Biologic properties of nano-hydroxyapatite: An in vivo study of calvarial defects, ectopic bone formation and bone implantation

OBJECTIVES/SPECIFIC AIMS: The study aims to further investigate how cyclophilin D (CypD), the key mPTP opening regulator, affects BMSCs fate and to determine potential regulatory mechanisms involved in CypD regulation during osteogenesis. METHODS/STUDY POPULATION: We evaluated CypD mRNA expression in mouse BMSCs and in osteogenic-like (OL) cells during the course of OB differentiation. CypD protein level was also probed. Moreover, BMSCs had their mPTP activity recorded during osteoinduction. We further analyzed the effect of CypD genetic deletion on osteogenesis in vitro and in vivo. For our in vivo model, we performed the ectopic bone formation assay to asses differences in ossicle formation when CypD KO BMSCs were transplanted compared to wild type littermate BMSCs. In our in vitro model, we transfected OL cells with either CypD gain of function or CypD loss of function vector and measured their osteogenic differentiation potential. Additionally, we treated BMSCs with CypD inhibitor and compare to non-treated BMSCs for mineralization level. To determine potential regulatory mechanisms involved in CypD regulation, we analyzed the CypD gene (Ppif) promoter for potential transcription factor (TF) binding sites and found multiple Smad-binding elements within this promoter. Smads (Smad1, 5, 8) are TFs downstream from Bone Morphogenic Protein (BMP) signaling pathway that transmit cell differentiation signaling, and exert either activating or inhibitory effects on a variety of genes. We also transfect OL cells with Smad1 vector and analyzed for CypD mRNA levels. RESULTS/ANTICIPATED RESULTS: - Our data showed that CypD mRNA levels decreased in both primary cells and OL cells at day 7 and day 14 in osteogenic media. - Osteogenic induction also decreased mPTP activity. - In vivo ectopic bone formation assay showed increased ossicle fo DISCUSSION/SIGNIFICANCE OF IMPACT: Our data suggest that downregulation of CypD increases OB differentiation due to improved OxPhos activity led by mPTP closure. Our results corroborate reports of CypD downregulation and mPTP closure during neuronal differentiation in developing rat brains as well as in cardiomyocyte differentiation in developing mouse hearts. Our studies also suggest a yet unknown mechanism linking differentiation signaling with mitochondrial function – BMP/Smad mediated downregulation of CypD transcription. As initially mentioned, in a previous study, our lab showed that CypD KO mice present higher mitochondrial function and osteogenicity in aged BMSCs and less osteoporosis burden. Taken together, these results suggest that CypD can be a potential target to prevent bone loss in aging.

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Hedgehog Signaling Inhibition by Smoothened Antagonist BMS-833923 Reduces Osteoblast Differentiation and Ectopic Bone Formation of Human Skeletal (Mesenchymal) Stem Cells

Stem Cells International ◽

10.1155/2019/3435901 ◽

2019 ◽

Vol 2019 ◽

pp. 1-12 ◽

Cited By ~ 3

Author(s):

Nihal AlMuraikhi ◽

Nuha Almasoud ◽

Sarah Binhamdan ◽

Ghaydaa Younis ◽

Dalia Ali ◽

...

Keyword(s):

Gene Expression ◽

Bone Formation ◽

Osteoblast Differentiation ◽

Global Gene Expression ◽

Ectopic Bone Formation ◽

Ectopic Bone ◽

In Vitro Mineralization ◽

Global Gene Expression Profiling

Background. Hedgehog (Hh) signaling is essential for osteoblast differentiation of mesenchymal progenitors during endochondral bone formation. However, the critical role of Hh signaling during adult bone remodeling remains to be elucidated. Methods. A Smoothened (SMO) antagonist/Hedgehog inhibitor, BMS-833923, identified during a functional screening of a stem cell signaling small molecule library, was investigated for its effects on the osteoblast differentiation of human skeletal (mesenchymal) stem cells (hMSC). Alkaline phosphatase (ALP) activity and Alizarin red staining were employed as markers for osteoblast differentiation and in vitro mineralization capacity, respectively. Global gene expression profiling was performed using the Agilent® microarray platform. Effects on in vivo ectopic bone formation were assessed by implanting hMSC mixed with hydroxyapatite-tricalcium phosphate granules subcutaneously in 8-week-old female nude mice, and the amount of bone formed was assessed using quantitative histology. Results. BMS-833923, a SMO antagonist/Hedgehog inhibitor, exhibited significant inhibitory effects on osteoblast differentiation of hMSCs reflected by decreased ALP activity, in vitro mineralization, and downregulation of osteoblast-related gene expression. Similarly, we observed decreased in vivo ectopic bone formation. Global gene expression profiling of BMS-833923-treated compared to vehicle-treated control cells, identified 348 upregulated and 540 downregulated genes with significant effects on multiple signaling pathways, including GPCR, endochondral ossification, RANK-RANKL, insulin, TNF alpha, IL6, and inflammatory response. Further bioinformatic analysis employing Ingenuity Pathway Analysis revealed significant enrichment in BMS-833923-treated cells for a number of functional categories and networks involved in connective and skeletal tissue development and disorders, e.g., NFκB and STAT signaling. Conclusions. We identified SMO/Hedgehog antagonist (BMS-833923) as a powerful inhibitor of osteoblastic differentiation of hMSC that may be useful as a therapeutic option for treating conditions associated with high heterotopic bone formation and mineralization.

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In vivo bioluminescence imaging study to monitor ectopic bone formation by luciferase gene marked mesenchymal stem cells

Journal of Orthopaedic Research® ◽

10.1002/jor.20582 ◽

2008 ◽

Vol 26 (7) ◽

pp. 901-909 ◽

Cited By ~ 31

Author(s):

Cristina Olivo ◽

Jacqueline Alblas ◽

Vivienne Verweij ◽

Anton-Jan Van Zonneveld ◽

Wouter J. A. Dhert ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Bone Formation ◽

Bioluminescence Imaging ◽

Imaging Study ◽

Ectopic Bone Formation ◽

Luciferase Gene ◽

Ectopic Bone ◽

In Vivo Bioluminescence Imaging

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Alginate Bead-Encapsulated PEDF Induces Ectopic Bone Formation In Vivo in the Absence of Co-Administered Mesenchymal Stem Cells

Current Drug Targets ◽

10.2174/1389450116666151001112455 ◽

2018 ◽

Vol 19 (5) ◽

pp. 467-478 ◽

Cited By ~ 4

Author(s):

Mina Elahy ◽

Michael R. Doschak ◽

Jeffery D. Hughes ◽

Swati Baindur-Hudson ◽

Crispin R. Dass

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Bone Formation ◽

Alginate Bead ◽

Ectopic Bone Formation ◽

Ectopic Bone

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Short-Term Application of Dexamethasone Enhances Bone Morphogenetic Protein-7-Induced Ectopic Bone Formation In Vivo: Erratum

Journal of Trauma and Acute Care Surgery ◽

10.1097/ta.0b013e318210fe59 ◽

2011 ◽

Vol 70 (2) ◽

pp. 525

Author(s):

&NA;

Keyword(s):

Bone Formation ◽

Bone Morphogenetic Protein ◽

Ectopic Bone Formation ◽

Short Term ◽

Bone Morphogenetic Protein 7 ◽

Morphogenetic Protein ◽

Ectopic Bone

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Comparison of Ectopic Bone Formation of Embryonic Stem Cells and Cord Blood Stem Cells In Vivo

Tissue Engineering Part A ◽

10.1089/ten.tea.2009.0546 ◽

2010 ◽

Vol 16 (8) ◽

pp. 2475-2483 ◽

Cited By ~ 28

Author(s):

Jörg Handschel ◽

Christian Naujoks ◽

Fabian Langenbach ◽

Karin Berr ◽

Rita A. Depprich ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Bone Formation ◽

Cord Blood ◽

Embryonic Stem ◽

Ectopic Bone Formation ◽

Ectopic Bone ◽

Blood Stem Cells ◽

Cord Blood Stem Cells

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In Vivo Ectopic Bone Formation by Devitalized Mineralized Stem Cell Carriers Produced Under Mineralizing Culture Condition

BioResearch Open Access ◽

10.1089/biores.2014.0050 ◽

2014 ◽

Vol 3 (6) ◽

pp. 265-277 ◽

Cited By ~ 4

Author(s):

Yoke Chin Chai ◽

Liesbet Geris ◽

Johanna Bolander ◽

Grzegorz Pyka ◽

Simon Van Bael ◽

...

Keyword(s):

Stem Cell ◽

Bone Formation ◽

Culture Condition ◽

Ectopic Bone Formation ◽

Ectopic Bone ◽

Cell Carriers

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Glycosaminoglycan Mimetic Associated to Human Mesenchymal Stem Cell-Based Scaffolds Inhibit Ectopic Bone Formation, but Induce Angiogenesis In Vivo

Tissue Engineering Part A ◽

10.1089/ten.tea.2012.0377 ◽

2013 ◽

Vol 19 (13-14) ◽

pp. 1641-1653 ◽

Cited By ~ 16

Author(s):

Guilhem Frescaline ◽

Thibault Bouderlique ◽

Leyya Mansoor ◽

Gilles Carpentier ◽

Brigitte Baroukh ◽

...

Keyword(s):

Stem Cell ◽

Mesenchymal Stem Cell ◽

Bone Formation ◽

Human Mesenchymal Stem Cell ◽

Ectopic Bone Formation ◽

Ectopic Bone

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BMPER Enhances Bone Formation by Promoting the Osteogenesis-Angiogenesis Coupling Process in Mesenchymal Stem Cells

Cellular Physiology and Biochemistry ◽

10.1159/000487969 ◽

2018 ◽

Vol 45 (5) ◽

pp. 1927-1939 ◽

Cited By ~ 6

Author(s):

Fei Xiao ◽

Chuandong Wang ◽

Chenglong Wang ◽

Yuan Gao ◽

Xiaoling Zhang ◽

...

Keyword(s):

Stem Cells ◽

Bone Formation ◽

Bone Repair ◽

Ectopic Bone Formation ◽

Bmp Signaling ◽

Coupling Process ◽

Ectopic Bone

Background/Aims: During bone repair and remodeling, osteogenesis is coupled with angiogenesis. Bone morphogenetic protein (BMP) antagonists are important modulators of BMP signaling and bone homeostasis. Several investigations have demonstrated that one ‘BMP antagonist’, BMP-binding endothelial cell precursor-derived regulator (BMPER), participates in the regulation of BMP signaling. In this study, we examined the role of BMPER in the osteogenesis-angiogenesis coupling process. Methods: Human bone mesenchymal stem cells (hBMSCs) and human umbilical vein endothelial cells (HUVECs) were used in this experiment. After overexpressing or silencing BMPER with lentiviruses or siRNA, hBMSCs were stimulated by BMP-2, and osteogenic differentiation activity was detected by alkaline phosphatase and alizarin red staining. VEGF and endostatin release were assessed by ELISA. HUVEC migration was detected by the cell scratch test and transwell migration assay, and in vitro angiogenesis was determined by the tube formation assay. Bone formation was assessed using in vivo femoral monocortical defect and ectopic bone formation models. Results: BMP-2 upregulated BMPER expression. Overexpression of BMPER remarkably enhanced BMP-2-induced osteogenic differentiation, while suppression of BMPER effectively inhibited this process both in vitro and in vivo. In addition, overexpression of BMPER promoted BMP-2-induced VEGF expression in vitro and vascularization in the ectopic bone formation model. Conclusion: BMPER functions as a positive regulator of the osteogenesis-angiogenesis coupling process in hBMSCs, suggesting a novel therapeutic role of BMPER in the regenerative capacity of bone repair.

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