Bioactivity Of Bone Morphogenetic Protein-2 Research Articles

Tuning the bioactivity of bone morphogenetic protein-2 with surface immobilization strategies

Bone morphogenetic protein-2 (BMP-2) involved therapy is of great potential for bone regeneration. However, its clinical application is restricted due to the undesirable bioactivity and relevant complications in vivo. Immobilization of recombinant BMP-2 (rhBMP-2) is an efficient strategy to mimic natural microenvironment and retain its bioactivity. Herein, we present evidences indicating that osteoinductive capacity of rhBMP-2 can be regulated via variant immobilizing approaches. Three representative superficial immobilizing models were employed to fabricate rhBMP-2-immobilized surfaces including physical adsorption (Au/rhBMP-2), covalent grafting (rhBMP-2-SAM-Au) and heparin binding (Hep-SAM-Au/rhBMP-2) (SAM: self-assembled monolayer). Loading capacity, releasing behavior, osteogenic differentiation and signaling pathways involved, as well as the cellular recognition of rhBMP-2 under various immobilization modes were systematically investigated. As a result, disparate immobilizing approaches not only have effects on loading capacity, but also lead to disparity of osteoinduction at the same dosage. Notably, heparin could reinforce the recognition between rhBMP-2 and its receptors (BMPRs) whereas weaken its binding to its antagonist Noggin. Owing to this “selective” binding feature, the favorable osteoinduction and maximum ectopic bone formation can be achieved with the heparin-binding approach. In particular, manipulation of orientation-mediated BMP-2-cell recognition efficiency may be a potential target to design more therapeutic efficient rhBMP-2 delivery system. Statement of SignificanceBone morphogenetic protein-2 (BMP-2) is crucial in bone regeneration. However, its clinical application is challenged due to its shorten half-life and supra-physiological dose associated complications. In this study, three representative superficial immobilizing patterns were fabricated through physical adsorption, covalent grafting and electrostatic interaction with heparin respectively. We provided evidences indicating an dose-dependent osteoinductive capacity of immobilized BMP-2. Further, a possible mechanism of rhBMP-2-cell recognition at the interface was presented, highlighting the superior effect of heparin on rhBMP-2 bioactivity. Finally, We proposed a dual mechanism of tuning the bioactivity of immobilized rhBMP-2 through surface immobilization approaches: regulation of the saturated loading capacity and orientation-mediated rhBMP-2-cell recognition. These results provide novel insights into designing criterion of efficient delivery vehicle for rhBMP-2.

Bone morphogenetic protein-2 release profile modulates bone formation in phosphorylated hydrogel.

The optimal release profile of locally delivered bone morphogenetic protein-2 (BMP-2) for safe and effective clinical application is unknown. In this work, the effect of differential BMP-2 release on bone formation was investigated using a novel biomaterial oligo[(polyethylene glycol) fumarate] bis[2-(methacryloyloxy) ethyl] phosphate hydrogel (OPF-BP) containing poly(lactic-co-glycolic acid) microspheres. Three composite implants with the same biomaterial chemistry and structure but different BMP-loading methods were created: BMP-2 encapsulated in microspheres (OPF-BP-Msp), BMP-2 encapsulated in microspheres and adsorbed on the phosphorylated hydrogel (OPF-BP-Cmb), and BMP-2 adsorbed on the phosphorylated hydrogel (OPF-BP-Ads). These composites were compared with the clinically used BMP-2 carrier, Infuse® absorbable collagen sponge (ACS). Differential release profiles of bioactive BMP-2 were achieved by these composites. In a rat subcutaneous implantation model, OPF-BP-Ads and ACS generated a large BMP-2 burst release (>75%), whereas a more sustained release was seen for OPF-BP-Msp and OPF-BP-Cmb (~25% and 50% burst, respectively). OPF-BP-Ads generated significantly more bone than did all other composites, and the bone formation was 12-fold higher than that of the clinically used ACS. Overall, this study clearly shows that BMP-2 burst release generates more subcutaneous bone than do sustained release in OPF-BP-microsphere composites. Furthermore, composites should not only function as a delivery vehicle but also provide a proper framework to achieve appropriate bone formation.

Design of hydrogels to stabilize and enhance bone morphogenetic protein activity by heparin mimetics

Although bone morphogenetic protein-2 (BMP-2) is known to be the most potent stimulator available for bone formation, a major barrier to widespread clinical use is its inherent instability and absence of an adequate delivery system. Heparin is being widely used in controlled release systems due to its strong binding ability and protective effect for many growth factor proteins. In this work, we developed a hydrogel surface that can mimic heparin to stabilize BMP-2 and to enhance osteogenesis by introducing heparin-mimicking sulfonated molecules such as poly-vinylsulfonic acid (PVSA) or poly-4-styrenesulfonic acid (PSS), into photo-crosslinkable hydrogel. Bioactivity of BMP-2 was well preserved in the presence of polysulfonates during exposure to various therapeutically relevant stressors. The heparin-mimicking sulfonated hydrogels were effective to bind BMP-2 compared to unmodified MeGC hydrogel and significantly enhanced osteogenic differentiation of encapsulated bone marrow stromal cells (BMSCs) without the addition of exogenous BMP-2. The sulfonated hydrogels were effective in delivering exogenous BMP-2 with reduced initial burst and increased BMSCs osteogenesis induced by BMP-2. These findings suggest a novel hydrogel platform for sequestering and stabilizing BMP-2 to enhance osteoinductive activity in bone tissue engineering. Statement of SignificanceAlthough bone morphogenetic protein-2 (BMP-2) is believed to be the most potent cytokine for bone regeneration, its clinical applications require supraphysiological BMP dosage due to its intrinsic instability and fast enzymatic degradation, leading to worrisome side effects. This study demonstrates a novel hydrogel platform that mimics a natural protector of BMPs, heparin, to sequester and stabilize BMP-2 for increased osteoinductive signaling. This study will achieve the stabilization of BMPs with prolonged bioactivity by a synthetic heparin mimic that has not been examined previously. Moreover, the heparin mimetic hydrogel surface can augment endogenous BMP activity by sequestering and localizing the cell-produced BMPs. The additional knowledge gained from this study may suggest basis for future development of material-based therapeutics for tissue engineering.

Harnessing macrophage-mediated degradation of gelatin microspheres for spatiotemporal control of BMP2 release

Biomaterials-based approaches to harnessing the immune and inflammatory responses to potentiate wound healing hold important promise. Bone fracture healing is characterized by an acute inflammatory phase, followed by a transition to a regenerative and repair phase. In this study, we developed genipin-crosslinked gelatin microspheres designed to be preferentially degraded by inflammatory (M1) macrophages. Highly crosslinked (>90%) microspheres allowed efficient incorporation of bioactive bone morphogenetic protein 2 (BMP2), a potent stimulator of osteogenesis in progenitor cells, via electrostatic interactions. Release of BMP2 was directly correlated with degradation of the gelatin matrix. Exposure of microspheres to polarized murine macrophages showed that degradation was significantly higher in the presence of M1 macrophages, relative to alternatively activated (M2) macrophages and unpolarized controls. Microsphere degradation in the presence of non-inflammatory cells resulted in very low degradation rates. The expression of matrix metalloproteinases (MMPs) and tissue inhibitors of MMP (TIMPs) by macrophages were consistent with the observed phenotype-dependent degradation rates. Indirect co-culture of BMP2-loaded microspheres and macrophages with isolated adipose-derived mesenchymal stem cells (MSC) showed that M1 macrophages produced the strongest osteogenic response, comparable to direct supplementation of the culture medium with BMP2. Controlled release systems that are synchronized with the inflammatory response have the potential to provide better spatiotemporal control of growth factor delivery and therefore may improve the outcomes of recalcitrant wounds.

Dose reduction of bone morphogenetic protein-2 for bone regeneration using a delivery system based on lyophilization with trehalose.

IntroductionTo induce sufficient new bone formation, high doses of bone morphogenetic protein-2 (BMP-2) are applied in regenerative medicine that often induce serious side effects. Therefore, improved treatment strategies are required. Here, we investigate whether the delivery of BMP-2 lyophilized in the presence of trehalose reduced the dose of BMP-2 required for bone regeneration.Materials and methodsA new growth factor delivery system was fabricated using BMP-2-loaded TiO2 nanotubes by lyophilization with trehalose (TiO2-Lyo-Tre-BMP-2). We measured BMP-2 release characteristics, bioactivity, and stability, and determined the effects on the osteogenic differentiation of bone marrow stromal cells in vitro. Additionally, we evaluated the ability of this formulation to regenerate new bone around implants in rat femur defects by micro-computed tomography (micro-CT), sequential fluorescent labelling, and histological analysis.ResultsCompared with absorbed BMP-2-loaded TiO2 nanotubes (TiO2-BMP-2), TiO2-Lyo-Tre-BMP-2 exhibited sustained release, consistent bioactivity, and higher stability of BMP-2, and resulted in greater osteogenic differentiation of BMSCs. Eight weeks post-operation, TiO2-Lyo-Tre-BMP-2 nanotubes, with various dosages of BMP-2, regenerated larger amounts of new bone than TiO2-BMP-2 nanotubes.ConclusionOur findings indicate that delivery of BMP-2 lyophilized with trehalose may be a promising method to reduce the dose of BMP-2 and avoid the associated side effects.

Yolk shell nanocomposite particles as bioactive bone fillers and growth factor carriers.

The efficient delivery of bioactive molecules via rationally designed nanoparticles is an important focus in regenerative medicine. The yolk shell nanocomposite particles described herein are composed of silk fibroin movable cores formed within voided calcium carbonate shells to load and control the release of labile cytokines. These particles are excellent carrier vehicles of potent molecules as they sustained the release of bioactive Bone Morphogenetic Protein 2 (BMP-2) for more than 28 days in vitro. Implantation into bone defects in rabbits corroborates the in vitro results and also reveals that upon contact with phosphate containing body fluids, implanted yolk shell particles agglomerate and transform into a filler that adapts to defect contour to further act as an absorbable hemostatic agent. Taken together, the fabrication of these yolk shell particle-based "bone fillers" could expand the horizon for the development of newer generations of advanced bioactive materials in tissue regeneration applications.

Silk-Hydroxyapatite Nanoscale Scaffolds with Programmable Growth Factor Delivery for Bone Repair.

Osteoinductive biomaterials are attractive for repairing a variety of bone defects, and biomimetic strategies are useful toward developing bone scaffolds with such capacity. Here, a multiple biomimetic design was developed to improve the osteogenesis capacity of composite scaffolds consisting of hydroxyapatite nanoparticles (HA) and silk fibroin (SF). SF nanofibers and water-dispersible HA nanoparticles were blended to prepare the nanoscaled composite scaffolds with a uniform distribution of HA with a high HA content (40%), imitating the extracellular matrix (ECM) of bone. Bone morphogenetic protein-2 (BMP-2) was loaded in the SF scaffolds and HA to tune BMP-2 release. In vitro studies showed the preservation of BMP-2 bioactivity in the composite scaffolds, and programmable sustained release was achieved through adjusting the ratio of BMP-2 loaded on SF and HA. In vitro and in vivo osteogenesis studies demonstrated that the composite scaffolds showed improved osteogenesis capacity under suitable BMP-2 release conditions, significantly better than that of BMP-2 loaded SF-HA composite scaffolds reported previously. Therefore, these biomimetic SF-HA nanoscaled scaffolds with tunable BMP-2 delivery provide preferable microenvironments for bone regeneration.

Size-Mediated Adsorption Dynamics, Conformation and Bioactivity of Bone Morphogenetic Protein-2 Onto Silica Nanoparticles.

Delivery of bone morphogenetic protein-2 (BMP-2) carries great promise for the field of bone regenerative medicine. But, how the size and curvature of matrix affect the way BMP-2 binding to the support and in turn influence its bioactivity remain poorly understood. In this contribution, silica nanoparticles (SNPs) with 20 nm, 60 nm and 100 nm (named as SNP20, SNP60, SNP100, respectively) were introduced as models. Based on the models, the nanoscale curvature-mediated adsorption dynamics, conformation, and bioactivity of recombinant human BMP-2 (rhBMP-2) were investigated. Our data showed that SNPs bound rapidly to and induced unfolding of rhBMP-2 molecules, which undermined their interactions with the corresponding receptors on the cell surface and decreased the bioactivities of adsorbed rhBMP-2. In contrast, rhBMP-2 showed increasingly stronger affinity to and lost less secondary structure on the larger SNPs, while better bioactivity was observed on the medium SNP60 surfaces. The results indicated that the size of the SNPs, perhaps because of the contribution of surface curvature, influences the structure and function of the adsorbed BMP-2. This study demonstrates the possibility to mediate the binding, conformation and bioactivity of BMP-2 by tailoring the nanoscale curvature, allowing fabrication of BMP-2-based bone tissue scaffolds with high osteoinductivity at low BMP-2 dosage.

Magnesium modification up-regulates the bioactivity of bone morphogenetic protein-2 upon calcium phosphate cement via enhanced BMP receptor recognition and Smad signaling pathway

Efficient presentation of growth factors is one of the great challenges in tissue engineering. In living systems, bioactive factors exist in soluble as well as in matrix-bound forms, both of which play an integral role in regulating cell behaviors. Herein, effect of magnesium on osteogenic bioactivity of recombinant human bone morphogenetic protein-2 (rhBMP-2) was investigated systematically with a series of Mg modified calcium phosphate cements (xMCPCs, x means the content of magnesium phosphate cement wt%) as matrix model. The results indicated that the MCPC, especially 5MCPC, could promote the rhBMP-2-induced in vitro osteogenic differentiation via Smad signaling of C2C12 cells. Further studies demonstrated that all MCPC substrates exhibited similar rhBMP-2 release rate and preserved comparable conformation and biological activity of the released rhBMP-2. Also, the ionic extracts of MCPC made little difference to the bioactivity of rhBMP-2, either in soluble or in matrix-bound forms. However, with the quartz crystal microbalance (QCM), we observed a noticeable enhancement of rhBMP-2 mass-uptake on 5MCPC as well as a better recognition of the bound rhBMP-2 to BMPR IA and BMPR II. In vivo results demonstrated a better bone regeneration capacity of 5MCPC/rhBMP-2. From the above, our results demonstrated that it was the Mg anchored on the underlying substrates that tailored the way of rhBMP-2 bound on MCPC, and thus facilitated the recognition of BMPRs to stimulate osteogenic differentiation. The study will guide the development of Mg-doped bioactive bone implants for tissue regeneration.

Facilitated receptor-recognition and enhanced bioactivity of bone morphogenetic protein-2 on magnesium-substituted hydroxyapatite surface.

Biomaterial surface functionalized with bone morphogenetic protein-2 (BMP-2) is a promising approach to fabricating successful orthopedic implants/scaffolds. However, the bioactivity of BMP-2 on material surfaces is still far from satisfactory and the mechanism of related protein-surface interaction remains elusive. Based on the most widely used bone-implants/scaffolds material, hydroxyapatite (HAP), we developed a matrix of magnesium-substituted HAP (Mg-HAP, 2.2 at% substitution) to address these issues. Further, we investigated the adsorption dynamics, BMPRs-recruitment, and bioactivity of recombinant human BMP-2 (rhBMP-2) on the HAP and Mg-HAP surfaces. To elucidate the mechanism, molecular dynamic simulations were performed to calculate the preferred orientations, conformation changes, and cysteine-knot stabilities of adsorbed BMP-2 molecules. The results showed that rhBMP-2 on the Mg-HAP surface exhibited greater bioactivity, evidenced by more facilitated BMPRs-recognition and higher ALP activity than on the HAP surface. Moreover, molecular simulations indicated that BMP-2 favoured distinct side-on orientations on the HAP and Mg-HAP surfaces. Intriguingly, BMP-2 on the Mg-HAP surface largely preserved the active protein structure evidenced by more stable cysteine-knots than on the HAP surface. These findings explicitly clarify the mechanism of BMP-2-HAP/Mg-HAP interactions and highlight the promising application of Mg-HAP/BMP-2 matrixes in bone regeneration implants/scaffolds.

Strontium attenuates rhBMP-2-induced osteogenic differentiation via formation of Sr-rhBMP-2 complex and suppression of Smad-dependent signaling pathway

Strontium (Sr2+) has pronounced effects on stimulating bone formation and inhibiting bone resorption in bone regeneration. In this current study, the effect and the underlying mechanism involved of Sr2+ on the biological activity of bone morphogenetic protein-2 (BMP-2) were studied in detail with pluripotent skeletal muscle myogenic progenitor C2C12 model cell line. The results indicated that Sr2+ could bind recombinant human BMP-2 (rhBMP-2) rapidly, even in the presence of Ca2+ and Mg2+, and inhibited rhBMP-2-induced osteogenic differentiation in vitro and osteogenetic efficiency in vivo. Further studies demonstrated that Sr2+ treatment undermined the binding capacity of rhBMP-2 with its receptor BMPRIA and thus attenuated Smad 1/5/8 phosphorylation without affecting their dephosphorylation in C2C12 cells. Furthermore, circular dichroism spectroscopy, fluorescence spectroscopy and X-ray photoelectron spectroscopy all revealed that the inhibitory effect of Sr2+ on the rhBMP-2 osteogenic activity was associated with the formation of Sr-rhBMP-2 complex and ensuing enhancement of β-sheet structure. Our work suggests the activity of rhBMP-2 to induce osteogenic differentiation was decreased by directly interaction with free Sr ions in solution, which should provide guide and assist for development of BMP-2-based materials for bone regeneration. Statement of SignificanceDue to easy denaturation and ensuing the reduced activity of rhBMP-2, preserving/enhancing the capacity of rhBMP-2 to induce osteogenic differentiation is of critical importance in developing the protein-based therapy. Cations as effective elements influence the conformation and thereby the bioactivity of protein. Strontium (Sr2+), stimulating bone formation and inhibiting bone resorption, has been incorporated into biomaterials/scaffold to improve the bioactivity for bone-regeneration applications. However, Sr2+-induced changes in the conformation and bioactivity of BMP-2 have never been investigated. In this study, the formation of Sr-rhBMP-2 complex inhibited the osteogenic differentiation in vitro and osteogenetic efficiency in vivo through the inhibition of BMP/Smad signaling pathway, providing guidance for development of Sr-containing BMP-2-based bone scaffold/matrice and other Sr-dopped protein therapy.

BMP-2-PLLA缓释微球的制备及其对BMP-2活性的维持

本研究探索载骨形态发生蛋白-2 (BMP-2)的左旋聚乳酸(PLLA)载药微球的制备条件，并检测BMP-2- PLLA微球中BMP-2的释放规律和生物活性。采用乳化法制备载牛血清白蛋白(BSA)的聚乳酸(PLLA)载药微球，考察PLLA分子量、PLLA浓度、PEG分子量对PLLA载药微球粒径、包封率和载药量的影响，选择优化条件制备载BMP-2的PLLA微球，通过细胞合成ALP检测BMP-2的活性。实验制备的BMP-2-PLLA微球表面光滑，粒径为4.815 μm，载药量为2.28 × 10−3%，包封率为70.88%，二氯甲烷残留量为0.0041%。BMP-2-PLLA微球有效实现了BMP-2长期稳定缓慢释。乳化过程未对BMP-2活性产生显著影响，且BMP-2-PLLA微球中释放的BMP-2对细胞合成ALP的诱导作用较BMP-2溶液更加显著。实验制备的PLLA微球可用于负载生长因子实现缓释，并能较好地维持其生物学活性。 The preparation conditions of poly-l-lactide (PLLA) microspheres, release characteristics and bi-oactivity of Bone Morphogenetic Protein-2 (BMP-2) were studied. Bovine serum albumin (BSA)- loaded PLLA microspheres were prepared with a double emulsion method. The effects of the mo-lecular weight and concentration of PLLA, the molecular weight of PEG on the particle size, loading capacity and entrapment efficiency were studied. Then, the optimal conditions were selected to prepare BMP-2-loaded PLLA microspheres, and the bioactivity of BMP-2 was investigated by pro-duction of cell Alkaline Phosphatase (ALP). The results showed that the BMP-2-PLLA microspheres were spherical shape with diameters of 4.815 μm. The encapsulation efficiency of the BMP-2 in PLLA microspheres was 70.88%, and the loading capacity was 2.28 × 10−3%. The residual amount of methylene chloride in BMP-2-PLLA microsphere was 0.0041%. BMP-2-loaded PLLA microspheres had excellent drug sustained-release performance, which would control release BMP-2. BMP-2 biological activity indicated that the biological activity was not effected after emulsion process. The bioactivity of growth factors released from PLLA microspheres was more durable and effective. PLLA microspheres prepared can be used to load growth factors to control their release and maintain their biological activity.

Heparin microparticles loaded with bone morphogenetic protein-2 (BMP-2) induce bone regeneration in a critically sized rat femoral defect model

Event Abstract Back to Event Heparin microparticles loaded with bone morphogenetic protein-2 (BMP-2) induce bone regeneration in a critically sized rat femoral defect model Marian Hettiaratchi1*, Catherine Chou1, Laxminarayanan Krishnan2, Mon-Tzu A. Li1, Johnna S. Temenoff1, 2, Todd C. Mcdevitt3, 4 and Robert E. Guldberg2, 5 1 Georgia Institute of Technology & Emory University, Wallace H. Coulter Department of Biomedical Engineering, United States 2 Georgia Institute of Technology, Parker H. Petit Institute for Bioengineering and Bioscience, United States 3 Gladstone Institute of Cardiovascular Disease, United States 4 University of California - San Francisco, Department of Bioengineering & Therapeutic Sciences, United States 5 Georgia Institute of Technology, George W. Woodruff School of Mechanical Engineering, United States Introduction: Bone morphogenetic protein-2 (BMP-2), an osteoinductive growth factor, is delivered clinically to stimulate endogenous healing after severe bone loss. However, existing biomaterials exhibit limited affinity for BMP-2, resulting in rapid diffusion of growth factor away from the injury site. Heparin, a negatively charged glycosaminoglycan, binds growth factors electrostatically and has high affinity for BMP-2. We have previously fabricated heparin microparticles (HMPs) that can retain large amounts of bioactive BMP-2 (>300 μg/mg HMPs)[1]. Thus, here we hypothesize that HMPs can improve BMP-2 delivery in vivo and enhance healing in a critically sized bone defect. Methods: HMPs were fabricated from heparin methacrylamide as previously described [1]. HMPs were loaded with BMP-2 overnight at 4°C, and bioactivity was evaluated via C2C12 myoblast growth and alkaline phosphatase (ALP) activity. For in vivo delivery, 2% RGD-alginate gels within polycaprolactone mesh tubes were cast with or without HMPs and/or BMP-2. BMP-2 release from constructs was evaluated in vitro at 37°C. Constructs with 1 mg of fluorescent HMPs were implanted subcutaneously in rats for 6 weeks to evaluate HMP retention in vivo. Constructs with 2.5 μg of BMP-2 loaded onto 1 mg of HMPs or mixed directly into the alginate were implanted in rat femoral defects to evaluate bone healing (Fig 2A). X-ray and micro-CT were performed at 4, 8, and 12 weeks post-surgery, histology was completed on 2, 4, and 12-week femurs, and biomechanical testing was conducted at 12 weeks. Results: C2C12 cells (20,000 cells/well) exposed to 50 ng of total BMP-2 demonstrated increased ALP activity and cell proliferation when ≥10 ng of BMP-2 were loaded onto the HMPs (Fig 1A-B). Alginate constructs with HMP-bound BMP-2 exhibited lower BMP-2 release over 21 days in vitro compared to alginate-bound BMP-2 (Fig 1C). Fluorescent imaging of subcutaneously implanted constructs revealed persistence of ~55% of the HMPs after 6 weeks in vivo (Fig 1D-E). Bone volume within femoral defects increased over 12 weeks in response to both HMP-bound and alginate-bound BMP-2 (Fig 2B). Bony bridging occurred in all alginate-bound BMP-2 samples and 60% of HMP-bound BMP-2 samples (Fig 2D). Functional restoration of maximum torque and stiffness was comparable (Fig 2C). New bone formation (Fig 2E-F, blue) was observed in close proximity to HMPs and alginate (red) at 4 and 12 weeks. Proliferating Ki-67+ cells (Fig 2G, brown) were localized with HMPs (red) at 2 weeks, suggesting HMP-mediated proliferation similar to that observed in vitro. Conclusions: BMP-2-loaded HMPs induce functional regeneration of large bone defects; however, optimization is required to achieve consistent bridging of newly formed bone. Since bioactivity assays have demonstrated the benefits of HMP-bound BMP-2, ongoing experiments are being performed to deliver a combination of alginate-bound and HMP-bound BMP-2 in vivo, in order to provide both rapid release and sustained presentation of growth factor. Overall, HMPs may provide a more effective method of spatially controlling the delivery of BMP-2 for in vivo tissue regeneration applications. Funding is provided by the National Institutes of Health (R01 AR062006) and Armed Forces Institute of Regenerative Medicine (AFIRM-II).; MHH is supported by a Natural Sciences and Engineering Research Council (NSERC) of Canada Postgraduate Scholarship and Philanthropic Education Organization (PEO) Scholar Award.; CC is supported by the Petit Undergraduate Research Scholars Program of the Georgia Institute of Technology.

The promotion of bone tissue regeneration by BMP2-derived peptide P24-loaded calcium phosphate cement microspheres

Calcium phosphate cement (CPC) scaffolds have been widely used as bone graft substitutes, but their clinical application is restricted by the undesirable osteoinductivity and slow degradability. We developed new CPC microspheres loaded with bone morphogenetic protein-2 (BMP-2)-derived peptide P24 (P24/CPC). The BMP2-derived peptide P24 was evenly pasted with CPC microspheres, which maintained the bioactivity of BMP-2. We evaluated the effect of the P24/CPC microspheres on osteogenesis and examined the underlying mechanism in vitro and in vivo. The in vitro studies showed that the bioactivity of P24 was preserved in the P24/CPC microspheres and the P24 release duration lasted up to 39 days. The P24/CPC microspheres with different proportions of P24 (2% and 4%) induced high proliferation of bone marrow stromal cells (BMSCs) and well supported cell adhesion on the sphere surfaces. Furthermore, the cells exhibited higher alkaline phosphatase activity and higher expression of osteogenic-specific genes (OCN and Runx2) in 4% P24/CPC than in the CPC and control groups (P<0.05). Therefore, these findings indicated the enhanced osteoblastic differentiation of BMSCs on P24/CPC microspheres. In vivo osteoinductive studies revealed that the degrees of ectopic osteogenesis in the dorsal muscle pocket of rat were significantly higher in the P24/CPC microspheres than in the CPC microspheres. Finally, the CPC or P24/CPC microspheres were implanted into rabbit femoral condyle defects and P24/CPC serial groups were observed for 4, 8, and 12 weeks. Computed tomography (CT) and histological and histomorphological studies proved that P24/CPC microspheres were biocompatible, bioabsorbable, and osteoinductive. In conclusion, this study demonstrated that P24/CPC microspheres can enhance osteogenesis in vitro and promote bone regeneration in vivo. Furthermore, our results confirmed that BMP2-derived peptide P24 can have a beneficial effect on the bone-generating properties of CPC microspheres, suggesting that P24/CPC microspheres are strong potential bone graft substitutes in bone tissue engineering.

Nanostructured hydroxyapatite surfaces-mediated adsorption alters recognition of BMP receptor IA and bioactivity of bone morphogenetic protein-2

Highly efficient loading of bone morphogenetic protein-2 (BMP-2) onto carriers with desirable performance is still a major challenge in the field of bone regeneration. Till now, the nanoscaled surface-induced changes of the structure and bioactivity of BMP-2 remains poorly understood. Here, the effect of nanoscaled surface on the adsorption and bioactivity of BMP-2 was investigated with a series of hydroxyapatite surfaces (HAPs): HAP crystal-coated surface (HAP), HAP crystal-coated polished surface (HAP-Pol), and sintered HAP crystal-coated surface (HAP-Sin). The adsorption dynamics of recombinant human BMP-2 (rhBMP-2) and the accessibility of the binding epitopes of adsorbed rhBMP-2 for BMP receptors (BMPRs) were examined by a quartz crystal microbalance with dissipation. Moreover, the bioactivity of adsorbed rhBMP-2 and the BMP-induced Smad signaling were investigated with C2C12 model cells. A noticeably high mass-uptake of rhBMP-2 and enhanced recognition of BMPR-IA to adsorbed rhBMP-2 were found on the HAP-Pol surface. For the rhBMP-2-adsorbed HAPs, both ALP activity and Smad signaling increased in the order of HAP-Sin<HAP<HAP-Pol. Furthermore, hybrid molecular dynamics and steered molecular dynamics simulations validated that BMP-2 tightly anchored on the HAP-Pol surface with a relative loosened conformation, but the HAP-Sin surface induced a compact conformation of BMP-2. In conclusion, the nanostructured HAPs can modulate the way of adsorption of rhBMP-2, and thus the recognition of BMPR-IA and the bioactivity of rhBMP-2. These findings can provide insightful suggestions for the future design and fabrication of rhBMP-2-based scaffolds/implants. Statement of SignificanceThis study provides strong evidences that nanoscaled HAPs yield extraordinary influence on the adsorption behaviors and bioactivity of rhBMP-2. It has been found that the surface roughness and crystallinity played a crucial role in governing the way of rhBMP-2 binding to HAPs, and thus the conformation, recognition of BMPR-IA and bioactivity of adsorbed rhBMP-2. It is also for the first time to correlate numerical modeling and experimental results of the bioactivity of rhBMP-2 on nanostructured HAPs. This work can pave an avenue for the wider uses of rhBMP-2 in clinical applications and arouse broad interests among researchers in the fields of nano-biotechnology, biomaterials and bone tissue engineering.

Calcium ion-induced formation of β-sheet/-turn structure leading to alteration of osteogenic activity of bone morphogenetic protein-2

Preserving bioactivity of bone morphogenetic protein 2 (BMP-2) still remains a challenge in protein-based therapy. It is not known how Ca2+ released from extracellular matrix or existing in physiological environment influences bioactivity in situ till now. Here, effects of extracellular Ca2+ on conformation and osteogenic bioactivity of recombinant human BMP-2 (rhBMP-2) were investigated systematically. In vitro results indicated that Ca2+ could bind rhBMP-2 rapidly and had no obvious effect on cell behaviors. Low concentration of Ca2+ (0.18 mM) enhanced rhBMP-2-induced osteogenic differentiation, while high Ca2+ concentration (>1.80 mM) exerted negative effect. In vivo ectopic bone formation exhibited similar trend. Further studies by circular dichroism spectroscopy, fluorescence spectroscopy, together with cell culture experiments revealed at low concentration, weak interaction of Ca2+ and rhBMP-2 slightly increased β-sheet/-turn content and facilitated recognition of BMP-2 and BMPRIA. But, high Ca2+ concentration (>1.8 mM) induced formation of Ca-rhBMP-2 complex and markedly increased content of β-sheet/-turn, which led to inhibition binding of rhBMP-2 and BMPRIA and thus suppression of downstream Smad1/5/8, ERK1/2 and p38 mitogen-associated protein kinase signaling pathways. Our work suggests osteogenic bioactivity of BMP-2 can be adjusted via extracellular Ca2+, which should provide guide and assist for development of BMP-2-based materials for bone regeneration.

Effect of selective heparin desulfation on preservation of bone morphogenetic protein-2 bioactivity after thermal stress.

Bone morphogenetic protein-2 (BMP-2) plays an important role in bone and cartilage formation and is of interest in regenerative medicine. Heparin can interact electrostatically with BMP-2 and thus has been explored for controlled release and potential stabilization of this growth factor in vivo. However, in its natively sulfated state, heparin has potent anticoagulant properties that may limit its use. Desulfation reduces anticoagulant properties, but may impact heparin's ability to interact and protect BMP-2 from denaturation. The goal of this study was to characterize three selectively desulfated heparin species (N-desulfated (Hep(-N)), 6-O,N-desulfated (Hep(-N,-6O)), and completely desulfated heparin (Hep(-))) and determine if the sulfation level of heparin affected the level of BMP-2 bioactivity after heat treatment at 65 °C. BMP-2 bioactivity was evaluated using the established C2C12 cell assay. The resulting alkaline phosphatase activity data demonstrated that native heparin maintained a significant amount of BMP-2 bioactivity and the effect appeared to be heparin concentration dependent. Although all three had the same molecular charge as determined by zeta potential measurements, desulfated heparin derivatives Hep(-N) and Hep(-N,-6O) were not as effective as native heparin in maintaining BMP-2 bioactivity (only ~35% of original activity remained in both cases). These findings can be used to better select desulfated heparin species that exhibit low anticoagulant activity while extending the half-life of BMP-2 in solution and in delivery systems.

Hydrolysis and sulfation pattern effects on release of bioactive bone morphogenetic protein-2 from heparin-based microparticles

Glycosaminoglycans (GAGs) such as heparin are promising materials for growth factor delivery due to their ability to efficiently bind positively charged growth factors including bone morphogenetic protein-2 (BMP-2) through their negatively charged sulfate groups. Therefore, the goal of this study was to examine BMP-2 release from heparin-based microparticles (MPs) after first, incorporating a hydrolytically degradable crosslinker and varying heparin content within MPs to alter MP degradation and second, altering the sulfation pattern of heparin within MPs to vary BMP-2 binding and release. Using varied MP formulations, it was found that the time course of MP degradation for 1 wt% heparin MPs was ~4 days slower than 10 wt% heparin MPs, indicating that MP degradation was dependent on heparin content. After incubating 100 ng BMP-2 with 0.1 mg MPs, most MP formulations loaded BMP-2 with ~50% efficiency and significantly more BMP-2 release (60% of loaded BMP-2) was observed from more sulfated heparin MPs (MPs with ~100% and 80% of native sulfation). Similarly, BMP-2 bioactivity in more sulfated heparin MP groups was at least four-fold higher than soluble BMP-2 and less sulfated heparin MP groups, as determined by an established C2C12 cell alkaline phosphatase (ALP) assay. Ultimately, the two most sulfated 10 wt% heparin MP formulations were able to efficiently load and release BMP-2 while enhancing BMP-2 bioactivity, making them promising candidates for future growth factor delivery applications.

Release and bioactivity of bone morphogenetic protein-2 are affected by scaffold binding techniques in vitro and in vivo

A low dose of 1μg rhBMP-2 was immobilised by four different functionalising techniques on recently developed poly(l-lactide)-co-(ε-caprolactone) [(poly(LLA-co-CL)] scaffolds. It was either (i) physisorbed on unmodified scaffolds [PHY], (ii) physisorbed onto scaffolds modified with nanodiamond particles [nDP-PHY], (iii) covalently linked onto nDPs that were used to modify the scaffolds [nDP-COV] or (iv) encapsulated in microspheres distributed on the scaffolds [MICS]. Release kinetics of BMP-2 from the different scaffolds was quantified using targeted mass spectrometry for up to 70days. PHY scaffolds had an initial burst of release while MICS showed a gradual and sustained increase in release. In contrast, NDP-PHY and nDP-COV scaffolds showed no significant release, although nDP-PHY scaffolds maintained bioactivity of BMP-2. Human mesenchymal stem cells cultured in vitro showed upregulated BMP-2 and osteocalcin gene expression at both week 1 and week 3 in the MICS and nDP-PHY scaffold groups. These groups also demonstrated the highest BMP-2 extracellular protein levels as assessed by ELISA, and mineralization confirmed by Alizarin red. Cells grown on the PHY scaffolds in vitro expressed collagen type 1 alpha 2 early but the scaffold could not sustain rhBMP-2 release to express mineralization. After 4weeks post-implantation using a rat mandible critical-sized defect model, micro-CT and Masson trichrome results showed accelerated bone regeneration in the PHY, nDP-PHY and MICS groups. The results demonstrate that PHY scaffolds may not be desirable for clinical use, since similar osteogenic potential was not seen under both in vitro and in vivo conditions, in contrast to nDP-PHY and MICS groups, where continuous low doses of BMP-2 induced satisfactory bone regeneration in both conditions. The nDP-PHY scaffolds used here in critical-sized bone defects for the first time appear to have promise compared to growth factors adsorbed onto a polymer alone and the short distance effect prevents adverse systemic side effects.

Bone morphogenetic protein-2 for bone regeneration – Dose reduction through graphene oxide-based delivery

High doses of bone morphogenetic protein-2 (BMP-2) are required for effective bone regeneration. However, these high doses often cause undesirable adverse effects, including bone overgrowth, osteolysis, and activation of the immune response, and raise treatment costs. In an effort to reduce the BMP-2 dose to avoid or diminish side effects, we investigated whether delivery of BMP-2 using graphene oxide (GO) can reduce the BMP-2 dose for bone regeneration. Delivery of BMP-2 using GO flakes suspended in fibrin gels (GO/F) resulted in significantly greater osteogenic differentiation of human bone marrow-derived mesenchymal stem cells in vitro, and at various doses of BMP-2, significantly greater amounts of bone regeneration in mouse calvarial defects occurred than when fibrin gel (F) alone was used. A half-dose of BMP-2 delivered by GO/F resulted in bone regeneration similar to that resulting from a full dose of BMP-2 delivered by F. The enhanced bone regeneration efficacy was likely due, at least in part, to the sustained release, higher structural stability, and higher bioactivity of BMP-2 delivered by GO/F compared to BMP-2 delivered by F. Therefore, GO may be an effective carrier for BMP-2 to reduce the BMP-2 dose and avoid adverse effects.