Recombinant Human Peptide Growth Factors, Bone Morphogenetic Protein-7 (rhBMP7), and Platelet-Derived Growth Factor-BB (rhPDGF-BB) for Osteoporosis Treatment in an Oophorectomized Rat Model.

Bone morphogenetic protein (BMP) and platelet-derived growth factor (PDGF) are known to regulate/stimulate osteogenesis, playing vital roles in bone homeostasis, rendering them strong candidates for osteoporosis treatment. We evaluated the effects of recombinant human BMP-7 (rhBMP7) and PDGF-BB (rhPDGF-BB) in an oophorectomy-induced osteoporosis rat model. Forty Sprague Dawley rats underwent oophorectomy surgery; treatments commenced on the 100th day post-surgery when all animals exhibited signs of osteoporosis. These peptide growth factors were administered intraocularly (iv) once or twice a week and the animals were monitored for a total of five weeks. Two weeks after the conclusion of the treatments, the animals were euthanized and tissues were collected for assessment of alkaline phosphatase, X-ray, micro-CT, and histology. The results indicate that the most promising treatments were 20 µg/kg rhPDGF-BB + 30 µg/kg rhBMP-7 twice a week and 30 µg/kg BMP-7 twice a week, showing significant increases of 15% (p < 0.05) and 13% (p < 0.05) in bone volume fraction and 21% (p < 0.05) and 23% (p < 0.05) in trabecular number, respectively. In conclusion, rhPDGF-BB and rhBMP-7 have demonstrated the ability to increase bone volume and density in this osteoporotic animal model, establishing them as potential candidates for osteoporosis treatment.

Combined effects of bone morphogenetic protein-7 and mineral trioxide aggregate on the proliferation, migration, and differentiation of human dental pulp stem cells.

BACKGROUND: Bioactive molecules present the potential to be used along with biomaterials in vital pulp therapy and regenerative endodontic treatment. OBJECTIVE: The aim of this study was to assess the effects of the combined use of bone morphogenetic protein-7 (BMP-7) and mineral trioxide aggregate (MTA) on the proliferation, migration, and differentiation of human dental pulp stem cells (DPSCs). METHODOLOGY: For the proliferation analysis, DPSCs were incubated with a growth medium and treated with MTA and/or BMP-7 at different concentrations. For the following analyses, DPSCs were incubated with a differentiation medium and treated with MTA and/or BMP-7. Moreover, there were groups in which DPSCs were incubated with the growth medium (control), the differentiation medium, or DMEM/F12 containing fetal bovine serum, and not treated with MTA or BMP-7. Cell proliferation was analyzed using the WST-1 assay. The odontogenic/osteogenic differentiation was evaluated by immunocytochemistry, alkaline phosphatase (ALP) activity assay, alizarin red staining, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Cell migration was evaluated using a wound-healing assay. Data were analyzed using analysis of variance and Tukey test (p=0.05). RESULTS: The use of BMP-7 with MTA presented no significant effect on cell proliferation in comparison with the treatment with MTA alone (p>0.05), but showed higher ALP activity, increased mineralization, and higher expression of DMP1 and DSPP when compared with other groups (p<0.05). Nestin expression was higher in the control group than in groups treated with MTA and/or BMP-7 (p<0.05). The cell migration rate increased after treatment with MTA when compared with other groups in all periods of time (p<0.05). At 72 hours, the wound area was smaller in groups treated with MTA and/or BMP-7 than in the control group (p<0.05). CONCLUSION: The use of BMP-7 with MTA increased odontogenic/osteogenic differentiation without adversely affecting proliferation and migration of DPSCs. The use of BMP-7 with MTA may improve treatment outcomes by increasing repair and regeneration capacity of DPSCs.

Tamoxifen and bone morphogenic protein-7 modulate fibrosis and inflammation in the peritoneal fibrosis model developed in uremic rats.

BACKGROUND: Peritoneal fibrosis (PF) represents a long-term complication of peritoneal dialysis (PD), affecting peritoneal membrane (PM) integrity and function. Understanding the mechanisms underlying PF development in an uremic environment aiming alternative therapeutic strategies for treating this process is of great interest. The aim of this study was to analyze the effects of tamoxifen (TAM) and recombinant BMP7 (rBMP7) in an experimental model of PF developed in uremic rats. METHODS: To mimic the clinical situation of patients on long-term PD, a combo model, characterized by the combination of PF and CKD with severe uremia, was developed in Wistar rats. PF was induced by intraperitoneal (IP) injections of chlorhexidine gluconate (CG), and CKD was induced by an adenine-rich diet. Uremia was confirmed by severe hypertension, increased blood urea nitrogen (BUN> 120 mg/dL) and serum creatinine levels (> 2 mg/dL). Uremic rats with PF were treated with TAM (10 mg/Kg by gavage) or BMP7 (30 µg/Kg, IP). Animals were followed up for 30 days. RESULTS: CG administration in uremic rats induced a striking increase in PM thickness, neoangiogenesis, demonstrated by increased capillary density, and failure of ultrafiltration capacity. These morphological and functional changes were blocked by TAM or rBMP7 treatment. In parallel, TAM and rBMP7 significantly ameliorated the PM fibrotic response by reducing α-SMA, extracellular matrix proteins and TGF-ß expression. TAM or rBMP7 administration significantly inhibited peritoneal Smad3 expression in uremic rats with PF, prevented Smad3 phosphorylation, and induced a remarkable up-regulation of Smad7, an intracellular inhibitor of TGFß/Smad signaling, contributing to a negative modulation of profibrotic genes. Both treatments were also effective in reducing local inflammation, possibly by upregulating IκB-α expression in the PM of uremic rats with PF. In vitro experiments using primary peritoneal fibroblasts activated by TGF-ß confirmed the capacity of TAM or rBMP7 in blocking inflammatory mediators, such as IL-1ß expression. CONCLUSIONS: In conclusion, these findings indicate important roles of TGF-ß/Smad signaling in PF aggravated by uremia, providing data regarding potential therapeutic approaches with TAM or rBMP7 to block this process.

Apoptosis-associated speck-like protein containing a caspase-1 recruitment domain (ASC) contributes to osteoblast differentiation and osteogenesis.

The role of apoptosis-associated speck-like protein containing a caspase-1 recruitment domain (ASC) in bone healing remains to be understood. To address this issue, we investigated the requirement of inflammasome-related genes in response to bone morphogenetic protein 7 (BMP7)-induced osteoblast differentiation in vitro. To validate the importance of ASC on osteogenesis, we subjected wild-type (WT) and ASC knockout C57BL/6 mice (ASC KO) to tibia defect to evaluate the bone healing process (up to 28 days). Our in vitro data showed that there is an involvement of ASC during BMP7-induced osteoblast differentiation, concomitant to osteogenic biomarker expression. Indeed, primary osteogenic cells from ASC KO presented a lower osteogenic profile than those obtained from WT mice. To validate this hypothesis, we evaluated the bone healing process of tibia defects on both WT and ASC KO mice genotypes and the ASC KO mice were not able to fully heal tibia defects up to 28 days, whereas WT tibia defects presented a higher bone de novo volume at this stage, evidencing ASC as an important molecule during osteogenic phenotype. In addition, we have shown a higher involvement of runt-related transcription factor 2 in WT sections during bone repair, as well as circulating bone alkaline phosphatase isoform when both were compared with ASC KO mice behavior. Altogether, our results showed for the first time the involvement of inflammasome during osteoblast differentiation and osteogenesis, which opens new avenues to understand the pathways involved in bone healing.

Role of rhBMP-2 and rhBMP-7 in the metabolism and differentiation of osteoblast-like cells cultured on chemically modified titanium surfaces.

This study analyzed the role of recombinant human bone morphogenetic protein 2 (rhBMP-2) and recombinant human bone morphogenetic protein 7 (rhBMP-7) in the adhesion and differentiation of rat osteoblast-like (osteo-1) cells cultured on chemically modified titanium surfaces. Osteo-1 cells were cultured on chemically modified (modified sandblasted and acid-etched) titanium surfaces in 3 different types of medium: control, medium supplemented with 20 ng/mL rhBMP-2, and medium supplemented with 20 ng/mL rhBMP-7. The following parameters were evaluated: cell adhesion after 24 hours; total protein content; collagen content and alkaline phosphatase (AP) activity after 7, 14, and 21 days; and calcified nodule formation after 21 days. The addition of rhBMP-2 or rhBMP-7 did not influence cell adhesion (P = .1175). Cell differentiation was influenced by rhBMP-2, as demonstrated by a significant increase in collagen content after 7 days of culture (P < .0001) and a significant decrease in AP activity after 21 days (P < .0001). The addition of rhBMP-7 only influenced AP activity, and a significant increase was observed after 21 days (P < .0001). Within the limitations of the study, we conclude that the presence of rhBMP-2 or rhBMP-7 did not influence cell adhesion to chemically modified titanium surfaces but provided an additional stimulus during the differentiation of rat osteo-1 cells cultured on this type of surface.

Design of a controlled release system of OP-1 and TGF-ß1 based in microparticles of sodium alginate and release characterization by HPLC-UV.

A new system for sustained release of growth factors, such as osteogenic protein 1 (OP-1) and transforming growth factor ß1 (TGF-ß1), intended to repair and promote dental tissue regeneration in rats was designed and characterized in this work. The release system was made with microparticles of sodium alginate, produced by ionic gelling dripping technique. The release profiles of OP-1 and TGF-ß1 from biopolymer matrix were determined by high-performance liquid chromatography (HPLC), and with this purpose, an HPLC-UV method was developed. About 80% of each growth factor was released in the first 24 h, reaching almost 100% in 168 h. The system was tested during the tissue repair in rat molars in comparison with calcium hydroxide and both growth factors not encapsulated. The dentin sialoprotein (DSP) was used as a repair marker. It was detected by immunohistochemistry, after 14- and 28-d post-treatment. X (2) test (p ≤ 0.001) and Fisher exact test (p ≤ 0.05) were applied for assessment of the amount of immunostaining. The treatment with encapsulated OP-1 showed an increased DSP immunostaining after 14 d and did not find any significant difference with the immunostaining observed for calcium hydroxide treatment. Treatment with TGF-ß1 did not show significant difference with calcium hydroxide. Treatment with both factors OP-1 and TGF-ß1 showed higher DSP immunostaining in comparison with calcium hydroxide treatment. In conclusion, the combination of both growth factors encapsulated showed more DSP immunostaining in comparison with each one separated, either encapsulated or not.

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.

Dentin-derived BMP-2 and odontoblast differentiation.

It is known that stem cells from exfoliated deciduous teeth (SHED) can be induced to differentiate into odontoblasts. However, the nature of dentin-derived morphogenic signals required for dental pulp stem cell differentiation remains unclear. The hypothesis underlying this work is that dentin-derived Bone Morphogenetic Proteins (BMP) are necessary for the differentiation of SHED into odontoblasts. We observed that SHED express markers of odontoblastic differentiation (DSPP, DMP-1, MEPE) when seeded in human tooth slice/scaffolds and cultured in vitro, or implanted subcutaneously into immunodeficient mice. In contrast, SHED cultured in deproteinized tooth slice/scaffolds, or scaffolds without a tooth slice, do not express these markers. SHED express the BMP receptors BMPR-IA, BMPR-IB, and BMPR-II. Notably, blockade of BMP-2 signaling inhibited the expression of markers of odontoblastic differentiation by SHED cultured in tooth slice/scaffolds. Collectively, this work demonstrates that dentin-derived BMP-2 is required to induce the differentiation of SHED into odontoblasts.