Bone morphogenetic protein-2 (BMP-2) is an osteoinductive growth factor used clinically to induce bone regeneration and fusion. Some complications associated with BMP-2 treatment have been attributed to rapid release of BMP-2 from conventional collagen scaffolds, motivating the development of tunable sustained-release strategies. We incorporated BMP-2-binding heparin microparticles (HMPs) into a hydrogel scaffold to improve spatiotemporal control of BMP-2 delivery to large bone defects. HMPs pre-loaded with BMP-2 were mixed into alginate hydrogels and compared to hydrogels containing BMP-2 alone. BMP-2 release from scaffolds in vitro, BMP-2 retention within injury sites in vivo, and bone regeneration in a critically sized femoral defect were evaluated. Compared to hydrogel delivery alone, BMP-2-loaded HMPs reduced BMP-2 release in vitro and increased early BMP-2 retention in the bone defect. BMP-2-loaded HMPs induced bone formation at both ectopic and orthotopic sites; however, the volume of induced bone was lower for defects treated with BMP-2-loaded HMPs compared to hydrogel delivery. To better understand the effect of HMPs on BMP-2 release kinetics, a computational model was developed to predict BMP-2 release from constructs in vivo. The model suggested that HMPs limited BMP-2 release into surrounding tissues, and that changing the HMP density could modulate BMP-2 release. Taken together, these experimental and computational results suggest the importance of achieving a balance of BMP-2 retention within the bone defect and BMP-2 release into surrounding soft tissues. HMP delivery of BMP-2 may provide a method of tuning BMP-2 release in vivo that can be further investigated to improve current methods of bone regeneration. Statement of Significance The development of effective biomaterials for sustained protein delivery is a crucial component of tissue engineering strategies. However, in most applications, including bone repair, the optimal balance between protein presentation in the injury site and protein release into the surrounding tissues is unknown. Herein, we introduced heparin microparticles (HMPs) into a tissue engineered construct to increase in vivo retention of bone morphogenetic protein-2 (BMP-2) and enhance healing in femoral defects. Although HMPs induced bone regeneration, no increase in bone volume was observed, leading to further experimental and computational analysis of the effect of HMP-BMP-2 interactions on protein retention and release. Ultimately, this work provides insight into designing tunable protein-material interactions and their implications for controlling BMP-2 delivery.
Background: In resting platelets, endothelial cell specific adhesion molecule (ESAM) is located in alpha granules, increasing its cell surface expression following platelet activation. However, the function of ESAM on platelets is unknown. Objective: To determine whether ESAM has a role in thrombus formation. Methods and results: We found that following platelet activation ESAM localizes to the junctions between adjacent platelets, suggesting a role for this protein in contact-dependent events that regulate thrombus formation. To test this hypothesis we examined the effect of ESAM deletion on platelet function. In vivo, ESAM-/- mice achieved more stable hemostasis than wild-type mice following tail transection, and developed larger thrombi following laser injury of cremaster muscle arterioles. In vitro, ESAM-/- platelets aggregated at lower concentrations of G protein-dependent agonists than wild-type platelets, and were more resistant to disaggregation. In contrast, agonist-induced calcium mobilization, αIIbβ3 activation, alpha-granule secretion and platelet spreading, were normal in ESAM-deficient platelets. To understand the molecular mechanism by which ESAM regulates platelet activity, we utilized a PDZ domain array to identify the scaffold protein NHERF-1 as an ESAM binding protein, and further demonstrated that it associates with ESAM in both resting and activated platelets. Conclusions: These findings support a model in which ESAM localizes to platelet contacts following platelet activation in order to limit thrombus growth and stability so that the optimal hemostatic response occurs following vascular injury.
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.