiaratchi Marian H MH Chou Catherine C Servies Nicholas N Smeekens Johanna M JM Cheng Albert A Esancy Camden C Wu Ronghu R McDevitt Todd C TC Guldberg Robert E RE Krishnan Laxminarayanan L

Tissue engineering. Part A 20170324 13-14

Tissue engineering strategies involving the in vivo delivery of recombinant growth factors are often limited by the inability of biomaterials to spatially control diffusion of the delivered protein within the site of interest. The poor spatiotemporal control provided by porous collagen sponges, which are used for the clinical delivery of bone morphogenetic protein-2 (BMP-2) for bone regeneration, has necessitated the use of supraphysiological protein doses, leading to inflammation and heterotopi ...[more]

PMID: 28338419

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Project description:ObjectiveBone regeneration involves a coordinated cascade of events that are regulated by several cytokines and growth factors, among which bone morphogenic protein-2 (BMP-2), vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2) play important roles. In this study, we investigated the effects of dual release of the three growth factors on bone regeneration in femur defects.MethodsA composite consisting of Gelatin microparticles loaded with VEGF/FGF-2 and poly(lactic-co-glycolic acid)-poly(ethylene glycol)-carboxyl (PLGA-PEG-COOH) microparticles loaded with BMP-2 encapsulated in a nano hydroxyapatite-poly actic-co-glycolic acid (nHA-PLGA) scaffold was prepared for the dual release of the growth factors.ResultsOn the 14th day, decreased release rate of BMP-2 compared with FGF-2 and VEGF was observed. However, after 14 days, compared to FGF-2 and VEGF, BMP-2 showed an increased release rate. Controlled dual release of BMP-2 and VEGF, FGF-2 resulted in a significant osteogenic differentiation of bone mesenchymal stem cells (BMSCs). Moreover, effects of the composite scaffold on functional connection of osteoblast-vascular cells during bone development were evaluated. The synergistic effects of dual delivery of growth factors were shown to promote the expression of VEGF in BMSCs. Increased secretion of VEGF from BMSCs promoted the proliferation and angiogenic differentiation of human umbilical vein endothelial cells (HUVECs) in the co-culture system. At 12 weeks after implantation, blood vessel and bone formation were analyzed by micro-CT and histology. The composite scaffold significantly promoted the formation of blood vessels and new bone in femur defects.ConclusionsThese findings demonstrate that dual delivery of angiogenic factors and osteogenic factors from Gelatin and PLGA-PEG-COOH microparticles-based composite scaffolds exerted an osteogenic-angiogenic coupling effect on bone regeneration. This approach will inform on the development of appropriate designs of high-performance bioscaffolds for bone tissue engineering.

Project description:Products of adipocytes control bone cells and may therefore lead to new anti-osteoporosis therapies. To address this issue we mated mice expressing the primate diphtheria toxin receptor (DTR), which is equivalent to Heparin Binding Epidermal Like Growth Factor (HB-EGF), under control of a Stop-flox cassette, to those expressing adiponectin-Cre (ADQ-Cre). Injected diphtheria toxin (DT) completely eliminates white and brown adipose tissue in these DTRADQ mice and ablates marrow adipocytes. Medullary bone of DTRADQ mice begins to accumulate, due to stimulated osteoblast recruitment and differentiation, within 4 days of injected DT. By 10-14 days of DT administration to DTRADQ mice, trabecular bone has increased 1000%. Two months after fat ablation, cortical thickness has markedly increased as has bone strength. To gain insight into the signaling events by which DT/DTRADQ adipocyte ablation promotes osteogenesis we performed RNAseq analysis of marrow prior to and after 1-3 days of DT injection. This exercise confirms ablation of marrow adipocytes, as adiponectin expression is markedly reduced while enhanced expression of Col1a1 and Col1a2 genes is in keeping with stimulated osteogenesis. RNAseq analysis also indicates that DT/DTRADQ-induced osteogenesis is mediated by the BMPR pathway. For example, there is suppressed genetic expression of the specific BMPR inhibitors, chordin-like1 (CHRDL1) and gremlin1 (GREM1). This adipocyte ablation-mediated increase in bone mass is due to cooperative activation of BMP and EGF receptor signaling as adipocyte-produced inhibitors are suppressed and downstream targets activated or expressed. Medicinal inhibition of either signaling pathway prevents induction of the osteosclerotic phenotype. DTR-induced post-natal osteogenesis does not represent the products of peripheral fat depots and is more likely due to those of marrow adipocytes. Thus, anabolic drugs activating both BMP and EGF receptor signaling may be a means of profoundly increasing bone mass and treating osteoporosis.

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Competitive Protein Binding Influences Heparin-Based Modulation of Spatial Growth Factor Delivery for Bone Regeneration.

Publications

Competitive Protein Binding Influences Heparin-Based Modulation of Spatial Growth Factor Delivery for Bone Regeneration.

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