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

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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Ectopic vascularized bone formation by human mesenchymal stem cell microtissues in a biocomposite scaffold

Colloids and Surfaces B Biointerfaces ◽

10.1016/j.colsurfb.2017.10.004 ◽

2017 ◽

Vol 160 ◽

pp. 661-670 ◽

Cited By ~ 12

Author(s):

Rajan Narayan ◽

Tarun Agarwal ◽

Debasish Mishra ◽

Somnath Maji ◽

Sujata Mohanty ◽

...

Keyword(s):

Stem Cell ◽

Mesenchymal Stem Cell ◽

Bone Formation ◽

Human Mesenchymal Stem Cell ◽

Vascularized Bone

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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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Combinatorial morphogenetic and mechanical cues to mimic bone development for defect repair

10.1101/561837 ◽

2019 ◽

Author(s):

S. Herberg ◽

A. M. McDermott ◽

P. N. Dang ◽

D. S. Alt ◽

R. Tang ◽

...

Keyword(s):

Stem Cell ◽

Bone Formation ◽

Mechanical Loading ◽

Endochondral Ossification ◽

Bone Development ◽

Human Mesenchymal Stem Cell ◽

Bone Defects ◽

Mechanical Environment ◽

Tgf Β1

AbstractEndochondral ossification during long bone development and natural fracture healing initiates by mesenchymal cell condensation and is directed by local morphogen signals and mechanical cues. Here, we aimed to mimic these developmental conditions for regeneration of large bone defects. We hypothesized that engineered human mesenchymal stem cell (hMSC) condensations with in situ presentation of transforming growth factor-β1 (TGF-β1) and/or bone morphogenetic protein-2 (BMP-2) from encapsulated microparticles would promote endochondral regeneration of critical-sized rat femoral bone defects in a manner dependent on the in vivo mechanical environment. Mesenchymal condensations induced bone formation dependent on morphogen presentation, with dual BMP-2 + TGF-β1 fully restoring mechanical bone function by week 12. In vivo ambulatory mechanical loading, initiated at week 4 by delayed unlocking of compliant fixation plates, significantly enhanced the bone formation rate in the four weeks after load initiation in the dual morphogen group. In vitro, local presentation of either BMP-2 alone or BMP-2 + TGF-β1 initiated endochondral lineage commitment of mesenchymal condensations, inducing both chondrogenic and osteogenic gene expression through SMAD3 and SMAD5 signaling. In vivo, however, endochondral cartilage formation was evident only in the BMP-2 + TGF-β1 group and was enhanced by mechanical loading. The degree of bone formation was comparable to BMP-2 soaked on collagen but without the ectopic bone formation that limits the clinical efficacy of BMP-2/collagen. In contrast, mechanical loading had no effect on autograft-mediated repair. Together, this study demonstrates a biomimetic template for recapitulating developmental morphogenic and mechanical cues in vivo for tissue engineering.One Sentence SummaryMimicking aspects of the cellular, biochemical, and mechanical environment during early limb development, chondrogenically-primed human mesenchymal stem cell condensations promoted functional healing of critical-sized femoral defects via endochondral ossification, and healing rate and extent was a function of the in vivo mechanical environment.

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