Int J Stem Cells.  2016 Nov;9(2):221-229. 10.15283/ijsc16024.

Assessment of the Role of Noni (Morinda citrifolia) Juice for Inducing Osteoblast Differentiation in Isolated Rat Bone Marrow Derived Mesenchymal Stem Cells

• Hussain S
• Tamizhselvi R
• George L
• Manickam V

• KMID: 2362516
• DOI: http://doi.org/10.15283/ijsc16024

Abstract

BACKGROUND AND OBJECTIVES
Morinda citrifolia (Noni), an important traditional medicinal plant still used in patients with bone fractures or dislocation to promote connective tissue repair and to reduce inflammation. However, the effects of Noni on bone metabolism and whether it influences the osteogenic differentiation is yet to be clarified. In this study, we investigated the effect of Morinda citrifolia (Noni) juice on the proliferation rate of rat bone marrow derived mesenchymal stem cells (BMSC) and the osteoblastic differentiation as shown by alkaline phosphatase (ALP), Runt-related transcription factor 2 (Runx2) and osteocalcin (OCN) mRNA expression in vitro.
METHODS AND RESULTS
Treatment with 200 μg/ml Noni juice enhanced the proliferation rate of the BMSC and also upregulated the osteogenic differentiation marker genes ALP and OCN, and Runx2 measured by RTPCR. Consistent with these results collagen scaffolds implanted in vivo, which were loaded with BMSC pre-exposed to Noni, showed increased bone density measured by computed tomography and histological analysis revealed neo-angiogenesis for bone formation.
CONCLUSIONS
These results suggest that Noni stimulates osteoblastogenesis and can be used as adjuvant natural medicine for bone diseases such as osteoporosis.

Keyword

Bone marrow derived mesenchymal stem cells; Morinda citrifolia; Alkaline phosphatase; Runt-related transcription factor 2; Osteocalcin

MeSH Terms

Alkaline Phosphatase
Animals
Bone Density
Bone Diseases
Bone Marrow*
Collagen
Connective Tissue
Dislocations
Fractures, Bone
Humans
In Vitro Techniques
Inflammation
Mesenchymal Stromal Cells*
Metabolism
Morinda*
Osteoblasts*
Osteocalcin
Osteogenesis
Osteoporosis
Plants, Medicinal
Rats*
RNA, Messenger
Transcription Factors
Alkaline Phosphatase
Collagen
Osteocalcin
RNA, Messenger
Transcription Factors

Figure

• 

  Fig. 1 Rat BMSC where cultured with varying doses of Noni (50, 75, 100, 125, 150, 175, 200 and 250 μg/ml) for 24 h. Noni at a concentration of 200μg/ml significantly increased the proliferation of BMSC. MTT assay was performed to measure the cell proliferation as described in Materials and Methods. The data are mean±SD, n=6. *p<0.01 and #p<0.001 as compared with BMSC (BMSC-bone marrow derived mesenchymal stem cells).

• 

  Fig. 2 Effect of Noni (200 μg/ml) on rat bone marrow derived stem cells after 5 days and before inducing with osteogenesis media. Morphology of cells (A) without and with (B) Noni on the fifth day (40× objectives). (C) Transcriptional analysis for the expression of mesenchymal stem cell marker genes CD 44 and CD 90 generated from 5 independent experiments that were normalized with β actin. (D) Growth curve of rat BMSC through 6 days. At day 1, 2, 3, 4, 5, 6 first passage BMSCs in DMEM with or without noni were plated in each well of a 96-well plate; cell number was then evaluated at different times via Thiazolyl blue (MTT) staining. Noni at a concentration of 200 μg/ml showed a significant increase in BMSC proliferation from 2nd through 5th day. MTT results are reported as percentages (BMSC-bone marrow derived mesenchymal stem cells).

• 

  Fig. 3 Transcriptional analysis for the expression of osteogenic marker genes in bone marrow derived stem cells after 7 days of differentiation in the presence of Noni (200 μg/ml). (A) Semiquantitative RT-PCR detection of ALP, runx2 and OCN mRNA expression in cells treated with and without Noni. Sample loading was normalized with GAPDH internal control. (B) A significant increase in ALP, Runx2 and OCN was observed by real-time analysis. The data are mean±SD. Results of 3 to 5 experiments are shown. *p<0.01 when compared with BMSC (BMSC-bone marrow derived mesenchymal stem cells).

• 

  Fig. 4 Histological appearance of bone regeneration by implanting BMSC with Noni (200 μg/ml). Surgical specimens 6 weeks after implantation of (A) Type I collagen with BMSC shows normal bone healing with single harvesian system. (B) Type I collagen with BMSC and Noni, arrow showing neo-vascularization with additional blood vessels and increased bone density. (C) Histogram shown the new bone formation 6 weeks after implantation of type I collagen with BMSC and Noni (n=6). The data are mean±SD. *p<0.01 when compared with type I collagen and BMSC group (BMSC-bone marrow derived mesenchymal stem cells).

Reference

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