Nicholas M. Radio

Melatonin enhances alkaline phosphatase activity in differentiating human adult mesenchymal stem cells grown in osteogenic medium via MT2 melatonin receptors and the MEK/ERK (1/2) signaling cascade

Abstract:  The goals of this study were to determine (a) if melatonin enhances human adult mesenchymal stem cell (hAMSC) differentiation into osteoblasts as assessed by measuring alkaline phosphatase (ALP) enzyme activity, and (b) identify potential signal transduction pathways that mediate this process. ALP activity significantly increased in hAMSCs following a 10‐day incubation in osteogenic medium, relative to hAMSCs incubated in basal growth medium alone. Melatonin (50 nm), added in combination with the osteogenic medium, significantly increased ALP activity relative to osteogenic medium alone. Co‐exposure of hAMSCs to osteogenic medium supplemented with melatonin and either pertussis toxin or the melatonin receptor antagonists, luzindole or 4P‐PDOT (MT2 receptor selective), inhibited the melatonin‐induced increase in ALP activity, indicating the involvement of melatonin receptors, in particular, MT2 receptors. Assessment of melatonin receptor function following exposure to osteogenic medium containing either vehicle or melatonin produced dichotomous results. That is, if the differentiation of hAMSCs into an osteoblast was induced by osteogenic medium alone, then 2‐[125I]‐iodomelatonin binding and melatonin receptor function increased. However, examination of melatonin receptor function following chronic melatonin exposure, an exposure that resulted in a 50% enhancement in ALP activity, revealed that these receptors were desensitized. This was reflected by a complete loss in specific 2‐[125I]‐iodomelatonin binding as well as melatonin efficacy to inhibit forskolin‐induced cAMP accumulation. Further characterization of the mechanisms underlying melatonins effects on these differentiation processes revealed that MEK (1/2) and ERK (1/2), epidermal growth factor receptors, metalloproteinase and clathrin‐mediated endocytosis were essential while PKA was not. Our results are consistent with a role for melatonin in osteoblast differentiation. If so, then, the decrease in plasma melatonin levels observed in humans during late adulthood may further enhance susceptibility to osteoporosis.

Therapeutic treatments potentially mediated by melatonin receptors: potential clinical uses in the prevention of osteoporosis, cancer and as an adjuvant therapy.

Abstract:  Melatonins therapeutic potential is grossly underestimated because its functional roles are diverse and its mechanism(s) of action are complex and varied. Melatonin produces cellular effects via a variety of mechanisms in a receptor independent and dependent manner. In addition, melatonin is a chronobiotic agent secreted from the pineal gland during the hours of darkness. This diurnal release of melatonin impacts the sensitivity of melatonin receptors throughout a 24‐hr period. This changing sensitivity probably contributes to the narrow therapeutic window for use of melatonin in treating sleep disorders, that is, at the light‐to‐dark (dusk) or dark‐to‐light (dawn) transition states. In addition to the cyclic changes in melatonin receptors, many genes cycle over the 24‐hr period, independent or dependent upon the light/dark cycle. Interestingly, many of these genes support a role for melatonin in modulating metabolic and cardiovascular physiology as well as bone metabolism and immune function and detoxification of chemical agents and cancer reduction. Melatonin also enhances the actions of a variety of drugs or hormones; however, the role of melatonin receptors in modulating these processes is not known. The goal of this review is to summarize the evidence related to the utility of melatonin as a therapeutic agent by focusing on its other potential uses besides sleep disorders. In particular, its use in cancer prevention, osteoporosis and, as an adjuvant to other therapies are discussed. Also, the role that melatonin and, particularly, its receptors play in these processes are highlighted.

Determination of the minimal melatonin exposure required to induce osteoblast differentiation from human mesenchymal stem cells and these effects on downstream signaling pathways.

Abstract:  The purpose of this study was to determine the critical time periods of melatonin treatment required to induce human mesenchymal stem cells (hAMSCs) into osteoblasts and to determine which osteogenic genes are involved in the process. The study design consisted of adding melatonin for different times (2, 5, 10, 14 or 21 days) toward the end of a 21‐day treatment containing osteogenic (OS+) medium or at the beginning of the 21‐day treatment and then withdrawn. The results show that a 21‐day continuous melatonin treatment was required to induce both alkaline phosphatase (ALP) activity and calcium deposition and these effects were mediated through MT2Rs. Functional analysis revealed that peak ALP levels induced by melatonin were accompanied by attenuation of melatonin‐mediated inhibition of forskolin‐induced cAMP accumulation. Immunoprecipitation and western blot analyses, respectively, showed that MT2R/β‐arrestin scaffolds complexed to Gi, MEK1/2 and ERK1/2 formed in these differentiated hAMSCs (i.e., when ALP levels were highest) where ERK1/2 resided primarily in the cytosol. It is hypothesized that these complexes form to modulate the subcellular localization of ERK1/2 to affect osteogenic gene expression. Using real‐time RT‐PCR, chronic melatonin exposure induced the expression of osteogenic genes RUNX‐2, osteocalcin and BMP‐2, through MT2Rs. No melatonin‐mediated changes in the mRNA expression of ALP, BMP‐6 or in the oxidative enzymes MtTFA, PGC‐1α, Polγ, NRF‐1, PDH, PDK and LDH occurred. These data show that a continuous 21‐day melatonin exposure is required to induce osteoblast differentiation from hAMSCs through the formation of MT2R/Gi/β‐arrestin/MEK/ERK1/2 complexes to induce osteogenesis.

Platelet-Rich Preparations to Improve Healing. Part II: Platelet Activation and Enrichment, Leukocyte Inclusion, and Other Selection Criteria

Multiple platelet-rich preparations have been reported to improve wound and bone healing, such as platelet-rich plasma (PRP) and platelet rich fibrin (PRF). The different methods employed during their preparation are important, as they influence the quality of the product applied to a wound or surgical site. Besides the general protocol for preparing the platelet-rich product (discussed in Part 1 of this review), multiple choices need to be considered during its preparation. For example, activation of the platelets is required for the release and enmeshment of growth factors, but the method of activation may influence the resulting matrix, growth factor availability, and healing. Additionally, some methods enrich leukocytes as well as platelets, but others are designed to be leukocyte-poor. Leukocytes have many important roles in healing and their inclusion in PRP results in increased platelet concentrations. Platelet and growth factor enrichment reported for the different types of platelet-rich preparations are also compared. Generally, TGF-β1 and PDGF levels were higher in preparations that contain leukocytes compared to leukocyte-poor PRP. However, platelet concentration may be the most reliable criterion for comparing different preparations. These and other criteria are described to help guide dental and medical professionals, in large and small practices, in selecting the best procedures for their patients. The healing benefits of platelet-rich preparations along with the low risk and availability of simple preparation procedures should encourage more clinicians to incorporate platelet-rich products in their practice to accelerate healing, reduce adverse events, and improve patient outcomes.

An Analysis of a Rapid, Simple, and Inexpensive Technique Used to Obtain Platelet-Rich Plasma for Use in Clinical Practice

The use of platelet-rich plasma (PRP) has become more generally accepted, and implant dentists are using PRP more frequently to promote the healing of oral surgical and/or periodontal wounds. Critical elements of PRP are thought to be growth factors contained within the concentrated platelets. These growth factors are known to promote soft-tissue healing, angiogenesis and osteogenesis. We present a rapid, simple, and inexpensive methodology for preparing PRP using the Cliniseal centrifuge method. This study demonstrates that platelets are concentrated approximately 6-fold without altering platelet morphology. Further we demonstrate that key growth factors, platelet-derived growth factor BB (PDGF-BB), transforming growth factor B (TGF-B1), vasculature endothelial growth factor (VEGF), and epidermal growth factor (EGF) are present in comparable or higher concentrations than those reported with the use of other techniques. Prolonged bench set time (>3 hours) after centrifugation resulted in decreased concentration of TGF-B1 but not decreased concentration of PDGF-BB, VEGF, or EGF. This study confirms the molecular aspects of PRP obtained using this inexpensive and efficient methodology.

Microtubules modulate melatonin receptors involved in phase-shifting circadian activity rhythms: in vitro and in vivo evidence

Abstract:  MT1 melatonin receptors expressed in Chinese hamster ovary (CHO) cells remain sensitive to a melatonin re‐challenge even following chronic melatonin exposure when microtubules are depolymerized in the cell, an exposure that normally results in MT1 receptor desensitization. We extended our findings to MT2 melatonin receptors using both in vitro and in vivo approaches. Using CHO cells expressing human MT2 melatonin receptors, microtubule depolymerization prevents the loss in the number of high potency states of the receptor when compared to melatonin‐treated cells. In addition, microtubule depolymerization increases melatonin‐induced PKC activity but not PI hydrolysis via Gi proteins similar to that shown for MT1Rs. Furthermore, microtubule depolymerization in MT2‐CHO cells enhances the exchange of GTP on Gi‐proteins using a photoaffinity analog of GTP. To test whether microtubules are capable of modulating melatonin‐induced phase‐shifts, microtubules are depolymerized specifically within the suprachiasmatic nucleus of the hypothalamus (SCN) of the Long Evans rat and the efficacy of melatonin to phase shift their circadian activity rhythms was assessed and compared to animals with intact SCN microtubules. We find that microtubule depolymerization in the SCN using either Colcemid or nocodazole enhances the efficacy of 10 pm melatonin to phase‐shift the activity rhythms of the Long Evans rat. No enhancement occurs in the presence of β‐lumicolchicine, the inactive analog of Colcemid. Taken together, these data suggest that microtubule dynamics can modulate melatonin‐induced phase shifts of circadian activity rhythms which may explain, in part, why circadian disturbances occur in individuals afflicted with diseases associated with microtubule disturbances.

Platelet-Rich Preparations to Improve Healing. Part I: Workable Options for Every Size Practice

Numerous studies have demonstrated that platelet-rich preparations applied to surgical sites, injuries, or wounds are a safe and effective way to promote soft tissue healing and bone growth. Various protocols have been developed for preparing platelet-rich preparations, with subtle but important differences between them. Unfortunately, only a minority of clinicians use platelet-rich preparations, such as platelet-rich plasma and platelet-rich fibrin, in their practice, possibly due to confusion about the different methods and their advantages and disadvantages. Therefore, the different types of preparations are described to help guide the selection of the best method for any size practice. Classic methods generally require large volumes of blood and can be expensive, complicated, and time-intensive. Simpler protocols have been developed recently, which require relatively inexpensive equipment and small blood volumes and, thus, may be more applicable for small clinical practices. Platelet-rich preparations accelerate healing at earlier time points to reduce discomfort and the potential for adverse outcomes, including infection, poor wound closure, and delays in forming strong bone for subsequent procedures (such as implants). However, platelet-rich preparations may also improve long-term outcomes in patients expected to have impaired healing, such as with lifestyle choices (eg, smoking), medications (eg, steroids), diseases (eg, diabetes, osteoporosis, atherosclerosis), and aging, by supplementing the deficient wound environment to restore proper healing. Therefore, both large and small clinical practices would benefit from utilizing platelet-rich preparations to enhance healing in their patients.

Modulation of melatonin receptors and G-protein function by microtubules

Abstract:  Chronic melatonin exposure produces microtubule rearrangements in Chinese hamster ovary (CHO) cells expressing the human MT1 melatonin receptor while at the same time desensitizing MT1 receptors. Because microtubule rearrangements parallel MT1 receptor desensitization, we tested whether microtubules modulate receptor responsiveness. We determined whether depolymerization of microtubules by Colcemid, which prevents melatonin‐induced outgrowths in MT1‐expressing CHO cells, also prevents MT1 receptor desensitization by affecting G‐GTP exchange on G‐proteins. In this study, we found that depolymerization of microtubules in MT1 receptor expressing cells, prevented melatonin‐induced receptor desensitization reflected by an increase in the number of high potency sites when compared with melatonin‐treated cells. Further examination of the mechanism(s) underlying this desensitization suggested that these effects occurred at the level of G‐proteins. Depolymerization of microtubules during melatonin‐induced desensitization, attenuated forskolin‐induced cAMP accumulation, the opposite of which usually occurs following melatonin exposure alone. Concomitant to this attenuation in the forskolin response was a reduction in the amount of Giα protein coupled to MT1 receptors and an increase in [32P] azidoanilido GTP incorporation into Gi proteins. These data are consistent with the findings that microtubule depolymerization did not affect MT1/Gq coupling nor did it affect melatonin‐induced phosphoinositide hydrolysis following melatonin exposure. However, interestingly, microtubule depolymerization enhanced melatonin‐induced protein kinase C activation that was blocked in the presence of pertussis toxin. These data demonstrate that microtubule dynamics can modulate melatonin receptor function through their actions on Gi proteins and impact on downstream signaling cascades.

Neuronal Cell Morphology in Primary Cerebellar Granule Cells Using High-Content Analysis

Neurite outgrowth, one of the underlying cellular processes that defines the development and functionality of the mammalian nervous system, is also a sensitive indicator of neuronal cell health. From screening libraries of putative neurotherapeutic compounds to analyzing the millions of environmental pollutants for which we have inadequate neurotoxicity safety data, the large volume of chemical compounds that require evaluation is a major obstacle for manual imaging and analysis methods. In this context, high-content analysis (HCA) has emerged as a sensitive and accurate method for detecting changes in neuronal cell morphology within a format applicable to screening large chemical libraries. Advances in HCA technologies have enabled the automated imaging and quantitative analysis of neurite outgrowth morphology within a 96-well plate in less than 5 min. Traditionally, neurite outgrowth assessment has been conducted on immortalized cell lines such as pheochromocytoma (PC-12) cells that differentiate into neuron-like cells upon culture with nerve growth factor. Unfortunately, they do not retain all the in vivo characteristics of physiological neuronal tissue, including lack of synapse formation. As researchers refine neurite outgrowth quantitative analysis using HCA, an emerging question is how to quantify this biology in more complex models that more faithfully recapitulate in vivo environments. Primary neurons provide several benefits relative to neuronal cell lines, including the elaboration of axons from secondary dendrites and formation of both pre- and postsynaptic junctions. This chapter reviews techniques for evaluating neurite outgrowth using the ArrayScan HCA platform within a model system of primary cultures of rodent cerebellar granule cells.

Impact Factors in Scientific Journals: Keeping a Balance for the JOI Readers

Editor-in-Chief: James L. Rutkowski, DMD, PhD* Senior Editor: Sheldon Winkler, DDS Editor Emeritus: Robert J. Buhite, DDS Senior Associate Editors: Sebastiano Andreana, DDS, MS; H. Dexter Barber, DDS; Nicholas Caplanis, DMD, MS; Dennis Flanagan, DDS; John Kern, PhD; Jaime Lozada, DDS; Craig M. Misch, DDS, MDS; Harold F. Morris, DDS, MS; Nicholas Radio, PhD; James Ference, DMD; David M. Dohan Ehrenfest, DDS, PhD* Literature Review Editor: Omar Bayati, MDS, BDS Editorial Board: Barry Bartee, DDS, MD; Kim Gowey, DDS; Richard Guaccio, DDS; Brian Jackson, DDS; Ahmad Kutkut, DDS, MS; Pankaj Narkhede, DDS, MDS, BDS; John Nase, DDS Guest: Lidia M. Dohan Ehrenfest, EPhil, EM