Amount Of Bone Morphogenetic Protein-2 Research Articles

Delivery of RANKL-Binding Peptide OP3-4 Promotes BMP-2–Induced Maxillary Bone Regeneration

Although bone morphogenetic protein 2 (BMP-2) is known to stimulate osteogenesis, there is evidence that high doses of BMP-2 can lead to side effects, including inflammation and carcinogenesis. The supplementation of other bone-augmenting agents is considered helpful in preventing such side effects by reducing the amount of BMP-2 required to obtain a sufficient amount of bone. We recently showed that a receptor activator of nuclear factor κB ligand (RANKL)–binding peptide promotes osteoblast differentiation. In the present study, we aimed to investigate whether OP3-4, a RANKL-binding peptide, promotes BMP-2–induced bone formation in the murine maxilla using an injectable gelatin hydrogel (GH) carrier. A GH carrier containing OP3-4 with BMP-2 was subperiosteally injected into the murine maxillary right diastema between the incisor and the first molar. The mice were sacrificed 28 d after the injections. The local bone formation in the OP3-4-BMP-2–injected group was analyzed in comparison to the carrier-injected, BMP-2–injected, and control-peptide-BMP-2–injected groups. The GH carrier containing OP3-4 with BMP-2 enlarged the radio-opaque area and increased the bone mineral content and density in the radiological analyses in comparison to the other experimental groups. Interestingly, fluorescence-based histological analyses revealed that the mineralization had started from the outside, then proceeded inward, suggesting that the size of the newly formed bone had already been set before calcification started and that the effects of OP3-4 might be involved in accelerating the early steps of osteogenesis. Actually, OP3-4 enhanced the BMP-2–induced 5-bromo-2′-deoxyuridine (BrdU)–positive cell numbers at the injected site on day 7 and the expression of Runx2 and Col1a1, which are early osteogenic cell markers, on day 10 after the subperiosteal injections. In summary, we demonstrated, for the first time, that the application of OP3-4 by subperiosteal injection promoted BMP-2–induced bone formation, which could lead to the development of an easy and noninvasive means of promoting alveolar ridge formation.

Induced differentiation of mesencymal precursor cells in hyaluronan hydrogel with covalently linked bisphosphonates

Event Abstract Back to Event Induced differentiation of mesencymal precursor cells in hyaluronan hydrogel with covalently linked bisphosphonates Dmitri Ossipov1*, Sujit Kootala1*, Yu Zhang1*, Vladimir Tolmachev2* and Jöns Hilborn1* 1 Uppsala University, Chemistry-Ångström Laboratory, Sweden 2 Uppsala University, Unit of Biomedical Radiation Sciences, Sweden Introduction: Repair or replacement of damaged tissues is often reliant on the ability of a biomaterial to control the delivery of growth factors[1]. Specific affinity interactions, which are engineered between the proteins and different matrices[2], exhibited capability to tune the rate of release while maintaining consistent levels of bioactivity. In this work we present an in situ cross-linkable hydrogel system that permits manipulation of entrapped cells in 3-D with the help of bisphosphonate (BP) ligands attached to the hyaluronic acid (HA) matrix and hence providing effective retention of bone morphogenetic protein-2 (BMP-2) in the matrix. Specifically, we demonstrate that (1) linking of predetermined amounts of BP ligands to HA matrix provide tailored degrees of BMP-2 release and that (2) BMP-2 sequestration caused by BPs provide a stable microenvironment for 3D entrapment of C2C12 myogenic progenitor cells in the hydrogel and promote their osteogenic differentiation (Fig. 1). Fig. 1. In situ encapsulation of myotube forming cells and BMP-2 in hyaluronic acid hydrogel to induce 3D differentiation of the cells through the controlled retention and release of the growth factor by the matrix-linked bisphosphonate ligands. Materials and Methods: HA-BP-SH derivative was synthesized by thiolation of the previously described HA-BP derivative[3] with 3,3'-dithiobis(propionic hydrazide). Results and Discussion: Three HA derivatives carrying 2-dithiopyridyl (HA-SSPy), thiol (HA-SH), and bisphosphonate and thiol groups (HA-BP-SH) were prepared to form two types of hydrogels by thiol-disulfide exchange cross-linking reaction[3]. The hydrogels were in situ formed by mixing of either HA-SSPy and HA-SH (giving control HA network) or HA-SSPy and HA-BP-SH (giving bisphosphonated HABP network). In both hydrogels, HA chains were cross-linked by disulfide bonds and equal cross-linking density was ensured by the same degree of substitution with the functional groups in all three derivatives. Loading of BMP-2 into the hydrogels was achieved in situ by pre-dissolving the growth factor in HA-SSPy solution. We observed that the release of BMP-2 from HA hydrogel was faster than from the HABP matrix. Moreover, the rate of the growth factor release could be tuned by changing the amount of BP groups in the matrix which was easily achieved by combination of HA-SH and HA-BP-SH components at different mass ratios. Next, C2C12 cells were encapsulated in the hydrogels together with the equal amount of BMP-2 and the obtained constructs were cultured for 5 days. Expression of alkaline phosphatase (ALP), a major late-stage osteogenic differentiation marker, was then measured. A 5-fold increase in ALP expression was detected for the cells cultured 3-D in HABP hydrogels as compared to HA hydrogels (Fig. 2). Fig. 2. ALP production by C2C12 cells cultured for 5 days in different hydrogels. Basing on the release results, we propose that the cells were exposed to BMP-2 for longer time and at higher concentrations in HABP hydrogel than in HA hydrogel. The retention of BMP-2 in HABP hydrogel was attributed to BP groups that bind and protect the growth factor. Conclusion: We present a novel hydrogel delivery system enabling sequestration of BMP-2 basing on use of in situ cross-linkable HA-BP conjugate. Application of this conjugate in combination with BMP-2 to cells in 3-D allows the cells to differentiate in osteogenic direction.

Accelerated bony defect healing by chitosan/silica hybrid membrane with localized bone morphogenetic protein-2 delivery

Therapeutic agents such as growth factors have been widely applied to promote bone regeneration with implantable biomaterials. In this study, a hybrid of chitosan–xerogel membrane was combined with bone morphogenetic protein-2 (BMP-2) for enhancing bone regeneration. The characteristics of the hybrid membrane as a carrier of BMP-2 were compared with that of the pure chitosan membrane, and the effect of combining the hybrid membrane with BMP-2 on its bone healing ability was evaluated using in vitro and in vivo animal tests. The amount of BMP-2 adsorbed on the pure chitosan membrane was less than that on the hybrid membrane and, therefore, the cellular behaviors of the pure chitosan membrane were only slightly improved by the protein treatment. In contrast, the hybrid membrane showed excellent affinity toward BMP-2, and the adsorption of BMP-2 elicited a dramatic improvement in the cellular responses of the hybrid membrane. Moreover, the in vivo study demonstrated that bone regeneration was significantly enhanced when using the hybrid membrane with BMP-2 as compared to the hybrid membrane without BMP-2. These results suggest that the hybrid membrane is very efficient in carrying BMP-2 and accelerates the bone healing process through the successful delivery of BMP-2 and its osteoconductive composition.

Polyelectrolyte Complex Carrier Enhances Therapeutic Efficiency and Safety Profile of Bone Morphogenetic Protein-2 in Porcine Lumbar Interbody Fusion Model.

Porcine lumbar interbody fusion model. This study evaluates the effect of polyelectrolyte complex (PEC) carrier in enhancing the therapeutic efficiency and safety profile of bone morphogenetic protein-2 (BMP-2) in a large animal model. Extremely large amounts of BMP-2 are administered to achieve consistent spinal fusion, which has led to complications. Heparin-modified PEC carrying reduced BMP-2 doses of 0.5 μg was demonstrated to achieve consistent spinal fusion with reduction of complications in rodent model. The purpose of this study was to evaluate whether PEC could improve the therapeutic efficiency of BMP-2 in porcine model. Three-segment (L3-L6) anterior lumbar interbody fusions with instrumentation were performed on 6 pigs using 3 different doses of BMP-2, namely, (1) 50 μg, (2) 150 μg, and (3) 300 μg. The BMP-2 was delivered using heparin-modified alginate microbeads loaded into biodegradable cage. Fusion performance was evaluated after 3 months. Manual palpation and micro-computed tomography showed consistent fusion in all experimental groups. Heterotopic bone formation beyond the cage implant area was more evident in group 2 and group 3 than in group 1. Similarly, superior bone microstructure was observed in the new bone with the lowered BMP-2 dose. Biomechanical evaluation revealed enhanced stiffness of the operated segments compared with nonoperated segments (P < 0.05). Mechanical stability was maintained despite dose reduction of BMP-2. Although the mineral apposition rate was higher in group 3, unsatisfactory bony microstructure with decreased trabecular number was observed in group 3 compared with group 1. PEC carrying low doses of BMP-2 achieved consistent interbody fusion. We observed dose-related reduction in heterotopic ossification without compromising the stability of the fused segments. PEC carrier reduces the efficacious doses of BMP-2. This could enhance the safety profile of BMP-2 and reduce dose- and carrier-related complications. N/A.

Macrophages support splenic erythropoiesis in 4T1 tumor-bearing mice.

Anemia is a common complication of cancer; a role of spleen in tumor-stress erythropoiesis has been suggested. However, the molecular mechanisms involved in the splenic erythropoiesis following tumor maintenance remain poorly understood. Here we show that tumor development blocks medullar erythropoiesis by granulocyte colony-stimulating factor (G-CSF) and then causes anemia in murine 4T1 breast tumor-bearing mice. Meanwhile, tumor-stress promotes splenic erythropoiesis. Splenectomy worsened tumor-induced anemia, and reduced tumor volume and tumor weight, indicating the essential role of spleen in tumor-stress erythropoiesis and tumor growth. Tumor progression of these mice led to increased amounts of bone morphogenetic protein 4 (BMP4) in spleen. The in vivo role of macrophages in splenic erythropoiesis under tumor-stress conditions was investigated. Macrophage depletion by injecting liposomal clodronate decreased the expression of BMP4, inhibited splenic erythropoiesis, aggravated the tumor-induced anemia and suppressed tumor growth. Our results provide insight that macrophages and BMP4 are positive regulators of splenic erythropoiesis in tumor pathological situations. These findings reveal that during the tumor-stress period, the microenvironment of the spleen is undergoing changes, which contributes to adopt a stress erythropoietic fate and supports the expansion and differentiation of stress erythroid progenitors, thereby replenishing red blood cells and promoting tumor growth.

Myoconductive and osteoinductive free-standing polysaccharide membranes

Free-standing (FS) membranes have increasing applications in the biomedical field as drug delivery systems for wound healing and tissue engineering. Here, we studied the potential of free-standing membranes made by the layer-by-layer assembly of chitosan and alginate to be used as a simple biomimetic system of the periosteum. The design of a periosteum-like membrane implies the elaboration of a thick membrane suitable for both muscle and bone formation. Our aim was to produce well-defined ∼50μm thick polysaccharide membranes that could be easily manipulated, were mechanically resistant, and would enable both myogenesis and osteogenesis in vitro and in vivo. The membranes were chemically crosslinked to improve their mechanical properties. Crosslinking chemistry was followed via Fourier transform infrared spectroscopy and the mechanical properties of the membranes were assessed using dynamic mechanical analysis. The loading and release of the potent osteoinductive growth factor bone morphogenetic protein 2 (BMP-2) inside and outside of the FS membrane was followed by fluorescence spectroscopy in a physiological buffer over 1month. The myogenic and osteogenic potentials of the membranes in vitro were assessed using BMP-2-responsive skeletal myoblasts. Finally, their osteoinductive properties in vivo were studied in a preliminary experiment using a mouse ectopic model. Our results showed that the more crosslinked FS membranes enabled a more efficient myoblast differentiation in myotubes. In addition, we showed that a tunable amount of BMP-2 can be loaded into and subsequently released from the membranes, depending on the crosslinking degree and the initial BMP-2 concentration in solution. Only the more crosslinked membranes were found to be osteoinductive in vivo. These polysaccharide-based membranes have strong potential as a periosteum-mimetic scaffold for bone tissue regeneration.

Endothelial microparticles mediate inflammation-induced vascular calcification.

Stimulation of endothelial cells (ECs) with TNF-α causes an increase in the expression of bone morphogenetic protein-2 (BMP-2) and the production of endothelial microparticles (EMPs). BMP-2 is known to produce osteogenic differentiation of vascular smooth muscle cells (VSMCs). It was found that EMPs from TNF-α-stimulated endothelial cells (HUVECs) contained a significant amount of BMP-2 and were able to enhance VSMC osteogenesis and calcification. Calcium content was greater in VSMCs exposed to EMPs from TNF-α-treated HUVECs than EMPs from nontreated HUVECs (3.56 ± 0.57 vs. 1.48 ± 0.56 µg/mg protein; P < 0.05). The increase in calcification was accompanied by up-regulation of Cbfa1 (osteogenic transcription factor) and down-regulation of SM22α (VSMC lineage marker). Inhibition of BMP-2 by small interfering RNA reduced the VSMC calcification induced by EMPs from TNF-α-treated HUVECs. Similar osteogenic capability was observed in EMPs from both patients with chronic kidney disease and senescent cells, which also presented a high level of BMP-2 expression. Labeling of EMPs with CellTracker shows that EMPs are phagocytized by VSMCs under all conditions (with or without high phosphate, control, and EMPs from TNF-α-treated HUVECs). Our data suggest that EC damage results in the release of EMPs with a high content of calcium and BMP-2 that are able to induce calcification and osteogenic differentiation of VSMCs.

The BMP2 antagonist inhibitor L51P enhances the osteogenic potential of BMP2 by simultaneous and delayed synergism

Bone morphogenetic protein 2 (BMP2) is a potent osteoinductive cytokine that plays crucial roles in bone repair. However, large amounts of BMP2 are required to induce sufficient bone formation in humans possibly due to a feedback response of BMP antagonists. The engineered BMP2 variant L51P is deficient in BMP receptor type I activation but maintains affinity for BMP antagonists and can allow for the inactivation of BMP antagonists, and eventually enhance BMP2 action. As hypothesized, simultaneous addition of L51P enhanced the BMP2-induced osteogenesis. To test the ability of L51P to competitively inactivate BMP antagonists, cell binding affinity of BMP2 ligands was investigated in the presence or absence of L51P. Because the BMP antagonists were highly expressed 3days after exogenous BMP2 stimulation, we collected supernatants from 3-day stimulated cell cultures and used as condition culture media (CM). The results showed a significant decrease in the cell binding of BMP2 ligands when cells were incubated with exogenous BMP2 and CM, whereas L51P addition competitively rescued the suppression of BMP2-to-cell binding induced by CM incubation. In a delayed experimental model, L51P was applied 3days after exogenous BMP2 stimulation and we could observe a striking enhancement of the BMP2-induced SMAD-1/5/8 phosphorylation and luciferase activity of the Id1 promoter compared to the simultaneous addition of the two factors. These findings provide a deeper insight into the cellular and molecular mechanisms involved in the effect of L51P in suppressing the BMP antagonists and enhancing BMP activity. Additionally, these results demonstrate that L51P is a promising down regulator of BMP-induced negative feedback, which could have a significant impact in future applications of BMP2 in research and clinical settings.

Chitosan–hyaluronic acid polyelectrolyte complex scaffold crosslinked with genipin for immobilization and controlled release of BMP-2

Polyelectrolyte complex (PEC) is formed when polymers with opposite charges are combined in solution. PECs are recently gaining attention as carriers for controlled release of drugs and proteins. Herein, bone morphogenetic protein-2 (BMP-2) was immobilized in a PEC of natural polymers, chitosan and hyaluronic acid. Charge-to-charge stoichiometry of the formed PEC was estimated based on turbidity of combined chitosan and hyaluronic acid solutions. Free amino groups in chitosan were crosslinked with different amounts of genipin. The degree of crosslinking, consequently its effects in vitro in terms of swelling, degradation and cytocompatibility were analyzed. Immobilization of three different amount of BMP-2 in chitosan–hyaluronic acid PEC scaffold resulted sustained release of the growth factor for more than 30 days. Immobilization efficacies varied from 61% to 76% depending on the amount of BMP-2. Finally effects in osteogenic differentiation of the PEC with BMP-2 to MC3T3-E1 cells were determined by reverse transcriptase PCR.

Mesenchymal stem cells ameliorate diabetic glomerular fibrosis in vivo and in vitro by inhibiting TGF-β signalling via secretion of bone morphogenetic protein 7.

To investigate whether mesenchymal stem cells (MSCs) could inhibit transforming growth factor beta (TGF-β) signalling pathway by paracrine action. Bone marrow-derived MSCs were transplanted to streptozotocin-induced diabetic rats via tail vein. MSC-conditioned media were used with a model of mesangial cell fibrosis induced by high glucose in vitro. At 8 weeks after MSC treatment, the renal function and the glomerulosclerosis as revealed by periodic acid Schiff stain was dramatically attenuated. The expression of collagen I, collagen IV and α-smooth muscle actin (SMA) in diabetic kidney was decreased, and E-cadherin increased after MSC treatment. The TGF-β signalling pathway was suppressed both in vivo and in vitro. MSCs secreted a significant amount of bone morphogenetic protein 7 (BMP7), in vitro, MSC-conditioned media inhibited TGF-β signalling stimulated by high glucose, and BMP7 neutralizing antibody blocked the inhibitory effect of MSC-conditioned media. MSCs ameliorated glomerular fibrosis in vivo and in vitro by inhibiting TGF-β/Smad signalling pathway via secretion of BMP7.

Low-Dose Bone Morphogenetic Protein-2/Stromal Cell-Derived Factor-1β Cotherapy Induces Bone Regeneration in Critical-Size Rat Calvarial Defects

Increasing evidence suggests that stromal cell-derived factor-1 (SDF-1/CXCL12) is involved in bone formation, though underlying molecular mechanisms remain to be fully elucidated. Also, contributions of SDF-1β, the second most abundant splice variant, as an osteogenic mediator remain obscure. We have shown that SDF-1β enhances osteogenesis by regulating bone morphogenetic protein-2 (BMP-2) signaling in vitro. Here we investigate the dose-dependent contribution of SDF-1β to suboptimal BMP-2-induced local bone formation; that is, a dose that alone would be too low to significantly induce bone formation. We utilized a critical-size rat calvarial defect model and tested the hypotheses that SDF-1β potentiates BMP-2 osteoinduction and that blocking SDF-1 signaling reduces the osteogenic potential of BMP-2 in vivo. In preliminary studies, radiographic analysis at 4 weeks postsurgery revealed a dose-dependent relationship in BMP-2-induced new bone formation. We then found that codelivery of SDF-1β potentiates suboptimal BMP-2 (0.5 μg) osteoinduction in a dose-dependent order, reaching comparable levels to the optimal BMP-2 dose (5.0 μg) without apparent adverse effects. Blocking the CXC chemokine receptor 4 (CXCR4)/SDF-1 signaling axis using AMD3100 attenuated the osteoinductive potential of the optimal BMP-2 dose, confirmed by qualitative histologic analysis. In conclusion, SDF-1β provides potent synergistic effects that support BMP-induced local bone formation and thus appears a suitable candidate for optimization of bone augmentation using significantly lower amounts of BMP-2 in spine, orthopedic, and craniofacial settings.

The effect of bone membrane on secretion of BMP-2 and bone healing in a rabbit model of bone defect

Objective To investigate the effect of bone membrane on secretion of bone morphogenetic protein-2(BMP-2) and bone healing in a rabbit model of bone defect.Methods Forty-eight New Zealand rabbits (4 months old,half male and half female) were randomly divided into 2 equal groups (n =24).A 0.5 cm bone defect of middle radius was created operatively under anesthesia in all the animals.The bone membrane around the defect was preserved in group A but exsected in group B.Six rabbits in each group were sacrificed each time at immediate,1,3 and 4 weeks post-operation.X-ray films of the bone defects were taken,gross observation of the tissue samples from the defect site was made,and semiquantitative detection of BMP-2 content by Western blotting was conducted at each time point.Quantitative assessment of bone healing was performed using Lane-Sandhu X-ray evaluation system.Results In group A,the amount of BMP-2 increased gradually at l,3,4 weeks postoperation,significantly higher than that at immediate postoperation (P ＜ 0.05).In group B,the amount of BMP-2 peaked at one week postoperation,significantly higher than that at immediate postoperation (P ＜ 0.05).At one week postoperation,there was no significant difference between the 2 groups in the amount of BMP-2 (P ＞ 0.05).At 3 and 4 weeks postoperation,the amount of BMP-2 in group A was significantly higher than in group B (P ＜ 0.05) and the mean Lane-Sandhu X-ray score in group A was also significantly higher than in group B (P ＜ 0.05).Conclusion Since preservation of bone membrane increases secretion of BMP-2 and promotes bone healing in the healing process of bone defect,we should try to preserve bone membrane when bone defects are treated. Key words: Bone morphogenetic proteins; Bone membrane; Bone healing; Bone defect

Long-term controlled release of 125I-tagged BMP-2 by mesoporous bioactive glass with ordered nanopores

The aim of this study was to investigate the ability of mesoporous bioactive glass with ordered nanopores (80S MBG) to adsorb and provide the delayed release of 125I-tagged bone morphogenetic protein-2 (BMP-2). A 50 mg piece of 80S MBG was produced, which comprised SiO2, CaO and P2O5 in a component molar ratio of 80:15:5. Each MBG piece adsorbed 30 μg 125I-BMP-2. Persistent radioactivity in the MBG was periodically measured in simulated body fluid. The total amount of BMP-2 released and the mean amount released per day were calculated. A delayed release curve of BMP-2 was constructed. SPSS 15.0 software was used to perform a statistical analysis. The amount of BMP-2 released in the first two days was one-quarter of the total load. A line equation, y = 490.55×1/2 + 7268.82, was obtained from the square root of protein release doses value at 3–94 days. The total amount of BMP-2 released over 94 days was 11.894 μg, which was ~39.6% of the total load. The half-life of the release time was 248 days. From the second week, the rate of BMP release had stabilized to a mean of 37.42±18.67 ng/day and the difference of the mean amount released per day had no statistical significance (P>0.05). High adsorption and delayed release effects of BMP-2 were observed in 80S MBG. The delayed release conforms to the Higuchi equation, which indicates possible applications in promoting bone healing.

Dual Delivery of rhPDGF-BB and Bone Marrow Mesenchymal Stromal Cells Expressing the BMP2 Gene Enhance Bone Formation in a Critical-Sized Defect Model

Bone tissue healing is a dynamic, orchestrated process that relies on multiple growth factors and cell types. Platelet-derived growth factor-BB (PDGF-BB) is released from platelets at wound sites and induces cellular migration and proliferation necessary for bone regeneration in the early healing process. Bone morphogenetic protein-2 (BMP-2), the most potent osteogenic differentiation inducer, directs new bone formation at the sites of bone defects. This study evaluated a combinatorial treatment protocol of PDGF-BB and BMP-2 on bone healing in a critical-sized defect model. To mimic the bone tissue healing process, a dual delivery approach was designed to deliver the rhPDGF-BB protein transiently during the early healing phase, whereas BMP-2 was supplied by rat bone marrow stromal cells (BMSCs) transfected with an adenoviral vector containing the BMP2 gene (AdBMP2) for prolonged release throughout the healing process. In in vitro experiments, the dual delivery of rhPDGF-BB and BMP2 significantly enhanced cell proliferation. However, the osteogenic differentiation of BMSCs was significantly suppressed even though the amount of BMP-2 secreted by the AdBMP2-transfected BMSCs was not significantly affected by the rhPDGF-BB treatment. In addition, dual delivery inhibited the mRNA expression of BMP receptor type II and Noggin in BMSCs. In in vivo experiments, critical-sized calvarial defects in rats showed enhanced bone regeneration by dual delivery of autologous AdBMP2-transfected BMSCs and rhPDGF-BB in both the amount of new bone formed and the bone mineral density. These enhancements in bone regeneration were greater than those observed in the group treated with AdBMP2-transfected BMSCs alone. In conclusion, the dual delivery of rhPDGF-BB and AdBMP2-transfected BMSCs improved the quality of the regenerated bone, possibly due to the modulation of PDGF-BB on BMP-2-induced osteogenesis.

Retroviral-mediated overexpression of human bone morphogenetic protein 2 affects human mesenchymal stem cells during monolayer proliferation: A cautionary note

Background: Retroviral vectors are commonly used for gene transfer applications and they represent an effective way to provide a sustained delivery of a bioactive factor in basic research and tissue engineering applications. Cells that have been transduced with retroviral vectors ex vivo are usually amplified on tissue culture plastic, for a prolonged period of time, in order to obtain sufficient cell numbers prior to the experiment of interest. However, the effect of the transgene product on the transduced cells, during this period of time, is rarely, if ever, investigated. The current study investigated if transduction with a VSG.G pseudotyped retroviral vector expressing human bone morphogenetic protein 2 influences the gene expression profile of human bone marrow-derived mesenchymal stem cells during monolayer proliferation. MSCs that have been transduced with a VSG.G pseudotyped retroviral vector expressing enhanced green fluorescent protein served as controls. Results: It was confirmed that Rv.BMP-2 transduced hMSCs produce detectable amounts of bone morphogenetic protein 2. Gene expression analysis revealed that the hypertrophic marker collagen X was down-regulated by approximately 50% and the chondrogenic marker Aggrecan was elevated almost 9-fold in Rv.BMP-2 transduced hMSCs if compared to Rv.eGFP transduced control cells. Interestingly, the same changes in gene expression were detected when hMSCs were exposed to 100 ng/ml of recombinant human BMP-2 and their gene expression profile was compared to control hMSC. Again, collagen X message was down-regulated and Aggrecan message was up-regulated. Conclusion: These results indicate that, when using integrating vectors and then expanding the cells after transduction, controls need to be carefully planned to ensure the results obtained during the 3D experiments are not due to artefacts created in response to the different cell proliferation conditions employed.

Minimally invasive mandibular bone augmentation using injectable hydrogels

Hyaluronic acid-based hydrogels are proven biocompatible materials and excellent carriers of bone morphogenetic protein-2 (BMP-2) that have been successfully tested for bone generation in vivo. Different formulations, with or without nanohydroxyapatite, have shown promise for craniofacial applications. In this study, 28 rats were used to investigate whether it is possible to achieve mandibular bone augmentation upon injection of novel hyaluronic acid-based hydrogels containing nanohydroxyapatite and different concentrations of BMP-2 (0, 5 and 150 µg/ml). The biomaterials were injected subperiosteally through fine needles into the innate mandibular diastema, imitating a clinical procedure for resorbed mandibles. No incisions, flaps or sutures were necessary. After 8 weeks the mandibles were evaluated by peripheral quantitative computed tomography (pQCT), micro-computed tomography (μCT), histology, immunohistochemistry and fluorochrome labelling. As a result, engineered bone was observed in all treated mandibles, with a statistically significant increase in mandibular bone volume correlated with the amount of BMP-2 loaded in the hydrogel formula. We therefore demonstrated that minimally invasive mandibular bone augmentation is possible upon injection in rats, when using the appropriate injectable scaffolds. This represents an attractive clinical alternative for oral implantology patients.

Bone marrow-derived heparan sulfate potentiates the osteogenic activity of bone morphogenetic protein-2 (BMP-2)

Lowering the efficacious dose of bone morphogenetic protein-2 (BMP-2) for the repair of critical-sized bone defects is highly desirable, as supra-physiological amounts of BMP-2 have an increased risk of side effects and a greater economic burden for the healthcare system. To address this need, we explored the use of heparan sulfate (HS), a structural analog of heparin, to enhance BMP-2 activity. We demonstrate that HS isolated from a bone marrow stromal cell line (HS-5) and heparin each enhances BMP-2-induced osteogenesis in C2C12 myoblasts through increased ALP activity and osteocalcin mRNA expression. Commercially available HS variants from porcine kidney and bovine lung do not generate effects as great as HS5. Heparin and HS5 influence BMP-2 activity by (i) prolonging BMP-2 half-life, (ii) reducing interactions between BMP-2 with its antagonist noggin, and (iii) modulating BMP2 distribution on the cell surface. Importantly, long-term supplementation of HS5 but not heparin greatly enhances BMP-2-induced bone formation in vitro and in vivo. These results show that bone marrow-derived HS effectively supports bone formation, and suggest its applicability in bone repair by selectively facilitating the delivery and bioavailability of BMP-2.

In vitroevaluation of an injectable chitosan gel for sustained local delivery of BMP‐2 for osteoblastic differentiation

We investigated the effect of sustained release of bone morphogenetic protein-2 (BMP-2) from an injectable chitosan gel on osteoblastic differentiation in vitro. We first characterized the release profile of BMP-2 from the gels, and then examined the cellular responses of preosteoblast mouse stromal cells (W-20-17) and human embryonic palatal mesenchymal (HEPM) cells to BMP-2. The release profiles of different concentrations of BMP-2 exhibited sustained releases (41% for 2 ng/mL and 48% for 20 ng/mL, respectively) from the chitosan gels over a three-week period. Both cell types cultured in the chitosan gels were viable and significantly proliferated for 3 days (p < 0.05). Chitosan gels loaded with BMP-2 enhanced ALP activity of W-20-17 by 3.6-fold, and increased calcium mineral deposition of HEPM by 2.8-fold at 14 days of incubation, compared to control groups initially containing the same amount of BMP-2. In addition, schitosan gels loaded with BMP-2 exhibited significantly greater osteocalcin synthesis of W-20-17 at seven days, and of HEPM at both 7 and 14 days compared with the control groups (p<0.05). This study suggests that the enhanced effects of BMP-2 released from chitosan gels on cell differentiation and mineralization are species and cell type dependent.

Amino-modified silica surfaces efficiently immobilize bone morphogenetic protein 2 (BMP2) for medical purposes

Due to its ability to induce de novo bone formation the differentiation factor bone morphogenetic protein 2 (BMP2) is often used to enhance the integration of bone implants. With the aim of reducing possible high dose side-effects and to lower the costs, in order to produce affordable implants, we developed a simple and fast method for the immobilization of BMP2 on silica-based surfaces using silane linkers which carry amino or epoxy functions. We put an especial emphasis on the influence of the nanoscale surface topography of the silica layer. Therefore, we chose glass (for control experiments) and Bioverit® II (as a typical implant base material) as support materials and coated these substrates with unstructured or nanoporous amorphous silica layers for comparison. Immobilized BMP2 was quantified by two different methods: by ELISA and by a cell-based assay for active BMP2. These tests probe for immunologically and biologically active BMP2, respectively. The results show that the amino functionalization is better suited for immobilizing the protein. Strikingly, a considerably higher amount of BMP2 could be immobilized on coated Bioverit® II surfaces compared with coated glass substrates, which was presumably due to the macroscopic roughness of the Bioverit® II substrates. In addition, it was found that the nanoporous silica coatings on Bioverit® II substrates were able to bind more BMP2 than the unstructured ones.

Amniotic Fluid-Derived Mesenchymal Stem Cells Lead to Bone Differentiation when Cocultured with Dental Pulp Stem Cells

Mesenchymal stem cells are present in many tissues of the human body, including amniotic fluid (AF) and dental pulp (DP). Stem cells of both AF and DP give rise to a variety of differentiated cells. In our experience, DP stem cells (DPSCs) display a high capacity to produce bone. Therefore, our aim was to investigate if AF-derived stem cells (AFSCs) were able to undergo bone differentiation in the presence of DPSCs. AFSCs were seeded under three different conditions: (i) cocultured with DPSCs previously differentiated into osteoblasts; (ii) cultured in the conditioned medium of osteoblast-differentiated DPSCs; (iii) cultured in the osteogenic medium supplemented with vascular endothelial growth factor and bone morphogenetic protein-2 (BMP-2). Results showed that AFSCs were positive for mesenchymal markers, and expressed high levels of Tra1-60, Tra1-80, BMPR1, BMPR2, and BMP-2. In contrast, AFSCs were negative for epithelial and hematopoietic/endothelial markers. When AFSCs were cocultured with DPSCs-derived osteoblasts, they differentiated into osteoblasts. A similar effect was observed when AFSCs were cultured in the presence of a conditioned medium originated from DPSCs. We found that osteoblasts derived from DPSCs released large amounts of BMP-2 and vascular endothelial growth factor into the culture medium and that those morphogens significantly upregulate RUNX-2 gene, stimulating osteogenesis. This study highlights the mechanisms of osteogenesis and strongly suggests that the combination of AFSCs with DPSCs may provide a rich source of soluble proteins useful for bone engineering purposes.