Spatial patterning of BMP-2 and BMP-7 on biopolymeric films and the guidance of muscle cell fate

In the cellular microenvironment, growth factor gradients are crucial in dictating cell fate. Towards developing materials that capture the native microenvironment we engineered biomimetic films that present gradients of matrix-bound bone morphogenetic proteins (BMP-2 and BMP-7). To this end layer-by-layer films composed of poly(l-lysine) and hyaluronan were combined in a simple microfluidic device enabling spatially controlled growth factor diffusion along the film. Linear long-range gradients of both BMPs induced the trans-differentiation of C2C12 myoblasts towards the osteogenic lineage in a dose dependent manner with a different signature for each BMP. The osteogenic marker alkaline phosphatase (ALP) increased in a linear manner for BMP-7 and non-linearly for BMP-2. Moreover, an increased expression of the myogenic marker troponin T was observed with decreasing matrix-bound BMP concentration, providing a substrate that it is both osteo- and myo-inductive. Lastly, dual parallel matrix-bound gradients of BMP-2 and -7 revealed a complete saturation of the ALP signal. This suggested an additive or synergistic effect of the two BMPs. This simple technology allows for determining quickly and efficiently the optimal concentration of matrix-bound growth factors, as well as for investigating the presentation of multiple growth factors in their solid-phase and in a spatially controlled manner.     

Enhanced bioactivity of bone morphogenetic protein-2 with low dose of 2-N, 6-O-sulfated chitosan in vitro and in vivo

Bone morphogenetic protein-2 (BMP-2) has been widely used as an effective growth factor in bone tissue engineering. However, large amounts of BMP-2 are required to induce new bone and the resulting side effects limit its clinical application. Sulfated polysaccharides, such as native heparin, and heparan sulfate have been found to modulate BMP-2 bioactivity and play pivotal roles in bone metabolism. Whereas the direct role of chitosan modified with sulfate group in BMP-2 signaling has not been reported till now. In the present study, several sulfated chitosans with different positions were synthesized by regioselective reactions firstly. Using C2C12 myoblast cells as in vitro models, the enhanced bioactivity of BMP-2 was attributed primarily to the stimulation from 6-O-sulfated chitosan (6SCS), while 2-N-sulfate was subsidiary group with less activation. Low dose of 2-N, 6-O-sulfated chitosan (26SCS) showed significant enhancement on the alkaline phosphatase (ALP) activity and the mineralization formation induced by BMP-2, as well as the expression of ALP and osteocalcin mRNA. Moreover, increased chain-length and further sulfation on 26SCS also resulted in a higher ALP activity. Dose-dependent effects on BMP-2 bioactivity were observed in both sulfated chitosan and heparin. Compared with native heparin, 26SCS showed much stronger simultaneous effects on the BMP-2 bioactivity at low dose. Stimulated secreted Noggin protein failed to block the function of BMP-2 in the presence of 26SCS. The BMP-2 ligand bound to its receptor was enhanced by low dose of 26SCS, whereas weakened by the increasing amounts of 26SCS. Furthermore, simultaneous administration of BMP-2 and 26SCS in vivo dose-dependently induced larger amounts of ectopic bone formation compared with BMP-2 alone. These findings clearly indicate that 26SCS is a more potent enhancer for BMP-2 bioactivity to induce osteoblastic differentiation in vitro and in vivo by promoting BMP-2 signaling pathway, suggesting that 26SCS could be used as the synergistic factor of BMP-2 for bone regeneration.     

In vitro effects of combined and sequential delivery of two bone growth factors

Bone formation and repair occur by a complex cascade involving numerous growth factors and cytokines. In this study, two-layered heterogeneously loaded and crosslinked gelatin coatings were used to obtain combined and sequential delivery of two bone growth factors, BMP-2 and IGF-I, in cell cultures. Peak release from the top and bottom layers was localized around 1 and 6 days, respectively. For comparison, cells were also treated with soluble growth factors directly added to the culture medium. Pluripotent C3H10T1/2 (C3H) cells responded to soluble growth factor treatments with the greatest specific alkaline phosphatase (AP) activity resulting from addition of BMP-2 followed by IGF-I or by BMP-2+IGF-I. Altered loading and subsequent release of BMP-2 and IGF-I from gelatin coatings also affected AP activity in C3H cultures, and the coatings influenced AP activity and incorporation of calcium in the extracellular matrix of bone marrow stromal cell cultures. Early delivery of BMP-2 followed by increased release of BMP-2 and IGF-I after 5 days resulted in the largest, as well as earliest, elevation of AP activity and mineralized matrix formation compared to controls and other treatments. Simultaneous release of both growth factors from both layers did not significantly change AP activity or matrix calcium content compared to control coatings. These results demonstrate that temporally varying delivery of multiple growth factors can significantly affect cell behavior.     

Dual growth factor delivery from bilayered,biodegradable hydrogel composites for spatially-guided osteochondral tissue repair

The present work investigated the use of biodegradable hydrogel composite scaffolds, based on the macromer oligo(poly(ethylene glycol) fumarate) (OPF), to deliver growth factors for the repair of osteochondral tissue in a rabbit model. In particular, bilayered OPF composites were used to mimic the structural layers of the osteochondral unit, and insulin-like growth factor-1 (IGF-1) and bone morphogenetic protein-2 (BMP-2) were loaded into gelatin microparticles and embedded within the OPF hydrogel matrix in a spatially controlled manner. Three different scaffold formulations were implanted in a medial femoral condyle osteochondral defect: 1) IGF-1 in the chondral layer, 2) BMP-2 in the subchondral layer, and 3) IGF-1 and BMP-2 in their respective separate layers. The quantity and quality of osteochondral repair was evaluated at 6 and 12 weeks with histological scoring and micro-computed tomography (micro-CT). While histological scoring results at 6 weeks showed no differences between experimental groups, micro-CT analysis revealed that the delivery of BMP-2 alone increased the number of bony trabecular islets formed, an indication of early bone formation, over that of IGF-1 delivery alone. At 12 weeks post-implantation, minimal differences were detected between the three groups for cartilage repair. However, the dual delivery of IGF-1 and BMP-2 had a higher proportion of subchondral bone repair, greater bone growth at the defect margins, and lower bone specific surface than the single delivery of IGF-1. These results suggest that the delivery of BMP-2 enhances subchondral bone formation and that, while the dual delivery of IGF-1 and BMP-2 in separate layers does not improve cartilage repair under the conditions studied, they may synergistically enhance the degree of subchondral bone formation. Overall, bilayered OPF hydrogel composites demonstrate potential as spatially-guided, multiple growth factor release vehicles for osteochondral tissue repair.     

Enhancement of ectopic bone formation by bone morphogenetic protein-2 released from a heparin-conjugated poly(L-lactic-co-glycolic acid) scaffold

In this study, a heparin-conjugated poly(l-lactic-co-glycolic acid) (HP-PLGA) scaffold was developed for the sustained delivery of bone morphogenetic protein-2 (BMP-2), and then used to address the hypothesis that BMP-2 delivered from this scaffold could enhance ectopic bone formation. We found the amount of heparin conjugated to the PLGA scaffolds could be increased up to 3.2-fold by using scaffolds made from star-shaped PLGA, as compared to scaffolds made from linear PLGA, and that the release of BMP-2 from the HP-PLGA scaffold was sustained for at least 14 days in vitro. The BMP-2 released from the HP-PLGA scaffold stimulated an increase in alkaline phosphatase (ALP) activity of osteoblasts for 14 days in vitro, suggesting that the HP-PLGA scaffold delivery system releases BMP-2 in a bioactive form for a prolonged period. By contrast, BMP-2 release from unmodified (no heparin) PLGA scaffolds induced a transient increase in ALP activity for the first 3 days and a decrease thereafter. In vivo bone formation studies showed the BMP-2-loaded HP-PLGA scaffolds induced bone formation to a much greater extent than did either BMP-2-loaded unmodified PLGA scaffolds or unloaded (no BMP-2) HP-PLGA scaffolds, with 9-fold greater bone formation area and 4-fold greater calcium content in the BMP-2-loaded HP-PLGA scaffold group compared to the BMP-2-loaded unmodified PLGA scaffold group. Collectively, these results demonstrate that the HP-PLGA delivery system is capable of potentiating the osteogenic efficacy of BMP-2, and underscore its importance as a possible bone regeneration strategy.     

BMP Stimulation of Alkaline Phosphatase Activity in Pluripotent Mouse C2C12 Cells is Inhibited by Dermatopontin,One of the Most Abundant Low Molecular Weight Proteins in Demineralized Bone Matrix

Demineralized bone matrix (DBM) is a complex mixture of osteoinductive bone morphogenetic proteins (BMPs), as well as BMP-binding proteins that regulate BMP bioactivity and localization. Our aim was to use modern proteomic methods to identify additional BMP-binding proteins in DBM, with initial emphasis on the most abundant. Relatively large, water-soluble noncollagenous proteins (NCPs) were preferentially extracted from DBM with alkalinized urea. The insoluble residue, which contained the BMP activity, was extracted with GuHCl/CaCl₂, dialyzed versus citrate, defatted, resuspended in GuHCl, dialyzed sequentially against Triton X-100 and water, pelleted, and lyophilized. The proteins in this pellet were fractionated by hydroxyapatite affinity chromatography. Proteins that copurified with BMP bioactivity were separated by SDS-PAGE. Distinct bands were excised, and the proteins in them were reduced and alkylated, digested with trypsin, eluted, and subjected to MALDI/ToF MS (matrix-assisted laser-desorption ionization time-of-flight mass spectrometry). Computer-assisted peptide fingerprint analysis of the MS profiles was used to identify C-terminal lysine-6-oxidase; dermatopontin (DPT); histones H2A2, H2A3, and H2B; and trace amounts of γ-actin. DPT is a 22-kDa, tyrosine-rich acidic matrix protein not previously recognized to be among the most abundant small proteins to copurify with BMP bioactivity in DBM. We tested the effects of DPT on BMP-2 stimulation of alkaline phosphatase (ALP) activity in C2C12 cells. BMP-2 stimulated ALP activity in C2C12 cells by 6.2-fold above basal levels. DPT alone had no effect on ALP activity in C2C12 cells. When added with BMP-2, DPT blocked 40% of the stimulatory effect of BMP-2 on ALP activity in C2C12 cells. DPT is an abundant protein in DBM, and it can inhibit the stimulatory effects of BMP-2 on ALP activity in C2C12 cells.     

Retention of in vitro and in vivo BMP-2 bioactivities in sustained delivery vehicles for bone tissue engineering

In this study, we investigated the in vitro and in vivo biological activities of bone morphogenetic protein 2 (BMP-2) released from four sustained delivery vehicles for bone regeneration. BMP-2 was incorporated into (1) a gelatin hydrogel, (2) poly(lactic-co-glycolic acid) (PLGA) microspheres embedded in a gelatin hydrogel, (3) microspheres embedded in a poly(propylene fumarate) (PPF) scaffold and (4) microspheres embedded in a PPF scaffold surrounded by a gelatin hydrogel. A fraction of the incorporated BMP-2 was radiolabeled with (125)I to determine its in vitro and in vivo release profiles. The release and bioactivity of BMP-2 were tested weekly over a period of 12 weeks in preosteoblast W20-17 cell line culture and in a rat subcutaneous implantation model. Outcome parameters for in vitro and in vivo bioactivities of the released BMP-2 were alkaline phosphatase (AP) induction and bone formation, respectively. The four implant types showed different in vitro release profiles over the 12-week period, which changed significantly upon implantation. The AP induction by BMP-2 released from gelatin implants showed a loss in bioactivity after 6 weeks in culture, while the BMP-2 released from the other implants continued to show bioactivity over the full 12-week period. Micro-CT and histological analysis of the delivery vehicles after 6 weeks of implantation showed significantly more bone in the microsphere/PPF scaffold composites (Implant 3, p<0.02). After 12 weeks, the amount of newly formed bone in the microsphere/PPF scaffolds remained significantly higher than that in the gelatin and microsphere/gelatin hydrogels (p<0.001), however, there was no statistical difference compared to the microsphere/PPF/gelatin composite. Overall, the results from this study show that BMP-2 could be incorporated into various bone tissue engineering composites for sustained release over a prolonged period of time with retention of bioactivity.     

A differential effect of bone morphogenetic protein-2 and vascular endothelial growth factor release timing on osteogenesis at ectopic and orthotopic sites in a large-animal model

In bone tissue engineering, growth factors are widely used. Bone morphogenetic proteins (BMPs) and vascular endothelial growth factor (VEGF) are the most well-known regulators of osteogenesis and angiogenesis. We investigated whether the timing of dual release of VEGF and BMP-2 influences the amount of bone formation in a large-animal model. Poly(lactic-co-glycolic acid) (PLGA) microparticles (MPs) were loaded with BMP-2 or VEGF to create sustained-release profiles, and rapidly degrading gelatin was loaded with either growth factor for fast-release profiles. To study in vivo osteogenicity, the two delivery vehicles were combined with biphasic calcium phosphate (BCP) scaffolds and implanted in 10 Beagle dogs for 9 weeks, at both ectopic (paraspinal muscles) and orthotopic sites (critical-size ulnar defect). The 9 ectopic groups contained combined or single BMP/VEGF dosage, in sustained- or fast-release profiles. In the ulnae of 8 dogs, fast VEGF and sustained BMP-2 were applied to one leg, and the other received the opposite release profiles. The two remaining dogs received bilateral control scaffolds. Bone growth dynamics was analyzed by fluorochrome injection at weeks 3, 5, and 7. Postoperative and posteuthanization X-rays of the ulnar implants were taken. After 9 weeks of implantation, bone quantity and bone growth dynamics were studied by histology, histomorphometry, and fluorescence microscopy. The release of the growth factors resulted in both enhanced orthotopic and ectopic bone formation. Bone formation started before 3 weeks and continued beyond 7 weeks. The ectopic BMP-2 fast groups showed significantly more bone compared to sustained release, independent of the VEGF profile. The ulna implants revealed no significant differences in the amount of bone formed. This study shows that timing of BMP-2 release largely determines speed and amount of ectopic bone formation independent of VEGF release. Furthermore, at the orthotopic site, no significant effect on bone formation was found from a timed release of growth factors, implicating that timed-release effects are location dependent.     

Heparin-decorated, hyaluronic acid-based hydrogel particles for the controlled release of bone morphogenetic protein 2

We are interested in developing hydrophilic particulate systems that are capable of sequestering growth factors, regulating their release and potentiating their biological functions. To this end heparin (HP)-decorated, hyaluronic acid (HA)-based hydrogel particles (HGPs) were synthesized using an inverse emulsion polymerization technique employing divinyl sulfone as the crosslinker. By varying the feed composition of the aqueous phase the amount of HP integrated in the particles can be systematically tuned. The resulting microscopic particles are spherical in shape and contain nanosized pores suitable for growth factor encapsulation. The covalently immobilized HP retained its ability to bind bone morphogenetic protein-2 (BMP-2) specifically, and its release kinetics can be adjusted by tuning the particle composition. Compared with pure HA particles the hybrid HA/HP HGPs show a higher BMP-2 loading capacity. While BMP-2 was released from HA HGPs with a significant initial burst, a near zero order release kinetics was observed from HA/HP hybrid particles with an optimized heparin content of 0.55 μg per mg HGPs. The ability of HA/HP hybrid particles to present BMP-2 in a controlled manner, combined with the innate bioactivity of HA, induced robust and consistent chondrogenic differentiation of murine mesenchymal stem cells, as shown by up-regulation of the mRNA levels of chondrogenic markers and the production of cartilage-specific extracellular matrix components. The simplicity of the particle synthesis, combined with the defined biological activities of the constituent building blocks, renders the HP-decorated, HA-based hydrogel particle system an attractive candidate for the sustained release of BMP-2, possibly for cartilage repair and regeneration.     

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.     

Hollow calcium phosphate microcarriers for bone regeneration: in vitro osteoproduction and ex vivo mechanical assessment

Synthetic grafting materials, such as calcium phosphates (hydroxyapatite, HA; tricalcium phosphate, TCP), polymers, or composites thereof, can be used as osteoconductive scaffolds and delivery vehicles for osteoinductive growth factors. Carrier materials must be engineered to deliver these factors in a controlled fashion at a rate and dose consistent with the biological need and responsiveness of the system to optimize bone formation and ingrowth. They should also simultaneously provide mechanical support and slowly resorb as new bone is formed. This investigation assessed the elution characteristics of BMP-7 (OP-1) from hollow calcium phosphate spheres of varying chemical composition (HA/beta-TCP) and porosity (dense/porous). The pharmacokinetics indicated a bimodal trend of protein release with protein elution peaking between fifteen and thirty minutes in solution (bolus release) and continuing through the eight-week time point (sustained release). Eluted OP-1 bioactivity was characterized over a three-week period using mesenchymal stem cell (MSC) cultures and included assessment of the protein's differential, proliferative, and calcified nodule forming abilities. Alkaline phosphatase enzyme (ALP) activity in MSCs peaked between 12 and 16 days post-OP-1 exposure. Elutant from the HA dense treatment group induced the highest degree of ALP expression while elutant from the beta-TCP treatment groups induced the formation of significantly higher numbers of calcified nodules in culture. The aggregate modulus of a clinically relevant 2 cc dose of carriers was quantified using custom designed testing fixtures to investigate the effects of carrier size, porosity, chemical composition, and the presence of a central hole on mechanical integrity. Significant increases in moduli were noted for carrier size and chemical composition (HA>beta-TCP). These preliminary in vitro and ex vivo results indicate the clinical potential of the hollow calcium phosphate carriers as successful load-bearing delivery vehicles for OP-1.     

Enhancement of the biological activity of BMP-2 by synthetic dextran derivatives

In the present study, we explored the binding capacity of synthetic heparin-like dextran derivatives to recombinant human bone morphogenetic protein 2 (BMP-2), a heparin-binding osteoinductive growth factor. Affinity electrophoresis analysis provided evidence that carboxymethylated dextran polymers grafted with high amounts of benzylamide groups (named DMCB) interact with BMP-2. The capability of such polysaccharides to potentiate the growth factor biological activity was then investigated. In vitro, DMCB dose-dependently promoted osteoblast differentiation induced by BMP-2 in C2C12 myoblasts more efficiently than heparin. A screening study provided evidence that the potentiating effects of the dextran derivatives on the BMP-2-induced alkaline phosphatase activity improved with their benzylamide groups content and, therefore, with their affinity for the growth factor. The biological activity of BMP-2 was monitored in the culture medium after 6 days using C2C12 cells (containing a BMP sensitive luciferase reporter gene). Like heparin, DMCB sustained the biological activity of the growth factor; this result suggests that the formation of the BMP-2/DMCB complex may protect the protein from being inactivated. In rats in vivo, DMCB also stimulated ectopic calcification mediated by BMP-2. These data indicate that dextran-based polysaccharides prolong the half-life of the growth factor and promote its biological activity.     

Incorporation of a sequential BMP-2/BMP-7 delivery system into chitosan-based scaffolds for bone tissue engineering

The aim of this study was to develop a 3-D construct carrying an inherent sequential growth factor delivery system. Poly(lactic acid-co-glycolic acid) (PLGA) nanocapsules loaded with bone morphogenetic protein BMP-2 and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) nanocapsules loaded with BMP-7 made the early release of BMP-2 and longer term release of BMP-7 possible. 3-D fiber mesh scaffolds were prepared from chitosan and from chitosan–PEO by wet spinning. Chitosan of 4% concentration in 2% acetic acid (CHI4–HAc2) and chitosan (4%) and PEO (2%) in 5% acetic acid (CHI4–PEO2–HAc5) yielded scaffolds with smooth and rough fiber surfaces, respectively. These scaffolds were seeded with rat bone marrow mesenchymal stem cells (MSCs). When there were no nanoparticles the initial differentiation rate was higher on (CHI4–HAc2) scaffolds but by three weeks both the scaffolds had similar alkaline phosphatase (ALP) levels. The cell numbers were also comparable by the end of the third week. Incorporation of nanoparticles into the scaffolds was achieved by two different methods: incorporation within the scaffold fibers (NP–IN) and on the fibers (NP–ON). It was shown that incorporation on the CHI4–HAc2 fibers (NP–ON) prevented the burst release observed with the free nanoparticles, but this did not influence the total amount released in 25 days. However NP–IN for the same fibers revealed a much slower rate of release; ca. 70% released at the end of incubation period. The effect of single, simultaneous and sequential delivery of BMP-2 and BMP-7 from the CHI4–HAc2 scaffolds was studied in vitro using samples prepared with both incorporation methods. The effect of delivered agents was higher with the NP–ON samples. Delivery of BMP-2 alone suppressed cell proliferation while providing higher ALP activity compared to BMP-7. Simultaneous delivery was not particularly effective on cell numbers and ALP activity. The sequential delivery of BMP-2 and BMP-7, on the other hand, led to the highest ALP activity per cell (while suppressing proliferation) indicating the synergistic effect of using both growth factors holds promise for the production of tissue engineered bone.     

Retention and activity of BMP-2 in hyaluronic acid-based scaffolds in vitro.

Bone morphogenetic protein-2 (BMP-2) delivered in a suitable implantable matrix has the potential to repair local skeletal defects by inducing new bone formation from undifferentiated pluripotent stem cells resident in host tissue. In this study, we examined in vitro the potential of a derivatized hyaluronic acid (Hyaff-11) scaffold as a delivery vehicle for recombinant human BMP-2 (rhBMP-2) in bone and cartilage repair therapies. Hyaff-11 scaffolds were fabricated using a phase inversion/particulate leaching method and soak-loaded with rhBMP-2. In vitro release kinetics of rhBMP-2, demonstrated using enzyme-linked immunosorbant assay and alkaline phosphatase (ALP) assay revealed a slow, sustained rhBMP-2 release during 28 days, with a cumulative release of 31.82% of the initial rhBMP-2 loaded. rhBMP-2 was released in bioactive form as demonstrated by ALP induction of pluripotent cell line, C3H10T1/2 (T1/2), down the osteoblast lineage when incubated with the release supernatants. rhBMP-2 retention in Hyaff-11 scaffolds was greater than that from collagen gels, which released most of the initially loaded rhBMP-2 by 14 days. rhBMP-2-loaded Hyaff-11 scaffolds were also seeded with T1/2 cells and evaluated at 3, 7, 14, and 28 days for viability and expression of osteoblast phenotype. Cells remained viable throughout the study and expressed a time- and dose-dependent ALP and osteocalcin expression in the rhBMP-2 groups. Based on these observations, Hyaff-11 scaffolds may be suitable delivery systems for rhBMP-2 in bone/cartilage repair because of their ability to retain rhBMP-2, release low levels of bioactive rhBMP-2 to the local environment in a sustained manner, and stimulate differentiation of pluripotent stem cells.     

Sequential growth factor delivery from complexed microspheres for bone tissue engineering

Aim of the study was to design a 3D tissue-engineering scaffold capable of sequentially delivering two bone morphogenetic proteins (BMP). The novel delivery system consisted of microspheres of polyelectrolyte complexes of poly(4-vinyl pyridine) (P(4)VN) and alginic acid loaded with the growth factors BMP-2 and BMP-7 which themselves were loaded into the scaffolds constructed of PLGA. Microspheres carrying the growth factors were prepared using polyelectrolyte solutions with different concentrations (4-10%) to control the growth factor release rate. Release kinetics was studied using albumin as the model drug and the populations that release their contents very early and very late in the release study were selected to carry BMP-2 and BMP-7, respectively. Foam porosity changed when the microspheres were loaded. Bone marrow derived stem cells (BMSC) from rats were seeded into these foams. Alkaline phosphatase (ALP) activities were found to be lowest and cell proliferation was highest at all time points with foams carrying both the microsphere populations, regardless of BMP presence. With the present doses used neither BMP-2 nor BMP-7 delivery had any direct effect on proliferation, however, they enhanced osteogenic differentiation. Co-administration of BMP enhanced osteogenic differentiation to a higher degree than with their single administration.     

Surface immobilization of bone morphogenetic protein 2 via a self-assembled monolayer formation induces cell differentiation

Bone extracellular matrix consists of a network of proteins in which growth factors, like bone morphogenetic protein 2 (BMP-2), are embedded and released upon matrix turnover and degradation. Recombinant human (rh)BMP-2 shows promise in enhancing bone fracture repair, although issues regarding finding a suitable delivery system still limit its extensive clinical use. The aim of this study is to determine which cell activities are triggered by the presentation of immobilized rhBMP-2. For this purpose gold surfaces were first decorated with a self-assembled monolayer consisting of a hetero-bifunctional linker. rhBMP-2 was covalently bound to the surfaces via this linker and used to investigate the cellular responses of C2C12 myoblasts. We show that covalently immobilized rhBMP-2 (iBMP-2) initiates short-term signaling events. Using a BMP-responsive reporter gene assay and western blotting to monitor phosphorylation of Smad1/5/8 we prove that iBMP-2 activates BMP-dependent signal transduction. Furthermore, we demonstrate that iBMP-2 suppresses myotube formation and promotes the osteoblast phenotype in C2C12 cells. The bioactivity of surface-bound rhBMP-2 presented in this study is not due to its release into the medium. As such, our simple approach paves the way for the controlled local presentation of immobilized growth factors, limiting degradation while still maintaining biological activity.     

JNK MAPK is involved in BMP-2-induced odontoblastic differentiation of human dental pulp cells

Bone morphogenetic protein-2 (BMP-2) is a multi-functional growth factor belonging to the transforming growth factor β superfamily that has a broad range of activities that affect many different cell types. BMP-2 induces odontoblastic differentiation of human dental pulp cells (DPCs), but the underlying mechanism remains unclear. In this study, we investigated the potential role of the JNK mitogen-activated protein kinases (MAPK) pathway in BMP-2-induced odontoblastic differentiation of DPCs. The levels of phosphorylated and unphosphorylated JNK MAPK were quantified by Western blot analysis following treatment with BMP-2 and the JNK inhibitor SP600125. The role of JNK MAPK in the BMP-2-induced odontoblastic differentiation of DPCs was determined by measuring alkaline phosphatase (ALP) activity and by examining the expression of odontoblastic markers using quantitative real-time polymerase chain reaction analysis. The effect of JNK MAPK silencing on odontoblastic differentiation was also investigated. BMP-2 upregulated the phosphorylation of JNK in DPCs in a dose- and time-dependent manner. Early markers of odontoblastic differentiation, including ALP activity, osteopontin and dentin matrix protein-1, were not inhibited by the JNK inhibitor. However, the JNK inhibitor, SP600125, significantly inhibited late-stage differentiation of odontoblasts, including the gene expression of osteocalcin, dentin sialophosphoprotein and bone sialoprotein, and also reduced the formation of mineralized nodules in BMP-2-treated DPCs. Consistent with this observation, silencing of JNK MAPK also decreased late-stage odontoblastic differentiation. Taken together, these findings suggest that JNK activity is required for late-stage odontoblastic differentiation induced by BMP-2.     

Hyaluronic acid-based hydrogels functionalized with heparin that support controlled release of bioactive BMP-2

Bone morphogenetic protein-2 (BMP-2) is a potent osteoinductive factor, yet its clinical use is limited by a short biological half-life, rapid local clearance and propensity for side effects. Heparin (HP), a highly sulfated glycosaminoglycan (GAG) that avidly binds BMP-2, has inherent biological properties that may circumvent these limitations. Here, we compared hyaluronan-based hydrogels formulated to include heparin (Heprasil?) with similar gels without heparin (Glycosil?) for their ability to deliver bioactive BMP-2 in vitro and in vivo. The osteogenic activity of BMP-2 released from the hydrogels was evaluated by monitoring alkaline phosphatase (ALP) activity and SMAD 1/5/8 phosphorylation in mesenchymal precursor cells. The osteoinductive ability of these hydrogels was determined in a rat ectopic bone model by 2D radiography, 3D μ-CT and histological analyses at 8 weeks post-implantation. Both hydrogels sustain the release of BMP-2. Importantly, the inclusion of a small amount of heparin (0.3% w/w) attenuated release of BMP-2 and sustained its osteogenic activity for up to 28 days. In contrast, hydrogels lacking heparin released more BMP-2 initially but were unable to maintain BMP-2 activity at later time points. Ectopic bone-forming assays using transplanted hydrogels emphasized the therapeutic importance of the initial burst of BMP-2 rather than its long-term osteogenic activity. Thus, tuning the burst release phase of BMP-2 from hydrogels may be advantageous for optimal bone formation.     

BMP stimulation of alkaline phosphatase activity in pluripotent mouse C2C12 cells is inhibited by dermatopontin, one of the most abundant low molecular weight proteins in demineralized bone matrix

Demineralized bone matrix (DBM) is a complex mixture of osteoinductive bone morphogenetic proteins (BMPs), as well as BMP-binding proteins that regulate BMP bioactivity and localization. Our aim was to use modern proteomic methods to identify additional BMP-binding proteins in DBM, with initial emphasis on the most abundant. Relatively large, water-soluble noncollagenous proteins (NCPs) were preferentially extracted from DBM with alkalinized urea. The insoluble residue, which contained the BMP activity, was extracted with GuHCl/CaCl2, dialyzed versus citrate, defatted, resuspended in GuHCl, dialyzed sequentially against Triton X-100 and water, pelleted, and lyophilized. The proteins in this pellet were fractionated by hydroxyapatite affinity chromatography. Proteins that copurified with BMP bioactivity were separated by SDS-PAGE. Distinct bands were excised, and the proteins in them were reduced and alkylated, digested with trypsin, eluted, and subjected to MALDI/ToF MS (matrix-assisted laser-desorption ionization time-of-flight mass spectrometry). Computer-assisted peptide fingerprint analysis of the MS profiles was used to identify C-terminal lysine-6-oxidase; dermatopontin (DPT); histones H2A2, H2A3, and H2B; and trace amounts of gamma-actin. DPT is a 22-kDa, tyrosine-rich acidic matrix protein not previously recognized to be among the most abundant small proteins to copurify with BMP bioactivity in DBM. We tested the effects of DPT on BMP-2 stimulation of alkaline phosphatase (ALP) activity in C2C12 cells. BMP-2 stimulated ALP activity in C2C12 cells by 6.2-fold above basal levels. DPT alone had no effect on ALP activity in C2C12 cells. When added with BMP-2, DPT blocked 40% of the stimulatory effect of BMP-2 on ALP activity in C2C12 cells. DPT is an abundant protein in DBM, and it can inhibit the stimulatory effects of BMP-2 on ALP activity in C2C12 cells.