Short Pretreatment with Calcitriol Is Far Superior to Continuous Treatment in Stimulating Proliferation and Osteogenic Differentiation of Human Adipose Stem Cells

Fatemeh Mokhtari-Jafari; Ghassem Amoabediny; Mohammad Mehdi Dehghan; Marco N Helder; Behrouz Zandieh-Doulabi; Jenneke Klein-Nulend

doi:10.22074/cellj.2020.6773

Short Pretreatment with Calcitriol Is Far Superior to Continuous Treatment in Stimulating Proliferation and Osteogenic Differentiation of Human Adipose Stem Cells

Cell J. 2020 Oct;22(3):293-301. doi: 10.22074/cellj.2020.6773. Epub 2019 Dec 15.

Authors

Fatemeh Mokhtari-Jafari^{1

2}, Ghassem Amoabediny^{3

2

4}, Mohammad Mehdi Dehghan^{5

6}, Marco N Helder⁴, Behrouz Zandieh-Doulabi^#⁷, Jenneke Klein-Nulend^#⁷

Affiliations

¹ School of Chemical Engineering, College of Engineering, University of Tehran, Tehran, Iran.
² Department of Biomedical Engineering, Research Center for New Technologies in Life Science Engineering, University of Tehran, Tehran, Iran.
³ School of Chemical Engineering, College of Engineering, University of Tehran, Tehran, Iran. Electronic Address:amoabediny@ut.ac.ir.
⁴ Amsterdam UMC-location VUMC and Academic Centre for Dentistry Amsterdam (ACTA), Vrije Universiteit Amsterdam, Department of Oral and Maxillofacial Surgery/Oral Pathology, Amsterdam Movement Sciences, Amsterdam, The Netherlands.
⁵ Department of Surgery and Radiology, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran.
⁶ Institute of Biomedical Research, University of Tehran, Tehran, Iran.
⁷ Department of Oral Cell Biology, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and Vrije Universiteit Amsterdam, Amsterdam Movement Sciences, Amsterdam, The Netherlands.

^# Contributed equally.

Abstract

Objective: This study investigated whether short stimulation (30 minutes) of human adipose stem cells (hASCs) with 1,25-dihydroxyvitamin D₃ (calcitriol or 1,25-(OH)₂VitD₃), fitting within the surgical procedure time frame, suffices to induce osteogenic differentiation, and compared this with continuous treatment with 1,25-(OH)₂VitD₃.

Materials and methods: In this experimental study, hASCs were pretreated with/without 10 nM calcitriol for 30 minutes, seeded on biphasic calcium phosphate (BCP), and cultured for 3 weeks with/without 1,25-(OH)₂VitD₃. Cell attachment was determined 30 minutes after cell seeding. AlamarBlue assay, alkaline phosphatase (ALP) assay, ALP staining, real-time polymerase chain reaction (PCR), and protein assay were used to evaluate the effect of short calcitriol pretreatment on proliferation and osteogenic differentiation of hASCs up to 3 weeks.

Results: Pretreatment with 1,25-(OH)₂VitD₃ enhanced the attachment of hASCs to BCP by 1.5-fold compared to nontreated cells and increased the proliferation by 3.5-fold at day 14, and 2.6-fold at day 21. In contrast, continuous treatment increased the proliferation by 1.7-fold only at day 14. After 2 weeks, ALP activity was increased by 18.5-fold when hASCs were pretreated with 1,25-(OH)₂VitD₃ for 30 minutes but increased only 2.6-fold when compared with its continuous counterpart. Moreover, after 14 days, pretreatment resulted in significant upregulation of the osteogenic markers RUNX2 and SPARC by 3.6-fold and 2.2-fold, respectively, while this was not observed upon continuous treatment. Finally, 30 minutes pretreatment of hASCs with 1,25-(OH)₂VitD₃ increased VEGF₁₈₉ expression, which may contribute to the process of angiogenesis.

Conclusion: This study is the first research showing that 30 minutes pretreatment of hASCs with 1,25-(OH)₂VitD₃, not only enhanced cell attachment to the scaffold at seeding time, but also promoted the proliferation and osteogenic differentiation of hASCs more strongly than continuous treatment, suggesting that short pre-treatment with 1,25-(OH)₂VitD₃ is a promising approach for the regeneration of bones in a one-step surgical procedure.

Keywords: 1,25-dihydroxy Vitamin D3; Adipose-Derived Stem Cells; Bone; Osteogenesis; Proliferation.