Journal of Dental Implants

: 2020  |  Volume : 10  |  Issue : 2  |  Page : 53--58

Effects of melatonin on adult human mesenchymal stem cells in osteoblastic differentiation

Rosy Raheja 
 Department of Prosthodontics, Crown & Bridges and Oral Implantology, Rama Dental College, Hospital and Research Centre, Kanpur, Uttar Pradesh, India

Correspondence Address:
Dr. Rosy Raheja
Department of Prosthodontics, Crown and Bridges and Implantology, Rama Dental College, Hospital and Research Centre, Kanpur, Uttar Pradesh


Among the numerous functions of melatonin, the control of survival and differentiation of mesenchymal stem cells (MSCs) has been recently proposed. MSCs are a heterogeneous population of multipotent elements resident in tissues such as bone marrow, muscle, and adipose tissue, which are primarily involved in developmental and regeneration processes, gaining thus increasing interest for tissue repair and restoration therapeutic protocols. Melatonin directly accelerated the differentiation of human stem cells into osteoblasts and also suggested that melatonin could be applied as a pharmaceutical agent to promote bone regeneration. Inflammatory cytokines and adipokines, proangiogenic/mitogenic stimuli, and other mediators that influence the differentiation processes may affect the survival and functional integrity of these mesenchymal precursor cells. In this scenario, melatonin seems to regulate signaling pathways that drive commitment and differentiation of MSC into osteogenic, chondrogenic, adipogenic, or myogenic lineages. Common pathways suggested to be involved as master regulators of these processes are the Wnt/β-catenin pathway, the mitogen-activated protein kinases and the, transforming growth factor-β signaling. In this respect, melatonin emerges a novel and potential modulator of MSC lineage commitment and adipogenic differentiation. This review recognizes and critically examines the available information on the effect of melatonin as a regulator of MSC differentiation and protection in different organs and tissues.

How to cite this article:
Raheja R. Effects of melatonin on adult human mesenchymal stem cells in osteoblastic differentiation.J Dent Implant 2020;10:53-58

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Raheja R. Effects of melatonin on adult human mesenchymal stem cells in osteoblastic differentiation. J Dent Implant [serial online] 2020 [cited 2021 Apr 16 ];10:53-58
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Melatonin (N-acetyl-5-methoxytryptamine) is an indolamine originally isolated from bovine pineal tissue.[1] Recent studies suggest that the biological and multifaceted effects of melatonin may also include a regulatory function on mesenchymal stem cell (MSC) differentiation, a process primarily involved in the development and regeneration of several tissues as bone, muscle, and the adipose tissue. In bone marrow (BM)-derived MSC, for instance, melatonin enhances osteogenesis and inhibits adipogenesis; as a result, melatonin can shift BM precursor cells from an adipocytic to osteoblastic differentiation that be of importance in bone repair technologies.[2],[3] Such early evidence, however, suggests implications in the physiological control of bone components, in which melatonin receptor-dependent and receptor-independent effects promote tissue formation while preventing deterioration.[4] Further inferences for a regulatory role of melatonin in MSC differentiation relate to the pathophysiology of adipose tissue.[5] Here, the control of oligopotent elements and then the terminal differentiation of preadipocytes to mature adipocytes remain poorly understood processes, which may have profound effects on the endocrine and paracrine functions as well as on metabolic control of this organ.[3]

 Mesenchymal Stem Cells: Biology and Classification

What are stem cells?

Stem cells, whether they occur in the body or in the laboratory, are defined by two cardinal properties: they can self-renew (generate perfect copies of themselves upon division) and differentiate (produce specialized cell types that perform specific functions in the body). The promise of stem cells as new tools for benefiting human health resides in these twin properties that, in principle, allow the production of unlimited quantities of defined cell types (e.g., for use in drug screening or transplantation). Beyond this primary definition, stem cells are classified into two major subtypes, based on the range of specialized cells they can generate.

Tissue (or adult) stem cells are found throughout the body, where they function to maintain the organ or tissue in which they reside, throughout the lifespan. Most rapidly, renewing tissues are maintained by stem cells, with the notable exception of the liver, which is maintained by specialized liver cells called hepatocytes. Under normal physiological conditions, each type of tissue stem cell only generates cells of the organ or tissue system to which it belongs: the blood (hematopoietic) stem cell generates blood, the skin stem cell generates skin, and so on. An exception is the MSC, which can generate bone, cartilage, and muscle (Bianco et al., 2013); however, while the MSC field has generated much valuable research, it has also attracted controversy.

Pluripotent stem cells, in contrast, have the potential to generate any type of cell found in the body. Pluripotent stem cells are generated in the laboratory by capturing or recreating cell types that exist only transiently during embryonic development and have not been identified in the adult body. There are currently three types of pluripotent stem cell, each generated by a different route.

Embryonic stem (ES) cells are derived from early-stage, pre-implantation embryos, and were the first type of pluripotent stem cells to be discovered: first in mice (Evans and Kaufman, 1981, Martin, 1981) and then in humans (Thomson et al., 1998) and several additional species.

Epiblast stem cells are a type of pluripotent mouse stem cells derived from a slightly later stage of embryonic development than mouse ES cells; they more closely resemble the hES cells (Tesar et al., 2007, Brons et al., 2007).

Induced pluripotent stem (iPS) cells were discovered in 2006 using mouse cells (Takahashi and Yamanaka, 2006); just a year later, this finding was replicated in human cells (Takahashi et al., 2007, Yu et al., 2007). iPS cells are generated from specialized cells by using a technique called “reprogramming.” This groundbreaking work was awarded the Nobel Prize in Physiology or Medicine in 2012. Researchers have rapidly adopted iPS cells for study, although there is ongoing discussion in the field about whether they are completely interchangeable with ES cells (Yamanaka, 2012).[ 6]

The International Society for Cellular Therapy has provided three minimal criteria to define MSC independent of their source: (i) Plastic adherence in standard culture conditions (ii) Expression of nonspecific markers CD105, CD90, and CD73 along with the lack of expression of CD34, CD45, CD14, or CD11b, CD79α or CD19, and class II major histocompatibility complex (MHC-II) molecules, mainly human leukocyte antigen-DR, and (iii) Differentiation into osteoblast, adipocytes, and chondroblasts under specific in vitro stimulation. However, it should be emphasized that the immunophenotype of MSC is dynamic and changes over the course of culturing; some of these changes may represent alterations in the biological features of MSC.[7] It has been reported that MSCs from different sources, for example, BM and adipose tissue, share a similar immunophenotype and capacity for in vitro multilineage differentiation, although some differences are present. For example, after expansion in culture, BM-derived MSCs express the surface marker CD29, CD73, CD90, CD105, CD106, CD140b, and CD166, but not CD31, CD45, CD34, CD133, or MHC class II. Cultured MSC from other sources, such as chorion-and amnion-derived cells of the placenta, adipose tissue, peripheral blood, umbilical cord blood, amniotic fluid, fetal hepatic and pulmonary tissue, skin, and prostate[7] also seems to be negative for CD31, CD45, CD80, but uniformly express CD9, CD10, CD13, CD29, CD73, CD90, CD105, and CD106, and additional tissue-specific expression of other surface antigens has also been reported. Moreover, only adipose tissue-derived MSCs express high levels of CD34, while amnion-derived MSCs are positive for stage-specific embryonic antigen-4 and tumor rejection antigen. In contrast, BM-derived MSCs, but not placenta-derived MSC, express CD271 as well as tissue-nonspecific alkaline phosphatase (ALP).

It is important to highlight, however, that MSC populations exhibit donor-to-donor heterogeneity. In fact, an analysis of BM-derived MSC from 17 healthy human donors revealed marked disparities in growth rate, ALP levels, and osteogenic potential.[8] Other studies have confirmed these findings and attributed this heterogeneity to several factors including sampling procedure during BM aspiration,[9] age of the donor,[10] and postharvesting methods used to expand MSC populations.[11]

Regardless of the origin and tissue source, MSCs are easily isolated, cultured, and manipulated for terminal differentiation, thus providing an excellent tool for research with a huge potential for clinical applications. As far as homeostatic and regeneration mechanisms of cultured-expanded MSC are concerned, it was originally thought that after the delivery of these SCs into the injured host, they would migrate to the site of injury and directly differentiate into the cells of an appropriate phenotype and function, thus contributing to repair of injured tissue. Furthermore, several studies demonstrated that MSC-conditioned media alone have therapeutic effects. Collectively, these data suggest that MSC could exert their reparative and physiological functions also, or possibly exclusively, through paracrine effects.[12]

Paracrine factors are expected to regulate the physiological role that membrane contact sites (MCSs) have in healthy tissues, and besides such a regulatory component, the differentiating potential of MCSs from various origins is under the influence of several other factors that include a series of metabolic and endocrine effectors that, in turn, integrate to influence developmental origin, maintenance, and differentiation potential of MSCs. The available knowledge on these aspects, along with the potential role of melatonin on the biological properties of MSC, will be discussed below.

 Mesenchymal Stem Cell Differentiation: Signaling and Biological Effects by Melatonin

MSCs are multipotent progenitor cells that can be differentiated under appropriate conditions into several cell types such as osteoblasts, chondrocytes and adipocytes.[8],[13],[14],[15] MSC differentiation is finely regulated by the action of mechanical and molecular signals from the extracellular environment. Emerging evidence suggests that melatonin may also be an important regulator of precursor cell commitment and differentiation. Therefore, the signaling pathways involved in these melatonin-dependent responses are discussed in the sections below [Figure 1].{Figure 1}

Early studies[16],[17] demonstrated the ability of melatonin to promote in vitro osteoblast differentiation and mineralization of matrix, thereby suggesting that this indolamine may play an essential role in the regulation of bone growth. In the study of Roth et al.,[18] MC3T3 cells grown in the presence of 50 nm melatonin underwent cell differentiation and mineralization by day 12 instead of day 21, the span usually required for cell growth in conventional media not supplemented with melatonin. Although not performed on MSC cells, this study identified the capability of melatonin of promoting bone growth through differentiation and functional competence of osteoblasts that was also supported by the analysis of the dose-dependent response of molecular players such as bone-secreted protein and the other BM proteins, including ALP and osteopontin. Nakade et al.[19] demonstrated that melatonin at concentrations between 50 and 100 μm acts directly on normal human bone cells and human osteoblastic cell line to affect osteogenic action in vitro. This study provided new evidence that melatonin stimulates the proliferation and Type I collagen synthesis in human bone cells.

Subsequent studies by Radio et al.[20] detailed for the first time the involvement of melatonin in MSC regulation, demonstrating that this neurohormone at a physiological concentration (50 nm) in combination with an osteogenic medium, significantly increased ALP, a biomarker of the enhanced proliferation and differentiation of osteoblasts. This was accompanied by gene expression of Type I collagen, osteopontin, bone sialoprotein, and osteocalcin. The melatonin-mediated increase in these biomarkers was blocked by the presence of the melatonin receptor inhibitor pertussis toxin and the antagonists luzindole and 4P-PDOT. The results showed that the MT2 receptor is the most probable receptor form involved in that osteogenic response. To further elucidate the mechanisms underlying an MT2 receptor-mediated effect on ALP activity, the signaling occurring through the receptor tyrosine kinase-and mitogen-activated pathways was assessed. Pharmacologic inhibitors of MEK and epidermal growth factor activity blocked the melatonin-induced increase in ALP levels, thus pointing to an involvement of MEK/extracellular signal-related kinase (ERK) (1/2) signaling. MEK activation by G-protein-coupled receptors can also be modulated by clathrin-mediated endocytosis. Indeed, the clathrin-coated pit inhibitor monodansyl-cadaverine prevents the melatonin-induced increase in ALP activity.[21] Therefore, EFG receptors, the MEK signal transduction cascade, as well as clathrin-mediated endocytosis, all have the potential to represent signaling components of the melatonin induce control of osteoblastic differentiation of MSC.

Sethi et al.[22] further confirmed the role of MT2 receptor in the bone formation by the selective MT2 antagonist 4P-DOT, first highlighting the importance of both timing and duration of melatonin treatment in inducing MSC differentiation to osteoblast lineage. In this study, an increase in differentiation rate was observed when the cells were exposed to melatonin either before the differentiation is initiated or at later stages of the differentiation induced by 19-day preexposure to osteogenic medium. On the contrary, the intermediate stage of differentiation that is featured by a sharp increase in proliferation was not under the influence of melatonin. These findings were similar to those of the study by Roth et al.,[18], which suggested that MC3T3 cells must first undergo differentiation before they became responsive to melatonin. Interestingly, the increase in ALP activity induced by melatonin was coupled to cytoplasmic localization of ERK1/2, which further supports a specific involvement of melatonin in cellular differentiation rather than in proliferation.[22]

Other components in the ostogenic and chondrogenic function of Wnt signaling are bone morphogenic proteins (BMPs), a unique group of proteins within the transforming growth factor-β (TGFβ) superfamily of genes, with a pivotal role in the regulation of heart, neural, cartilage, and bone development.[23] The main biological function is, however, the promotion of bone mineralization. Indeed, these proteins were originally identified in bone-inductive extracts of demineralized bone. The role that these proteins play in MSC differentiation to osteoblasts via Wnt signaling activation has been demonstrated.[24] In fact, the impairment of Wnt signaling results in a significant reduction in the capacity of BMP-2 to induce ALP activity in MSC. BMP-2 and BMP-6 strongly promote osteogenesis in MSC. Downstream propagators of BMP signals involve the Smad family of proteins (Smad-1, -5, and -8), which induce runt-related transcription factor 2 (RUNX2) gene expression, and Smads interact physically with the RUNX2 protein to induce osteoblast differentiation.[25] RUNX2/Cbfa1/Pebp2aA is a global regulator of osteogenesis and is crucial for regulating the expression of bone-specific genes, and it is a major target of the BMP pathway. RUNX2 is known to represent a key regulator of chondroblast and osteoblast differentiation, and of bone development in vivo, influencing the expression of major extracellular matrix genes of chondroblasts and osteoblasts such as ALP, osteopontin, osteocalcin, Type I collagen, and type X collagen.[26]

Several observations[27] reported that RUNX2 identified a possible target of Wnt signaling for the early specification of the osteoblast lineage. With respect to promoting chondrogenesis, the most potent inducers are the TGF-β family of mediators, including TGF-β1, TGF-β2, and TGF-β3, as well as BMPs. For the human, MSC, TGF-β2, and TGF-β3 were shown to be more active than TGF-β1 in promoting chondrogenesis, and although cellular content is similar after culture, significantly more proteoglycans and collagen Type II can be produced after stimulation with the former two inducers.[28] BMPs, known for their involvement in cartilage formation, can act alone or in concert with other growth factors to induce or enhance MSC chondrogenic differentiation. For example, BMP-2, BMP-4, or BMP-6, combined with TGF-β3, induced chondrogenic phenotype in cultured human BM-derived MSC pellets, with BMP-2 having the most pronounced effect.[29]

The role of melatonin in the osteogenesis was also investigated in the differentiation of mouse osteoblastic MC3T3-E1 cells by Park et al.[2] Melatonin promotes cell differentiation in these cells through the BMP/ERK/Wnt signaling pathway. At the same time, interruption of BMP/SMAD signaling is reported to allow neural induction,[30] and further studies have shown that such an inductive effect of melatonin on neural progenitor phenotype also involves an increase in nestin gene expression and the stimulation of AKT1. These findings point to a role of a melatonin-sensitive pathway as a sort of switch in MSC differentiation at the crossroads between bone and neural compartments.[31],[32]

The role of melatonin in the chondrogenic differentiation of human MSC was investigated by Go et al.[33] Cells were induced during chondrogenic differentiation through high-density micromass culture in chondrogenic medium containing vehicle or 50 nm melatonin. Histological examination and quantitative analysis of glycosaminoglycan (GAG) showed induced cartilage tissues to be larger and richer in GAG, collagen isotypes in the melatonin group than in the untreated controls. Besides the collagen type II (COL2A1) and X (COL10A1), the genes involved in chondrogenic differentiation up-regulated by the melatonin treatment included aggrecan, sex-determining region Y, Sox 9, RUNX2, and the potent inducer of chondrogenic differentiation BMP2.


A number of positive effects of melatonin on the MSCs and its potential therapeutic roles on bone have been documented. Melatonin shows promise in the management of bone regeneration, oral implantology, and preventative dentistry. Implants made up of titanium coated with melatonin will have a direct effect on osseoinntegration. However, more clinical as well as animal studies, are required to ascertain the use of melatonin in the clinical setting.

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Conflicts of interest

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