Effects of melatonin-pretreated adipose-derived mesenchymal stem cells (MSC) in an animal model of spinal cord injury.

BACKGROUND: One of the most serious nervous system diseases is spinal cord injury(SCI), which is increasing for various reasons. Although no definitive treatment has yet been identified for SCI, one possible treatment is adipose-derived stem cells(ADSCs). However, a key issue in transplantation is improving cells' survival and function in the target tissue. Melatonin(MT) hormone with antioxidant properties can prolong cell survival and improve cell function. This study investigates the pre-conditioning of ADSCs with melatonin for enhancing the engraftment and neurological function of rats undergoing SCI. METHODS: 42 male Sprague-Dawley rats were divided into six groups, including Control, Sham, Model, Vehicle, and Lesion treatments A and B. After acquiring white adipose tissue, stem cells were evaluated by flow cytometry. SCI was then applied in Model, Vehicle, A, and B groups. Group A and B received ADSCs and ADSCs + melatonin, respectively, 1 week after SCI, but the vehicle received only an intravenous injection for simulation; The other groups were recruited for the behavioral test. Immunohistochemistry(IHC) was used to assess the engraftment and differentiation of ADSCs in the SCI site. Basso, Beattie, and Bresnahan's score was used to evaluate motor function between the six groups. RESULTS: Histological studies and cell count confirmed ADSCs implantation at the injury site, which was higher in the MT-ADSCs (P < 0.001). IHC revealed the differentiation of ADSCs and MT-ADSCs into neurons, astrocytes, and oligodendrocyte lineage cells, which were higher in MT-ADSCs. Functional improvement was observed in SCI + ADSCs and SCI + MT-ADSCs groups. CONCLUSION: The pre-conditioning of ADSCs with melatonin positively affects engraftment and neuronal differentiation in SCI but does not impact performance improvement compared to the ADSCs.

Mesenchymal stem cells versus their conditioned medium in the treatment of ischemia/reperfusion injury: Evaluation of efficacy and hepatic specific gene expression in mice.

Objectives: The mechanisms underlying the beneficial effects of MSCs on hepatic I/R injury are still poorly described, especially the changes in hepatocyte gene expression. In this study, the effect of bone marrow-derived mesenchymal stem cells (BMSCs) and adipose tissue-derived mesenchymal stem cells (AMSCs) and their conditioned medium on hepatocyte gene expression resulted by I/R shock were investigated. Materials and Methods: Liver ischemia models were induced by clamping in experimental groups. Experimental groups received MSCs or conditioned medium treatments and the control group received Dulbecco's Modified Eagle Medium (DMEM). During 1, 24 hr, and 1 week after treatment, the serum levels of alanine aminotransferase (ALT), aspartate transaminase (AST) and lactate dehydrogenase (LDH) enzymes and tissue catalase activity (CAT) were measured. Gene expression of a number of hepatocyte-specific genes (Alb, Afp, and Ck8) and Icam-1 which is upregulated under inflammatory conditions were also evaluated in 5, 24 hr, and 1-week intervals after I/R insult. Results: In this study, liver enzymes showed a much more shift in the control group than treated groups and it was more noticeable 5 hr post-treatment. Moreover, gene expression pattern of the control group underwent changes after I/R injury. However, treated groups gene expression analysis met a steady trend after I/R insult. Conclusion: Our finding shows that stem cell treatment has better curative effects than conditioned medium. BMSCs, AMSCs or BMSC and AMSC-derived bioactive molecules injection have potential to be considered as a therapeutic approach for treating acute liver injury.

Promotion of nerve regeneration by biodegradable nanofibrous scaffold following sciatic nerve transection in rats.

Peripheral nerve injuries (PNIs) are one of the common causes of morbidity and disability worldwide. Autograft is considered the gold standard treatment for PNIs. However, due to the complications associated with autografts, other sources are considered as alternatives. Recently, electrospun nanofibrous scaffolds have received wide attention in the field of tissue engineering. Exogenous tubular constructs with uniaxially aligned topographical cues to enhance the axonal re-growth are needed to bridge large nerve gaps between proximal and distal ends. Although several studies have used PLGA/PCL, but few studies have been conducted on developing a two-layer scaffold with aligned fibers properly orientated along the axis direction of the sciatic nerve to meet the physical properties required for suturing, transplantation, and nerve regeneration. In this study, we sought to design and develop PLGA-PCL-aligned nanofibers. Following the conventional examinations, we implanted the scaffolds into 7-mm sciatic nerve gaps in a rat model of nerve injury. Our in vivo evaluations did not show any adverse effects, and after eight weeks, an acceptable improvement was noted in the electrophysiological, functional, and histological analyses. Thus, it can be concluded that nanofiber scaffolds can be used as a reliable approach for repairing PNIs. However, further research is warranted.

Anti-proliferative and apoptotic effects of hull-less pumpkin extract on human papillary thyroid carcinoma cell line.

Papillary thyroid carcinoma (PTC) is one of the most common cancers of the endocrine system. Previous studies have shown that the extract of hull-less pumpkin seed (HLPS) has a significant anti-cancer effect. The aim of this study was to evaluate the effect of this plant extract on the proliferation of PTC cells. In this study, an extract of this plant was prepared by soxhlet extraction method and analyzed by Gas Chromatography-Mass Spectrometry. The cytotoxicity of PTX and plant extract was investigated using the methylthiazol tetrazolium (MTT) method. For careful investigation of morphological alteration, we used hematoxylin and eosin and Giemsa stinging. Based on MTT assay test, the IC50 value of paclitaxel (PTX) was significantly less than the hydro-alcoholic extract of HLPS at all of the incubation time. Our results of histological staining showed that HLPS and PTX induced significant morphological alteration in the PTC cultured cell that consistent with cell death. Comparing the groups treated by PTX or HLPS with control group showed significant differences. It seems that HLPS extract has an apparent effect on treatment of PTC, at least in laboratory condition, albeit for realistic decision about the effect of HLPS on PTC, more molecular investigations are necessary.

Decellularized peripheral nerve grafts by a modified protocol for repair of rat sciatic nerve injury.

Studies have shown that acellular nerve xenografts do not require immunosuppression and use of acellular nerve xenografts for repair of peripheral nerve injury is safe and effective. However, there is currently no widely accepted standard chemical decellularization method. The purpose of this study is to investigate the efficiency of bovine-derived nerves decellularized by the modified Hudson's protocol in the repair of rat sciatic nerve injury. In the modified Hudson's protocol, Triton X-200 was replaced by Triton X-100, and DNase and RNase were used to prepare accelular nerve xenografts. The efficiency of bovine-derived nerves decellularized by the modified Hudson's protocol was tested in vitro by hematoxylin & eosin, Alcian blue, Masson's trichrome, and Luxol fast blue staining, immunohistochemistry, and biochemical assays. The decellularization approach excluded cells, myelin, and axons of nerve xenografts, without affecting the organization of nerve xenografts. The decellularized nerve xenograft was used to bridge a 7 mm-long sciatic nerve defect to evaluate its efficiency in the repair of peripheral nerve injury. At 8 weeks after transplantation, sciatic function index in rats subjected to transplantation of acellular nerve xenograft was similar to that in rats undergoing transplantation of nerve allograft. Morphological analysis revealed that there were a large amount of regenerated myelinated axons in acellular nerve xenograft; the number of Schwann cells in the acellular nerve xenograft was similar to that in the nerve allograft. These findings suggest that acellular nerve xenografts prepared by the modified Hudson's protocol can be used for repair of peripheral nerve injury. This study was approved by the Research Ethics Committee, Research and Technology Chancellor of Guilan University of Medical Sciences, Iran (approval No. IR.GUMS.REC.1395.332) on February 11, 2017.

The methanolic extract of Cinnamomum zeylanicum bark improves formaldehyde-induced neurotoxicity through reduction of phospho-tau (Thr231), inflammation, and apoptosis.

Accumulation of formaldehyde (FA) in the brain is linked to age-related neurodegenerative disorders, as it accelerates memory impairment through tau protein aggregation, inflammation, and nuclear damage. This study aimed to assess the possible effects of methanolic cinnamon extract (CE) on FA-induced neurotoxicity in rats. The animals were treated with CE (100, 200, and 400 mg/kg, P.O.) for 30 days following FA administration (60 mg/kg, I.P.) for 30 days. Briefly, spatial and inhibitory memory were examined by Morris water maze (MWM) and passive avoidance (PA) tasks, respectively. The Nissl, Hoechst, and Bielschowsky silver staining methods were also used to assess apoptosis and neurofibrillary tangles (NFTs) in the hippocampal CA1 region, respectively. Brain tissues were probed with an anti-phospho-tau (Thr231) monoclonal antibody to assess tau hyperphosphorylation. Inflammatory cytokines (IL-1ß, IL-6, and TNF-α) were also measured by ELISA assay. Western blotting was performed to quantify the amount of phospho-tau (Thr231), caspase-8, and caspase-9. The results showed that FA injection significantly caused tau hyperphosphorylation at Thr231 residue, which in turn disturbed the MWM performance. The ratio of apoptotic to intact neurons increased following FA treatment. The results of Western blotting indicated that the hippocampal levels of phospho-tau (Thr231) and caspase-8 were significantly higher in the FA group compared to the control group. The hippocampal levels of IL-1ß, IL-6, and TNF-α in the FA group were also higher than the control group. Administration of 200 mg/kg of CE significantly improved the rats' MWM performance, decreased the levels of phospho-tau (Thr231), caspase-8, IL-6, and TNF-α, and reduced the ratio of apoptotic to intact neurons. Overall, cinnamon improved cognitive performance in FA-treated rats by eliminating tau hyperphosphorylation, inflammatory cytokines, and nuclear damage.

Cinnamaldehyde Enhanced Functional Recovery after Sciatic Nerve Crush Injury in Rats.

Peripheral nerve injury is a common clinical issue induced by trauma, tumor, and damage caused by treatment. Such factors create chemical and inflammatory alterations at the injury site, which increase nerve deterioration. Thus, minimizing these modifications can lead to nerve protection after injury. The present study sought to evaluate the possible improvement in nerve regeneration and enhancement of functional outcomes by cinnamaldehyde (Cin) administration following sciatic nerve crush in a rat model. Rats (n = 48) were distributed into 6 groups, including sham, injury, DMSO (vehicle group), and Cin groups (10, 30, and 90 mg/kg/day). Using small hemostatic forceps, crush injury was induced in the left sciatic nerve. Thereafter, Cin was administered for 28 successive days. Weekly records were taken for sciatic functional index (SFI) measurements. Further assessments including electrophysiological and histomorphometric evaluations, gastrocnemius muscle wet weight measurements, and estimation of the serum total oxidant status were performed. According to the results, Cin could accelerate sciatic nerve recovery after crush injury, and the dose of 30 mg/kg/day of Cin had better impacts on SFI recovery, muscle mass ratio, and myelin content. The current research demonstrated that Cin positively affects peripheral nerve restoration. Therefore, Cin therapy could be considered as a potential treatment method for peripheral nerve regeneration and its functional recovery. However, more investigations are required to further validate the study results and evaluate the optimal dose of Cin.

Comparison of melatonin and curcumin effect at the light and dark periods on regeneration of sciatic nerve crush injury in rats.

Being one of the acute clinical problems, peripheral nerve injury can bring about a number of consequences including severe disability, reduced Quality of life (QOL) and immense costs. Currently, melatonin and curcumin are widely applied because of their immunomodulatory, anti-inflammatory, neuro-protective and antioxidant properties. The present study aims to compare the effects of melatonin and curcumin during light and dark periods on sciatic nerve crush injury repair. Accordingly, rats received IP injections of curcumin (100 mg/kg) and melatonin (10 mg/kg) over two periods of light (9:00 a.m.) and dark (9:00 p.m.) for 4 weeks. In order to evaluate rats, functional (walking track analysis and electrophysiological measurements), histomorphometric and gastrocnemius muscle mass investigations were administered. No statistically significant difference was identified between dark and light curcumin groups while curcumin groups displayed better results than did melatonin groups. In addition, dark melatonin group displayed better results than the light melatonin. On the whole, this study found that melatonin and curcumin can be used to quicken neural recovery and help treat nerve injury. It was also found that better neuroregeneration or nerve regeneration was induced when rats were treated by melatonin during the dark period while effects and injection time did not correlate in curcumin application.

Engineered substrates with imprinted cell-like topographies induce direct differentiation of adipose-derived mesenchymal stem cells into Schwann cells.

Differentiation of stem cells to Schwann is considered efficient way for nerve regeneration since the sources of human Schwann cells are limited for clinical application. It is demonstrated that mimicking micromechanical forces or micro/nanotopographical environments that stem cells are experienced in vivo could control their fate. Here, the potency of substrates with imprinted cell-like topographies for direct differentiation of adipose-derived mesenchymal stem cells (ADSCs) into Schwann cells (SCs) is reported. For the preparation of substrates with imprinted SC-Like topographies, SCs are isolated from the sciatic nerve, grown, fixed, and then SC morphologies are transferred to polydimethylsiloxane (PDMS) substrates by mold casting. Subsequently, mesenchymal stem cells (MSCs) are seeded on the SC-imprinted substrates and their differentiation to SCs is evaluated by immunocytochemistry, real-time PCR, and western blotting. Analysis of morphology and expression of SC-specific markers show that MSCs cultured on the imprinted substrates have the typical SC-like morphology and express SC-specific markers including S100b, p75NTR, and Sox10. It is believed that specific cell-like topographies and related micromechanical cues can be sufficient for direct differentiation of ADSCs into Schwann cells by cell-imprinting method as a physical technique.

Advances in stem cell treatment for sciatic nerve injury.

INTRODUCTION: The sciatic nerve is one of the peripheral nerves that is most prone to injuries. After injury, the connection between the nervous system and the distal organs is disrupted, and delayed treatment results in distal organ atrophy and total disability. Regardless of great advances in the fields of neurosurgery, biological sciences, and regenerative medicine, total functional recovery is yet to be achieved. AREAS COVERED: Cell-based therapy for the treatment of peripheral nerve injuries (PNIs) has brought a new perspective to the field of regenerative medicine. Having the ability to differentiate into neural and glial cells, stem cells enhance neural regeneration after PNIs. Augmenting axonal regeneration, remyelination, and muscle mass preservation are the main mechanisms underlying stem cells' beneficial effects on neural regeneration. EXPERT OPINION: Despite the usefulness of employing stem cells for the treatment of PNIs in pre-clinical settings, further assessments are still needed in order to translate this approach into clinical settings. Mesenchymal stem cells, especially adipose-derived stem cells, with the ability of autologous transplantation, as well as easy harvesting procedures, are speculated to be the most promising source to be used in the treatment of PNIs.

Estrogen Attenuates Local Inflammasome Expression and Activation after Spinal Cord Injury.

17-estradiol (E2) is a neuroprotective hormone with a high anti-inflammatory potential in different neurological disorders. The inflammatory response initiated by spinal cord injury (SCI) involves the processing of interleukin-1beta (IL-1b) and IL-18 mediated by caspase-1 which is under the control of an intracellular multiprotein complex called inflammasome. We recently described in a SCI model that between 24 and 72 h post-injury, most of inflammasome components including IL-18, IL-1b, NLRP3, ASC, and caspase-1 are upregulated. In this study, we investigated the influence of E2 treatment after spinal cord contusion on inflammasome regulation. After contusion of T9 spinal segment, 12-week-old male Wistar rats were treated subcutaneously with E2 immediately after injury and every 12 h for the next 3 days. Behavioral scores were significantly improved in E2-treated animals compared to vehicle-treated groups. Functional improvement in E2-treated animals was paralleled by the attenuated expression of certain inflammasome components such as ASC, NLRP1b, and NLRP3 together with IL1b, IL-18, and caspase-1. On the histopathological level, microgliosis and oligodendrocyte injury was ameliorated. These findings support and extend the knowledge of the E2-mediated neuroprotective function during SCI. The control of the inflammasome machinery by E2 might be a missing piece of the puzzle to understand the anti-inflammatory potency of E2.

Stem cell therapy for nerve injury.

Peripheral nerve injury has remained a substantial clinical complication with no satisfactory treatment options. Despite the great development in the field of microsurgery, some severe types of neural injuries cannot be treated without causing tension to the injured nerve. Thus, current studies have focused on the new approaches for the treatment of peripheral nerve injuries. Stem cells with the ability to differentiate into a variety of cell types have brought a new perspective to this matter. In this review, we will discuss the use of three main sources of mesenchymal stem cells in the treatment of peripheral nerve injuries.

Evaluation of the effect of adipose tissue-derived stem cells on the quality of bone healing around implants.

PURPOSE/AIM: This study evaluates the efficacy of grafted adipose-derived stem cells (ADSCs) on blade-type implants in improving osseointegration in rat femurs using a low-density bone model. MATERIALS AND METHODS: After isolating and expanding ADSCs, twice-passaged cells were seeded on blade-type implants on culture plates. Osteogenic induction of grafted cells began after attaching cells to the prepared titanium surfaces and it continued for 4 days. The scaffolds were then implanted in the femurs of Wistar rats. Osteogenic differentiation of these cells was confirmed using polymerase chain reaction (PCR) and alizarin red staining of the mineralized extracellular matrix. After 8 weeks, histological and histomorphometric evaluations of undecalcified resin sections (bone-implant contact [BIC] % and bone mineral index [BMI]) were performed using light microscopy and scanning electron microscopy. RESULTS: Alizarin red staining in conjunction with gene expression results confirmed osteogenic differentiation. Histomorphometric assessment using scanning electron microscopy demonstrated improved BIC% and BMI near the treated surface compared with the untreated surface. CONCLUSIONS: The complex of differentiated grafted ADSCs and extracellular matrix and the macrodesign and microdesign of the implant can improve osseointegration in low-density bone.

A biosynthetic nerve guide conduit based on silk/SWNT/fibronectin nanocomposite for peripheral nerve regeneration.

As a contribution to the functionality of nerve guide conduits (NGCs) in nerve tissue engineering, here we report a conduit processing technique through introduction and evaluation of topographical, physical and chemical cues. Porous structure of NGCs based on freeze-dried silk/single walled carbon nanotubes (SF/SWNTs) has shown a uniform chemical and physical structure with suitable electrical conductivity. Moreover, fibronectin (FN) containing nanofibers within the structure of SF/SWNT conduits produced through electrospinning process have shown aligned fashion with appropriate porosity and diameter. Moreover, fibronectin remained its bioactivity and influenced the adhesion and growth of U373 cell lines. The conduits were then implanted to 10 mm left sciatic nerve defects in rats. The histological assessment has shown that nerve regeneration has taken places in proximal region of implanted nerve after 5 weeks following surgery. Furthermore, nerve conduction velocities (NCV) and more myelinated axons were observed in SF/SWNT and SF/SWNT/FN groups after 5 weeks post implantation, indicating a functional recovery for the injured nerves. With immunohistochemistry, the higher S-100 expression of Schwann cells in SF/SWNT/FN conduits in comparison to other groups was confirmed. In conclusion, an oriented conduit of biocompatible SF/SWNT/FN has been fabricated with acceptable structure that is particularly applicable in nerve grafts.

Transplantation of schwann cells differentiated from adipose stem cells improves functional recovery in rat spinal cord injury.

BACKGROUND: When the spinal cord is damaged, medical procedures are vital to prevent of improvement of the lesion. Because of poor regeneration ability of central nervous tissue, the most injuries are irreversible. One of encouraging interventions for treatment of spinal cord injury is Schwann cell transplantation. However, isolation of Schwann cell for clinical interventions is complicated approach with low cells yield and purity. Thus, easily accessed sources like Adipose mesenchymal stem cells have been taken notice. Therefore, this study was planned to assess the effect of adipose stromal cell-derived Schwann cell transplantation in functional recovery after lateral hemisection in adult rats. METHODS: After isolation, adipose stem cells were differentiated to Schwann cells. The differentiation was verified by immunocytochemistry and reverse transcription-polymerase chain reaction (RT-PCR). Then, we loaded the cells into collagen scaffolds with parallel aligned canals and transplanted into rats with 3 mm lesions at T9 - T10 level. Motor and sensory improvement were evaluated by open field locomotor scale, narrow beam, and tail flick tests for 60 days. Subsequently, conventional histology and immunohistochemistry were performed. RESULTS: In vitro results revealed that mesenchymal stem cells after differentiation gained Schwann cells morphology and markers. Schwann cell-grafted group had significantly higher locomotor and sensory scores in comparison with the control and scaffold without cell groups. Histological observations showed differentiated cells have the ability to improve axonal regeneration and remyelination. CONCLUSION: Our study proved that adipose tissue- derived Schwann cells can change the rough environment of damaged spinal cord and support axon regeneration and enhance functional recovery, and possibly be helpful for people suffering from spinal cord injuries.

Mesenchymal stem cells as an alternative for Schwann cells in rat spinal cord injury.

BACKGROUND: Spinal cord has a limited capacity to repair; therefore, medical interventions are necessary for treatment of injuries. Transplantation of Schwann cells has shown a great promising result for spinal cord injury (SCI). However, harvesting Schwann cell has been limited due to donor morbidity and limited expansion capacity. Furthermore, accessible sources such as bone marrow stem cells have drawn attentions to themselves. Therefore, this study was designed to evaluate the effect of bone marrow-derived Schwann cell on functional recovery in adult rats after injury. METHODS: Mesenchymal stem cells were cultured from adult rats' bone marrow and induced into Schwann cells in vitro. Differentiation was confirmed by immunocytochemistry and RT-PCR. Next, Schwann cells were seeded into collagen scaffolds and engrafted in 3 mm lateral hemisection defects. For 8 weeks, motor and sensory improvements were assessed by open field locomotor scale, narrow beam, and tail flick tests. Afterwards, lesioned spinal cord was evaluated by conventional histology and immunohistochemistry. RESULTS: In vitro observations showed that differentiated cells had Schwann cell morphology and markers. In this study, we had four groups (n = 10 each): laminectomy, control, scaffold and scaffold + Schwann cells. Locomotor and sensory scores of cell grafted group were significantly better than control and scaffold groups. In histology, axonal regeneration and remyelination were better than control and scaffold groups. CONCLUSION: This study demonstrates that bone marrow-derived Schwann cells can be considered as a cell source for Schwann cells in SCI treatment.

Osteogenic differentiation of rat mesenchymal stem cells from adipose tissue in comparison with bone marrow mesenchymal stem cells: melatonin as a differentiation factor.

BACKGROUND: Adipose-derived stem cells (ADSC) could be an appealing alternative to bone marrow stem cells (BMSC) for engineering cell-based osteoinductive grafts. Meanwhile, prior studies have demonstrated that melatonin can stimulate osteogenic differentiation. Therefore, we assayed and compared the melatonin effect on osteogenic differentiation of BMSC with that of ADSC. METHODS: Mesenchymal stem cells (MSC) were isolated from the bone marrow and fat of adult rats. Both cell types were cultured in osteogenic medium in the absence and presence of melatonin at physiological concentrations (20-200 pg/ml). After 4 weeks, the expression of osteocalcin gene was analyzed by reverse transcription-PCR, alkaline phosphatase (ALP) activity was assayed and alizarin red S and von Kossa staining were done. Cell viability and apoptosis were also assayed by 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide, a tetrazole (MTT) and flow cytometry, respectively. RESULTS: The osteoblastic differentiation of ADSC as demonstrated by ALP activity was less than that of BMSC. The amount of matrix mineralization has shown by alizarin red S and von Kossa staining also showed statistical differences between the two MSC. The incidence of apoptotic cells was higher among ADSC than BMSC. The flow cytometry proves that cell growth reduction is due to a decrease in the number of the cells entering the S phase of the cell cycle. MTT assay indicated that viable cells were fewer among ADSC than BMSC in control groups. CONCLUSION: The results of the study suggest that BMSC have greater osteogenic potential than ADSC and that melatonin promotes osteogenic differentiation to BMSC, but has a negative effect on ADSC osteogenic differentiation.

Effects of melatonin on the proliferation and differentiation of rat adipose-derived stem cells.

BACKGROUND: Osteogenesis driven by adipose-derived stem cells (ADSCs) is regulated by physiological and pathological factors. Accumulating evidence from in vitro and in vivo experiments suggests that melatonin may have an influence on bone formation. However, little is known about the effects of melatonin on osteogenesis, which thus remains to be elucidated. This study was performed to determine whether melatonin at physiological concentrations (0.01-10 nM) could affect the in vitro proliferation and osteogenic differentiation of rat ADSCs. MATERIALS AND METHODS: ADSCs were isolated from the fat of adult rats. After cell expansion in culture media and through three passages, osteogenesis was induced in a monolayer culture using osteogenic medium with or without melatonin at physiological concentrations (0.01-10 nM). After four weeks, the cultures were examined for mineralization by Alizarin Red S and von Kossa staining and for alkaline phosphatase (ALP) activity using an ALP kit. Cell viability and apoptosis were also assayed by 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTT) assay and flow cytometry, respectively. RESULTS: The results indicated that at physiological concentrations, melatonin suppressed proliferation and differentiation of ADSCs. These data indicate that ADSCs exposed to melatonin, had a lower ALP activity in contrast to the cells exposed to osteogenic medium alone. Similarly, mineral deposition (calcium level) also decreased in the presence of melatonin. Flow cytometry confirmed that cell growth had decreased and that the numbers of apoptotic cells had increased. CONCLUSION: These results suggest that the physiological concentration of melatonin has a negative effect on ADSC osteogenesis.