Sustained Release Of Bone Morphogenetic Protein-2 Research Articles

Improvement of osteoblast functions by sustained release of bone morphogenetic protein-2 (BMP-2) from heparin-coated chitosan scaffold

The aim of this study was to investigate the improvement in osteoblast functions by using bone morphogenetic protein-2 (BMP-2) immobilized heparin-coated chitosan scaffolds and comparing it with that using chitosan scaffold or BMP-2/chitosan scaffold in vitro. BMP-2 was released from the heparin-coated chitosan scaffold in a sustained manner compared to that released from the chitosan scaffold. The osteoblast functions of MG-63 cells grown on the chitosan scaffold, the BMP-2/chitosan scaffold, the BMP-2/Hep/chitosan scaffold were investigated by assessing cell proliferation, alkaline phosphatase (ALP) activity, calcium deposition, and gene expression. The results of the in vitro studies demonstrated that MG-63 cells grown on the BMP-2/Hep/chitosan scaffold showed a significant increment in ALP activity, and calcium deposition as compared to those grown on the chitosan scaffold by sustained release of BMP-2 due to the influence of heparin. Therefore, BMP-2 immobilized heparin-coated chitosan scaffolds are promising materials for improving osteoblast functions through sustained release of BMP-2.

The influence of collagen and hyaluronan matrices on the delivery and bioactivity of bone morphogenetic protein-2 and ectopic bone formation

Bone morphogenetic protein-2 (BMP-2) is known to enhance fracture healing when delivered via a bovine collagen sponge. However, collagen rapidly releases BMP-2 with a high burst phase that is followed by a low sustained phase. As a result, supra-physiological doses of BMP-2 are often required to successfully treat bone defects. High BMP-2 dosing can introduce serious side effects that include edema, bone overgrowth, cyst-like bone formation and significant inflammation. As the release behavior of BMP-2 carriers significantly affects the efficacy of fracture healing, we sought to compare the influence of two BMP-2 delivery matrices with contrasting release profiles on BMP-2 bioactivity and ectopic bone formation. We compared a thiol-modified hyaluronan (Glycosil™) hydrogel that exhibits a low burst followed by a sustained release of BMP-2 to a collagen sponge for the delivery of three different doses of BMP-2, the bioactivities of released BMP-2 and ectopic bone formation. Analysis of bone formation by micro-computed tomography revealed that low burst followed by sustained release of BMP-2 from a hyaluronan hydrogel induced up to 456% more bone compared to a BMP-2 dose-matched collagen sponge that has a high burst and sustained release. This study demonstrates that BMP-2 released with a low burst followed by a sustained release of BMP-2 is more desirable for bone formation. This highlights the therapeutic potential of hydrogels, particularly hyaluronan-based, for the delivery of BMP-2 for the treatment of bone defects and may help abrogate the adverse clinical effects associated with high dose growth factor use.

Trehalose Maintains Bioactivity and Promotes Sustained Release of BMP-2 from Lyophilized CDHA Scaffolds for Enhanced Osteogenesis In Vitro and In Vivo

Calcium phosphate (Ca-P) scaffolds have been widely employed as a supportive matrix and delivery system for bone tissue engineering. Previous studies using osteoinductive growth factors loaded Ca-P scaffolds via passive adsorption often experience issues associated with easy inactivation and uncontrolled release. In present study, a new delivery system was fabricated using bone morphogenetic protein-2 (BMP-2) loaded calcium-deficient hydroxyapatite (CDHA) scaffold by lyophilization with addition of trehalose. The in vitro osteogenesis effects of this formulation were compared with lyophilized BMP-2/CDHA construct without trehalose and absorbed BMP-2/CDHA constructs with or without trehalose. The release characteristics and alkaline phosphatase (ALP) activity analyses showed that addition of trehalose could sufficiently protect BMP-2 bioactivity during lyophilization and achieve sustained BMP-2 release from lyophilized CDHA construct in vitro and in vivo. However, absorbed BMP-2/CDHA constructs with or without trehalose showed similar BMP-2 bioactivity and presented a burst release. Quantitative real-time PCR (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA) demonstrated that lyophilized BMP-2/CDHA construct with trehalose (lyo-tre-BMP-2) promoted osteogenic differentiation of bone marrow stromal cells (bMSCs) significantly and this formulation could preserve over 70% protein bioactivity after 5 weeks storage at 25°C. Micro-computed tomography, histological and fluorescent labeling analyses further demonstrated that lyo-tre-BMP-2 formulation combined with bMSCs led to the most percentage of new bone volume (38.79% ±5.32%) and area (40.71% ±7.14%) as well as the most percentage of fluorochrome stained bone area (alizarin red S: 2.64% ±0.44%, calcein: 6.08% ±1.37%) and mineral apposition rate (4.13±0.62 µm/day) in critical-sized rat cranial defects healing. Biomechanical tests also indicated the maximum stiffness (118.17±15.02 Mpa) and load of fracture (144.67±16.13 N). These results lay a potential framework for future study by using trehalose to preserve growth factor bioactivity and optimize release profile of Ca-P based delivery system for enhanced bone regeneration.

Combined delivery of BMP-2 and bFGF from nanostructured colloidal gelatin gels and its effect on bone regeneration in vivo

During the process of bone regeneration, a multitude of morphogenetic signaling factors regulate cellular behavior and ultimately tissue response. These factors are presented to cells under strong spatial and temporal control, which stresses the relevance of controlled delivery of multiple growth factors for bone tissue regeneration. This demand for biomimetic delivery has prompted the development of a novel generation of biomaterials that is capable of delivering multiple growth factors in a controlled manner. Therefore, the current study has exploited the strong capacity of colloidal gels solely made of oppositely charged gelatin nanospheres to obtain controlled release of angiogenic and osteogenic growth factors. The release kinetics of dual delivery of osteogenic bone morphogenetic protein-2 (BMP-2) and angiogenic basic fibroblast growth factor (bFGF) were investigated in vitro by radiolabeling the respective growth factors and monitoring their release in vitro. Furthermore, the effect of single or dual delivery of BMP-2 and bFGF on bone regeneration was evaluated in vivo using a rat femoral condyle defect model. The in vitro results confirmed that the delivery kinetics of BMP-2 and/or bFGF are more dependent on the degree of crosslinking than on the type of gelatin. Sequential release characterized by rapid release of angiogenic bFGF and more sustained release of BMP-2 was obtained by loading bFGF onto cationic nanospheres of low crosslinking density and BMP-2 onto anionic nanospheres of high crosslinking density. The in vivo study demonstrated the biocompatibility and biodegradability of bare colloidal gelatin gels, and did not show any adverse effects on the process of bone healing after 4week of implantation since the volumes of new bone formation were comparable to empty control defects. An obvious stimulatory effect on bone regeneration was observed for the colloidal gels loaded with BMP-2, whereas bFGF-loaded colloidal gelatin gels did not influence the rate of bone regeneration. In contrast, the combined delivery of BMP-2 and bFGF resulted into an inhibitory effect on osteogenesis under the current experimental conditions. Summarizing, the current study proved that nanostructured colloidal gelatin gels are suitable carriers for programmed and sustained release of multiple therapeutic proteins for tissue regeneration.

Fabrication of porous PLGA microspheres with BMP-2 releasing polyphosphate-functionalized nano-hydroxyapatite for enhanced bone regeneration

This paper introduces a novel bone-regenerative scaffold that is based on the systematic combination of porous polymer microspheres, nano-hydroxyapatite, and bone morphogenetic protein-2 (BMP-2), where each component was rationally incorporated to express its intrinsic activity in bone tissue formation. Poly(lactide-co-glycolide) (PLGA) microspheres, with interconnected pore structures, were fabricated by a gas-forming method in a water-in-oil-in-water double emulsion and solvent evaporation process. Polyphosphate-functionalized nano-hydroxyapatite (PP-n-HAp) was employed as a main component and was immobilized on the pore surface of the PLGA microspheres to controllably incorporate and release BMP-2. The surface polyphosphate functionalities of PP-n-HAp enabled the stable chemical immobilization of nano-hydroxyapatite (n-HAp) on the amine-treated pore surface of the PLGA microspheres. Field-emission scanning electron microscopy (FE-SEM) and X-ray photoelectron spectroscopy (XPS) confirmed the nano-level exposure of n-HAp on the pore surface of the PLGA microspheres. BMP-2 with a positive charge was bound at a high efficiency onto the anionic phosphates of surface-immobilized PP-n-HAp and was controllably released for approximately 1 month. The release rate was manipulated by adjusting the amount of loaded BMP-2. The osteogenic differentiation and proliferation of human adipose-derived stem cells (hADSCs) within the n-HAp/BMP-2-incorporated microspheres were monitored in a dynamic 3D cell culture system. Histological, immunohistochemical, and quantitative real-time polymerase chain reaction (PCR) analyses showed that the PP-n-HAp-immobilized surface promoted cell adhesion/proliferation and osteoconduction. With its osteoinductive property, the sustained release of BMP-2 further enhanced the bone tissue regenerative activity of the porous microspheres. Open image in new window

Dual-Purpose Bone Grafts Improve Healing and Reduce Infection

To determine if a dual-purpose bone graft can regenerate bone and reduce infection in highly contaminated bone critical size defects in rats. Biodegradable polyurethane (PUR) scaffolds were loaded with recombinant human bone morphogenetic protein-2 (BMP-2) and vancomycin (Vanc). The release kinetics of the BMP-2 were tuned to take advantage of its mechanism of action (ie, an initial burst to recruit cells and sustained release to induce differentiation of the migrating cells). The Vanc release kinetics were designed to protect the graft from contamination until it is vascularized by having a burst for a week and remaining well over the minimum inhibitory concentration for Staphylococcus aureus for 2 months. The bone regeneration and infection reduction capability of these dual-purpose grafts (PUR+Vanc+BMP-2) were compared with collagen sponges loaded with BMP-2 (collagen+BMP-2) and PUR+BMP-2 in infected critical size rat femoral segmental defects. The dual-delivery approach resulted in substantially more new bone formation and a modest improvement in infection than PUR+BMP-2 and collagen+BMP-2 treatments. The PUR bone graft is injectable, provides a more sustained release of BMP-2 than the collagen sponge, and can release antibiotics for more than 8 weeks. Thus, the dual-delivery approach may improve patient outcomes of open fractures by protecting the osteoinductive graft from colonization until vascularization occurs. In addition, the more optimal release kinetics of BMP-2 may reduce nonunions and the amount of growth factor required.

Hydroxyapatite nanoparticles as a controlled-release carrier of BMP-2: absorption and release kinetics in vitro

Recently, nanoparticles have been extensively developed as controlled-release carriers; however, there has been little research on hydroxyapatite nanoparticles (HANPs) and their potential applications. In this study, HANPs were investigated as a controlled-release carrier of bone morphogenetic protein-2 (BMP-2), the absorption and release kinetics of which were analyzed in vitro. Different concentrations of BMP-2 solution were used to evaluate the adsorptive properties of HANPs. It was observed that the amount of BMP-2 adsorbed onto HANPs could be as high as 70 mug/mg and that adsorption rate was highly correlated with the concentration of BMP-2 solution used. After absorption, the suspension of HANPs absorbed BMP-2 (HANPs/BMP-2) was incubated at 37 degrees C for 15 days and the release kinetics of BMP-2 from HANPs/BMP-2 was determined daily. The release profile showed sustained release of BMP-2 over the period of the investigation. Collectively, these results suggest that HANPs has the potential to function as a carrier for drug delivery systems and as a scaffold material in bone tissue engineering.

Chondrogenic Differentiation on Perlecan Domain I, Collagen II, and Bone Morphogenetic Protein-2–Based Matrices

Extracellular matrix (ECM) molecules in cartilage cooperate with growth factors to regulate chondrogenic differentiation and cartilage development. Domain I of perlecan (Pln) bears heparan sulfate chains that bind and release heparin binding growth factors (HBGFs). We hypothesized that Pln domain I (PlnDI) might be complexed with collagen II (P-C) fibrils to improve binding of bone morphogenetic protein-2 (BMP-2) and better support chondrogenesis and cartilage-like tissue formation in vitro. Our results showed that P-C fibrils bound more BMP-2 than collagen II fibrils alone, and better sustained BMP-2 release. Polylactic acid (PLA)-based scaffolds coated with P-C fibrils immobilized more BMP-2 than either PLA scaffolds or PLA scaffolds coated with collagen II fibrils alone. Multipotential mouse embryonic mesenchymal cells, C3H10T1/2, were cultured on 2-dimensional P-C fibrils or 3-dimensional P-C/BMP-2-coated (P-C-B) PLA scaffolds. Chondrogenic differentiation was indexed by glycosaminoglycan (GAG) production, and expression of the pro-chondrogenic transcription factor, Sox9, as well as cartilaginous ECM proteins, collagen II, and aggrecan. Immunostaining for aggrecan, perlecan, tenascin, and collagen X revealed that both C3H10T1/2 cells and primary mouse embryonic fibroblasts cultured on P-C-B fibrils showed the highest expression of chondrogenic markers among all treatment groups. Safranin O-Fast Green staining indicated that cartilage-like tissue was formed in the P-C-B scaffolds, while no obvious cartilage-like tissue formed in other scaffolds. We conclude that P-C fibrils provide an improved biomimetic material for the binding and retention of BMP-2 and support chondrogenic differentiation.

Enhancement of bone growth by sustained delivery of recombinant human bone morphogenetic protein-2 in a polymeric matrix.

The purpose of this study was to develop a polymeric sustained delivery system for recombinant human bone morphogenetic protein-2 (BMP-2) and to evaluate local bone growth induced by the sustained release of BMP-2 in an animal model. BMP-2 was incorporated in biodegradable poly(D,L-lactide-co-glycolide) (PLGA) microspheres to obtain different release rates. Two sustained and an immediate release implants were produced by suspending the BMP-2 loaded PLGA microspheres in aqueous sodium carboxymethylcellulose (CMC), lyophilizing, and cutting the dried materials to the size of the animal bone defects. The local in vivo release at the implantation site in rat calvarial defects was determined by gamma scintigraphy using radiolabeled BMP-2. The local bone induction in the critical size of rabbit calvarial defects was evaluated six weeks post implantation. The immediate release implant showed about 65% initial drug release within 24 h and the remaining BMP-2 quickly exhausted from the implantation site within 7 days. The sustained release implants, showing 45-55% initial release followed by a prolonged release for 21 days, released a greater amount of BMP-2 at the implantation site and maintained higher serum BMP-2 for the longer period of time compared to the immediate release implant. Significant bone growth was observed in all BMP-2 treated defects while the defects without treatment or with BMP-2-free implant showed minimal bone healing. 75-79% of rabbit calvarial defect area was healed with newly induced bone matrix by the sustained release implants in 6 weeks as compared to 45% recovery from the immediate release implant. The sustained delivery of BMP-2 based on the biodegradable PLGA microsphere system resulted in faster and more complete bone healing in the animal model.