Association between Bone Morphogenetic Protein 2 Gene Polymorphisms and Skeletal Fluorosis of The Brick-tea Type Fluorosis in Tibetans and Kazakhs, China.

To investigate the potential association between BMP2 single nucleotide polymorphisms (SNPs) and brick-tea-type skeletal fluorosis risk in cross-sectional case-control study conducted in Sinkiang and Qinghai, China, a total of 598 individuals, including 308 Tibetans and 290 Kazakhs, were enrolled. Using the standard WS/192-2008 (China), 221 skeletal fluorosis cases were diagnosed, including 123 Tibetans and 98 Kazakhs. Logistic regressions 2 analysis did not find the association between SNPs (Rs235764, Rs235739 and Rs996544) and skeletal fluorosis. Genetic models, linkage disequilibrium (LD) and haplotype analysis were not found to be associated with risk of skeletal fluorosis after adjustment by age and sex (P>0.05).Our data suggested that Rs 235764, Rs 235739 and Rs 996544 were not linked susceptibility for skeletal fluorosis in our cross-sectional case-control study.

Long noncoding RNA C21orf121/bone morphogenetic protein 2/microRNA-140-5p gene network promotes directed differentiation of stem cells from human exfoliated deciduous teeth to neuronal cells.

Previous studies have revealed that long noncoding RNA (lncRNA) and microRNA play a crucial role in autism, which is a childhood neurodevelopmental disorder with complicated genetic origins. Hence, the study concerns whether lncRNA C21orf121/bone morphogenetic proteins 2 (BMP2)/miR-140-5p gene network affects directed differentiation of stem cells from human exfoliated deciduous teeth (SHED) to neuronal cells in rats with autism. Autism models were successfully established. The neuron cells that differentiated from SHED cell were identified. The expression of lncRNA C21orf121, miR-140-5p, BMP2, Nestin, ßIII-tubulin, and microtubule-associated protein 2 (MAP2) and the expression of neuron-specific enolase (NSE) were examined. Besides, the gap junction (GJ) function of SHED, the intracellular free Ca 2+ concentration, and the social behavior and repetitive stereotyped movements of rats in autism were detected. The target relationship between lncRNA C21orf121 and miR-140-5p and that between miR-140-5p and BMP2 were also verified. Firstly, we successfully isolated SHED and identified the differentiated neurons of SHED. Besides, the expression of BMP2, MAP2, Nestin, ßIII-tubulin, NSE positive rate, GJ function, and intracellular free Ca 2+ concentration were increased with the upregulation of C21orf121 and downregulation of miR-140-5p, and accumulated time of repetitive stereotyped movements decreased and the frequency of social behavior increased. The results indicate that lncRNA C21orf121 as a competing endogenous RNA competes with BMP2 binding to miR-140-5p, thereby promoting SHED to differentiate into neuronal cells via upregulating BMP2 expression.

Secreted phosphoprotein 24 kD (Spp24) inhibits growth of human pancreatic cancer cells caused by BMP-2.

The emerging role of bone morphogenetic proteins (BMPs) in the initiation and progression of multiple cancers has drawn great attention in cancer research. In this study, we report that BMP-2 can promote the proliferation of the pancreatic tumor cell line, PANC-1. Secreted phosphoprotein 24 kD (Spp24), a BMP binding protein, did not affect the proliferation of the cells but promoted the apoptosis of the cells in vitro. In a xeneograft tumor model using PANC-1 cells, BMP-2 dramatically promoted tumor growth, while Spp24 not only abolished the effect of BMP-2, but also dramatically induced tumor shrinking when used alone. Activation of Smad1/5/8 participated in this process as demonstrated by immunohistochemical staining of phosphorylated Smad 1/5/8. We conclude that Spp24 can be developed into a therapeutic agent that could be employed in clinical situations where the inhibition of BMPs and related proteins is advantageous.

Enhanced chondrogenesis through specific growth factors in a buffalo embryonic stem cell model.

Chondrogenic differentiation of embryonic stem cells (ESCs) via embryoid bodies (EBs) is an established model to investigate chondrogenesis signaling pathways and molecular mechanisms in vitro. Our aim has been to improve upon the number of differentiated cells needed for the in vitro development of functional cartilage. Chondrogenic differentiation of buffalo ESCs was modulated by bone morphogenetic protein 2 (BMP-2), fibroblast growth factor 10 (FGF-10), transforming growth factor-beta1 (TGF-ß1 ) individually and their combination. ESCs differentiation into chondrocytes was characterized by the appearance of Alcian blue-stained nodules and the expression of cartilage-associated genes (RT-PCR) and protein (immunocytochemistry). BMP-2 or FGF-10 treatment enhanced chondrogenic differentiation, whereas TGF-ß1 treatment inhibited buffalo ESC-derived chondrogenesis. The combination of BMP-2 and FGF-10 was the most effective treatment. This treatment resulted in a higher number of Alcian blue-positive nodules by 15.2-fold, expression of the mesenchymal cell marker scleraxis gene by 3.25-fold, and the cartilage matrix protein collagen II gene and protein 1.9- and 7-fold, respectively, compared to the untreated control group. Chondrogenesis was also recapitulated from mesenchymal and chondrogenic progenitor cells, resulting in the establishment of mature chondrocytes. Thus, buffalo ESCs can be successfully triggered in vitro to differentiate into chondrocyte-like cells by specific growth factors, which may provide a novel in vitro model for further investigation of the regulatory mechanism(s) involved.

BMP-2 promotes fracture healing by facilitating osteoblast differentiation and bone defect osteogenesis.

OBJECTIVE: To investigate the role of bone morphogenetic protein-2 (BMP-2) in promoting fracture healing in animal models. METHODS: Mouse models with muscle bag heterotopic osteogenesis (HO) were divided into a HO control group (not implanted with 250 µg rhBMP-2 bone repairing material), and a HO observation group (implanted with 250 µg rhBMP-2 bone repairing material); while rat models with bone defect (BD) were divided into a BD control group (not implanted with 250 µg rhBMP-2 bone repairing material) and a BD observation group (implanted with 250 µg rhBMP-2 bone repairing material). At 4 weeks after HO establishment, the new bone formation at the operation site was observed through visual inspections and X-ray scanning. The content of serum alkaline phosphatase (ALP) was detected by automatic biochemical analyzer. The formation of new bone at the operative sites was observed by Hematoxylin and eosin staining and Masson staining. At 0, 2, 4 and 8 weeks after operation, the growth of the defect area and its surrounding callus were observed by X-ray scanning. At 4 and 8 weeks after bone defect establishment in the mouse models, the histological changes and osteogenesis of the bone defect site were observed. RESULTS: The heterotopic osteogenesis experiment showed that at 4 weeks after operation, the mass at the muscle bag in the HO observation group became larger in contrast to the HO control group. X-ray scanning showed that there was obvious irregular bone shadow at the back muscle bag of mice from the HO observation group. The content of serum ALP in the HO observation group was significantly higher than that in the HO control group (all P<0.05). The muscle pocket in the HO observation group showed higher ectopic osteogenic activity comparing with the HO control group. Histological staining showed that bone tissue structure was visible in the newly regenerated bone, forming bone trabeculae and bone marrow tissue. Under the microscope, a large number of osteoblasts arranged neatly in a cubic shape presented at the edge of the new bone, and there were bone lacunae formed, and the bone tissue was in a relatively mature stage. In the rat bone defect models, X-ray scanning showed that the high-density development area was further increased. There was a large amount of callus formation in the bone defect area of the BD observation group, while the BD control group still had no high-density development. At 8 weeks after operation, the high-density development area decreased, indicating that there was partial absorption of callus, while there was still no high-density development in the BD control group. The callus of the bone defect area in the BD observation group was reduced and the defect area was gradually repaired, while the bone defect in the BD control group was still obvious and the bone repair was not completed. CONCLUSIONS: BMP-2 could promote osteoblast differentiation and bone defect osteogenesis in vivo. Thus, it is worthy of clinical application.

Remodeling of the osteoimmune microenvironment after biomaterials implantation in murine tibia: Single-cell transcriptome analysis.

Osseointegration seems to be a foreign body reaction equilibrium due to the complicated interactions between the immune and skeletal systems. The heterogeneity of the osteoimmune microenvironment in the osseointegration of implant materials remains elusive. Here, a single-cell study involving 40043 cells is conducted, and a total of 10 distinct cell clusters are identified from five different groups. A preliminary description of the osteoimmune microenvironment revealed the diverse cellular heterogeneity and dynamic changes modulated by implant properties. The increased immature neutrophils, Ly6C + CCR2hi monocytes, and S100a8hi macrophages induce an aggressive inflammatory response and eventually lead to the formation of fibrous capsule around the stainless steel implant. The enrichment of mature neutrophils, FcgR1hi and differentiated immunomodulatory macrophages around the titanium implant indicates favorable osseointegration under moderate immune response. Neutrophil-depletion mice are conducted to explore the role of neutrophils in osseointegration. Neutrophils may improve bone formation by enhancing the recruitment of BMSCs via the CXCL12/CXCR3 signal axis. These findings contribute to a better knowledge of osteoimmunology and are valuable for the design and modification of 'osteoimmune-smart' biomaterials in the bone regeneration field.

Bone marrow-derived mesenchymal stem cells expressing BMP2 suppress glioma stem cell growth and stemness through Bcl-2/Bax signaling.

Objectives: To find an effective molecule that controls glioma stem cell (GSC) proliferation and differentiation for the development of future therapeutic interventions against glioblastoma. Material and Methods: Bone marrow-derived mesenchymal stem cells (BMSCs) were infected with a lentiviral vector to express BMP2. Cell viability, cell counting, and tumor sphere formation assays, as well as flow cytometry, immunofluorescence staining, and Western blotting were used to investigate the effects of BMSC-BMP2 on GSCs. Results: The results of flow cytometry and the CKK-8 assay showed that BMSC-BMP2 induced GSC apoptosis while inhibiting proliferation. BMSC-BMP2 decreased GSC neurosphere formation and neurospheres' transverse and vertical diameter. Meanwhile, BMSC-BMP2 downregulated GSC Nanog and OCT4 expression levels, suggesting stemness inhibition. Western blotting showed that BMSC-BMP2 increased Bax protein expression and significantly decreased Bcl-2 protein expression. Accordingly, the Bcl-2/Bax ratio increased. Conclusion: BMSC-BMP2 could effectively inhibit GSC proliferation, induce GSC apoptosis, and decrease GSC stemness, thereby providing a novel strategy for treating malignant glioma.

Fabricating a novel HLC-hBMP2 fusion protein for the treatment of bone defects.

Treating serious bone trauma with an osteo-inductive agent such as bone morphogenetic proteins (BMPs) has been considered as an optimized option when delivered via a collagen sponge (CS). Previous works have shown that the BMP concentration and release rate from approved CS carriers is difficult to control with precision. Here we presented the fabrication of a recombinant fusion protein from recombinant human-like collagen (HLC) and human BMP-2 (hBMP2). The fusion protein preserved the characteristic of HLC allowing the recombinant protein to be expressed in Yeast (such as Pichia pastoris GS115) and purified rapidly and easily with mass production after methanol induction. It also kept the stable properties of HLC and hBMP2 in the body fluid environment with good biocompatibility and no cytotoxicity. Moreover, the recombinant fusion protein fabricated a vertical through-hole structure with improved mechanical properties, and thus facilitated migration of bone marrow mesenchymal stem cells (MSCs) into the fusion materials. Furthermore, the fusion protein degraded and released hBMP-2 in vivo allowing osteoinductive activity and the enhancement of utilization rate and the precise control of the hBMP2 release. This fusion protein when applied to cranial defects in rats was osteoinductively active and improved bone repairing enhancing the repairing rate 3.5- fold and 4.2- fold when compared to the HLC alone and the control, respectively. There were no visible inflammatory reactions, infections or extrusions around the implantation sites observed. Our data strongly suggests that this novel recombinant fusion protein could be more beneficial in the treatment of bone defects than the simple superposition of the hBMP2/collagen sponge.

A comparison of BMP2 delivery by coacervate and gene therapy for promoting human muscle-derived stem cell-mediated articular cartilage repair.

BACKGROUND: Osteoarthritis and cartilage injury treatment is an unmet clinical need. Therefore, development of new approaches to treat these diseases is critically needed. Previous work in our laboratory has shown that murine muscle-derived stem cells (MDSCs) can efficiently repair articular cartilage in an osteochondral and osteoarthritis model. However, the cartilage repair capacity of human muscle-derived stem cells has not been studied which prompt this study. METHOD: In this study, we tested the in vitro chondrogenesis ability of six populations of human muscle-derived stem cells (hMDSCs), before and after lenti-BMP2/GFP transduction using pellet culture and evaluated chondrogenic differentiation of via histology and Raman spectroscopy. We further compared the in vivo articular cartilage repair of hMDSCs stimulated with BMP2 delivered through coacervate sustain release technology and lenti-viral gene therapy-mediated gene delivery in a monoiodoacetate (MIA)-induced osteoarthritis (OA) model. We used microCT and histology to evaluate the cartilage repair. RESULTS: We observed that all hMDSCs were able to undergo chondrogenic differentiation in vitro. As expected, lenti-BMP2/GFP transduction further enhanced the chondrogenic differentiation capacities of hMDSCs, as confirmed by Alcian blue and Col2A1staining as well as Raman spectroscopy analysis. We observed through micro-CT scanning, Col2A1 staining, and histological analyses that delivery of BMP2 with coacervate could achieve a similar articular cartilage repair to that mediated by hMDSC-LBMP2/GFP. We also found that the addition of soluble fms-like tyrosine kinase-1 (sFLT-1) protein further improved the regenerative potential of hMDSCs/BMP2 delivered through the coacervate sustain release technology. Donor cells did not primarily contribute to the repaired articular cartilage since most of the repair cells are host derived as indicated by GFP staining. CONCLUSIONS: We conclude that the delivery of hMDSCs and BMP2 with the coacervate technology can achieve a similar cartilage repair relative to lenti-BMP2/GFP-mediated gene therapy. The use of coacervate technology to deliver BMP2/sFLT1 with hMDSCs for cartilage repair holds promise for possible clinical translation into an effective treatment modality for osteoarthritis and traumatic cartilage injury.

BMP-2 induces angiogenesis by provoking integrin α6 expression in human endothelial progenitor cells.

Bone morphogenetic protein-2 (BMP-2) is a multifunctional cytokine, capable of governing several cellular functions, including proliferation, motility, differentiation, and angiogenesis. Circulating endothelial progenitor cells (EPCs) have been shown to facilitate tissue repair, postnatal neovascularization, and tumor associated angiogenesis. Nevertheless, the impact of BMP-2 on angiogenesis of human EPCs has largely remained a mystery. In this study, we found that BMP-2 promoted cell migration and tube formation of EPCs in a concentration-dependent manner, indicating BMP-2 induced in vitro angiogenesis in human EPCs. Furthermore, BMP-2 significantly increased microvessel formation in Matrigel plug assay, and BMP-2 antagonist noggin prevented BMP-2-induced in vivo angiogenesis. Mechanistic investigations showed BMP-2 profoundly induced the expression of Id-1 and integrin α6 as well as EPCs angiogenesis by activating PI3K/Akt and MEK/ERK signaling pathways. Moreover, knockdown of Id-1 and integrin α6 by siRNA transfection obviously attenuated BMP-2-indueced tube formation of EPCs. These results suggest that BMP-2 promotes angiogenesis in human EPCs through the activation of PI3K/Akt, MEK/ERK, and Id-1/integrin α6 signaling cascades. This is the first demonstration that BMP-2 exhibits the angiogenesis property on human EPCs. BMP-2 might serve as the potential therapeutic target for treatment of angiogenesis-related diseases.

Effect of Polyhydroxybutyrate/Chitosan/Bioglass nanofiber scaffold on proliferation and differentiation of stem cells from human exfoliated deciduous teeth into odontoblast-like cells.

Scaffolds and their characteristics play a central role in tissue engineering. The purpose of this study was to determine the effects of Polyhydroxybutyrate (PHB)/Chitosan/nano-bioglass (nBG) nanofiber scaffold made using the electrospinning method, on the proliferation and differentiation of stem cells obtained from human exfoliated deciduous teeth into odontoblast-like cells. In this experimental study, the pulps of the molten deciduous teeth were isolated, thereafter, the stem cells from human exfoliated deciduous teeth (SHED) were extracted and then the 3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine the cell viability percentage. The expression of some stem cell genes was studied by flowcytometry. These cells were then subjected to odontoblast by using the bone morphogenetic proteins-2 (BMP2) growth factor in the differentiation medium and for the expression of their specific genes. Primers of collagen type-I, dentin sialophosphoprotein (DSPP) and alkaline phosphatase (ALP) were used and the percentage of differentiation to odontoblast cells in induction scaffolds was investigated using real-time PCR and immunohistochemistry methods. The results revealed a 6-fold increase in the expression of DSPP genes and collagen type-I, and a 2-fold increase in the expression of ALP in scaffold with BMP2 group compared to the scaffold as control group which according to the immunohistochemical test results, showed the extracted SHED to have been differentiated into dentin odontoblast-like cells. As a result, this scaffold can be used as a suitable substrate to apply in dentin tissue engineering.

Evaluation of the presence of MMP-2, TIMP-2, BMP2/4, and TGFß3 in the facial tissue of children with cleft lip and palate.

Cleft lip and palate (CLP) is the most common defect affecting the face. The treatment consists of surgical reconstruction of the anatomical structures of the cleft. Part of the surgical treatment is reconstruction of the alveolar bone by means of autogenic bone grafting (osteoplasty). This study aimed to evaluate the levels of expression of extracellular matrix remodeling factors in the facial tissue of children with a complete unilateral (CU) and a complete bilateral (CB) CLP to assess whether the wound healing process is adequate. Twenty-two CLP patients were enrolled in this study. Tissue samples were collected during alveolar osteoplasty for unilateral (n = 12) or bilateral (n = 10) cleft palate, (age range from 6 years 8 months to 12 years 2 months). Control material was obtained in the case of tooth extraction (age range from 6 years 9 months to 14 years 5 months). Immunohistochemistry was used to assess the levels of matrix metalloproteinase-2 (MMP-2), tissue inhibitor of metalloproteinase-2 (TIMP-2), bone morphogenetic proteins 2 and 4 (BMP2/4), and transforming growth factor ß3 (TGFß3). Numbers of positively stained cells were graded semi-quantitatively. Data were analysed using the Kraskel-Wallis rank test and the Bonferroni correction. The total number of MMP2-positive cells was significantly lower in the CBCLP and in the control group than in the CUCLP (p < 0.001 after the Bonferroni correction). The total number of TIMP2-positive cells was significantly higher in the CUCLP than in the CBCLP and in the control group (p < 0.001; p < 0.003 after the Bonferroni correction). The overall number of BMP2/4, TGFß3-positive cells was significantly higher in the CUCLP than in the CBCLP and in the control group (p < 0.001 after the Bonferroni correction). The decrease of the relative amount of statistically significant BMP2/4, TGFß3, MMP-2, TIMP-2 containing bone cells in CBCLP patients identifies affected alveolar bone regeneration and remodeling process.

Ex vivo bone morphogenetic protein 2 gene delivery using periodontal ligament stem cells for enhanced re-osseointegration in the regenerative treatment of peri-implantitis.

Peri-implantitis is a chronic inflammatory process with advanced bone loss and impaired healing potential. For peri-implantitis treatment, tissue engineering can be applied to enhance bone regeneration of peri-implant defects. This study aimed to evaluate ex vivo bone morphogenetic protein 2 (BMP2) gene delivery using canine periodontal ligament stem cells (PDLSCs) for regeneration of peri-implantitis defects. Canine PDLSCs were transduced with adenoviral vectors containing BMP2 (BMP2/PDLSCs). After peri-implantitis was induced by ligature placement in six beagle dogs, regenerative procedures were performed; hydroxyapatite (HA) particles and collagen gel with autologous canine PDLSCs (PDLSC group) or BMP2/PDLSCs (BMP/PDLSC group) or without cells (control group) were grafted into the defects and covered by an absorbable membrane. Three months later, the animals were sacrificed. In vitro, BMP2/PDLSCs showed similar levels of stem cell properties to PDLSCs, such as colony-forming efficiency and expression of MSC markers STRO-1 and CD 146. BMP2/PDLSCs produced BMP-2 until day 21 at a concentration of 4-8 ng/mL. In vivo, the BMP2/PDLSC group showed significantly more new bone formation and re-osseointegration in peri-implantitis defects compared to the other groups. In conclusion, ex vivo BMP2 gene delivery using PDLSCs enhanced new bone formation and re-osseointegration in peri-implantitis defects.