Bone morphogenetic protein-2 as a novel biomarker for refractory chronic rhinosinusitis with nasal polyps.

BACKGROUND: Bone morphogenetic proteins (BMPs), which are members of the TGF-ß superfamily, regulate bone remodeling by stimulating osteoblasts and osteoclasts. Although the association between osteitis and poor surgical outcomes is well known in patients with chronic rhinosinusitis (CRS), BMPs have not been fully investigated as potential biomarkers for the prognosis of CRS. OBJECTIVE: Our aim was to investigate the role of BMPs in osteitis in patients with CRS with nasal polyps (NPs) (CRSwNPs), as well as associations between BMPs and inflammatory markers in sinonasal tissues from patients with CRSwNP. METHODS: We investigated the expression of 6 BMPs (BMP-2, BMP-4, BMP-6, BMP-7, BMP-9, and BMP-10) and their cellular origins in NPs of human subjects by using immunohistochemistry and ELISA of NP tissues. Exploratory factor analysis was performed to identify associations between BMPs and inflammatory markers. Air-liquid interface cell culture of human nasal epithelial cells was performed to evaluate the induction of the epithelial-mesenchymal transition by BMPs. RESULTS: Of the 6 BMPs studied, BMP-2 and BMP-7 were associated with refractoriness. Only BMP-2 concentrations were higher in patients with severe osteitis and advanced disease extent according to the computed tomography findings. Eosinophils and some macrophages were identified as cellular sources of BMP-2 in immunofluorescence analysis. An in vitro experiment revealed that BMP-2 induced epithelial-mesenchymal transition in air-liquid interface-cultured human nasal epithelial cells, particularly in a TH2 milieu. CONCLUSION: BMP-2 could reflect the pathophysiology of mucosa and bone remodeling and may be a novel biomarker for refractory CRSwNP.

Functionalized Scaffold and Barrier Membrane with Anti-BMP-2 Monoclonal Antibodies for Alveolar Ridge Preservation in a Canine Model.

INTRODUCTION: The aim of this study was to investigate the ability of anti-bone morphogenetic protein 2 monoclonal antibody (anti-BMP-2 mAb) to functionalize scaffolds to mediate bone regeneration in a canine model. MATERIALS AND METHODS: The mandibular right premolar 4 (PM4) was extracted in eight beagle dogs and grafted with anti-BMP-2 mAb+anorganic bovine bone mineral with 10% collagen (ABBM-C) and porcine bilayer native collagen membrane (CM). The ABBM-C and CM were functionalized with either anti-BMP-2 mAb (test group) or an isotype matched control mAb (control group). Animals were euthanized at 12 weeks for radiographic, histologic, and histomorphometric analyses. Outcomes were compared between groups. RESULTS: 3D imaging using cone beam computed tomography (CBCT) revealed that sites treated with ABBM-C and CM functionalized with anti-BMP-2 mAb exhibited significantly more remaining bone width near the alveolar crest, as well as buccal bone height, compared with control groups. Histologic and histomorphometric analyses demonstrated that in anti-BMP-2 mAb-treated sites, total tissue volume was significantly higher in the coronal part of the alveolar bone crest compared with control sites. In anti-BMP-2 mAb-treated sites, bone formation was observed under the barrier membrane. CONCLUSION: Functionalization of the ABBM-C scaffold and CM appeared to have led to bone formation within healing alveolar bone sockets.

Bone Morphogenetic Protein-2 Induces Non-Canonical Inflammatory and Oxidative Pathways in Human Retinal Endothelial Cells.

The mechanisms of diabetic retinopathy (DR), are not yet fully understood. We previously demonstrated an upregulation of retinal bone morphogenetic protein-2 (BMP2) in experimental diabetes and in retinas of diabetic human subjects. The purpose of current study was to investigate the role of non-canonical inflammatory pathway in BMP2-induced retinal endothelial cell (REC) barrier dysfunction. For this purpose, we used RT-PCR and western blotting to evaluate the levels of BMP2 signaling components (BMP2, BMP4, BMP receptors), VEGF, phosphorylated p38 MAPK and NFκB, and oxidative stress markers in cultured human retinal endothelial cells (HRECs) subjected to BMP2 (50ng/ml) for up to 24 h. Also, effect of high glucose (HG, 30mM D-glucose) on the expression of BMP2 and its downstream genes was examined in HRECs. H2-DCF is a fluorogenic dye that measures the levels of cellular reactive oxygen species (ROS) was used to measure the pro-oxidative effect of BMP2. Moreover, we evaluated the effect of inhibiting p38 and VEGF signaling on BMP2-induced HRECs barrier dysfunction by measuring the trans-endothelial cell electrical resistance (TER) using electric cell-substrate impedance sensing (ECIS). We also tested the effect of HG on the integrity of HRECs barrier in the presence or absence of inhibitors of BMP2 signaling. Our data reveals that BMP2 and high glucose upregulates BMP components of the BMP signaling pathway (SMAD effectors, BMP receptors, and TGFß ligand itself) and induces phosphorylation of p38 MAPK and NFκB with nuclear translocation of NFκB. Inhibition of p38 or NFκB attenuated BMP2-induced VEGF expression and barrier dysfunction in HRECs. Also, inhibition of VEGFR2 attenuated BMP2-induced barrier dysfunction. Moreover, BMP2 induces generation of ROS and endothelial nitric oxide synthase (eNOS) expression and activity in HRECs. Finally, HG upregulated BMP2 and its downstream genes (SMAD, BMP4, ALKs, and TGF-ß) in HRECs and BMP2 inhibitors attenuated HG-induced HRECs barrier dysfunction. Our results suggest that in addition to the regular canonical SMAD signaling BMP2 induces non-canonical inflammatory pathway in HRECs via activation of p38/NFκB pathway that causes the upregulation of VEGF and the disruption of HRECs. Inhibition of BMP2 signaling is a potential therapeutic intervention to preserve endothelial cell barrier function in DR.

Bacterial cellulose membrane functionalized with hydroxiapatite and anti-bone morphogenetic protein 2: A promising material for bone regeneration.

Bone tissue engineering seeks to adequately restore functions related to physical and biological properties, aiming at a repair process similar to natural bone. The use of compatible biopolymers, such as bacterial cellulose (BC), as well as having interesting mechanical characteristics, presents a slow in vivo degradation rate, and the ability to be chemically modified. To promote better bioactivity towards BC, we synthesized an innovative BC membrane associated to hydroxyapatite (HA) and anti-bone morphogenetic protein antibody (anti-BMP-2) (BC-HA-anti-BMP-2). We present the physical-chemical, biological and toxicological characterization of BC-HA-anti-BMP-2. Presence of BC and HA components in the membranes was confirmed by SEM-EDS and FTIR assays. No toxic potential was found in MC3T3-E1 cells by cytotoxicity assays (XTT Assay and Clonogenic Survival), genotoxicity (Comet Assay) and mutagenicity (Cytokinesis-blocked micronucleus Test). The in vitro release kinetics of anti-BMP-2 antibodies detected gradually reducing antibody levels, reducing approximately 70% in 7 days and 90% in 14 days. BC-HA-anti-BMP-2 increased SPP1, BGLAP, VEGF, ALPL, RUNX2 and TNFRSF11B expression, genes involved in bone repair and also increased mineralization nodules and phosphatase alcalin (ALP) activity levels. In conclusion, we developed BC-HA-anti-BMP-2 as an innovative and promising biomaterial with interesting physical-chemical and biological properties which may be a good alternative to treatment with commercial BMP-2 protein.

Silencing SOCS3 Markedly Deteriorates Spondyloarthritis in Mice Induced by Minicircle DNA Expressing IL23.

Objective: Despite extensive studies, the precise mechanism underlying spondyloarthritis, especially ankylosing spondylitis, remains elusive. This study aimed to develop an ideal animal model for an insight into mechanism of spondyloarthritis and functional relevance of SOCS3 in spondyloarthritis. Methods: Since SOCS3 is a major regulator of IL23-STAT3 signaling, we generated SOCS3 knockdown transgenic (TG) mice for development of an animal model of spondyloarthritis. A hydrodynamic delivery method was employed to deliver minicircle DNA expressing IL23 (mc-IL23) into wild-type (WT) and the TG mice. Knockdown/overexpression systems mediated by lentivirus and retrovirus were used to determine whether SOCS3 regulated osteoblast differentiation. Results: Forced expression of IL23 induced severe joint destruction and extensive bone loss in SOCS3 knockdown TG mice, while this treatment only caused moderate symptoms in WT mice. Furthermore, severe spondyloarthritis was found in IL23-injected TG mice as compared to mild disease observed in WT controls under same condition. Moreover, our studies showed that IL23 promoted osteoblast differentiation via activation of STAT3 pathway and disruption of SOCS3 expression greatly increased phosphorylation of STAT3. In addition, silencing SOCS3 resulted in enhanced osteoblast differentiation through activation of Smad1/5/9 signaling, as evidenced by elevated phosphorylation level of Smad1/5/9. Experiments further demonstrated that SOCS3 interacted with Smad1 and thus suppressed the BMP2-Smad signaling. Conclusions: The results reveal that SOCS3 is involved in IL23-induced spondyloarthritis and acts as a key regulator of osteoblast differentiation, and suggest that SOCS3 knockdown TG mice may be an ideal animal model for further studies of spondyloarthritis.

Collagen sponge functionalized with chimeric anti-BMP-2 monoclonal antibody mediates repair of nonunion tibia defects in a nonhuman primate model: An exploratory study.

Recombinant human bone morphogenetic protein (BMP)-2 is an FDA-approved therapy for nonunion tibia fracture, though it has a number of biological and practical disadvantages. Our research group has developed a novel tissue engineering strategy termed antibody-mediated osseous regeneration. This entails application of anti-BMP-2 monoclonal antibodies (mAbs) to capture endogenous BMP's to mediate in vivo bone formation. This has been documented in a number of animal models. The present exploratory study sought to investigate the application of antibody-mediated osseous regeneration for repair of nonunion tibia defect in a nonhuman primate model. A 20 mm segmental osteotomy was performed in tibia of 6 Macaca fascicularis and was implanted with absorbable collagen sponge that was functionalized with chimeric anti-BMP-2 or isotype matched control mAb. Cone beam computed tomography (CBCT), histologic and histomorphometric analyses were performed 12 weeks post-operatively. CBCT analyzed by quantitative 3D volumetric analysis revealed that sites implanted with absorbable collagen sponge functionalized with anti-BMP-2 mAb demonstrated numerically higher mineralized tissue (408 ± 127 mm3) compared with sites implanted with isotype matched control mAb (214 ± 81 mm3), though the difference was not statistically significant ( p = 0.09). Histologic and histomorphometric analysis showed de novo bone formation with greater ( p < 0.01) percentage of bone volume in sites implanted with anti-BMP-2 (41.3 ± 4.4%), compared with isotype matched control mAb (14.6 ± 5.6%). Results from the present exploratory study provide evidence for the potential of anti-BMP-2 mAb to mediate repair of a large segmental tibia defects in a nonhuman primate model. Therapeutic antibodies have generally been shown to have great safety and efficacy profile, though their application in tissue engineering has been limited in the past. Following further investigation, anti-BMP-2 mAbs immobilized on appropriate scaffold may have application in repair of large skeletal defects without the need for exogenous growth factors.

Collagen Sponge Functionalized with Chimeric Anti-BMP-2 Monoclonal Antibody Mediates Repair of Critical-Size Mandibular Continuity Defects in a Nonhuman Primate Model.

Antibody-mediated osseous regeneration (AMOR) has been introduced by our research group as a tissue engineering approach to capture of endogenous growth factors through the application of specific monoclonal antibodies (mAbs) immobilized on a scaffold. Specifically, anti-Bone Morphogenetic Protein- (BMP-) 2 mAbs have been demonstrated to be efficacious in mediating bone repair in a number of bone defects. The present study sought to investigate the application of AMOR for repair of mandibular continuity defect in nonhuman primates. Critical-sized mandibular continuity defects were created in Macaca fascicularis locally implanted with absorbable collagen sponges (ACS) functionalized with chimeric anti-BMP-2 mAb or isotype control mAb. 2D and 3D analysis of cone beam computed tomography (CBCT) imaging demonstrated increased bone density and volume observed within mandibular continuity defects implanted with collagen scaffolds functionalized with anti-BMP-2 mAb, compared with isotype-matched control mAb. Both CBCT imaging and histologic examination demonstrated de novo bone formation that was in direct apposition to the margins of the resected bone. It is hypothesized that bone injury may be necessary for AMOR. This is evidenced by de novo bone formation adjacent to resected bone margins, which may be the source of endogenous BMPs captured by anti-BMP-2 mAb, in turn mediating bone repair.

Encapsulation of Adenovirus BMP2-Transduced Cells with PEGDA Hydrogels Allows Bone Formation in the Presence of Immune Response.

Gene therapy approaches have been difficult to implement due to pre-existing immunity against the virus used for delivery. To circumvent this problem, a cell-based approach was developed that avoided the use of free virus within the animal. However, even cells transduced in vitro with E1- to E3-deleted adenovirus encoding bone morphogenetic protein 2 (AdBMP2) resulted in the production of virus-neutralizing antibodies in mice. Furthermore, when mice received an intramuscular injection of nonencoding adenovirus (AdEmpty)-transduced cells, AdBMP2-transduced cells were unable to launch bone formation when an intramuscular injection of these BMP2-producing cells was delivered 1 week later. This phenomenon was not observed in NOD/SCID mice, and could be overcome in C57BL/6 mice by encapsulating the adenovirus-transduced cells in a nondegradable hydrogel poly(ethylene glycol) diacrylate (PEGDA). Data collectively suggest that PEGDA hydrogel encapsulation of AdBMP2-transduced cells prevents pre-existing immunity from suppressing BMP2-induced bone formation.

Immunoexpression of BMP-2 and BMP-4 and their receptors, BMPR-IA and BMPR-II, in ameloblastomas and adenomatoid odontogenic tumors.

OBJECTIVES: The present study evaluated the immunohistochemical expression of BMP-2 and BMP-4 and of their receptors (BMPR-IA and BMPR-II) in solid ameloblastoma (SA), unicystic ameloblastoma (UA) and adenomatoid odontogenic tumor (AOT) in order to obtain a better understanding of their role in the development and biological behavior of these tumors. DESIGN: This study analyzed these proteins in 30 cases of SA, 10 cases of UA, and 30 cases of AOT. Immunoexpression was evaluated in the parenchyma and stroma by attributing the following scores: 0, no stained cells; 1, ≤10%; 2, >10% and ≤25%; 3, >25% and ≤50%; 4, >50% and ≤75%.; 5, >75% stained cells. RESULTS: In SAs, positive correlations were observed between the stromal and parenchymal expression of BMP-2 (p<0.001) and between the stromal expression of BMP-2 and BMP-4 (p=0.020), as well as between the stromal expression of BMPR-II and BMP-4 (p=0.001) and the stromal and parenchymal expression of BMPR-II (p<0.001). In UAs, correlations were detected between the stromal and parenchymal expression of BMP-4 (p=0.035) and between the stromal expression of BMP-4 and BMPR-IA (p=0.022). In AOTs, analysis of immunoexpression in the parenchyma revealed positive correlations between all proteins. CONCLUSION: BMPs and their receptors play an important role in the differentiation and development of ameloblastomas and AOTs, but may not explain the different biological behaviors of these lesions. The positive correlation observed in AOTs might be related to the formation of mineralized material in this tumor.

Modulating macrophage polarization with divalent cations in nanostructured titanium implant surfaces.

Nanoscale topographical modification and surface chemistry alteration using bioactive ions are centrally important processes in the current design of the surface of titanium (Ti) bone implants with enhanced bone healing capacity. Macrophages play a central role in the early tissue healing stage and their activity in response to the implant surface is known to affect the subsequent healing outcome. Thus, the positive modulation of macrophage phenotype polarization (i.e. towards the regenerative M2 rather than the inflammatory M1 phenotype) with a modified surface is essential for the osteogenesis funtion of Ti bone implants. However, relatively few advances have been made in terms of modulating the macrophage-centered early healing capacity in the surface design of Ti bone implants for the two important surface properties of nanotopography and and bioactive ion chemistry. We investigated whether surface bioactive ion modification exerts a definite beneficial effect on inducing regenerative M2 macrophage polarization when combined with the surface nanotopography of Ti. Our results indicate that nanoscale topographical modification and surface bioactive ion chemistry can positively modulate the macrophage phenotype in a Ti implant surface. To the best of our knowledge, this is the first demonstration that chemical surface modification using divalent cations (Ca and Sr) dramatically induces the regenerative M2 macrophage phenotype of J774.A1 cells in nanostructured Ti surfaces. In this study, divalent cation chemistry regulated the cell shape of adherent macrophages and markedly up-regulated M2 macrophage phenotype expression when combined with the nanostructured Ti surface. These results provide insight into the surface engineering of future Ti bone implants that are harmonized between the macrophage-governed early wound healing process and subsequent mesenchymal stem cell-centered osteogenesis function.

Adipose-Derived Stem Cells Decrease Bone Morphogenetic Protein Type 2-Induced Inflammation In Vivo.

PURPOSE: Recombinant human bone morphogenetic protein type 2 (rhBMP-2) has been used to promote bone regeneration. In contrast, some reports have suggested rhBMP-2 does not provide advantages over autogenous bone grafting owing to the undesirable postoperative symptoms of this growth factor. Because the undesirable symptoms of rhBMP-2 are usually promoted by inflammation, this study evaluated the in vivo effect of human adipose-derived stem cells (ASCs) incorporated into polylactic co-glycolic acid (PLGA) scaffolds in decreasing the inflammatory response induced by a low dose of rhBMP-2. MATERIALS AND METHODS: PLGA scaffolds were characterized and loaded with rhBMP-2 1, 2.5, or 5 µg per scaffold (n = 6) and the in vitro released protein amounts were quantified at 7 hours and 1, 7, and 21 days after loading (n = 3). The muscle tissue of 6 beagles received the following treatments: PLGA, PLGA plus rhBMP-2 (2.5 µg), and PLGA plus rhBMP-2 plus ASCs (1 × 10(6) ASCs). The samples were evaluated 45 days after surgery. Statistical analyses were performed and the P value was set at .05. RESULTS: PLGA plus rhBMP-2 plus ASCs yielded the smallest number of inflammatory foci (P < .001) and giant cells (P < .001) and the largest number of angiogenesis sites (P < .001). CONCLUSIONS: Human ASCs administered in vivo into PLGA scaffolds with a low dose of rhBMP-2 decrease tissue inflammation and increase angiogenesis in muscular sites.

Targeted Mutation of Nuclear Bone Morphogenetic Protein 2 Impairs Secondary Immune Response in a Mouse Model.

We recently identified a nuclear variant of the BMP2 growth factor, called nBMP2. In an effort to understand the function of this variant protein, we generated a mouse line in which BMP2 is expressed and functions normally, but nBMP2 is excluded from the nucleus. This novel mutation allows the study of nBMP2 without compromising BMP2 function. To determine whether nBMP2 plays a role in immune function, we performed a series of experiments in which we compared mouse survival, organ weights, immune cells numbers, and bacterial load in wild type and nBmp2NLS(tm) mice following primary and secondary challenges with Staphylococcus aureus. Following primary challenge with S. aureus, wild type and nBmp2NLS(tm) mice showed no differences in survival or bacterial load and generated similar numbers and types of leukocytes, although mutant spleens were smaller than wild type. Secondary bacterial challenge with S. aureus, however, produced differences in survival, with increased mortality seen in nBmp2NLS(tm) mice. This increased mortality corresponded to higher levels of bacteremia in nBmp2NLS(tm) mice and to a reduced enlargement of mutant spleens in response to the secondary infection. Together, these results suggest that the recently described nuclear variant of BMP2 is necessary for efficient secondary immune responses.

Effective Inhibition of Bone Morphogenetic Protein Function by Highly Specific Llama-Derived Antibodies.

Bone morphogenetic proteins (BMP) have important but distinct roles in tissue homeostasis and disease, including carcinogenesis and tumor progression. A large number of BMP inhibitors are available to study BMP function; however, as most of these antagonists are promiscuous, evaluating specific effects of individual BMPs is not feasible. Because the oncogenic role of the different BMPs varies for each neoplasm, highly selective BMP inhibitors are required. Here, we describe the generation of three types of llama-derived heavy chain variable domains (VHH) that selectively bind to either BMP4, to BMP2 and 4, or to BMP2, 4, 5, and 6. These generated VHHs have high affinity to their targets and are able to inhibit BMP signaling. Epitope binning and docking modeling have shed light into the basis for their BMP specificity. As opposed to the wide structural reach of natural inhibitors, these small molecules target the grooves and pockets of BMPs involved in receptor binding. In organoid experiments, specific inhibition of BMP4 does not affect the activation of normal stem cells. Furthermore, in vitro inhibition of cancer-derived BMP4 noncanonical signals results in an increase of chemosensitivity in a colorectal cancer cell line. Therefore, because of their high specificity and low off-target effects, these VHHs could represent a therapeutic alternative for BMP4(+) malignancies.

Effects of the orientation of anti-BMP2 monoclonal antibody immobilized on scaffold in antibody-mediated osseous regeneration.

Recently, we have shown that anti-BMP2 monoclonal antibodies (mAbs) can trap endogenous osteogenic BMP ligands, which can in turn mediate osteodifferentiation of progenitor cells. The effectiveness of this strategy requires the availability of the anti-BMP-2 monoclonal antibodies antigen-binding sites for anti-BMP-2 monoclonal antibodies to bind to the scaffold through a domain that will leave its antigen-binding region exposed and available for binding to an osteogenic ligand. We examined whether antibodies bound to a scaffold by passive adsorption versus through Protein G as a linker will exhibit differences in mediating bone formation. In vitro anti-BMP-2 monoclonal antibodies was immobilized on absorbable collagen sponge (ACS) with Protein G as a linker to bind the antibody through its Fc region and implanted into rat calvarial defects. The biomechanical strength of bone regenerated by absorbable collagen sponge/Protein G/anti-BMP-2 monoclonal antibodies immune complex was compared to ACS/anti-BMP-2 monoclonal antibodies or ACS/Protein G/isotype mAb control group. Results demonstrated higher binding of anti-BMP-2 monoclonal antibodies/BMPs to C2C12 cells, when the mAb was initially attached to recombinant Protein G or Protein G-coupled microbeads. After eight weeks, micro-CT and histomorphometric analyses revealed increased bone formation within defects implanted with absorbable collagen sponge/Protein G/anti-BMP-2 monoclonal antibodies compared with defects implanted with absorbable collagen sponge/anti-BMP-2 monoclonal antibodies (p < 0.05). Confocal laser scanning microscopy (CLSM) confirmed increased BMP-2, -4, and -7 detection in sites implanted with absorbable collagen sponge/Protein G/anti-BMP-2 monoclonal antibodies in vivo. Biomechanical analysis revealed the regenerated bone in sites with Protein G/anti-BMP-2 monoclonal antibodies had higher mechanical strength in comparison to anti-BMP-2 monoclonal antibodies. The negative control group, Protein G/isotype mAb, did not promote bone regeneration and exhibited significantly lower mechanical properties (p < 0.05). Altogether, our results demonstrated that application of Protein G as a linker to adsorb anti-BMP-2 monoclonal antibodies onto the scaffold was accompanied by increased in vitro binding of the anti-BMP-2 mAb/BMP immune complex to BMP-receptor positive cell, as well as increased volume and strength of de novo bone formation in vivo.

Application of AMOR in craniofacial rabbit bone bioengineering.

Endogenous molecular and cellular mediators modulate tissue repair and regeneration. We have recently described antibody mediated osseous regeneration (AMOR) as a novel strategy for bioengineering bone in rat calvarial defect. This entails application of anti-BMP-2 antibodies capable of in vivo capturing of endogenous osteogenic BMPs (BMP-2, BMP-4, and BMP-7). The present study sought to investigate the feasibility of AMOR in other animal models. To that end, we examined the efficacy of a panel of anti-BMP-2 monoclonal antibodies (mAbs) and a polyclonal Ab immobilized on absorbable collagen sponge (ACS) to mediate bone regeneration within rabbit calvarial critical size defects. After 6 weeks, de novo bone formation was demonstrated by micro-CT imaging, histology, and histomorphometric analysis. Only certain anti-BMP-2 mAb clones mediated significant in vivo bone regeneration, suggesting that the epitopes with which anti-BMP-2 mAbs react are critical to AMOR. Increased localization of BMP-2 protein and expression of osteocalcin were observed within defects, suggesting accumulation of endogenous BMP-2 and/or increased de novo expression of BMP-2 protein within sites undergoing bone repair by AMOR. Considering the ultimate objective of translation of this therapeutic strategy in humans, preclinical studies will be necessary to demonstrate the feasibility of AMOR in progressively larger animal models.

BMP-2 and TGF-ß1 mediate biglycan-induced pro-osteogenic reprogramming in aortic valve interstitial cells.

UNLABELLED: Biglycan accumulates in aortic valves affected by calcific aortic valve disease (CAVD), and soluble biglycan upregulates BMP-2 expression in human aortic valve interstitial cells (AVICs) via Toll-like receptor (TLR) 2 and induces AVIC pro-osteogenic reprogramming, characterized by elevated pro-osteogenic activities. We sought to identify the factors responsible for biglycan-induced pro-osteogenic reprogramming in human AVICs. Treatment of AVICs with recombinant biglycan induced the secretion of BMP-2 and TGF-ß1, but not BMP-4 or BMP-7. Biglycan upregulated TGF-ß1 expression in a TLR4-dependent fashion. Neutralization of BMP-2 or TGF-ß1 attenuated the expression of alkaline phosphatase (ALP), osteopontin, and runt-related transcription factor 2 (Runx2) in cells exposed to biglycan. However, neutralization of both BMP-2 and TGF-ß1 abolished the expression of these osteogenic biomarkers and calcium deposition. Phosphorylated Smad1 and Smad3 were detected in cells exposed to biglycan, and knockdown of Smad1 or Smad3 attenuated the effect of biglycan on the expression of osteogenic biomarkers. While BMP-2 and TGF-ß1 each upregulated the expression of osteogenic biomarkers, an exposure to BMP-2 plus TGF-ß1 induced a greater upregulation and results in calcium deposition. We conclude that concurrent upregulation of BMP-2 and TGF-ß1 is responsible for biglycan-induced pro-osteogenic reprogramming in human AVICs. The Smad 1/3 pathways are involved in the mechanism of AVIC pro-osteogenic reprogramming. KEY MESSAGE: Biglycan upregulates BMP-2 and TGF-ß1 in human aortic valve cells through TLRs. Both BMP-2 and TGF-ß1 are required for aortic valve cell pro-osteogenic reprogramming. Smad signaling pathways are involved in mediating the pro-osteogenic effects of biglycan.

A unique tool to selectively detect the chondrogenic IIB form of human type II procollagen protein.

Type II collagen, the major fibrillar collagen of cartilage, is synthesized as precursor forms (procollagens) containing N- and C-terminal propeptides. Three splice variants are thought to be translated to produce procollagen II isoforms (IIA/D and IIB) which differ in their amino propeptide parts. The IIA and IID are transient embryonic isoforms that include an additional cysteine-rich domain encoded by exon 2. The IIA and IID transcripts are co-expressed during chondrogenesis then decline and the IIB isoform is the only one expressed and synthesized in fully differentiated chondrocytes. Additionally, procollagens IIA/D can be re-expressed by dedifferentiating chondrocytes and in osteoarthritic cartilage. Therefore, it is an important point to determine which isoform(s) is (are) synthesized in vivo in normal and pathological situations and in vitro, to fully assess the phenotype of cells producing type II collagen protein. Antibodies directed against the cysteine-rich extra domain found in procollagens IIA and IID are already available but antibodies detecting only the chondrogenic IIB form of type II procollagen were missing so far. A synthetic peptide encompassing the junction between exon 1 and exon 3 of the human sequence was used as immunogen to produce rabbit polyclonal antibodies to procollagen IIB. After affinity purification on immobilized peptide their absence of crossreaction with procollagens IIA/D and with the fibrillar procollagens I, III and V was demonstrated by Western blotting. These antibodies were used to reveal at the protein level that the treatment of dedifferentiated human chondrocytes by bone morphogenic protein (BMP)-2 induces the synthesis of the IIB (chondrocytic) isoform of procollagen II. In addition, immunohistochemical staining of bovine cartilage demonstrates the potential of these antibodies in the analysis of the differential spatiotemporal distribution of N-propeptides of procollagens IIA/D and IIB during normal development and in pathological situations.

Functionalization of scaffolds with chimeric anti-BMP-2 monoclonal antibodies for osseous regeneration.

Recent studies have demonstrated the ability of murine anti-BMP-2 monoclonal antibodies (mAb) immobilized on an absorbable collagen sponge (ACS) to mediate de novo bone formation, a process termed antibody-mediated osseous regeneration (AMOR). The objectives of this study were to assess the efficacy of a newly generated chimeric anti-BMP-2 mAb in mediating AMOR, as well as to evaluate the suitability of different biomaterials as scaffolds to participate in AMOR. Chimeric anti-BMP-2 mAb was immobilized on 4 biomaterials, namely, titanium microbeads (Ti), alginate hydrogel, macroporous biphasic calcium phosphate (MBCP) and ACS, followed by surgical implantation into rat critical-size calvarial defects. Animals were sacrificed after 8 weeks and the degree of bone fill was assessed using micro-CT and histomorphometry. Results demonstrated local persistence of chimeric anti-BMP-2 mAb up to 8 weeks, as well as significant de novo bone regeneration in sites implanted with chimeric anti-BMP-2 antibody immobilized on each of the 4 scaffolds. Ti and MBCP showed the highest volume of bone regeneration, presumably due to their resistance to compression. Alginate and ACS also mediated de novo bone formation, though significant volumetric shrinkage was noted. In vitro assays demonstrated cross-reactivity of chimeric anti-BMP-2 mAb with BMP-4 and BMP-7. Immune complex of anti-BMP-2 mAb with BMP-2 induced osteogenic differentiation of C2C12 cells in vitro, involving expression of RUNX2 and phosphorylation of Smad1. The present data demonstrated the ability of chimeric anti-BMP-2 mAb to functionalize different biomaterial with varying characteristics to mediate osteogenesis.

Co-encapsulation of anti-BMP2 monoclonal antibody and mesenchymal stem cells in alginate microspheres for bone tissue engineering.

Recently, it has been shown that tethered anti-BMP2 monoclonal antibodies (mAbs) can trap BMP ligands and thus provide BMP inductive signals for osteo-differentiation of progenitor cells. The objectives of this study were to: (1) develop a co-delivery system based on murine anti-BMP2 mAb-loaded alginate microspheres encapsulating human bone marrow mesenchymal stem cells (hBMMSCs); and (2) investigate osteogenic differentiation of encapsulated stem cells in alginate microspheres in vitro and in vivo. Alginate microspheres of 1 ± 0.1 mm diameter were fabricated with 2 × 10(6) hBMMSCs per mL of alginate. Critical-size calvarial defects (5 mm diameter) were created in immune-compromised mice and alginate microspheres preloaded with anti-BMP mAb encapsulating hBMMSCs were transplanted into defect sites. Alginate microspheres pre-loaded with isotype-matched non-specific antibody were used as the negative control. After 8 weeks, micro CT and histologic analyses were used to analyze bone formation. In vitro analysis demonstrated that anti-BMP2 mAbs tethered BMP2 ligands that can activate the BMP receptors on hBMMSCs. The co-delivery system described herein, significantly enhanced hBMMSC-mediated osteogenesis, as confirmed by the presence of BMP signal pathway-activated osteoblast determinants Runx2 and ALP. Our results highlight the importance of engineering the microenvironment for stem cells, and particularly the value of presenting inductive signals for osteo-differentiation of hBMMSCs by tethering BMP ligands using mAbs. This strategy of engineering the microenvironment with captured BMP signals is a promising modality for repair and regeneration of craniofacial, axial and appendicular bone defects.

Revision surgery after interbody fusion with rhBMP-2: a cautionary tale for spine surgeons.

Recombinant human bone morphogenetic protein-2 (rhBMP-2) promotes the induction of bone growth and is widely used in spine surgery to enhance arthrodesis. Recombinant human BMP-2 has been associated with a variety of complications including ectopic bone formation, adjacent-level fusion, local bone resorption, osteolysis, and radiculitis. Some of the complications associated with rhBMP-2 may be the result of rhBMP-2 induction of the inflammatory host response. In this paper the authors report on a patient with prior transforaminal lumbar interbody fusion (TLIF) using an interbody cage packed with rhBMP-2, in which rhBMP-2 possibly contributed to vascular injury during an attempted anterior lumbar interbody fusion. This 63-year-old man presented with a 1-year history of worsening refractory low-back pain and radiculopathy caused by a Grade 1 spondylolisthesis at L4-5. He underwent an uncomplicated L4-5 TLIF using an rhBMP-2-packed interbody cage. Postoperatively, he experienced marginal improvement of his symptoms. Within the next year and a half the patient returned with unremitting low-back pain and neurogenic claudication that failed to respond to conservative measures. Radiological imaging of the patient revealed screw loosening and pseudarthrosis. He underwent an anterior retroperitoneal approach with a plan for removal of the previous cage, complete discectomy, and placement of a femoral ring. During the retroperitoneal approach the iliac vein was adhered with scarring and fibrosis to the underlying previously operated L4-5 interbody space. During mobilization the left iliac vein was torn, resulting in significant blood loss and cardiac arrest requiring chest compression, defibrillator shocks, and blood transfusion. The patient was stabilized, the operation was terminated, and he was transferred to the intensive care unit. He recovered over the next several days and was discharged at his neurological baseline. The authors propose that the rhBMP-2-induced host inflammatory response partially contributed to vessel fibrosis and scarring, resulting in the life-threatening vascular injury during the reoperation. Spine surgeons should be aware of this potential inflammatory fibrosis in addition to other reported complications related to rhBMP-2.