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      Evaluation of BMP-2 Enhances the Osteoblast Differentiation of Human Amnion Mesenchymal Stem Cells Seeded on Nano-Hydroxyapatite/Collagen/Poly(l-Lactide)

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          Abstract

          Background: The aim of this study is to evaluate the effects of recombinant human bone morphogenetic protein 2 (rhBMP-2), human amnion mesenchymal stem cells (hAMSCs), and nanohydroxyapatite/collagen/poly( l-lactide) (nHAC/PLA) in tissue engineering to provide potential approaches for periodontal bone regeneration. Methods: hAMSCs were isolated from discarded amniotic membrane samples and cultured in vitro. Alkaline phosphatase (ALP) staining and alizarin red staining were performed to evaluate the osteoblast (OB) differentiation ability of hAMSCs. Three groups were divided: the experimental group (cells transfected with pcDNA3.1-rhBMP-2), the blank group (cells without gene transfection), and the control group (cells transfected with empty plasmid). RT-PCR and western blot were used to examine whether rhBMP-2 has been successfully expressed. 3-(4,5)-dimethylthiahiazol(-z-y1)-3,5-di-phenytetrazo-liumromide assay (MTT) was done to detect the effect of rhBMP-2 on hAMSCs seeded on nHAC/PLA. ALP activity, mineral formation assay, calcium, phosphate and osteocalcin (OCN) content, and OCN and RUNX2 expression of hAMSCs were detected to evaluate osteogenic differentiation capability of rhBMP-2 on hAMSCs seeded on nHAC/PLA. Results: hAMSCs exhibited intense ALP staining, obvious calcium deposition, and mineralization nodules, and rhBMP-2 were highly expressed in the experimental group. The proliferation of the hAMSCs with rhBMP-2 on nHAC/PLA was significantly higher than the cells without rhBMP-2, and the cells all increased in a time-dependent manner. rhBMP-2 significantly increased the OCN and phosphate content, mineral formation, ALP activity, osteogenic biomarkers OCN, and Runx2, and decreased calcium content in hAMSCs seeded on the nHAC/PLA scaffold. Conclusions: This finding demonstrated that hAMSCs has an ideal OB differentiation ability. rhBMP-2 facilitates the proliferation and osteogenesis of hAMSCs. The nHAC/PLA could act as a good scaffold for hAMSCs seeding, proliferation, and osteogenic differentiation. The application of rhBMP-2, nHAC/PLA, and hAMSCs in tissue engineering may offer promising possibilities for periodontal bone regeneration.

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          Novel regulators of bone formation: molecular clones and activities.

          J Wozney, R Kriz, R Hewick … (1988)
          Protein extracts derived from bone can initiate the process that begins with cartilage formation and ends in de novo bone formation. The critical components of this extract, termed bone morphogenetic protein (BMP), that direct cartilage and bone formation as well as the constitutive elements supplied by the animal during this process have long remained unclear. Amino acid sequence has been derived from a highly purified preparation of BMP from bovine bone. Now, human complementary DNA clones corresponding to three polypeptides present in this BMP preparation have been isolated, and expression of the recombinant human proteins have been obtained. Each of the three (BMP-1, BMP-2A, and BMP-3) appears to be independently capable of inducing the formation of cartilage in vivo. Two of the encoded proteins (BMP-2A and BMP-3) are new members of the TGF-beta supergene family, while the third, BMP-1, appears to be a novel regulatory molecule.
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            Birth and Death of Bone Cells: Basic Regulatory Mechanisms and Implications for the Pathogenesis and Treatment of Osteoporosis

            S C Manolagas (2000)
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              BMP signaling is required for RUNX2-dependent induction of the osteoblast phenotype.

              Renny Franceschi, Mattabhorn Phimphilai, Heidi Boules … (2006)
              RUNX2 expression in mesenchymal cells induces osteoblast differentiation and bone formation. BMP blocking agents were used to show that RUNX2-dependent osteoblast differentiation and transactivation activity both require BMP signaling and, further, that RUNX2 enhances the responsiveness of cells to BMPs. BMPs and the RUNX2 transcription factor are both able to stimulate osteoblast differentiation and bone formation. BMPs function by activating SMAD proteins and other signal transduction pathways to stimulate expression of many target genes including RUNX2. In contrast, RUNX2 induces osteoblast-specific gene expression by directly binding to enhancer regions in target genes. In this study, we examine the interdependence of these two factors in controlling osteoblast differentiation in mesenchymal progenitor cells. C3H10T1/2 mesenchymal cells and primary cultures of marrow stromal cells were transduced with a RUNX2 adenovirus and treated with BMP blocking antibodies or the natural antagonist, NOGGIN. Osteoblast differentiation was determined by assaying alkaline phosphatase and measuring osteoblast-related mRNA using quantitative RT/PCR. Activation of BMP-responsive signal transduction pathways (SMAD, extracellular signal-regulated kinase [ERK], p38, and c-jun-N-terminal kinase [JNK]) was assessed on Western blots. C3H10T1/2 cells constitutively synthesize BMP2 and 4 mRNA and protein, and this BMP activity is sufficient to activate basal levels of SMAD phosphorylation. Inhibition of BMP signaling was shown to disrupt the ability of RUNX2 to stimulate osteoblast differentiation and transactivate an osteocalcin gene promoter-luciferase reporter in C3H10T1/2 cells. BMP blocking antibodies also inhibited RUNX2-dependent osteoblast differentiation in primary cultures of murine marrow stromal cells. Conversely, RUNX2 expression synergistically stimulated BMP2 signaling in C3H10T1/2 cells. However, RUNX2 did not increase the ability of this BMP to activate SMAD, ERK, p38, and JNK pathways. This study shows that autocrine BMP production is necessary for the RUNX2 transcription factor to be active and that BMPs and RUNX2 cooperatively interact to stimulate osteoblast gene expression.
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                Author and article information

                Journal
                Journal ID (nlm-ta): Int J Mol Sci
                Journal ID (iso-abbrev): Int J Mol Sci
                Journal ID (publisher-id): ijms
                Title: International Journal of Molecular Sciences
                Publisher: MDPI
                ISSN (Electronic): 1422-0067
                Publication date (Electronic): 25 July 2018
                Publication date Collection: August 2018
                Volume: 19
                Issue: 8
                Electronic Location Identifier: 2171
                Affiliations
                [1 ]College of Stomatology, Chongqing Medical University, Chongqing 401147, China; shuhongwu069@ 123456gmail.com (S.W.); jinlinss973@ 123456gmail.com (J.S.); minlimin2006@ 123456gmail.com (M.L.); wenhuali000@ 123456gmail.com (W.L.)
                [2 ]Chongqing Key Laboratory of Oral Diseases and Sciences, Chongqing 401147, China
                [3 ]Chongqing Municipal Key Laboratory of Oral Biomedical Engineering of Higher Education, Chongqing 401147, China
                Author notes
                [* ]Correspondence: xiaozhili1973@ 123456gmail.com ; Tel.: +86-138-6173-5176
                Article
                Publisher ID: ijms-19-02171
                DOI: 10.3390/ijms19082171
                PMC ID: 6122075
                PubMed ID: 30044394
                SO-VID: a518470b-7aa2-4a5e-99a6-70151a8fd836
                Copyright © © 2018 by the authors.
                License:

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

                History
                Date received : 15 June 2018
                Date accepted : 20 July 2018
                Categories
                Subject: Article

                ScienceOpen disciplines: Molecular biology
                Keywords: human amnion mesenchymal stem cells,bone morphogenetic protein 2,nano-hydroxyapatite/collagen/poly(l-lactide) (nhac/pla),osteoblasts
                Data availability:
                ScienceOpen disciplines: Molecular biology
                Keywords: human amnion mesenchymal stem cells, bone morphogenetic protein 2, nano-hydroxyapatite/collagen/poly(l-lactide) (nhac/pla), osteoblasts

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