BMP6 binding to heparin and heparan sulfate is mediated by N-terminal and C-terminal clustered basic residues.

BACKGROUND: The bone morphogenetic protein 6 (BMP6) is a crucial inducer of hepcidin, the peptide hormone that regulates the iron availability in our body. Hepcidin expression is influenced by hepatic heparan sulfate (HS) and by heparin administration, suggesting BMP6 interaction with heparin/HS. The BMP2/4 subfamily has been deeply characterized to have a N-terminal heparin/HS binding domain (HBD), whose basic residues contact the sulfate groups on heparin and HS. Such detailed characterization is still required for other, structurally different BMPs, including BMP6. METHODS: BMP6 peptides encompassing potential HBDs were analysed on heparin-functionalized plates and microcantilevers, and on membrane HS expressing CHO-K1 cells. Monomeric wild-type BMP6 and mutants were produced, substituting the basic residues with non-charged ones, and their affinity to the heparin-column was measured. The BMP6-heparin interaction was also predicted at atomic level by in silico molecular dynamics. RESULTS: N-terminal and C-terminal BMP6 peptides showed high heparin affinity in solid-phase assays. The mutation of the two sites (R5L, R6S, R7L and K126N, K127N, R129S) abolished the heparin-binding activity of the recombinant monomeric BMP6. Monomeric BMP6 and peptides specifically bound to membrane HS of CHO-K1 cells through the same domains. Molecular dynamic studies supported the role of the two HBDs, suggesting a cooperative behaviour. CONCLUSIONS: In BMP6, N-terminal (R5, R6, R7) and C-terminal (K126, K127, R129) domains mediate the interaction with heparin and HS. GENERAL SIGNIFICANCE: This study provides the molecular mechanism supporting the use of heparin to sequester BMP6 and inhibit hepcidin expression, a novel clinical approach for high-hepcidin iron disorders.

Copresentation of BMP-6 and RGD Ligands Enhances Cell Adhesion and BMP-Mediated Signaling.

We report on the covalent immobilization of bone morphogenetic protein 6 (BMP-6) and its co-presentation with integrin ligands on a nanopatterned platform to study cell adhesion and signaling responses which regulate the transdifferentiation of myoblasts into osteogenic cells. To immobilize BMP-6, the heterobifunctional linker MU-NHS is coupled to amine residues of the growth factor; this prevents its internalization while ensuring that its biological activity is maintained. Additionally, to allow cells to adhere to such platform and study signaling events arising from the contact to the surface, we used click-chemistry to immobilize cyclic-RGD carrying an azido group reacting with PEG-alkyne spacers via copper-catalyzed 1,3-dipolar cycloaddition. We show that the copresentation of BMP-6 and RGD favors focal adhesion formation and promotes Smad 1/5/8 phosphorylation. When presented in low amounts, BMP-6 added to culture media of cells adhering to the RGD ligands is less effective than BMP-6 immobilized on the surfaces in inducing Smad complex activation and in inhibiting myotube formation. Our results suggest that a local control of ligand density and cell signaling is crucial for modulating cell response.

Hyaluronan hydrogels delivering BMP-6 for local targeting of malignant plasma cells and osteogenic differentiation of mesenchymal stromal cells.

Multiple myeloma is a malignant disease characterized by accumulation of clonal plasma cells in the bone marrow. Uncoupling of bone formation and resorption by myeloma cells leads to osteolytic lesions. These are prone to fracture and represent a possible survival space for myeloma cells under treatment causing disease relapse. Here we report on a novel approach suitable for local treatment of multiple myeloma based on hyaluronic acid (HA) hydrogels mimicking the physical properties of the bone marrow. The HA hydrogels are complexed with heparin to achieve sustained presentation and controlled release of bone morphogenetic protein 6 (BMP-6). Others and we have shown that BMP-6 induces myeloma cell apoptosis and bone formation. Using quartz crystal microbalance and enzyme-linked immunosorbent assay, we measured an initial surface density of 400 ng BMP6/cm2, corresponding to two BMP-6 per heparin molecule, with 50% release within two weeks. HA-hydrogels presenting BMP-6 enhanced the phosphorylation of Smad 1/5 while reducing the activity of BMP-6 antagonist sclerostin. These materials induced osteogenic differentiation of mesenchymal stromal cells and decreased the viability of myeloma cell lines and primary myeloma cells. BMP-6 functionalized HA-hydrogels represent a promising material for local treatment of myeloma-induced bone disease and residual myeloma cells within lesions to minimize disease relapse or fractures. STATEMENT OF SIGNIFICANCE: Multiple myeloma is a hematological cancer characterized by the accumulation of clonal plasma cells in the bone marrow and local suppression of bone formation, resulting in osteolytic lesions and fractures. Despite recent advances in systemic treatment of multiple myeloma, it is rare to achieve a targeted suppression of myeloma cells and healing of bone lesions. Here we present hydrogels which mimic the physico-chemical properties of the bone marrow, consisting of hyaluronic acid with crosslinked heparin for the controlled presentation of bioactive BMP-6. The hydrogels decrease the viability of myeloma cell lines and primary myeloma cells and induces osteogenic differentiation of mesenchymal stromal cells. The presentation of BMP-6 in the hyaluronan hydrogels enhances the phosphorylation of Smad1/5 while reducing the activity of the BMP-6 antagonist sclerostin. As such, BMP-6 functionalized hyaluronan hydrogels represent a promising material for the localized eradication of myeloma cells.

Reduction of BMP6-induced bone formation by calcium phosphate in wild-type compared with nude mice.

Nude mice have been extensively used to investigate the potency of tissue engineering strategies for bone repair. However, the contribution of pro-inflammatory and proregenerative stimuli of the host for the process of new bone formation and integration remains poorly understood. In this study, ectopic bone formation was investigated in nude (Nu) versus wild-type (WT) mice. Calcium phosphate (CaP) scaffolds (CopiOs [Zimmer] and Bio-Oss [Geistlich]) were loaded with different concentrations of rhBMP6 (40, 120, and 240 ng/mm3 rhBMP6) and implanted subcutaneously in Nu (BALB/c and NMR1) and WT (BALB/c and c57BL/6) mice. CaP scaffolds loaded with rhBMP6 did not form bone in WT mice. However, in Nu mice, 40 ng/mm3 rhBMP6 was sufficient to generate relevant volumes of new bone at 6 weeks after implantation. Looking into potential underlying mechanisms, TNF-α blocking antibodies were injected intraperitoneally but could not restore bone formation. Also, mouse periosteal cells (mPDCs) seeded in CopiOs loaded with rhBMP6 did not significantly improve the outcome. Abrogation of bone formation was associated with dense cellular infiltration, in particular with the presence of CD3+ T-lymphocytes. To probe a correlation between calcium ions and impaired bone formation in WT mice, type 1 collagen gels were loaded with rhBMP6 and calcium chloride and injected subcutaneously. These gels generated new bone in WT mice despite the increased percentage of CD3+ cells at Day 3 after implantation as compared with control gels. Overall, this study illustrated the negative effect of the inflammatory host response on the bone-forming capacity of rhBMP6 coated on bioceramic scaffolds.

Enhanced selective cellular proliferation by multi-biofunctionalization of medical implant surfaces with heterodimeric BMP-2/6, fibronectin, and FGF-2.

Increasing cell adhesion on implant surfaces is an issue of high biomedical importance. Early colonization with endogenous cells reduces the risk of bacterial contamination and enhances the integration of an implant into the diverse cellular tissues surrounding it. In vivo integration of implants is controlled by a complex spatial and temporal interplay of cytokines and adhesive molecules. The concept of a multi-biofunctionalized TiO2 surface for stimulating bone and soft tissue growth is presented here. All supramolecular architectures were built with a biotin-streptavidin coupling system. Biofunctionalization of TiO2 with immobilized FGF-2 and heparin could be shown to selectively increase the proliferation of fibroblasts while immobilized BMP-2 only stimulated the growth of osteoblasts. Furthermore, TiO2 surfaces biofunctionalized with either the BMP-2 or BMP-2/6 growth factor and the cell adhesion-enhancing protein fibronectin showed higher osteoblast adhesion than a TiO2 surface functionalized with only one of these proteins. In conclusion, the presented immobilization strategy is applicable in vivo for a selective surface coating of implants in both hard and connective tissue. The combined immobilization of different extracellular proteins on implants has the potential to further influence cell-specific reactions. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2910-2922, 2018.

Hydrolytically Labile Linkers Regulate Release and Activity of Human Bone Morphogenetic Protein-6.

Release of growth factors while simultaneously maintaining their full biological activity over a period of days to weeks is an important issue in controlled drug delivery and in tissue engineering. In addition, the selected strategy to immobilize growth factors largely determines their biological activity. Silica surfaces derivatized with glycidyloxy propyl trimethoxysilane and poly(glycidyl methacrylate) brushes yielded epoxide-functionalized surfaces onto which human bone morphogenetic protein-6 (hBMP-6) was immobilized giving stable secondary amine bonds. The biological activity of hBMP-6 was unleashed by hydrolysis of the surface siloxane and ester bonds. We demonstrate that this type of labile bonding strategy can be applied to biomaterial surfaces with relatively simple and biocompatible chemistry, such as siloxane, ester, and imine bonds. Our data indicates that the use of differential hydrolytically labile linkers is a versatile method for functionalization of biomaterials with a variety of growth factors providing control over their biological activity.

Bone morphogenetic protein-2 is a stronger inducer of osteogenesis within muscle tissue than heterodimeric bone morphogenetic protein-2/6 and -2/7: Implications for expedited gene-enhanced bone repair.

BACKGROUND: Bone morphogenetic protein (BMP)-2 gene-activated muscle tissue fragments can regenerate large bone defects in preclinical animal models. The use of tissue fragments instead of isolated cells expedites gene-enhanced tissue engineering and may increase the possibility of clinical translation. The present in vitro study investigated whether the osteoinductive effect of BMP-2 on muscle tissue fragments can be enhanced using the heterodimers BMP-2/6 or BMP-2/7. METHODS: Skeletal muscle tissue fragments from rats were cultured in vitro for up to 20 days in normal medium, osteogenic medium or osteogenic medium supplemented with either a low (50 ng/ml) or high (200 ng/ml) concentration of recombinant human BMP-2, BMP-2/6 or BMP-2/7. Osteoinduction was evaluated by a quantitative reverse transcriptase-polymerase chain reaction, Alizarin red S staining, immunohistology and histomorphometry. RESULTS: Interestingly, BMP-2 was a significantly stronger inducer of osteogenic differentiation within muscle tissue than both heterodimers. Even the low concentration of BMP-2 elicited significantly higher levels of calcium deposition, bone-specific gene expression and protein production than the high concentration of both heterodimers. At the high concentration, BMP-2/7 had a significantly stronger osteogenic effect on muscle than BMP-2/6. CONCLUSIONS: The homodimer BMP-2 induced osteoblastogenesis in muscle faster, at a lower concentration and with a higher potency than the heterodimers BMP-2/6 or BMP-2/7. The findings of this in vitro study encourage bone repair by muscle implants in combination with BMP-2 single growth factor delivery, which might be beneficial with respect to clinical translation.

Zein nanoparticle as a novel BMP6 derived peptide carrier for enhanced osteogenic differentiation of C2C12 cells.

Zein nanoparticles as a carrier system for BMP6-derived peptide were prepared by liquid-liquid phase separation procedure and characterized with SEM, DLS, FTIR and thermogravimetric methods. After peptide encapsulation, nanoparticle size increased from 236.3 ± 92.2 nm to 379.4 ± 116.8 nm. The encapsulation efficiency of peptide was 72.6% and the release of peptide from Zein nanoparticles was partly sustained in trypsin containing phosphate buffered saline (pH 7.4) for up to 14 days. Peptide-loaded nanoparticles showed similar cell viability compared with blank ones. ALP activity of C2C12 cells treated with peptide-loaded nanoparticles (500 µg/mL) was evaluated 7, 14, 21 and 28 days after culture. In peptide-loaded nanoparticles, ALP activity was significantly higher (p < .05) compared with other groups at day 14. Alizarin Red S staining showed, C2C12 cells behind peptide-loaded nanoparticles had significantly (p < .05) higher calcium deposition at day 21. The results of RT-qPCR show that the BMP-6 peptide activated expression of RUNX2 as a transcription factor. In turn, RUNX2 regulates SPP1 and BGLAP gene expression, as osteogenic marker genes. The results confirm that the peptide-loaded Zein nanoparticles, as osteoinductive material, may be used to repair small area of bone defects, with low load bearing.

Synergistic effects of BMP-2, BMP-6 or BMP-7 with human plasma fibronectin onto hydroxyapatite coatings: A comparative study.

Design of new osteoinductive biomaterials to reproduce an optimized physiological environment capable of recruiting stem cells and instructing their fate towards the osteoblastic lineage has become a priority in orthopaedic surgery. This work aims at evaluating the bioactivity of BMP combined with human plasma fibronectin (FN/BMP) delivered in solution or coated onto titanium-hydroxyapatite (TiHA) surfaces. Herein, we focus on the comparison of in vitro osteogenic efficacy in mouse C2C12 pre-osteoblasts of three BMP members, namely: BMP-2, BMP-6 and BMP-7. In parallel, we evaluated the molecular binding strength between each BMP with FN using the Surface Plasmon Resonance (SPR) technology. The affinity of BMPs for FN was found totally different and dependent on BMP type. Indeed, the combination of FN with BMP-2 on TiHA surfaces potentiates the burst of gene-mediated osteogenic induction, while it prolongs the osteogenic activity of BMP-6 and surprisingly annihilates the BMP-7 one. These results correlate with FN/BMP affinity for TiHA, since BMP-6>BMP-2>BMP-7. In addition, by analyzing the osteogenic activity in the peri-implant environment, we showed that osteoinductive paracrine effects were significantly decreased upon (FN/BMP-6), as opposed to (FN/BMP-2) coatings. Altogether, our results support the use of FN/BMP-6 to develop a biomimetic microenvironment capable to induce osteogenic activity under physiological conditions, with minimum paracrine signalization. STATEMENT OF SIGNIFICANCE: The originality of our paper relies on the first direct comparison of the in vitro osteogenic potential of three osteogenic BMPs (BMP-2, -6 and -7) combined with native human plasma fibronectin delivered in solution or coated by laser transfer onto titanium hydroxyapatite surfaces. We confirm that BMP association with fibronectin enhances the osteogenic activity of BMP-2, -6 and -7, but with essential discrepancies, depending on the BMP member, and in agreement with the affinity of BMPs for fibronectin. Moreover, we bring elements to explain the origin of the BMP-2 medical life-threatening side-effects by analyzing in vitro paracrine effects. Finally, this work supports the alternative use of FN/BMP-6 to induce osteogenic activity under physiological conditions, with minimum side effects.

Identification of new BMP6 pro-peptide mutations in patients with iron overload.

Hereditary Hemochromatosis (HH) is a genetically heterogeneous disorder caused by mutations in at least five different genes (HFE, HJV, TFR2, SLC40A1, HAMP) involved in the production or activity of the liver hormone hepcidin, a key regulator of systemic iron homeostasis. Nevertheless, patients with an HH-like phenotype that remains completely/partially unexplained despite extensive sequencing of known genes are not infrequently seen at referral centers, suggesting a role of still unknown genetic factors. A compelling candidate is Bone Morphogenetic Protein 6 (BMP6), which acts as a major activator of the BMP-SMAD signaling pathway, ultimately leading to the upregulation of hepcidin gene transcription. A recent seminal study by French authors has described three heterozygous missense mutations in BMP6 associated with mild to moderate late-onset iron overload (IO). Using an updated next-generation sequencing (NGS)-based genetic test in IO patients negative for the classical HFE p.Cys282Tyr mutation, we found three BMP6 heterozygous missense mutations in four patients from three different families. One mutation (p.Leu96Pro) has already been described and proven to be functional. The other two (p.Glu112Gln, p.Arg257His) were novel, and both were located in the pro-peptide domain known to be crucial for appropriate BMP6 processing and secretion. In silico modeling also showed results consistent with their pathogenetic role. The patients' clinical phenotypes were similar to that of other patients with BMP6-related IO recently described. Our results independently add further evidence to the role of BMP6 mutations as likely contributing factors to late-onset moderate IO unrelated to mutations in the established five HH genes.

Comparative analysis of zebrafish bone morphogenetic proteins 2, 4 and 16: molecular and evolutionary perspectives.

BMP2, BMP4 and BMP16 form a subfamily of bone morphogenetic proteins acting as pleiotropic growth factors during development and as bone inducers during osteogenesis. BMP16 is the most recent member of this subfamily and basic data regarding protein structure and function, and spatio-temporal gene expression is still scarce. In this work, insights on BMP16 were provided through the comparative analysis of structural and functional data for zebrafish BMP2a, BMP2b, BMP4 and BMP16 genes and proteins, determined from three-dimensional models, patterns of gene expression during development and in adult tissues, regulation by retinoic acid and capacity to activate BMP-signaling pathway. Structures of Bmp2a, Bmp2b, Bmp4 and Bmp16 were found to be remarkably similar; with residues involved in receptor binding being highly conserved. All proteins could activate the BMP-signaling pathway, suggesting that they share a common function. On the contrary, stage- and tissue-specific expression of bmp2, bmp4 and bmp16 suggested the genes might be differentially regulated (e.g. different transcription factors, enhancers and/or regulatory modules) but also that they are involved in distinct physiological processes, although with the same function. Retinoic acid, a morphogen known to interact with BMP-signaling during bone formation, was shown to down-regulate the expression of bmp2, bmp4 and bmp16, although to different extents. Taxonomic and phylogenetic analyses indicated that bmp16 diverged before bmp2 and bmp4, is not restricted to teleost fish lineage as previously reported, and that it probably arose from a whole genomic duplication event that occurred early in vertebrate evolution and disappeared in various tetrapod lineages through independent events.

Poly(lactic-co-glycolide) polymer constructs cross-linked with human BMP-6 and VEGF protein significantly enhance rat mandible defect repair.

We have previously shown that the combined delivery of mesenchymal stem cells (MSCs), vascular endothelial growth factor (VEGF) and bone morphogenetic protein 6 (BMP-6) induces significantly more bone formation than that induced by the delivery of any single factor or a combination of any two factors. We now determine whether the exogenous addition of VEGF and BMP-6 is sufficient for bone healing when MSCs are not provided. Poly(lactic-co-glycolic acid) (PLAGA) microsphere-based three-dimensional scaffolds (P) were fabricated by thermal sintering of PLAGA microspheres. The scaffolds were chemically cross-linked with 200 ng recombinant human VEGF (P(VEGF)) or BMP-6 (P(BMP-6)) or both (P(VEGF+BMP-6)) by the EDC-NHS-MES method. Release of the proteins from the scaffolds was detected for 21 days in vitro which confirmed their comparable potential to supply the proteins in vivo. The scaffolds were delivered to a critical-sized mandibular defect created in 32 Sprague Dawley rats. Significant bone regeneration was observed only in rats with P(VEGF+BMP-6) scaffolds at weeks 2, 8 and 12 as revealed by micro-computer tomography. Vascular ingrowth was higher in the P(VEGF+BMP-6) group as seen by microfil imaging than in other groups. Trichrome staining revealed that a soft callus formed in P(VEGF), P(BMP-6) and P(VEGF+BMP-6) but not in P. MSCs isolated from rat femurs displayed expression of the bone-specific marker osteocalcin when cultured with P(VEGF), P(BMP-6), or P(VEGF+BMP-6) but not with P. Robust mineralization and increased alkaline phosphatase gene expression were seen in rat MSCs when cultured on P(VEGF+BMP-6) but not on P, P(VEGF), or P(BMP-6). Thus, unlike the delivery of VEGF or BMP-6 alone, the combined delivery of VEGF and BMP-6 to the bone defect significantly enhanced bone repair through the enhancement of angiogenesis and the differentiation of endogenously recruited MSCs into the bone repair site.

A supramolecular host-guest carrier system for growth factors employing V(H)H fragments.

A supramolecular strategy is presented for the assembly of growth factors employing His6-tagged single-domain antibodies (VHH). A combination of orthogonal supramolecular interactions of ß-cyclodextrin (ßCD)-adamantyl (Ad) host-guest and N-nitrilotriacetic acid (NTA)-histidine (His) interactions was employed to generate reversible and homogeneous layers of growth factors. A single-domain antibody V(H)H fragment was identified to bind to the human bone morphogenetic protein-6 (hBMP6) growth factor and could be recombinantly expressed in E. coli. The V(H)H fragment was equipped with a C-terminal hexahistidine (His6) tether to facilitate the assembly on ßCD surfaces using a linker that contains an Ad group to bind to the ßCD receptors and an NTA moiety to interact with the His6-tag upon cocomplexation of Ni(2+) ions. After exploring the thermodynamic and kinetic stability of the V(H)H assemblies on ßCD surfaces using a variety of experimental techniques including microcontact printing (µCP), surface plasmon resonance (SPR), microscale thermophoresis (MST), and theoretical models for determining the thermodynamic behavior of the system, hBMP6 was assembled onto the V(H)H-functionalized surfaces. After analyzing the immobilized hBMP6 using immunostaining, the biological activity of hBMP6 was demonstrated in cell differentiation experiments. Early osteogenic differentiation was analyzed in terms of alkaline phosphatase (ALP) activity of KS483-4C3 mouse progenitor cells, and the results indicated that the reversibly immobilized growth factors were functionally delivered to the cells. In conclusion, the supramolecular strategy used here offers the necessary affinity, reversibility, and temporal control to promote biological function of the growth factors that were delivered by this strategy.

Repulsive guidance molecule (RGM) family proteins exhibit differential binding kinetics for bone morphogenetic proteins (BMPs).

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor beta superfamily that exert their effects via type I and type II serine threonine kinase receptors and the SMAD intracellular signaling pathway to regulate diverse biologic processes. Recently, we discovered that the repulsive guidance molecule (RGM) family, including RGMA, RGMB, and RGMC/hemojuvelin (HJV), function as co-receptors that enhance cellular responses to BMP ligands. Here, we use surface plasmon resonance to quantitate the binding kinetics of RGM proteins for BMP ligands. We show that among the RGMs, HJV exhibits the highest affinity for BMP6, BMP5, and BMP7 with K(D) 8.1, 17, and 20 nM respectively, versus 28, 33, and 166 nM for RGMB, and 55, 83, and 63 nM for RGMA. Conversely, RGMB exhibits preferential binding to BMP4 and BMP2 with K(D) 2.6 and 5.5 nM respectively, versus 4.5 and 9.4 nM for HJV, and 14 and 22 nM for RGMA, while RGMA exhibits the lowest binding affinity for most BMPs tested. Among the BMP ligands, RGMs exhibit the highest relative affinity for BMP4 and the lowest relative affinity for BMP7, while none of the RGMs bind to BMP9. Thus, RGMs exhibit preferential binding for distinct subsets of BMP ligands. The preferential binding of HJV for BMP6 is consistent with the functional role of HJV and BMP6 in regulating systemic iron homeostasis. Our data may help explain the mechanism by which BMPs exert cell-context specific effects via a limited number of type I and type II receptors.

Different requirements for proteolytic processing of bone morphogenetic protein 5/6/7/8 ligands in Drosophila melanogaster.

Bone morphogenetic proteins (BMPs) are synthesized as proproteins that undergo proteolytic processing by furin/subtilisin proprotein convertases to release the active ligand. Here we study processing of BMP5/6/7/8 proteins, including the Drosophila orthologs Glass Bottom Boat (Gbb) and Screw (Scw) and human BMP7. Gbb and Scw have three functional furin/subtilisin proprotein convertase cleavage sites; two between the prodomain and ligand domain, which we call the Main and Shadow sites, and one within the prodomain, which we call the Pro site. In Gbb each site can be cleaved independently, although efficient cleavage at the Shadow site requires cleavage at the Main site, and remarkably, none of the sites is essential for Gbb function. Rather, Gbb must be processed at either the Pro or Main site to produce a functional ligand. Like Gbb, the Pro and Main sites in Scw can be cleaved independently, but cleavage at the Shadow site is dependent on cleavage at the Main site. However, both Pro and Main sites are essential for Scw function. Thus, Gbb and Scw have different processing requirements. The BMP7 ligand rescues gbb mutants in Drosophila, but full-length BMP7 cannot, showing that functional differences in the prodomain limit the BMP7 activity in flies. Furthermore, unlike Gbb, cleavage-resistant BMP7, although non-functional in rescue assays, activates the downstream signaling cascade and thus retains some functionality. Our data show that cleavage requirements evolve rapidly, supporting the notion that changes in post-translational processing are used to create functional diversity between BMPs within and between species.

Low melting point amphiphilic microspheres for delivery of bone morphogenetic protein-6 and transforming growth factor-ß3 in a hydrogel matrix.

Low melting-point poly(1,3-trimethylene carbonate-co-ε-caprolactone)-b-poly(ethylene glycol)-b-poly(1,3-trimethylene carbonate-co-ε-caprolactone), P(TMC-CL)(2)-PEG, was employed to fabricate microspheres for sustained growth factor delivery in a photocrosslinked N-methacrylate glycol chitosan hydrogel matrix. The P(TMC-CL)(2)-PEG had a melting range such that it was solid at 10°C, yet liquid with a low degree of crystallinity at 37°C. The in vitro degradation of P(TMC-CL)(2)-PEG microspheres was slow, regardless of the triblock copolymer molecular weight and so did not influence protein release. The size of protein loaded P(TMC-CL)(2)-PEG microspheres manufactured using a low-temperature electrospray technique was between 65 and 85µm. Initial formulation work was done with the model protein lysozyme, co-lyophilized with trehalose and encapsulated as approximately 2µm particles within P(TMC-CL)(2)-PEG microspheres. This work indicated a sustained release could be achieved with high trehalose content (90% w/w) in the particles. Under these conditions, the release rate of bone morphogenetic protein-6 was more sustained than that of the excipient bovine serum albumin (BSA) and closely followed that of lysozyme. On the other hand, transforming growth factor-ß3 and the stabilizing agent BSA generated similar release profiles. This difference in release was proposed to be linked to the protein isoelectric point, with positively charged proteins possibly being more strongly adsorbed to the P(TMC-CL)(2)-PEG. Both growth factors were released in highly bioactive form, indicating the potential of the release approach.

BMP-2/6 heterodimer is more effective than BMP-2 or BMP-6 homodimers as inductor of differentiation of human embryonic stem cells.

BACKGROUND: Bone Morphogenetic Protein (BMP) signaling pathways are involved in differentiation of stem cells into diverse cell types, and thus BMPs can be used as main guidance molecules for in vitro differentiation of human stem cells. METHODOLOGY/PRINCIPAL FINDINGS: We have analyzed the ability for inducing differentiation of the heterodimer BMP-2/BMP-6 (BMP-2/6) compared to the homodimers BMP-2 or BMP-6, using human embryonic stem (hES) cells H9 as model system. When incubated in a medium with high concentration of basic fibroblastic growth factor (FGF2), 100 ng/ml of human recombinant BMPs induced morphological changes and differentiation of hES cells in 24 to 48 hours. After 5 days, expression of differentiation markers was induced and quantified by quantitative PCR (qPCR) and flow cytometry. BMP-2/6 exhibited stronger activity for the induction of the expression of trophectodermal (CDX2) and endodermal (SOX17, GATA4, AFP) markers than BMP-2 or BMP-6 homodimers. BMP-2/6 also induced the expression of BMPR2 gene more effectively than BMP-2 or BMP-6 when used at the same concentration and time. Moreover, the percentage of cells expressing the surface endodermal marker CXCR4 was also increased for the heterodimer when compared to both homodimers. BMP-2/6 was a more potent activator of Smad-dependent (SMAD1/5) and Smad-independent signaling (mitogen-activated protein kinases ERK and p38) than BMP-2 and BMP-6, and the activation of these pathways might play a role in its increased potency for inducing hES cell differentiation. CONCLUSIONS/SIGNIFICANCE: Therefore, we conclude that BMP-2/6 is more potent than BMP-2 or BMP-6 for inducing differentiation of hES cells, and it can be used as a more powerful substitute of these BMPs in in vitro differentiation guidance.