OBJECTIVE: To measure the concentration of bone morphogenetic protein 2 (BMP-2) in demineralized bone matrix (DBM) prepared from different long bones and to evaluate the osteoinductivity of different DBM on MC3T3-E1 cells. METHODS: Different bones from the same cadaver donor were used as the initial materials for making DBM, which were divided into ulna group (uDBM), humerus group (hDBM), tibia group (tDBM), and femur group (fDBM) according to the origins, and boiled DBM (cDBM) was taken as the control group. The proteins of DBM were extracted by guanidine hydrochloride, and the concentrations of BMP-2 were determined by ELISA assay. Then the DBM were co-cultured with MC3T3-E1 cells, the proliferation of MC3T3-E1 cells was observed by cell counting kit 8 (CCK-8) assay. The osteogenic differentiation ability of MC3T3-E1 cells was qualitatively observed by alizarin red, alkaline phosphatase (ALP), and Van Gieson staining, and the osteogenic differentiation ability of MC3T3-E1 cells was quantitatively analyzed by ALP content. Linear regression was used to analyze the effect of BMP-2 concentration in DBM on ALP synthesis. RESULTS: There were significant differences in the concentration of BMP-2 among the DBM groups (P<0.05). The concentrations of BMP-2 in the lower limb long bone were higher than those in the upper limb long bone, and the concentration of BMP-2 in the fDBM group was about 35.5 times that in the uDBM group. CCK-8 assay showed that the cells in each group continued to proliferate within 5 days of co-culture, and the absorbance (A) values at different time points were in the order of cDBM group2 concentration in DBM. The order of ALP content from low to high was cDBM groupP<0.05). Linear regression analysis showed that y^=0.361+0.017x, the effect of BMP-2 concentration in DBM on cellular ALP content was significant (t=3.552, P=0.005); for every 1 ng/g increase in BMP-2 concentration, ALP content would increase by 0.017 [95%CI (0.006, 0.027)] U/100 mL. CONCLUSION The concentration of natural BMP-2 in different long bones varies greatly, and the lower limb long bone is higher than the upper limb long bone. The harvested location of bone material was an important factor affecting the osteoinductivity of DBM.
Alkaline Phosphatase ; Bone Matrix ; Bone Morphogenetic Protein 2 ; Cell Count ; Coloring Agents ; Osteogenesis ; Animals ; Mice

OBJECTIVE: To explore the mechanism of the Notch1 signaling pathway in regulating osteogenic factors and influencing lumbar disc calcification. METHODS: Primary annulus fibroblasts from SD rats were isolated and subcultured in vitro. The calcification-inducing factors bone morphogenetic protein-2 (BMP-2) and basic fibroblast growth factor (b-FGF) were added to separate groups to induce calcification, which were referred to as the BMP-2 group and the b-FGF group, respectively. A control group was also set up, which was cultured in normal medium. Subsequently, cell morphology and fluorescence identification, alizarin red staining, ELISA, and quantitative real-time polymerase chain reaction (QRT-PCR) were performed to determine the effect of calcification induction. Cell grouping was performed again, including the control group, the calcification group (adding the inducer BMP-2), the calcification + LPS group(adding the inducer BMP-2 and the Notch1 pathway activator LPS), and the calcification + DAPT group (adding the inducer BMP-2 and the Notch1 pathway inhibitor DAPT). Alizarin red staining and flow cytometry were used to detect cell apoptosis, ELISA was used to detect the content of osteogenic factors, and Western blot was used to detect the expression of BMP-2, b-FGF, and Notch1 proteins. RESULTS: The induction factor screening results showed that the number of mineralized nodules in fibroannulus cells in BMP-2 group and b-FGF group was significantly increased, and the increase was greater in the BMP-2 group Meanwhile, ELISA and Western blot results showed that BMP-2, b-FGF and mRNA expression levels of BMP-2, b-FGF and Notch1 in the induced group were significantly increased (P<0.01). The results of the mechanism of Notch1 signaling pathway affecting lumbar disc calcification showed that compared to calcified group, the number of fibroannulus cell mineralization nodules, apoptosis rate, BMP-2, b-FGF content, the expression levels of BMP-2, b-FGF, and Notch1 proteins were further increased significantly However, the number of mineralization nodules, apoptosis rate, BMP-2 and b-FGF levels, BMP-2, b-FGF and Notch1 protein expression levels were decreased in the calcified +DAPT group (P<0.05 or P<0.01). CONCLUSION Notch1 signaling pathway promotes lumbar disc calcification through positive regulation of osteogenic factors.
Animals ; Rats ; Bone Morphogenetic Protein 2/metabolism* ; Calcinosis ; Cell Differentiation ; Cells, Cultured ; Lipopolysaccharides ; Osteogenesis ; Rats, Sprague-Dawley ; Receptor, Notch1/genetics* ; Signal Transduction

Bone regeneration remains a great clinical challenge. Low intensity near-infrared (NIR) light showed strong potential to promote tissue regeneration, offering a promising strategy for bone defect regeneration. However, the effect and underlying mechanism of NIR on bone regeneration remain unclear. We demonstrated that bone regeneration in the rat skull defect model was significantly accelerated with low-intensity NIR stimulation. In vitro studies showed that NIR stimulation could promote the osteoblast differentiation in bone mesenchymal stem cells (BMSCs) and MC3T3-E1 cells, which was associated with increased ubiquitination of the core circadian clock protein Cryptochrome 1 (CRY1) in the nucleus. We found that the reduction of CRY1 induced by NIR light activated the bone morphogenetic protein (BMP) signaling pathways, promoting SMAD1/5/9 phosphorylation and increasing the expression levels of Runx2 and Osterix. NIR light treatment may act through sodium voltage-gated channel Scn4a, which may be a potential responder of NIR light to accelerate bone regeneration. Together, these findings suggest that low-intensity NIR light may promote in situ bone regeneration in a CRY1-dependent manner, providing a novel, efficient and non-invasive strategy to promote bone regeneration for clinical bone defects.
Animals ; Rats ; Bone Morphogenetic Protein 2/metabolism* ; Bone Regeneration ; Cell Differentiation ; Circadian Clocks ; Cryptochromes/metabolism* ; Osteoblasts/metabolism* ; Osteogenesis ; Transcription Factors/metabolism*

Objective To determine whether the signaling activation of bone morphogenetic protein 2(BMP2)can induce myeloid-derived suppressor cells(MDSC)to secret transforming growth factor β(TGF-β),further enhancing the differentiation and infiltration of regulatory T lymphocytes(Treg)into tumor tissue. Methods The BMP2-induced mRNA and protein expression of TGF-β in MDSC was detected by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay(ELISA),respectively.The effect of BMP2-induced TGF-β secretion by MDSC on Treg differentiation was then determined by flow cytometry.Finally,we implanted the recombined human bone morphogenetic protein 2(rhBMP2)collagen gels into tumor-burdened mice to examine the role of BMP2 in Treg differentiation via MDSC-secreted TGF-β
Animals ; Bone Morphogenetic Protein 2 ; Cell Differentiation ; Mice ; Myeloid-Derived Suppressor Cells ; Neoplasms ; T-Lymphocytes, Regulatory ; Transforming Growth Factor beta

OBJECTIVES: To evaluate the repairing ability of nano-pearl powder bone substitute in rabbit with defect of distal femur bone. METHODS: Thirty-two New Zealand rabbits were randomly divided into four groups: a nano-pearl powder/recombinant human bone morphogenetic protein 2 (rhBMP-2)/hyaluronic acid group, a nano-pearl powder/hyaluronic acid group, a nano-pearl powder group and a blank control group (=8 in each group). A defect with the diameter of 7 mm and height of 10 mm was prepared at the distal femoral metaphysis line of the rabbit.Different bone substitutes were planted, and the effect of repair was evaluated by macroscopic observation, imaging examination, and histopathological examination. RESULTS: The results of imageology showed that: the bone repairing effect in the nano-pearl powder/rhBMP-2/hyaluronic acid group was better than that in the pure pearl powder group and the nano-pearl powder/hyaluronic acid group, and which in the 3 experimental groups was better than that in the blank control group; The results of histology showed that: at the 4th, 8th and 12th weeks after the modeling operation, the speed of bone repair in the nano-pearl powder/rhBMP-2/hyaluronic acid group was faster than that in the pure pearl powder group and the nano-pearl powder/hyaluronic acid group, and which in the blank control group was far slower than that in the 3 experimental groups. The results of immunohistochemistry staining for osteocalcin antibody showed that: the osteogenic effect in the nano-pearl powder/rhBMP-2/hyaluronic acid group was better than that in the pure pearl powder group and the nano-pearl powder/hyaluronic acid group (both <0.05); there was no significant difference between the nano-pearl powder/hyaluronic acid group and the pure pearl powder group (>0.05); however, there was significant difference between the pure pearl powder group and the blank control group (<0.05). According to the staining results of Type I collagen antibody, there was no significant difference in the osteogenic effect between the nano-pearl powder/rhBMP-2/hyaluronic acid group and the nano-pearl powder/hyaluronic acid group (>0.05), but the osteogenic effect in the nano-pearl powder/hyaluronic acid group was better than that in the pure pearl powder group and the blank control group (both <0.05). CONCLUSIONS Nano-pearl powder and its bone substitute can promote the repair of bone defect, and the nano-pearl powder which contains rhBMP-2 has better osteogenic and repairing effect on defect.
Animals ; Bone Morphogenetic Protein 2 ; Bone Substitutes ; Collagen ; Femur ; Humans ; Osteogenesis ; Powders ; Rabbits ; Recombinant Proteins ; Transforming Growth Factor beta

OBJECTIVE: To explore the effect of lentivirus-mediated BMP-2 overexpression plasmid transfection into bone marrow mesenchymal stem cells and silk fibroin scaffold on osteoblast transformation. METHODS: The lentivirus BMP-2 overexpression vector was constructed, bone marrow mesenchymal stem cells were cultured, and the combined culture system of nuclear scaffolds was constructed. Alizarin red staining and alkaline phosphatase staining were used to detect the osteogenic transformation of bone marrow mesenchymal stem cells in vitro. Ten New Zealand white rabbits, weighing 3.2 to 4.5 kg(averaging 3.9 kg), aged (2.89±0.45) years old, were selected to construct the rabbit tibial defect model by drilling a conical tibial defect (5 mm in length, 2 mm in width and 3 mm in depth) with an oral drill. The repair of the tibial defect in the animal model was observed by HE staining. The experimental group was implanted with silk fibroin scaffold + BMP-2 overexpression vector bone marrow mesenchymal stem cell complex, while the negative control group was implanted with silk fibroin scaffold+non-transfected bone marrow mesenchymal stem cell complex. RESULTS: Compared with the control group(silk fibroin scaffold+non-transfected bone marrow mesenchymal stem cells), the number of adherent cells on the surface of the scaffold in the experimental group(silk fibroin scaffold+transfected BMP-2 overexpression vector BMP-2 complex) increased significantly. Compared with the control group, the ECM secretion in the experimental group increased significantly. EDX analysis showed that the content of calcium ion was 0.22% in the control group and 0.86% in the experimental group, which showed that the ability of inducing calcium ion formation in the experimental group was stronger than that in the control group. Alizarin red staining of calcium nodules showed that there was no obvious change in the naked eye of the control group, and a small amount of calcium nodules could be seen under the microscope. In the experimental group, obvious red area staining was observed by naked eye, and a large number of calcium nodules were observed by microscopy. The results of alkaline phosphatase staining showed that there was no obvious change in the naked eye of the control group, and no obvious change in the microscopic observation. In the experimental group, purple area staining was observed by naked eyes, and ALP staining was strongly positive by microscopy. The combined culture system of silk fibroin scaffold and bone marrow mesenchymal stem cells can repair cartilage defects. The repair effect of BMP-2 bone marrow mesenchymal stem cells after transfection is obviously better than that of non-transfection group. HE staining showed that inflammatory cells decreased and scaffolds disappeared slightly in the control group. In the experimental group, inflammatory cells were significantly reduced, scaffolds disappeared and angiogenesis was observed. CONCLUSIONS Lentivirus-mediated BMP-2 overexpression plasmid can promote BMSC to differentiate into osteocytes and secrete more extracellular matrix containing Ca²⁺ to promote bone defect repair.
Animals ; Bone Marrow Cells ; Bone Morphogenetic Protein 2 ; Cells, Cultured ; Fibroins ; Lentivirus ; Mesenchymal Stem Cells ; Osteoblasts ; Osteogenesis ; Plasmids ; Rabbits ; Transfection

BACKGROUND: Silica particles (SPs) induce cell proliferation and osteogenic differentiation. We reported that SPs in the scaffold induced early stage osteogenic differentiation. METHODS: A polycaprolactone (PCL) scaffold was fabricated with a 10 wt% SPs. The surface of PCL scaffold was coated with a 10 µg/mL collagen solution. Next, the scaffold was conjugated with 2 µM SPs, 2 µg/mL bone morphogenetic protein 2 (BMP2), or 2 µM BMP2-conjugated SPs (BCSPs). Green fluorescent protein-coupled BMP2 was applied to fabricate the scaffold. The fluorescence intensity was analyzed by confocal microscopy. The mRNA levels of the early osteogenic differentiation marker, alkaline phosphatase (ALP), were analyzed by real-time quantitative polymerase chain reaction. Levels of BMP2, RUNX2, ERK1/2, and AKT were assessed by western blotting. RESULTS: ALP mRNA levels were significantly higher in the BCSP-conjugated scaffold than in the other scaffolds. In the early stage of osteogenic differentiation, the protein levels of BMP2, RUNX2, ERK1/2, and AKT in cells were significantly higher in the BCSP-conjugated scaffold than in other scaffolds. Thus, the BCSP composite scaffold induced rapid osteogenic differentiation. CONCLUSION: These results suggest that BCSP composite can be used to promote early stage osteogenic differentiation and show promise as a material for use in scaffolds for bone regeneration.
Alkaline Phosphatase ; Blotting, Western ; Bone Morphogenetic Protein 2 ; Bone Morphogenetic Proteins ; Bone Regeneration ; Cell Proliferation ; Collagen ; Fluorescence ; Microscopy, Confocal ; Polymerase Chain Reaction ; RNA, Messenger ; Silicon Dioxide ; Stem Cells

PURPOSE: The aim of this study was to evaluate the enhancement of osteogenic potential of biphasic calcium phosphate (BCP) bone substitute coated with Escherichia coli-derived recombinant human bone morphogenetic protein-2 (ErhBMP-2) and epigallocatechin-3-gallate (EGCG). METHODS: The cell viability, differentiation, and mineralization of osteoblasts was tested with ErhBMP-2-/EGCG solution. Coated BCP surfaces were also investigated. Standardized, 6-mm diameter defects were created bilaterally on the maxillary sinus of 10 male New Zealand white rabbits. After removal of the bony windows and elevation of sinus membranes, ErhBMP-2-/EGCG-coated BCP was applied on one defect in the test group. BCP was applied on the other defect to form the control group. The animals were sacrificed at 4 or 8 weeks after surgery. Histologic and histometric analyses of the augmented graft and surrounding tissue were performed. RESULTS: The 4-week and 8-week test groups showed more new bone (%) than the corresponding control groups (P<0.05). The 8-week test group showed more new bone (%) than the 4-week test group (P<0.05). CONCLUSIONS: ErhBMP-2-/EGCG-coated BCP was effective as a bone graft material, showing enhanced osteogenic potential and minimal side effects in a rabbit sinus augmentation model.
Animals ; Bone Morphogenetic Protein 2 ; Bone Substitutes ; Calcium ; Cell Survival ; Escherichia ; Humans ; In Vitro Techniques ; Male ; Maxillary Sinus ; Membranes ; Miners ; Osteoblasts ; Rabbits ; Transplants

PURPOSE: The purpose of this study was to evaluate the synergistic effect of adjunctive hyperbaric oxygen (HBO) therapy on new bone formation and angiogenesis after 8 weeks of healing. METHODS: Sprague-Dawley rats (n=28) were split into 2 groups according to the application of adjunctive HBO therapy: a group that received HBO therapy (HBO group [n=14]) and another group that did not receive HBO therapy (NHBO group [n=14]). Each group was divided into 2 subgroups according to the type of bone graft material: a biphasic calcium phosphate (BCP) subgroup and an Escherichia coli-derived recombinant human bone morphogenetic protein-2-/epigallocatechin-3-gallate-coated BCP (mBCP) subgroup. Two identical circular defects with a 6-mm diameter were made in the right and left parietal bones of each rat. One defect was grafted with bone graft material (BCP or mBCP). The other defect was not grafted. The HBO group received 2 weeks of adjunctive HBO therapy (1 hour, 5 times a week). The rats were euthanized 8 weeks after surgery. The specimens were prepared for histologic analysis. RESULTS: New bone (%) was higher in the NHBO-mBCP group than in the NHBO-BCP and control groups (P<0.05). Blood vessel count (%) and vascular endothelial growth factor staining (%) were higher in the HBO-mBCP group than in the NHBO-mBCP group (P<0.05). CONCLUSIONS: HBO therapy did not have a positive influence on bone formation irrespective of the type of bone graft material applied after 8 weeks of healing. HBO therapy had a positive effect on angiogenic activity.
Animals ; Blood Vessels ; Bone Morphogenetic Protein 2 ; Bone Substitutes ; Calcium ; Escherichia ; Humans ; Hyperbaric Oxygenation ; Osteogenesis ; Oxygen ; Parietal Bone ; Rats ; Rats, Sprague-Dawley ; Transplants ; Vascular Endothelial Growth Factor A

OBJECTIVE: To screen for BMP2 mutation with functional impact in patients with congenital tooth agenesis and to make oral and skeletal phenotype record and functional analysis with in vitro experiments. METHODS: We enrolled eighteen patients with congenital tooth agenesis. The medical and dental history was collected,and clinical and dental examinations including the X-ray examination of oral-facial and skeletal bone were performed for the phenotypic analysis. Blood samples were collected to extract DNA and whole exome sequencing was conducted. The genes involved in oral-facial development and congenital skeletal diseases were investigated for mutation screening. The mutations with functional impact were then investigated. In one patient, the BMP2 mutation with putative functional impact was selected for functional analysis. Wild type and mutant BMP2 plasmids with green fluorescent protein (GFP) tag were constructed and transfected into HEK293T cells. Subcellular protein distribution was observed under laser scanning confocal microscope. The activation of downstream SMAD1/5/9 phosphorylation by BMP2 was detected by Western blotting to investigate the functional impact and genetic pathogenicity. RESULTS: BMP2 mutation NM_001200.3:c.393A>T (p.Arg131Ser), rs140417301 was detected in one patient with congenital tooth agenesis, while for other genes involved in oral-facial development and congenital skeletal diseases, no functionally significant mutation was found. The proband's parents didn't carry this mutation. The father had normal dentition, while the mother lacked one premolar, and both the parents showed normal palate and maxilla. The patient also had maxillary hypoplasia in both sagittal and coronal planes, palatal dysmorphology, and malocclusion, and was diagonsed with osteopenia after the X-ray examnination of his skeletal bone. Functional analysis showed this mutation had normal subcelluar localization but reduced phosphorylation of SMAD1/5/9 (reduction by 32%, 22%, and 27% in three independent replicates). Taken together with family co-segregation, this mutaion was considered as "likely pathogenic". CONCLUSION BMP2 mutation c.393A>T (p. Arg131Ser) affects bone morphogenetic protein signaling activity, and may affect the number of teeth, growth of maxilla and palate, and bone mineral density.
Bone Morphogenetic Protein 2/genetics* ; HEK293 Cells ; Humans ; Mutation ; Phenotype ; Plasmids ; Tooth