Expression, Purification, Bioactivity, and Partial Characterization of a Recombinant Human Bone Morphogenetic Protein-7 Produced in Human 293T Cells

Bone morphogenetic protein-7 (BMP-7) is a secreted multifunctional growth factor of the TGF-beta superfamily, which is predominantly known for its osteoinductive properties and emerging potential for treatment of kidney diseases. The mature 34-38 kDa disulfide-linked homodimer protein plays a key role in the differentiation of mesenchymal cells into bone and cartilage. In this study, the full-length sequence of hBMP-7 was amplified and, then, cloned, expressed, and purified from the conditioned medium of 293T cells stably transfected with a lentiviral vector. The mature protein dimer form was properly secreted and recognized by anti-BMP-7 antibodies, and the protein was shown to be glycosilated by treatment with exoglycosidase, followed by western blotting. Moreover, the activity of the purified protein was demonstrated both in vitro, by alkaline phosphatase activity in C2C12 cells, and in vivo by induction of ectopic bone formation in Balb/c Nude mice after 21 days, respectively. This recombinant protein platform may be very useful for expression of different human cytokines and other proteins for medical applications.

Modulation of Bone Morphogenetic Protein-9 Expression and Processing by Insulin, Glucose, and Glucocorticoids: Possible Candidate for Hepatic Insulin-Sensitizing Substance

Bone morphogenetic protein 9 (BMP-9), a member of the TGF-beta superfamily predominantly expressed in nonparenchymal liver cells, has been demonstrated to improve glucose homeostasis in diabetic mice. Along with this therapeutic effect, BMP-9 was proposed as a candidate for the hepatic insulin-sensitizing substance ( HISS). Whether BMP-9 plays a physiological role in glucose homeostasis is still unknown. In the present study, we show that BMP-9 expression and processing is severely reduced in the liver of insulin-resistant rats. BMP-9 expression and processing was directly stimulated by in situ exposition of the liver to the combination of glucose and insulin and oral glucose in overnight fasted rats. Additionally, prolonged fasting ( 72 h) abrogated refeeding-induced BMP-9 expression and processing. Previous exposition to dexamethasone, a known inductor of insulin resistance, reduced BMP-9 processing stimulated by the combination of insulin and glucose. Finally, we show that neutralization of BMP-9 with an anti-BMP-9 antibody induces glucose intolerance and insulin resistance in 12-h fasted rats. Collectively, the present results demonstrate that BMP-9 plays an important role in the control of glucose homeostasis of the normal rat. Additionally, BMP-9 is expressed and processed in an HISS-like fashion, which is impaired in the presence of insulin resistance. BMP-9 regulation according to the feeding status and the presence of diabetogenic factors reinforces the hypothesis that BMP-9 might exert the role of HISS in glucose homeostasis physiology. ( Endocrinology 149: 6326-6335, 2008)

Functional Analysis of saxophone, the Drosophila Gene Encoding the BMP Type I Receptor Ortholog of Human ALK1/ACVRL1 and ACVR1/ALK2

In metazoans, bone morphogenetic proteins (BMPS) direct a myriad of developmental and adult homeostatic evens through their heterotetrameric type I and type II receptor complexes. We examined 3 existing and 12 newly generated mutations in the Drosophila type I receptor gene, saxophone (sax), the ortholog of the human Activin Receptor-Like. Kinasel and -2 (ALK1/ACVR1 and ALK2/ACVR1) genes. Our genetic analyses identified two distinct classes of sax alleles. The first class consists of homozygous viable gain-of-function (GOF) alleles that exhibit (1) synthetic lethality in combination with mutations in BMP pathway components, and (2) significant maternal effect lethality that can be rescued by an increased dosage of the BMP encoding gene, dpp(+). In contrast, the second class consists of alleles that are recessive lethal and do not exhibit lethality in combination with mutations in other BMP pathway components. The alleles in this second class are clearly loss-of-function (LOF) with both complete and partial loss-of-function mutations represented. We find that one allele in the second class of recessive lethals exhibits dominant-negative behavior, albeit distinct from the GOF activity of the first class of viable alleles. On the basis of the fact that the first class of viable alleles can be reverted to lethality and on our ability to independently generate recessive lethal sat mutations, our analysis demonstrates that sax is an essential gene. Consistent with this conclusion, we find that a normal sax transcript is produced by sax(P), a viable allele previously reported to be mill, and that this allele can be reverted to lethality. Interestingly, we determine that two mutations in the first: class of sax alleles show the same amino acid substitutions as mutations in the human receptors ALK1/ACVR1-1 and ACVR1/ALK2, responsible for cases of hereditary hemorrhagic telangiectasia type 2 (HHT2) and fibrodysplasia ossificans progressiva (FOP), respectively. Finally, the data presented here identify different functional requirements for the Sax receptor, support the proposal that Sax participates in a heteromeric receptor complex, and provide a mechanistic framework for future investigations into disease states that arise from defects in BMP/TGF-beta signaling.

NUCEL (Cell and Molecular Therapy Center): A multidisciplinary center for translational research in Brazil

Social and economical development is closely associated with technological innovation and a well-developed biotechnological industry. In the last few years, Brazil`s scientific production has been steadily increasing; however, the number of patents is lagging behind, with technological and translational research requiring governmental incentive and reinforcement. The Cell and Molecular Therapy Center (NUCEL) was created to develop activities in the translational research field, addressing concrete problems found in biomedical and veterinary areas and actively searching for solutions by employing a genetic engineering approach to generate cell lines over-expressing recombinant proteins to be transferred to local biotech companies, aiming at furthering the development of a national competence for local production of biopharmaceuticals of widespread use and of life-saving importance. To this end, mammalian cell engineering technologies were used to generate cell lines over-expressing several different recombinant proteins of biomedical and biotechnological interest, namely, recombinant human Amylin/IAPP for diabetes treatment, human FVIII and FIX clotting factors for hemophilia, human and bovine FSH for fertility and reproduction, and human bone repair proteins (BMPs). Expression of some of these proteins is also being sought with the baculovirus/insect cell system (BEVS) which, in many cases, is able to deliver high-yield production of recombinant proteins with biological activity comparable to that of mammalian systems, but in a much more cost-effective manner. Transfer of some of these recombinant products to local Biotech companies has been pursued by taking advantage of the Sao Paulo State Foundation (FAPESP) and Federal Government (FINEP, CNPq) incentives for joint Research Development and Innovation partnership projects.

Influence of rhBMP-2 on bone formation and osseointegration in different implant systems after sinus-floor elevation. An in vivo study on sheep

Background Several studies have reported certain bone morphogenic proteins (BMPs) to have positive effects on bone generation Although some investigators have studied the effects of human recombinant BMP (rhBMP-2) in sinus augmentation in sheep, none of these studies looked at the placement of implants at the time of sinus augmentation Furthermore, no literature could be found to report on the impact that different implant systems, as well as the positioning of the implants had on bone formation if rhBMP-2 was utilized in sinus-lift procedures Purpose The aim of this study was to compare sinus augmentation with rhBMP-2 on a poly-D, L-lactic-co-glycolic acid gelatine (PLPG) sponge with sinus augmentation with autologous pelvic cancellous bone in the maxillary sinus during the placement of different dental Implants Materials and methods Nine adult female sheep were submitted to bilateral sinus-floor elevation In one side (test group) the sinus lift was performed with rhBMP-2 on a PLPG-sponge, while the contralateral side served as the control by using cancellous bone from the iliac crest Three different implants (Branemark (R), 31 (R) and Straumann (R)) were inserted either simultaneously with the sinus augmentation or as a two staged procedure 6 weeks later The animals were sacrificed at 6 and 12 weeks for histological and histomorphometrical evaluations during which bone-to-implant contact (BIC) and bone density (BD) were evaluated Results BD and BIC were significantly higher at 12 weeks in the test group if the Implants were placed at the time of the sinus lift (p < 0 05) No difference was observed between the different implant systems or positions Conclusions The use of rhBMP-2 with PLPG-sponge increased BIC as well as BD in the augmented sinuses if compared to autologous bone Different implant systems and positions of the implants had no effect on BIC or BD (C) 2010 European Association for Cranio-Maxillo-Facial Surgery

Bone marrow mononuclear cells attenuate fibrosis development after severe acute kidney injury

One of the early phases that lead to fibrosis progression is inflammation. Once this stage is resolved, fibrosis might be prevented. Bone marrow mononuclear cells (BMMCs) are emerging as a new therapy for several pathologies, including autoimmune diseases, because they enact immunosuppression. In this study we aimed to evaluate the role of BMMC administration in a model of kidney fibrosis induced by an acute injury. C57Bl6 mice were subjected to unilateral severe ischemia by clamping the left renal pedicle for 1 h. BMMCs were isolated from femurs and tibia, and after 6 h of reperfusion, 1 x 10(6) cells were administrated intraperitoneally. At 24 h after surgery, treated animals showed a significant decrease in creatinine and urea levels when compared with untreated animals. Different administration routes were tested. Moreover, interferon (IFN) receptor knockout BMMCs were used, as this receptor is necessary for BMMC activation. Labeled BMMCs were found in ischemic kidney on FACS analysis. This improved outcome was associated with modulation of inflammation in the kidney and systemic modulation, as determined by cytokine expression profiling. Despite non-amelioration of functional parameters, kidney mRNA expression of interleukin (IL)-6 at 6 weeks was lower in BMMC-treated animals, as were levels of collagen 1, connective tissue growth factor (CTGF), transforming growth factor-beta (TGF-beta) and vimentin. Protective molecules, such as IL-10, heme oxygenase 1 (HO-1) and bone morphogenetic 7 (BMP-7), were increased in treated animals after 6 weeks. Moreover, Masson and Picrosirius red staining analyses showed less fibrotic areas in the kidneys of treated animals. Thus, early modulation of inflammation by BMMCs after an ischemic injury leads to reduced fibrosis through modulation of early inflammation.

Immunohistochemical, tomographic and histological study on onlay iliac grafts remodeling

The information concerning the molecular events taking place in onlay bone grafts are still incipient. The objective of the present study is to correlate the effects of perforation of resident bone bed on (1) the timing of onlay autogenous graft revascularization; (2) the maintenance of volume/density of the graft (assessed through tomography); and (3) the occurrence of bone remodeling proteins (using immunohistochemistry technique) delivered in the graft. Thirty-six New Zealand White rabbits were subjected to iliac crest onlay bone grafting on both sides of the mandible. The bone bed was drill-perforated on one side aiming at accelerating revascularization, whereas on the other side it was kept intact. After grafts fixation and flaps suture all animals were submitted to tomography on both mandible sites. Six animals were sacrificed, respectively, at 3, 5, 7, 10, 20 and 60 days after surgery. A second tomography was taken just before sacrifice. Histological slides were prepared from each grafted site for both immunohistochemistry analysis [osteopontin, osteocalcin, type I collagen and vascular endothelial growth factor (VEGF) anti-bodies] and histometric analysis. The values on bone volume measured on tomography showed no statistic significance (P >= 0.05) between perforated and intact sites. Grafts placed on perforated beds showed higher bone density values compared with non-perforated ones at 3 days (P <= 0.05). This correlation was inverted at 60 days postoperatively. The findings from VEGF labeling revealed a tendency for earlier revascularization in the perforated group. The early revascularization of bone grafts accelerated the bone remodeling process (osteocalcin, type I collagen and osteopontin) that led to an increased bone deposition at 10 days. The extended osteoblast differentiation process at intermediate stages in the perforated group cooperated for a denser bone at 60 days.

Smad5 regulates Akt2 expression and insulin-induced glucose uptake in L6 myotubes

Insulin-induced glucose uptake by skeletal muscle results from Akt2 activation and is severely impaired during insulin resistance Recently, we and others have demonstrated that BMP9 improves glucose homeostasis in diabetic and non-diabetic rodents. However, the mechanism by which BMP9 modulates insulin action remains unknown. Here we demonstrate that Smad5. a transcription factor activated by BMP9, and Akt2. are upregulated in differentiated L6 myotubes. Smad5, rather than Smad1/8, is downregulated ""in vivo"" and ""in vitro"" by dexamethasone Smad5 knockdown decreased Akt2 expression and serine phosphorylation and insulin-induced glucose uptake, and increased the expression of the lipid phosphatase Ship2. Additionally, binding of Smad5 to Akt2 gene is decreased in dexamethasone-treated rats and Increased in L6 myotubes compared to myoblasts The present study indicates that Smad5 regulates glucose uptake in skeletal muscle by controlling Akt2 expression and phosphorylation These finding reveals Smad5 as a potential target for the therapeutic of type 2 diabetes. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

Immunohistochemical, tomographic and histological study on onlay bone graft remodeling. Part II: calvarial bone

Objectives Little information is available on the molecular events that occur during graft incorporation over time. The calvarial bone (Cb) grafts have been reported to produce greater responses compared with other donor regions in maxillofacial reconstructions, but the scientific evidences for this are still lacking. The objectives of this study are (1) to study the morphological pattern of Cb onlay bone grafts and compare them with the biological events through immunohistochemical responses and (2) to establish the effects of perforations in maintaining the volume and bone density of the receptor bed. Material and methods Sixty New Zealand White rabbits were submitted to Cb onlay bone grafts on the mandible. In 30 rabbits, the receptor bed was perforated (perforated group), while for the remaining animals the bed was kept intact (non-perforated group). Six animals from each group were sacrificed at 5, 7, 10, 20 and 60 days after surgery. Histological sections from the grafted area were prepared for immunohistochemical and histological analyses. Immuno-labeling was found for proteins Osteoprotegerin (OPG), receptor activator of nuclear factor-kappa beta ligand (RANKL), alkaline phosphatase (ALP), osteopontin (OPN), vascular endothelial growth factor (VEGF), tartrate-resistant acid phosphatase (TRAP), Type I collagen (COL I) and osteocalcin (OC). The tomography examination [computerized tomography (CT) scan] was conducted just after surgery and at the sacrifice. Results The histological findings revealed that the perforations contributed to higher bone deposition during the initial stages at the graft-receptor bed interface, accelerating the graft incorporation process. The results of the CT scan showed lower resorption for the perforated group (P < 0.05), and both groups showed high bone density rates at 60 days. This set of evidences is corroborated by the immunohistochemical outcomes indicating that proteins associated with revascularization and osteogenesis (VEGF, OPN, TRAP and ALP) were found in higher levels in the perforated group. Conclusions These findings indicate that the bone volume of calvarial grafts is better maintained when the receptor bed is perforated, probably resulting from more effective graft revascularization and greater bone deposition. The process of bone resorption peaked between 20 and 60 days post-operatively in both groups although significantly less in the perforated group. To cite this article:Pedrosa Jr WF, Okamoto R, Faria PEP, Arnez MFM, Xavier SP, Salata LA. Immunohistochemical, tomographic and histological study on onlay bone grafts remodeling. Part II: calvarial bone.Clin. Oral Impl. Res. 20, 2009; 1254-1264.doi: 10.1111/j.1600-0501.2009.01747.x.

Effect of meloxicam and diclofenac sodium on peri-implant bone healing in rats

Background: This study evaluated the effects of diclofenac sodium and meloxicam on peri-implant bone healing. Methods: Thirty male rats were divided into three groups: the control group (CG) received no drug; the diclofenac sodium group (DSG) received 1.07 mg/kg twice a day for 5 days; and the meloxicam group (MG) received 0.2 mg/kg daily for 5 days. A screw-shaped titanium implant was placed in the tibia. Fluorochromes, oxytetracycline (OxT), calcein (CA), and alizarin (AL), were injected at 7, 14, and 21 days, respectively, after implantation, and the animals were sacrificed 28 days after implant placement. The percentages of OxT-, CA-, and AL-labeled bone as well as the percentages of bone-to-implant contact (BIC), cortical bone area (CBA), and trabecular bone area (TBA) within the implant threads were evaluated. Results: Bone healing was delayed in the DSG during the first 14 days after implant placement (OxT-labeled bone: DSG: 5.3% +/- 7.3% versus CG: 13.2% +/- 9.8%, P= 0.002, and versus MG: 14.4% +/- 13.1%, P = 0.05). The percentages of BIC (DSG: 49.6% +/- 21.9%; MG: 67.1% +/- 22.8%; and CG: 68.1% +/- 22.8%) and CBA (DSG: 63.7% +/- 21.2%; MG: 82.7% +/- 12.4%; CG: 84.9% +/- 10.6%) were lower in the DSG compared to the MG and CG (P<0.001). The percentage of TBA was significantly greater in the DSG compared to the MG and CG (DSG: 36.3% +/- 21.2% versus MG: 17.3% +/- 12.7% and versus CG: 15.1% +/- 10.6%; P<0.001). Conclusion: Diclofenac sodium seemed to delay peri-implant bone healing and to decrease BIC, whereas meloxicam had no negative effect on peri-implant bone healing.