This study investigated the effects of bonemorphogeneticprotein 6 (BMP-6) on in vitro primordial follicle development in goats. Samples of goat ovarian cortex were cultured in vitro for 1 or 7 days in Minimum Essential Medium (control medium) supplemented with different concentrations of BMP-6. Follicle survival, activation and growth were evaluated through histology and transmission electron microscopy (TEM). After 7 days of culture, histological analysis demonstrated that BMP-6 enhanced the percentages of atretic primordial follicles when compared to fresh control (day 0). Nevertheless, BMP-6 increased follicular and oocyte diameter during both culture periods. As the culture period progressed from day 1 to day 7, a significant increase in follicle diameter was observed with 1 or 50ng/ml BMP-6. However, on the contrary to that observed with the control medium TEM revealed that follicles cultured for up to 7 days with 1 or 50ng/ml BMP-6 had evident signs of atresia. In conclusion, this study demonstrated that BMP-6 negatively affects the survival and ultrastructure of goat primordial follicles. INDEX TERMS: TGF-ß superfamily, folliculogenesis, germ cell, growth factors, goats.
To identify the signaling pathway through which BMP- 2/PELA/VEG promotes the differentiation of rMSCs into osteoblasts, cells were cultured for 3, 7, and 14 days in the presence of control, BMP-2/PELA/VEGF, BMP-2/ PELA or PELA/VEGF scaffolds. The level of phosphory- lated ERK1/2 was also examined (Figures 3A and 4). The level of phosphorylated ERK1/2 was higher from days 3 to 14 in cells cultured in the presence of BMP-2/PELA/ VEGF scaffolds than in those cultured in the presence of control scaffolds. There were no changes in the levels Figure 2. Results of the MTT assay after incubation of cells with PELA scaffolds. Cells cultured in the absence of scaffolds served as the positive control. Data are reported as means ± SD. BMP-2: bonemorphogeneticprotein-2; PELA: polylactide-poly (ethylene glycol)-polylac- tide; VEGF: vascular endothelial growth factor.
Several studies have shown that bonemorphogeneticprotein (BMP) and basic fibroblast growth factor (bFGF) induce bone formation in ectopic and orthotopic sites in vivo (1,2). In addition, systemic coadministration of BMP and bFGF in preclinical models has been shown to stimulate bone deposition in skeletal tissues (3,4). BMP2 is an important stimulator of bone formation by controlling the proliferation and differentiation of osteoblasts (5,6). A recent study reported that BMP2 could modulate osteogenic differentiation of adipose-derived stem cells (7). Notably, BMP2 and bFGF exhibited a synergistic effect on promotion of bone repair and healing (8). The osteogenic activity of bone marrow stromal cells (BMSCs) induced by both BMP2 and bFGF was greater than that induced by either bFGF or BMP2 alone (9).
Wear particles are phagocytosed by macrophages and other inflammatory cells, resulting in cellular activation and release of proinflammatory factors, which cause periprosthetic osteolysis and subsequent aseptic loosening, the most common causes of total joint arthroplasty failure. During this pathological process, tumor necrosis factor-alpha (TNF-a) plays an important role in wear-particle-induced osteolysis. In this study, recombination adenovirus (Ad) vectors carrying both target genes [TNF-a small interfering RNA (TNF-a-siRNA) and bonemorphogeneticprotein 2 (BMP-2)] were synthesized and transfected into RAW264.7 macrophages and pro-osteoblastic MC3T3-E1 cells, respectively. The target gene BMP-2, expressed on pro-osteoblastic MC3T3-E1 cells and silenced by the TNF-a gene on cells, was treated with titanium (Ti) particles that were assessed by real- time PCR and Western blot. We showed that recombinant adenovirus (Ad-siTNFa-BMP-2) can induce osteoblast differentiation when treated with conditioned medium (CM) containing RAW264.7 macrophages challenged with a combination of Ti particles and Ad-siTNFa-BMP-2 (Ti-ad CM) assessed by alkaline phosphatase activity. The receptor activator of nuclear factor-kB ligand was downregulated in pro-osteoblastic MC3T3-E1 cells treated with Ti-ad CM in comparison with conditioned medium of RAW264.7 macrophages challenged with Ti particles (Ti CM). We suggest that Ad-siTNFa-BMP-2 induced osteoblast differentiation and inhibited osteoclastogenesis on a cell model of a Ti particle-induced inflammatory response, which may provide a novel approach for the treatment of periprosthetic osteolysis.
All-trans retinoic acid (ATRA) plays an important role in ocular development. Previous stud- ies found that retinoic acid could influence the metabolism of scleral remodeling by promot- ing retinal pigment epithelium (RPE) cells to secrete secondary signaling factors. The purpose of this study was to investigate whether retinoic acid affected secretion of bonemorphogeneticprotein 2 (BMP-2) and matrix metalloproteinase 2 (MMP-2) and to explore the signaling pathway of retinoic acid in cultured acute retinal pigment epithelial 19 (ARPE- 19) cells.
Transplantation of mesenchymal stem cells (MSCs) with electrotransferred bonemorphogeneticprotein-2 (BMP- 2) transgene is an attractive therapeutic modality for the treatment of large bone defects: it provides both stem cells with the ability to form bone and an effective bone inducer while avoiding viral gene transfer. The objective of the present study was to determine the inluence of the promoter driving the human BMP-2 gene on the level and duration of BMP-2 expression after transgene electrotransfer into rat MSCs. Cytomegalovirus, elongation factor-1α, glyceraldehyde 3-phosphate dehydrogenase, and beta-actin promoters resulted in a BMP-2 secretion rate increase of 11-, 78-, 66- and 36-fold over respective controls, respectively. In contrast, the osteocalcin promoter had predictable weak activity in undifferentiated MSCs but induced the strongest BMP-2 secretion rates in osteoblastically-differentiated MSCs. Regardless of the promoter driving the transgene, a plateau of maximal BMP-2 secretion persisted for at least 21 d after the hBMP-2 gene electrotransfer. The present study demonstrates the feasibility of gene electrotransfer for eficient BMP-2 transgene delivery into MSCs and for a three-week sustained BMP-2 expression. It also provides the irst in vitro evidence for a safe alternative to viral methods that permit eficient BMP-2 gene delivery and expression in MSCs but raise safety concerns that are critical when considering clinical applications.
13. Glansbeek HL, van Beuningen HM, Vitters EL, Morris EA, van der Kraan PM, van den Berg WB. Bonemorphogeneticprotein 2 stimulates articular cartilage proteoglycan synthesis in vivo but does not counteract interleukin-1alpha effects on proteoglycan synthesis and content. Arthritis Rheum 1997; 40(6): 1020-8. 14. Zheng LW, Wong MC, Rabie AB, Cheung LK. Evaluation of recombinant human bonemorphogenetic
Developmental competence of in vitro matured (IVM) oocytes needs to be improved and this can potentially be achieved by adding recombinant bonemorphogeneticprotein 15 (BMP15) or growth differentiation factor (GDF9) to IVM. The aim of this study was to determine the effect of a purified pro-mature complex form of recombinant human BMP15 versus the commercially available bioactive forms of BMP15 and GDF9 (both isolated mature regions) during IVM on bovine embryo development and metabolic activity. Bovine cumulus oocyte complexes (COCs) were matured in vitro in control medium or treated with 100 ng/ml pro-mature BMP15, mature BMP15 or mature GDF9 +/2 FSH. Metabolic measures of glucose uptake and lactate production from COCs and autofluorescence of NAD(P)H, FAD and GSH were measured in oocytes after IVM. Following in vitro fertilisation and embryo culture, day 8 blastocysts were stained for cell numbers. COCs matured in medium +/2 FSH containing pro-mature BMP15 displayed significantly improved blastocyst development (57.763.9%, 43.564.2%) compared to controls (43.362.4%, 28.963.7%) and to mature GDF9+FSH (36.163.0%). The mature form of BMP15 produced intermediate levels of blastocyst development; not significantly different to control or pro-mature BMP15 levels. Pro-mature BMP15 increased intra-oocyte NAD(P)H, and reduced glutathione (GSH) levels were increased by both forms of BMP15 in the absence of FSH. Exogenous BMP15 in its pro-mature form during IVM provides a functional source of oocyte-secreted factors to improve bovine blastocyst development. This form of BMP15 may prove useful for improving cattle and human artificial reproductive technologies.
Sequências de genes específicos que controlam a neoformação óssea: bonemorphogeneticprotein-2 e bonemorphogeneticprotein-4 foram clonadas a partir de tecidos ósseos humanos em regeneração e utilizadas para construir dois vetores de alta expressão em mamíferos regulados pelo promotor constitutivo de citomegalovirus: (pCMV-BMP2 e pCMV-BMP4). Para avaliar a eficácia destes vetores foram utilizados fibroblastos bovinos em cultura que foram transfectados atráves de lipossomos. As expressões foram analisadas na amplificação por RT-PCR em gel de agarose. Os resultados mostraram ser possível reproduzir estes fatores de crescimento em células de mamíferos que poderão futuramente ser utilizadas como vias de liberação controlada de BMPs em sítios do esqueleto lesionados que necessitam de neoformação óssea.
Bone homeostasis seems to be controlled by delicate and subtle ‘‘cross talk’’ between the nervous system and ‘‘osteo- neuromediators’’ that control bone remodeling. The purpose of this study was to evaluate the effect of interactions between neuropeptides and human bonemorphogeneticprotein 2 (hBMP2) on human osteoblasts. We also investigated the effects of neuropeptides and hBMP2 on gap junction intercellular communication (GJIC). Osteoblasts were treated with neuropeptide Y (NPY), substance P (SP), or hBMP2 at three concentrations. At various intervals after treatment, cell viability was measured by the MTT assay. In addition, cellular alkaline phosphatase (ALP) activity and osteocalcin were determined by colorimetric assay and radioimmunoassay, respectively. The effects of NPY, SP and hBMP on GJIC were determined by laser scanning confocal microscopy. The viability of cells treated with neuropeptides and hBMP2 increased significantly in a time-dependent manner, but was inversely associated with the concentration of the treatments. ALP activity and osteocalcin were both reduced in osteoblasts exposed to the combination of neuropeptides and hBMP2. The GJIC of osteoblasts was significantly increased by the neuropeptides and hBMP2. These results suggest that osteoblast activity is increased by neuropeptides and hBMP2 through increased GJIC. Identification of the GJIC-mediated signal transduction capable of modulating the cellular activities of bone cells represents a novel approach to studying the biology of skeletal innervation.
Male Sprague Dawley rats (150–175 g) were obtained from Taconic farms, Charles River Laboratories (Raleigh, NC) and acclimated prior to surgery. The surgery was performed under general anesthesia by weight-adapted intraperitoneal injection of Xylazine 2% (Medistar; 12 mg/kg body weight) and Ketamine hydrochloride (Ketaset; 100 mg/mL; 80 mg/kg body weight). The thorax and abdomen were shaved and scrubbed with Betadine and alcohol. Using aseptic technique, two 5 mm incisions were made at the midline so that two subcutaneous pouches were prepared by blunt dissection. Collagen gel (200 m l) containing 2 m g rhBMP-2 with or without collagen-BMP bifunc- tional peptide (BC-1) was injected into the left or the right side of the subcutaneous region. Each rat received two injections and a total of 20 animals were used in the study. The BMP-2 dose (2 mg) used in this experiment had been determined from preliminary experiments (data not shown). In those experiments, rats were implanted with collagen gel containing either 1 or 5 mg of BMP-2. Collagen gel without peptide resulted in bone formation at the 5 mg dose but not at the 1 mg dose. Collagen gel with peptide resulted in bone formation at both doses (1 and 5 mg), and a dose of 2 mg BMP-2 was selected for the experiment.
Bonemorphogenetic proteins (BMPs) are multifunctional growth factors that belong to the TGFβ superfamily and form a subfamily with more than 20 members (Bragdon et al., 2011). BMP2 was first identified in bone and later associated with the control of osteogenesis and chondrogenesis through BMP signaling pathway (reviewed by Carreira et al., 2014; Rosen, 2009). Beside its critical role during skeletogenesis, BMP2 is also involved in many other physiological processes, such as embryonic patterning and organogenesis (reviewed in Asahina, 2014; Hogan, 1996). BMP2 gene is flanked by regions classified as gene deserts (long regions without nearby genes) that may contain important regulatory elements, and the presence of long-range elements controlling BMP2 transcription was reported in mammals (Chandler et al., 2007; Dathe et al., 2009). The remarkable conservation of protein structure and function (Carreira et al., 2014) conjugated with its crucial role during development, maintained throughout vertebrate evolution, suggest that BMP2 transcription may be tightly controlled (Sugiura, 1999). The conservation of BMP2 gene, in particular its promoter region, has been reported in mammals (i.e. mouse and human; Abrams et al., 2004; Sugiura, 1999) and binding sites for several bone- and cartilage-related transcription factors (TFs), such as RUNX and SOX9, were predicted. Although the activation of human BMP2 promoter by RUNX2 has not been proved (Helvering et al., 2000), RUNX2 was shown to effectively increase BMP2 gene transcription while BMP2 was also able to regulate RUNX2 transcription in a feedback regulatory mechanism (Choi et al., 2005). Surprisingly, not much more is known about transcriptional regulation of BMP2 by bone- and cartilage-related TFs and thus much remains to be done regarding this question.
recombinant human BMP-2 (for 2 hours at the indicated doses). Groups were compared using a 2-way ANOVA. Both WT and MGP -/- VSMCs exhibited similar Id1 mRNA levels, both at baseline and in response to exogenous BMP-2. (B) Cultured aortic VSMCs from wild-type mice were transfected with either scrambled siRNA (siSC) or siRNA targeting MGP (siMGP) at 20 nM. RNA was isolated from cells after 4 days. siMGP decreased MGP mRNA levels in WT VSMCs by >95% compared with siSC-treated cells. However, depletion of MGP in WT VSMCs did not alter Id1 mRNA levels. **P<0.0001 compared to siSC-treated VSMCs. (C) VSMCs isolated from wild-type mice were treated with 20 nM of either scrambled siRNA (siSC) or siRNA specific for MGP (siMGP). Cells were incubated with or without BMP-2 (20 ng/mL) for 1 h prior to protein harvest. Western blots were probed with antibodies specific for phosphorylated Smad 1/5 (P-Smad 1/5) and total Smad 1. Depletion of MGP in WT VSMCs did not alter the ratio of P-Smad 1/5 levels to total Smad 1 levels, both at baseline and in response to exogenous BMP-2.
Bone and cartilage regeneration has become a hot research topic in the past decade (Puppi et al., 2010). The high prevalence of degenerative diseases as well as accidental fractures prompted the search of alternatives to the autogenous and allogeneic grafts. Tissue engineering approaches aim to develop scaffolds that can induce and guide cell proliferation, differentiation and new tissue formation at the site of tissue injury or defect. The scaffolds contain cells or biomolecules that can attract cells and growth factors – small molecules or peptides that promote cell survival and tissue regeneration (Kim and Valentini, 2002; Schoichet, 2010). Bonemorphogenetic proteins (BMPs) from bone matrix regulate the differentiation and function of the cells involved in bone and cartilage formation, including osteoblasts and chondrocytes (Urist, 1965; Cowan et al., 2005; Reddi, 2005). Most clinical trials have focused on the use of human recombinant BMP-2 (rhBMP-2) since it is one of the most potent inductors of bone formation in vivo (Cheng et al., 2003; Takahashi et al., 2005; Bishop and Einhorn, 2007; Bessa et al., 2008a). In several types of cell cultures, BMP-2 increases the expression of alkaline phosphatase (ALP), osteopontin, osteocalcin, and collagen type I (Jeon et al., 2007). However, BMP-2 administered in solution is quite unstable, rapidly loses its bioactivity and is not retained at the application site long enough for the effective healing of fractures in large animals (Seeherman et al., 2004). The elimination half-life of BMP-2 in nonhuman primates has been found to be about 6-7 min (Poynton and Lane, 2002). Therefore, the design of delivery systems able to protect growth factors from premature degradation, to retain them at the bone injury or defect, and to sustain their release is a clinical requirement (Hsieh et al., 2006; Jeon et al., 2007; Bishop and Einhorn, 2007). An ideal BMP delivery system should be biocompatible, biodegradable or bioeliminable, malleable, easy to handle and sterilizable and should act as a structural template to ﬁ ll the tissue lesion (Geiger et al., 2003; Seeherman and Wozney, 2005; Issa et al., 2008).
growth or mRNA for BMP receptors (Silva et al. 2004a) are not translated into protein by goat primordial follicles. Nevertheless, Lee et al. (2001) demonstrated in vivo, that direct injection of BMP-7 in the mouse ovarian burse leads to an increase in the number of developing follicles, reducing the number of primordial ones. These contradictory results can be explained by differences among species and protocols tested. Recently, in vitro studies showed that 100ng/ml of BMP-7, in the presence of FSH, promoted the transition from primordial to primary follicles in mouse, suggesting that BMP-7 can play a role in follicular activation when associated with FSH (Lee et al. 2004).
BMPR2 is a type II serine/threonine kinase receptor, which transduces signals from BMPs by forming heteromeric complexes with type I receptors. In our current study, BMPR2 and BMPR1a mRNA levels increased in the mouse pancreas with CP induction, along with BMP2 and BMP4, but not BMP7, implicating the potential roles of BMPRs in CP. To investigate the role of BMP signaling in the pancreas, we used a mouse line with a genetic deletion of BMPR2 in this study. BMPR2 deletion in homozygous Figure 6. BMPR2 deficiency increased Smad2 phosphorylation in PSCs. (A) Wild-type and BMPR2 +/2 PSCs were treated with 250 ng/ml of BMP2 for the indicated time points. The whole cell lysates were subjected to Western blotting with anti-pSmad1/5/8 and total Smad1 antibodies. (B) Quantification of the Western blots. The protein levels were normalized against GAPDH and quantified as fold of 0 min. *P,0.05 compared with 0 min, #
During routine clinical dentistry procedures, it is possible to expose the pulp tissue and proceed to pulp capping to preserve pulp vitality and promote healing and function. Numerous factors may influence the healing process of the dental pulp, including the condition of the pulp itself, the restorative material manipulation, the applied capping material, and so forth. The most commonly used materials for dental pulp capping are various forms of calcium hydroxyde by their odontogenetic effect (1). The application of calcium hydroxide is a treatment option to encourage hard tissue bridging after dental pulp capping. However, this material is not considered ideal because is not able to really induce new tissue formation in most cases. Other material such as dentin adhesives, hydroxyapatite, mineral trioxide aggregate, tricalcium phosphate, allogenic dentin matrix, bonemorphogeneticprotein (BMP) and calcium hydroxide cements have been studied with regard to hard tissue barrier formation after pulp exposition (2,3). It has been suggested that tissue engineering techniques can offer a solution for this problem (4). One approach is the development of a conductive scaffold to induce the resident cells in the dental pulp to regenerate the new tooth tissue.
I njectable bone substitutes and techniques have been developed for use in minimally invasive procedures for bone augmentation. Objective: To develop a novel injectable thermo-sensitive alginate hydrogel (TSAH) as a scaffold to induce bone regeneration, using a minimally invasive tunnelling technique. Material and Methods: An injectable TSAH was prepared from a copolymer solution of 8.0 wt% Poly(N-isopropylacrylamide) (PNIPAAm) and 8.0 wt% AAlg-g-PNIPAAm. In vitro properties of the material, such as its microstructure and the sustained release of recombinant human bonemorphogeneticprotein-2 (rhBMP-2), were investigated. Then, with the subperiosteal tunnelling technique, this material, carrying rhBMP-2, was injected under the labial periosteum of the maxillary anterior alveolar ridge in a rabbit model. New bone formation was evaluated by means of X-ray, micro-computed tomography (micro-CT), luorescence labelling, histological study, and immunohistochemistry study. Results: The material exhibited good injectability and thermo-irreversible properties. SEM showed an interconnected porous microstructure of the TSAH. The result of ALP activity indicated sustained delivery of BMP-2 from the TSAH from days 3 to 15. In a rabbit model, both TSAH and TSAH/rhBMP-2 induced alveolar ridge augmentation. The percentage of mineralised tissue in the TSAH/rhBMP-2 group (41.6±3.79%) was signiicantly higher than in the TSAH group (31.3±7.21%; p<0.05). The density of the regenerating tissue was higher in the TSAH/rhBMP-2 group than in the other groups (TSAH group, positive control, blank control; p<0.05). Conclusions: The TSAH provided convenient handling properties for clinical application. To some extent, TSAH could induce ridge augmentation and mineral deposition, which can be enhanced when combined with rhBMP-2 for a minimally invasive tunnelling injection.
Brazilian Santa Inês sheep are very well-adapted to the tropical conditions of Brazil and are an important source of animal protein. A high rate of twin births was reported in some SI flocks. The Growth and Differentiation Factor 9 (GDF-9) and BoneMorphogeneticProtein 15 (BMP-15) are the first two genes expressed by the oocyte that are associated with an increased ovulation rate in sheep. All GDF-9 and BMP-15 variants characterized, up until now, present the same phenotype: the heterozygote ewes have an increased ovulation rate and the mutated homozygotes are sterile. In this study we have found a new allele of GDF-9, named FecGE (Embrapa), which leads to a substitution of a phenylalanine by a cysteine in a conservative position of the mature peptide. Homozygote ewes presenting the FecGE allele have shown an increase in their ovulation rate (82%), and prolificacy (58%). This new phenotype can be very useful in better understanding the genetic control of follicular development; the mechanisms involved in the control of ovulation rate in mammals; and for the improvement of sheep production.
The sample size was calculated with the results of previous studies that investigated the levels of fetuin-A (4), dickkopf-1 (DKK-1) (6) and bonemorphogeneticprotein-7 (BMP-7) (7) based on a = 0.05 and a power of 80%. At least 39 patients were required per group. We excluded subjects with renal impairment (serum creatini- ne.1.4 mg/dl) and patients who were treated with gluco- corticoids during the previous four weeks. We consecutively enrolled 45 AS patients with syndesmophytes and 49 AS patients without syndesmophytes. All patients met the 1984 modified New York criteria for AS (8). To assess the disease activity, functional ability and spinal mobility, we used the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) (9), Functional Index (BASFI) (10) and Metrology Index (BASMI) (11), respectively. There were 68 healthy subjects who served as a control group. The controls were the relatives of the health professionals and blood donors without inflammatory back pain. Data regarding cardiovascular risk factors, such as smoking status (defined as ever/never smokers), hypertension (HT) and diabetes mellitus (DM) were collected. The symptom duration and past or current treatment for AS were also recorded. Local ethical committee approval was obtained and all patients signed informed consent forms.