Heparin microparticle effects on presentation and bioactivity of bone morphogenetic protein-2
Introduction
Recombinant growth factor delivery has been effective for a number of tissue engineering applications. In particular, bone morphogenetic proteins (BMPs), which are potent osteoinductive growth factors, have been used extensively to treat bone defects in both research and clinical settings [1], [2], [3]. However, current treatment strategies require supraphysiological levels of recombinant proteins, such as BMPs, in order to stimulate endogenous mechanisms of repair. This inefficient use of growth factor is largely due to the inability of biomaterial delivery vehicles to provide adequate sustained and localized presentation of growth factors necessary to stimulate repair over long periods of time. Current biomaterial delivery vehicles have major limitations, such as the rapid release of molecular cargo upon deployment, causing low retention of soluble factors at the site of interest [4], [5], [6], or alternatively, reliance upon growth factor tethering strategies that can significantly reduce growth factor bioactivity [7], [8]. Thus, materials with the ability to strongly, but reversibly, interact with their molecular payload are necessary, and may significantly decrease the amount of growth factor required for therapies, while improving physiological response.
Recently, glycosaminoglycan-containing biomaterials have become an attractive delivery method for recombinant growth factors, due to their ability to strongly bind a variety of growth factors in a reversible manner. Glycosaminoglycans (GAGs) are linear polysaccharide chains that bind positively charged growth factors primarily through their negatively charged sulfate groups and exist both as free chains and covalently-linked components of glycosylated proteins known as proteoglycans [9], [10]. GAGs such as heparin, heparan sulfate, and chondroitin sulfate are ubiquitous components of natural extracellular matrices (ECM) that are involved in sequestering and immobilizing growth factors within the cellular microenvironment [11], [12], [13]. Thus, GAG-based materials present the opportunity to harness the natural growth factor binding capacity of the ECM and deliver growth factors in a biomimetic manner with spatiotemporal control. Heparin, in particular, is highly negatively charged and has a strong affinity for a class of positively charged growth factors known as “heparin-binding growth factors,” for which specific growth factor binding sequences on heparin chains have been identified [14], [15], [16]. The non-covalent, reversible interactions between heparin and heparin-binding growth factors ensure that binding occurs with minimal impact on growth factor structure. Heparin-binding growth factors such as transforming growth factor β (TGF-β), vascular endothelial growth factor (VEGF), fibroblast growth factors (FGFs), insulin-like growth factors (IGFs), and bone morphogenetic proteins (BMPs), are especially influential in many developmental and regeneration processes, and it is thought that heparin itself may play an influential role in the preservation and presentation of molecules through electrostatic interactions [17], [18].
The use of heparin and heparin-containing biomaterials for BMP-2 delivery, as well as the delivery of several other growth factors, including FGF-2, VEGF, and TGF-β2, has been widely explored in both in vitro and in vivo test beds [19], [20], [21], [22], [23], [24]. Although several studies have investigated heparin-BMP-2 interactions, the effects of heparin-BMP-2 binding on protein bioactivity have been inconsistent and depend largely on the amount of heparin and method of heparin immobilization. Previous studies have demonstrated that co-delivery of soluble heparin with BMP-2 can enhance BMP-2-mediated osteogenesis or, contrastingly, interfere with BMP-2 and BMP receptor binding to inhibit osteogenesis, depending on the cell type and culture conditions [25], [26], [27], [28], [29], [30], [31]. Nevertheless, the addition of heparin to biomaterials, including microparticles and bulk gels, has previously resulted in improvement in growth factor retention and BMP-2-induced osteogenesis [32], [33], [34], [35], [36]. Heparin-mediated delivery of BMP-2 has also resulted in a wide range of effects in vivo, with studies demonstrating variable amounts of mineralization in both ectopic and orthotopic sites [25], [37], [38], [39], reflecting an inconsistent ability to form functional bone. Furthermore, the majority of these materials consist of relatively small amounts of heparin immobilized within a larger bulk material [23], [24], [40], [41], [42], [43], which may attenuate heparin's ability to effectively bind and present growth factors. As a result, previous reports on heparin-containing biomaterials may significantly underestimate the amount of BMP-2 that can be delivered via heparin-binding. Thus, improving the growth factor binding ability of heparin-containing biomaterials may enable consistent delivery of highly localized BMP-2 concentrations necessary to stimulate more effective bone formation.
Herein, we present a method of fabricating pure heparin microparticles from a modified heparin methacrylamide species that can be thermally cross-linked. Physical and chemical characterization of heparin microparticles was performed, and growth factor binding and release were quantified with different BMP-2 loading concentrations. Additionally, growth factor bioactivity was evaluated by introducing BMP-2 laden heparin microparticles to cultures of C2C12 cells and measuring BMP-2-induced alkaline phosphatase activity, as well as changes in DNA content. Overall, this study marks a crucial first step in developing heparin microparticles as a versatile delivery vehicle and therapeutic platform for growth factor-stimulated tissue engineering, by investigating their capacity to efficiently capture and present BMP-2 to induce a potent functional cell response.
Section snippets
Heparin methacrylamide modification
Heparin ammonium salt from porcine intestinal mucosa (17–19 kDa; Sigma–Aldrich, St. Louis, MO) was conjugated with N-(3-Aminopropyl)methacrylamide (APMAm; Polysciences, Warrington, PA) using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC; Thermoscientific, Rockford, IL) and N-hydroxysulfosuccinimide (Sulfo-NHS; Thermoscientific, Rockford, IL) as described in previous protocols [44], [45] (Fig. 1A). EDC/Sulfo-NHS chemistry causes activation of the carboxyl groups on heparin and subsequent
Heparin methacrylamide microparticle characterization
1H NMR analysis indicated that approximately 50% of the carboxyl groups of heparin were conjugated with methacrylamide groups (Fig. 1B). Heparin microparticle fabrication produced microparticles with an average diameter of 5.6 ± 4.0 μm (Fig. S1), and approximately 1.9 × 107 microparticles per mg of heparin methacrylamide polymer. SEM images of the microparticles depicted a smooth, spherical morphology (Fig. 1C), while brightfield images revealed that the microparticles stained positively with
Discussion
In this study, the ability of heparin methacrylamide microparticles to bind, retain, and present bioactive growth factors in vitro was investigated. Heparin microparticles bound considerable amounts of several positively charged heparin-binding growth factors (BMP-2, VEGF, FGF-2), including high quantities of BMP-2 that exceeded the maximum reported growth factor binding capacity of other heparin-containing biomaterials by >1000-fold [23], [24], [36], [38], [53], [54] and surpassed the BMP-2
Conclusions
The results of this study demonstrate that heparin microparticles can be used to sequester and retain large amounts of bioactive BMP-2, and that sustained presentation of BMP-2 via heparin microparticles can elicit a comparable cellular response to soluble BMP-2 treatment. Heparin microparticles offer a versatile platform for growth factor delivery, as loaded microparticles can be directly injected and retained in a tissue defect site, providing a much higher surface area for efficient growth
Acknowledgments
This work was supported by a Transformative Research Award from the National Institutes of Health (TR01 AR062006) and a grant from the National Science Foundation (NSF DMR 1207045). MHH is supported by funding from the Natural Science and Engineering Research Council (NSERC) of Canada. The authors would like to thank Ms. Shalini Saxena for her assistance with scanning electron microscopy, and Ms. Marissa Cooke for her assistance with manuscript preparation.
References (69)
- et al.
Bone morphogenic protein and its application in trauma cases: a current concept update
Injury
(2007) - et al.
Poly(ethylene glycol) hydrogels formed by conjugate addition with controllable swelling, degradation, and release of pharmaceutically active proteins
J Control Release
(2005) - et al.
Effect of the covalent modification of horseradish peroxidase with poly (ethylene glycol) on the activity and stability upon encapsulation in polyester microspheres
J Pharm Sci
(2005) - et al.
Molecular engineering of glycosaminoglycan chemistry for biomolecule delivery
Acta Biomater
(2014) - et al.
Specific molecular interactions of oversulfated chondroitin sulfate E with various heparin-binding growth factors: implications as a physiological binding partner in the brain and other tissues
J Biol Chem
(2002) - et al.
Prediction of heparin binding sites in bone morphogenetic proteins (BMPs)
Biochim Biophys Acta
(2012) - et al.
Fibroblast growth factor receptor signalling is dictated by specific heparan sulphate saccharides
Curr Biol
(1999) - et al.
Heparin-regulated release of growth factors in vitro and angiogenic response in vivo to implanted hyaluronan hydrogels containing VEGF and bFGF
Biomaterials
(2006) - et al.
Collagen and heparin matrices for growth factor delivery
J Control Release
(1997) - et al.
Preparation, structure and BMP-2 controlled release of heparin-conjugated hyaluronan microgels
Carbohyd Polym
(2011)
Heparin-decorated, hyaluronic acid-based hydrogel particles for the controlled release of bone morphogenetic protein 2
Acta Biomater
Heparin potentiates the in vivo ectopic bone formation induced by bone morphogenetic protein-2
J Biol Chem
Dual effects of heparin on BMP-2-induced osteogenic activity in MC3T3-E1 cells
Pharmacol Rep
The effect of heparin on osteoblast differentiation and activity in primary cultures of bovine aortic smooth muscle cells
Atherosclerosis
Heparan sulfate proteoglycans (HSPGs) modulate BMP2 osteogenic bioactivity in C2C12 cells
J Biol Chem
Sulfated polysaccharides enhance the biological activities of bone morphogenetic proteins
J Biol Chem
Hyaluronic acid-based hydrogels functionalized with heparin that support controlled release of bioactive BMP-2
Biomaterials
Enhancement of ectopic bone formation by bone morphogenetic protein-2 released from a heparin-conjugated poly (l-lactic-co-glycolic acid) scaffold
Biomaterials
Bone marrow-derived heparan sulfate potentiates the osteogenic activity of bone morphogenetic protein-2 (BMP-2)
Bone
Enhanced osteogenic activity of bone morphogenetic protein-2 by 2-O-desulfated heparin
Acta Biomater
Photopolymerized hyaluronic acid-based hydrogels and interpenetrating networks
Biomaterials
Development of nano-and microscale chondroitin sulfate particles for controlled growth factor delivery
Acta Biomater
Human tumor necrosis factor. Production, purification, and characterization
J Biol Chem
Affinity-based growth factor delivery using biodegradable, photocrosslinked heparin-alginate hydrogels
J Control Release
Perlecan domain I-conjugated, hyaluronic acid-based hydrogel particles for enhanced chondrogenic differentiation via BMP-2 release
Biomaterials
Accelerated tissue regeneration through incorporation of basic fibroblast growth factor-impregnated gelatin microspheres into artificial dermis
Biomaterials
Enhancement of cell growth on growth factor-immobilized polymer film
Biomaterials
Tethered-TGF-β increases extracellular matrix production of vascular smooth muscle cells
Biomaterials
Dorsomorphin and LDN-193189 inhibit BMP-mediated Smad, p38 and Akt signalling in C2C12 cells
Int J Biochem Cell Biol
Activation of mitogen-activated protein kinase cascades is involved in regulation of bone morphogenetic protein-2-induced osteoblast differentiation in pluripotent C2C12 cells
Bone
Prevalence, complications, and hospital charges associated with use of bone-morphogenetic proteins in spinal fusion procedures
JAMA
Bone morphogenetic proteins in clinical applications
ANZ J Surg
Temporally regulated delivery of VEGF in vitro and in vivo
J Biomed Mater Res A
Characterization of rhBMP-2 pharmacokinetics implanted with biomaterial carriers in the rat ectopic model
J Biomed Mater Res
Cited by (92)
Materials-based nanotherapeutics for injured and diseased bone
2023, Progress in Materials ScienceMicrospheres in bone regeneration: Fabrication, properties and applications
2022, Materials Today AdvancesAffibody-mediated controlled release of fibroblast growth factor 2
2022, Journal of Controlled ReleaseMicrogels based on Infernan, a glycosaminoglycan-mimetic bacterial exopolysaccharide, as BMP-2 delivery systems
2022, Carbohydrate PolymersCitation Excerpt :Microdroplets are solidified to form gelled microparticles or microgels by chemical, photochemical or physical methods directly into the microfluidic device or in the collecting bath. Contrary to other emulsification techniques, where the encapsulation of active compounds, such as growth factors, is ensured by their adsorption inside microdroplets through their diffusion from solution (Hettiaratchi et al., 2014; Hettiaratchi et al., 2020; Tellier et al., 2015), microfluidic offers a unique possibility to formulate microdroplets and encapsulate the totality of the active compound in only one step process. In this context, the objective of the present study was to further exploit physico-chemical (gelling) and biological (GAG-mimetic) properties of Infernan and its derivatives to develop EPS-based microgels appropriate for bone healing purposes.