Defense Date

3-16-2018

Graduation Date

Spring 5-11-2018

Availability

Immediate Access

Submission Type

dissertation

Degree Name

PhD

Department

Pharmacology

School

School of Pharmacy

Committee Chair

Paula A. Witt-Enderby

Committee Member

David A. Johnson

Committee Member

Lauren A. O'Donnell

Committee Member

Frank D'Amico

Committee Member

Holly C. Lassila

Keywords

Menopause, Osteopenia, Melatonin, Strontium citrate, BMD, Bone marker, FRAX, Quality of Life (QoL), Osteoblast/Osteoclast Co-culture, ERK/OPG/RANKL

Abstract

Objective: The purpose of this study was to assess if a novel combination of melatonin and three other natural bone-aiding micronutrients: strontium citrate, vitamins D3 and K2 (MSDK) could improve bone health by modulating the activity of osteoblasts and osteoclasts in favor of balanced bone remodeling and by improving the overall health-related quality of life in postmenopausal osteopenic women.

Methods: The Melatonin-micronutrients Osteopenia Treatment Study (MOTS) is a translational research study that used both clinical and in vitro approaches to assess the efficacy of MSDK on bone health in women and to identify potential mechanisms for its effects. The clinical component of this study was designed as a one-year double-blind, placebo-controlled randomized trial, which assessed the effects of nightly MSDK supplementation containing 5 mg melatonin, 450 mg strontium citrate, 2000 IU vitamin D3 and 60 mcg vitamin K2 (MK7) on bone mineral density (BMD), bone marker turnover and quality of life (QOL) in postmenopausal osteopenic women. A total of 22 women (ages 49–75) were randomized to receive either MSDK (n = 11) or placebo (n = 11) p.o. nightly for 12 months. Bone mineral density (BMD) was measured by dual-energy X-ray absorptiometry (DXA) and Achilles ultrasound. Bone turnover markers total procollagen type 1 amino-terminal propeptide (P1NP), osteocalcin (OC; both intact and N-terminal mid-fragments) and collagen type I c-telopeptide (CTx) were assessed at months 0, 6 and 12 in serum. Participants’ serum vitamin D3 and C-reactive protein (CRP) levels were measured at months 0, 6 and 12. Nocturnal urinary melatonin levels were measured at month 12. Quality of life questionnaires measuring menopausal symptoms (MENQOL), anxiety (STAI), stress (PSS) and depression (CES-D) were administered at months 0, 6 and 12. Participants were given a daily diary to keep track of their pill intake, sleep duration, exercise, supplement usage and other information relevant to their general health and mood throughout the study.

The in vitro component of this translational study focused on identifying potential mechanisms underlying MSDK’s effect on bone cell differentiation and activity using two co-culture systems containing human adult mesenchymal stem cells (hMSCs) and human peripheral blood monocytes (hPBMCs). Using a novel in vitro treatment paradigm that closely mimics the in vivo condition, hMSCs/hPBMCs were co-cultured for 21 days either separately using transwell culture dishes (transwell co-culture) or by seeding hPBMCs directly on top of differentiating hMSCs (layered co-culture). The effect of MSDK on the differentiation and activity of bone cells was measured via alizarin red staining assay for osteoblast activity and TRAP and resorption pit assays for osteoclast activity, respectively. This study further assessed various signaling cascades underlying MSDK’s effects on osteoblastogenesis and osteoclastogenesis that included: OPG/RANKL, ERK1/2 and 5, RUNX2, INTEGRIN β1, NFκB, PPARγ, GLUT4 and INSULIN Rβ.

Results: One-year of MSDK treatment significantly increased lumbar spine BMD (4.3%), left femoral neck BMD (2.2%), with an upward trend for total left hip BMD (5.03% vs. 2.2% in placebo; p=.069) in postmenopausal osteopenic women taking MSDK compared to placebo. MSDK also decreased the ten-year probability of vertebral fracture risk by 6.48% compared to the 10.8% increase observed in placebo. MSDK reduced bone turnover (¯CTx:P1NP ratio) primarily by increasing the serum bone formation marker P1NP (vs. placebo; p = 0.023 and p = 0.004 at months 6 and 12, respectively); the bone resorption marker, CTx remained constant throughout the study. Serum OC levels also did not change with MSDK throughout the study. Serum CRP levels showed a downward trend, suggesting potentially positive effects of MSDK on one’s inflammatory status. MSDK produced no effect on height, weight and lean body mass; however, MSDK resulted in less variability in weight gain or loss compared to women taking placebo which could positively contribute to bone health. MSDK exhibited beneficial effects on the quality of life, perhaps by lessening the sexual symptoms of menopause (not significant vs. placebo) and showing some improvements with respect to sleep quality. MSDK did not produce adverse effects psychologically or physically in our cohort and there was a high compliance rate (92.4%).

MSDK-exposed human mesenchymal stem cells (hMSCs) and human peripheral blood monocytes (hPBMCs) plated in transwells or layered co-cultures demonstrated increases in osteoblastogenesis, decreases in osteoclastogenesis, increases in the ratio of OPG:RANKL by both increasing OPG and decreasing RANKL expression in osteoblasts. In transwell osteoblasts, MSDK increased pERK1/2 and RUNX2 levels; decreased ERK5; and did not affect the expression of NFκB and INTEGRIN β1. In layered osteoblasts, MSDK also decreased expression of the metabolic proteins PPARγ and GLUT4. These findings demonstrate that MSDK may be a novel, safe and efficacious therapy for treating those afflicted with osteopenia.

Language

English

Additional Citations

Maria, S., M. H. Swanson, L. T. Enderby, F. D'Amico, B. Enderby, R. M. Samsonraj, A. Dudakovic, A. J. van Wijnen and P. A. Witt-Enderby (2017). "Melatonin-micronutrients Osteopenia Treatment Study (MOTS): a translational study assessing melatonin, strontium (citrate), vitamin D3 and vitamin K2 (MK7) on bone density, bone marker turnover and health related quality of life in postmenopausal osteopenic women following a one-year double-blind RCT and on osteoblast-osteoclast co-cultures." Aging (Albany NY) 9(1): 256.

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