Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Mar 14:10:802699.
doi: 10.3389/fcell.2022.802699. eCollection 2022.

Selective Retinoic Acid Receptor γ Antagonist 7C is a Potent Enhancer of BMP-Induced Ectopic Endochondral Bone Formation

Daisuke Tateiwa  1 , Takashi Kaito  1 , Kunihiko Hashimoto  2 , Rintaro Okada  3 , Joe Kodama  4 , Junichi Kushioka  1 , Zeynep Bal  1 , Hiroyuki Tsukazaki  5 , Shinichi Nakagawa  1 , Yuichiro Ukon  1 , Hiromasa Hirai  1 , Hongying Tian  4 , Ivan Alferiev  6 , Michael Chorny  6 , Satoru Otsuru  4 , Seiji Okada  1 , Masahiro Iwamoto  4
Affiliations

Selective Retinoic Acid Receptor γ Antagonist 7C is a Potent Enhancer of BMP-Induced Ectopic Endochondral Bone Formation

Daisuke Tateiwa et al. Front Cell Dev Biol. .

Abstract

Bone morphogenetic proteins (BMPs) have been clinically applied for induction of bone formation in musculoskeletal disorders such as critical-sized bone defects, nonunions, and spinal fusion surgeries. However, the use of supraphysiological doses of BMP caused adverse events, which were sometimes life-threatening. Therefore, safer treatment strategies for bone regeneration have been sought for decades. Systemic administration of a potent selective antagonist of retinoic acid nuclear receptor gamma (RARγ) (7C) stimulated BMP-induced ectopic bone formation. In this study, we developed 7C-loaded poly lactic nanoparticles (7C-NPs) and examined whether local application of 7C enhances BMP-induced bone regeneration. The collagen sponge discs that absorbed recombinant human (rh) BMP-2 were implanted into the dorsal fascia of young adult mice to induce ectopic bone. The combination of rhBMP-2 and 7C-NP markedly increased the total bone volume and thickness of the bone shell of the ectopic bone in a dose-dependent manner compared to those with rhBMP-2 only. 7C stimulated sulfated proteoglycan production, expression of chondrogenic marker genes, and Sox9 reporter activity in both chondrogenic cells and MSCs. The findings suggest that selective RARγ antagonist 7C or the related compounds potentiate the bone inductive ability of rhBMP-2, as well as support any future research to improve the BMP-2 based bone regeneration procedures in a safe and efficient manner.

Keywords: Bmp/Smad signaling; RARγ inverse agonist; bone morphogenetic protein; bone regeneration; endochondral bone formation; retinoic acid receptor γ.

PubMed Disclaimer

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
A mouse model of BMP-2-induced ectopic bone formation. (A) BMP-2 pellets 14 days after implantation. (B) The experiment outline. (C) Histology and μCT images of the BMP-2 pellets on Days 7, 10, and 14 (scale bar = 1 mm or 100 μm for whole view and the magnified view, respectively). (D) The BMP-2 pellet on Day 10. Cartilage tissue was replaced by new bone (OCN, red) and vasculature (αSMA, green).
FIGURE 2
FIGURE 2
Analysis of bone induced in the BMP-2 pellets on Day 14. (A) Ex vivo 3D micro-CT images of the ectopic bone (scale bar = 1 mm). (B) In vivo micro-CT images of ectopic bones (scale bar = 2 mm). (C) The BV of the ectopic bone in the 7C and NRX groups. The BV was significantly higher in the 7C groups and lower in the NRX groups in a dose-dependent manner compared to the control value (control; n = 25, 7C-0.3; n = 17, 7C-1.0; n = 17, 7C-5.0; n = 25, NRX-1.0; n = 7, and NRX-5.0; n = 7; *p < 0.05, ***p < 0.001, and ****p < 0.0001). (D) Comparison of microstructural bone parameters between the control and 7C groups. n = 5, *p < 0.05, and **p < 0.01. (E) Histological sections (H&E staining) of BMP-2 pellets (whole view, scale bar = 1 mm; magnified view, scale bar = 400 μm). 7C group formed thicker bone shell. (F) Comparison of bone shell thickness. n = 5, *p < 0.05 and **p < 0.01.
FIGURE 3
FIGURE 3
Evaluation of cartilage components in BMP pellets on Day 7. (A) Macroscopic appearance and wet weight of the BMP-2 pellets with 7C-NPs (7C) and blank-NP (Control) (scale bar = 5 mm). (B) The sGAG amount in the BMP-2 pellets. (C) Safranin O staining of the BMP-2 pellets (whole view, scale bar = 1 mm; magnified view, scale bar = 200 μm). (D) Comparison of cartilage tissue area. The red stained area was measured using ImageJ (version 1.52q, U. S. National Institutes of Health; https://imagej.nih.gov/ij/). n = 8 per group, **p < 0.01.
FIGURE 4
FIGURE 4
Immunohistochemical staining of BMP-2 pellets on Day 7. (A) p-Smad1, p-Smad2, and Sox9 positive cells in the cartilage tissue of the BMP-2 pellets with blank-NPs (Control) and 7C-NPs (7C). (B) Quantitative analysis of positive cells. The immunopositive cells for p-Smad1, p-Smad2, and Sox9 were counted only in cartilaginous tissue formed in BMP-2 pellets. n = 8 per group, *p < 0.05, and **p < 0.01.
FIGURE 5
FIGURE 5
Effect of 7C on inflammatory response. (A) Representative microscopic images of the inflammatory zone stained with H&E in the pellets; the inflammatory zone expanded depending on the BMP amount, and the pellets without BMP (containing only blank-NP or 7C-NP) showed a small inflammatory zone. Whole view, scale bar = 1 mm; magnified view, scale bar = 200 μm. (B) Histological analysis of the inflammatory zone. The tissue (inflammatory zone) surrounding the collagen sponge (CS) was infiltrated with inflammatory cells such as macrophages (yellow arrow), fibroblasts (white arrow), leukocytes (black arrow) and lymphocytes (red arrow). Immunohistochemistry for CD163 that is positive in histiocytes. (C) The analysis of the inflammatory zone. *p < 0.05, ****p < 0.0001, and ns, not significant. (D) The mRNA expression levels of inflammatory cytokines (TNF-α, IL1-β, and IL-6) in the BMP-2 (1.5 μg) pellets with or without 7C-NPs. n = 12 per group. ns, not significant.
FIGURE 6
FIGURE 6
Effect of RARγ agonist/antagonist on ATDC5 cells and MSCs. (A) Alcian blue staining of ATDC5 cells in micromass (scale bar = 1 mm). The blue stained area was extracted using ImageJ (version 1.52q, U. S. National Institutes of Health; https://imagej.nih.gov/ij/) (n = 4 per group). (B) Quantitative RT-PCR analysis of ATDC5 cells cultured in chondrogenic medium supplemented with BMP-2 (20 ng/ml) and 7C (50 nM) or NRX (100 nM) (n = 4 per group). (C) Alcian blue staining of MSCs in micromass (scale bar = 1 mm). The blue stained area was extracted using ImageJ (n = 4 per group). (D) The gene expression level of Sox9 in MSCs cultured in chondrogenic medium. Sox9 expression was upregulated by 7C, even in the absence of BMP-2 (n = 3 per group). *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 and ns, not significant.
FIGURE 7
FIGURE 7
Effect of RARγ antagonists and agonists on BMP signaling. (A) Id1-luc reporter activity in ATDC5 cells treated with RARγ antagonists (CD2665 and 7C) with or without 10 ng/ml rhBMP-2. n = 4 per group. **p < 0.01, ***p < 0.001, ****p < 0.0001, and ns, not significant. (B) Immunoblotting analysis with p-Smad1 antibody. The ATDC5 cells were treated with BMP-2 and increasing concentrations of RARγ antagonists (CD2665 and 7C) and RARγ agonists (NRX204647 and CD1530). The cell lysates were prepared 45 min after treatment and subjected to immunoblot analysis for p-Smad1 and GAPDH.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Amarilio R., Viukov S. V., Sharir A., Eshkar-Oren I., Johnson R. S., Zelzer E. (2007). HIF1α Regulation of Sox9 Is Necessary to Maintain Differentiation of Hypoxic Prechondrogenic Cells during Early Skeletogenesis. Development (Cambridge, England) 134 (21), 3917–3928. 10.1242/dev.008441 - DOI - PubMed
    1. Betz R. R. (2002). Limitations of Autograft and Allograft: New Synthetic Solutions. Orthopedics 25 (5 Suppl. l), s561–70. 10.3928/0147-7447-20020502-04 - DOI - PubMed
    1. Burkus J. K., Transfeldt E. E., Kitchel S. H., Watkins R. G., Balderston R. A. (2002). Clinical and Radiographic Outcomes of Anterior Lumbar Interbody Fusion Using Recombinant Human Bone Morphogenetic Protein-2. Spine 27 (21), 2396–2408. 10.1097/00007632-200211010-00015 - DOI - PubMed
    1. Cahill K. S., Chi J. H., Day A., Claus E. B. (2009). Prevalence, Complications, and Hospital Charges Associated with Use of Bone-Morphogenetic Proteins in Spinal Fusion Procedures. Jama 302 (1), 58–66. 10.1001/jama.2009.956 - DOI - PubMed
    1. Chakkalakal S. A., Uchibe K., Convente M. R., Zhang D., Economides A. N., Kaplan F. S., et al. (2016). Palovarotene Inhibits Heterotopic Ossification and Maintains Limb Mobility and Growth in Mice with the HumanACVR1R206HFibrodysplasia Ossificans Progressiva (FOP) Mutation. J. Bone Miner Res. 31 (9), 1666–1675. 10.1002/jbmr.2820 - DOI - PMC - PubMed