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
. 2017 Nov 15;18(11):2422.
doi: 10.3390/ijms18112422.

Enhancement of Bone Marrow-Derived Mesenchymal Stem Cell Osteogenesis and New Bone Formation in Rats by Obtusilactone A

Affiliations

Enhancement of Bone Marrow-Derived Mesenchymal Stem Cell Osteogenesis and New Bone Formation in Rats by Obtusilactone A

Yi-Hsiung Lin et al. Int J Mol Sci. .

Abstract

The natural pure compound obtusilactone A (OA) was identified in Cinnamomum kotoense Kanehira & Sasaki, and shows effective anti-cancer activity. We studied the effect of OA on osteogenesis of bone marrow-derived mesenchymal stem cells (BMSCs). OA possesses biocompatibility, stimulates Alkaline Phosphatase (ALP) activity and facilitates mineralization of BMSCs. Expression of osteogenesis markers BMP2, Runx2, Collagen I, and Osteocalcin was enhanced in OA-treated BMSCs. An in vivo rat model with local administration of OA via needle implantation to bone marrow-residing BMSCs revealed that OA increased the new bone formation and trabecular bone volume in tibias. Micro-CT images and H&E staining showed more trabecular bone at the needle-implanted site in the OA group than the normal saline group. Thus, OA confers an osteoinductive effect on BMSCs via induction of osteogenic marker gene expression, such as BMP2 and Runx2 expression and subsequently elevates ALP activity and mineralization, followed by enhanced trabecular bone formation in rat tibias. Therefore, OA is a potential osteoinductive drug to stimulate new bone formation by BMSCs.

Keywords: Cinnamomum kotoense; bone formation; bone marrow derived mesenchymal stem cells (BMSCs); obtusilactone A; osteogenesis.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Determining the cytotoxic effects of pure compounds on BMSCs: (A) structures of the extracted pure compounds from the leaves of Cinnamomum kotoense; (B) treatment time-line for analyzing the osteoinductive effects of extracted pure compounds from C. kotoense on BMSCs, P-Day: day post OIM replacement; (C) cytotoxicity analysis of BMSCs after treatment with 0.01% DMSO as a control group or a variety of pure compounds at concentrations of 1 μM and 10 μM for one day via LDH assays; and (D) MTT assays were used to determine cell viability after treatment with pure compounds extracted from C. kotoense for three days. ** p < 0.01.
Figure 2
Figure 2
Obtusilactone A induced osteogenic differentiation of BMSCs: (A) pre-screening of the mineralization stimulatory ability of the natural pure compounds extracted from C. kotoense in BMSCs based on a mineralization assay; (B) determination of the ALP activity of BMSCs after treatment with 0.01% DMSO (control) or 1 and 10 μM OA for three days; (C) mineralization assay of BMSCs after OA treatment; and (D) quantified results of the mineralization assay. Data are presented as the mean of three independent experiments. The data shown are the means ± SD of 3 independent experiments. * p <0.05, ** p <0.01 for compounds-treated versus control groups.
Figure 3
Figure 3
OA enhances osteogenic marker gene expression in BMSCs. The osteogenic marker gene expression levels of: (A) Runx2; (B) BMP2; (C) Collagen I; and (D) Osteocalcin in OA treated-BMSCs were examined by Q-PCR analysis. (E) Western blot of the protein expression of BMP2, Collagen I, Osteocalcin and β-actin (internal control) in BMSCs after treatment with 10 μM OA for 2 days. * p < 0.05 and ** p < 0.01.
Figure 4
Figure 4
Evaluation of new bone formation via needle implantation of OA into the proximal tibial metaphysis of rats. OA (30 μM, 30 μL, once/two day) was locally administered into the tibia via a needle. (A) The cut tip of the needle (4 mm) and the posteriolateral side of the proximal tibial metaphysis in both hindlimbs. (B) Schematic view and X-ray film of the needle implantation side. (C) H&E staining of the needle implantation site in the OA-treated right tibia (right panel; enlarged ad) and normal saline-treated left tibia (left panel; enlarged ad) in rats after 14 days administration (green dotted line: the original size of the needle defect; red arrow: the new bone formation). (D) Quantified analysis of new bone formation inside the region of interest we selected as the original bone defect. *** p < 0.005.
Figure 5
Figure 5
3D reconstructed micro-CT images show enhanced trabecula bone formation in OA-treated rat tibias. The original and flip 3D micro-CT images around the ROI of the tibia (4-mm radius around 1.0-mm up and down from the region of the needle implantation site; 100 cuts) in OA-treated right tibias (right panel) and normal saline-treated left tibias (left panel) of rats after 14 days of administration (red arrow head: the needle implantation site; yellow arrow: the new-formed trabeculae).
Figure 6
Figure 6
The 3D morphometric parameters show increased trabecular bone volume in OA-treated rat tibias. (A) Defined ROI for the trabeculae volume (inside the red dotted line) and cortical (between the red dotted line) bone volume analysis. (B) Cortical bone; and (C) trabecular bone volume measurement using micro-CT analysis CTAn software. Bone mass index of trabecular bone evaluation including: (D) trabecular separation (Tb.Sp).; (E) trabecular thickness (Tb.Th).; and (F) trabecular number (Tb.N). * p < 0.05.

References

    1. Chen C.H., Lo W.L., Liu Y.C., Chen C.Y. Chemical and cytotoxic constituents from the leaves of Cinnamomum kotoense. J. Nat. Prod. 2006;69:927–933. doi: 10.1021/np060107l. - DOI - PubMed
    1. Wang H.M., Cheng K.C., Lin C.J., Hsu S.W., Fang W.C., Hsu T.F., Chiu C.C., Chang H.W., Hsu C.H., Lee A.Y. Obtusilactone A and (-)-sesamin induce apoptosis in human lung cancer cells by inhibiting mitochondrial Lon protease and activating DNA damage checkpoints. Cancer Sci. 2010;101:2612–2620. doi: 10.1111/j.1349-7006.2010.01701.x. - DOI - PMC - PubMed
    1. Cheng K.C., Hsueh M.C., Chang H.C., Lee A.Y., Wang H.M., Chen C.Y. Antioxidants from the leaves of Cinnamomum kotoense. Nat. Prod. Commun. 2010;5:911–912. - PubMed
    1. Potier E., Noailly J., Ito K. Directing bone marrow-derived stromal cell function with mechanics. J. Biomech. 2010;43:807–817. doi: 10.1016/j.jbiomech.2009.11.019. - DOI - PubMed
    1. Rosset P., Deschaseaux F., Layrolle P. Cell therapy for bone repair. Orthop. Traumatol. Surg. Res. 2014;100:S107–S112. doi: 10.1016/j.otsr.2013.11.010. - DOI - PubMed

MeSH terms

LinkOut - more resources