Heparin microparticle effects on presentation and bioactivity of bone morphogenetic protein-2

Abstract

Biomaterials capable of providing localized and sustained presentation of bioactive proteins are critical for effective therapeutic growth factor delivery. However, current biomaterial delivery vehicles commonly suffer from limitations that can result in low retention of growth factors at the site of interest or adversely affect growth factor bioactivity. Heparin, a highly sulfated glycosaminoglycan, is an attractive growth factor delivery vehicle due to its ability to reversibly bind positively charged proteins, provide sustained delivery, and maintain protein bioactivity. This study describes the fabrication and characterization of heparin methacrylamide (HMAm) microparticles for recombinant growth factor delivery. HMAm microparticles were shown to efficiently bind several heparin-binding growth factors (e.g. bone morphogenetic protein-2 (BMP-2), vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (FGF-2)), including a wide range of BMP-2 concentrations that exceeds the maximum binding capacity of other common growth factor delivery vehicles, such as gelatin. BMP-2 bioactivity was assessed on the basis of alkaline phosphatase (ALP) activity induced in skeletal myoblasts (C2C12). Microparticles loaded with BMP-2 stimulated comparable C2C12 ALP activity to soluble BMP-2 treatment, indicating that BMP-2-loaded microparticles retain bioactivity and potently elicit a functional cell response. In summary, our results suggest that heparin microparticles stably retain large amounts of bioactive BMP-2 for prolonged periods of time, and that presentation of BMP-2 via heparin microparticles can elicit cell responses comparable to soluble BMP-2 treatment. Consequently, heparin microparticles present an effective method of delivering and spatially retaining growth factors that could be used in a variety of systems to enable directed induction of cell fates and tissue regeneration.

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.