Photoactivation of Bone Marrow Mesenchymal Stromal Cells With Diode Laser: Effects and Mechanisms of Action MARCO GIANNELLI, 1 FLAMINIA CHELLINI, 2 CHIARA SASSOLI, 2 FABIO FRANCINI, 3 ALESSANDRO PINI, 4 ROBERTA SQUECCO, 3 DANIELE NOSI, 2 DANIELE BANI, 2 SANDRA ZECCHI-ORLANDINI, 2 AND LUCIA FORMIGLI 2 * 1 Odontostomatologic Laser Therapy Center, Florence, Italy 2 Department of Human Anatomy, Histology and Forensic Medicine, University of Florence, Florence, Italy 3 Department of Physiological Sciences, University of Florence, Florence, Italy 4 Department of Preclinical and Clinical Pharmacology, University of Florence, Florence, Italy Mesenchymal stromal cells (MSCs) are a promising cell candidate in tissue engineering and regenerative medicine. Their proliferative potential can be increased by low-level laser irradiation (LLLI), but the mechanisms involved remain to be clarified. With the aim of expanding the therapeutic application of LLLI to MSC therapy, in the present study we investigated the effects of 635 nm diode laser on mouse MSC proliferation and investigated the underlying cellular and molecular mechanisms, focusing the attention on the effects of laser irradiation on Notch-1 signal activation and membrane ion channel modulation. It was found that MSC proliferation was significantly enhanced after laser irradiation, as judged by time lapse videomicroscopy and EdU incorporation. This phenomenon was associated with the up-regulation and activation of Notch-1 pathway, and with increased membrane conductance through voltage-gated K þ , BK and Kir, channels and T- and L-type Ca 2þ channels. We also showed that MSC proliferation was mainly dependent on Kir channel activity, on the basis that the cell growth and Notch-1 up-regulation were severely decreased by the pre-treatment with the channel inhibitor Ba 2þ (0.5 mM). Interestingly, the channel inhibition was also able to attenuate the stimulatory effects of diode laser on MSCs, thus providing novel evidence to expand our knowledge on the mechanisms of biostimulation after LLLI. In conclusions, our findings suggest that diode laser may be a valid approach for the preconditioning of MSCs in vitro prior cell transplantation. J. Cell. Physiol. 228: 172–181, 2013. ß 2012 Wiley Periodicals, Inc. Mesenchymal stromal cells (MSCs) have been shown to be a promising source of adult stem cells for cell transplantation and tissue engineering (Quattrocelli et al., 2010; Wang et al., 2011). These cells, in fact, can be easily isolated from different sources of the adult body, typically the bone-marrow, and are capable of providing significant functional benefits after implantation in the damaged tissue (Doorn et al., 2012). They have the potential to acquire lineage of any-mesenchymal-derived tissue in vitro. However, there is a general consensus that their beneficial effects on tissue repair/regeneration do not require differentiation of MSCs at target sites and mostly depend on their ability to secrete a broad panel of growth factors and cytokines, which instruct the neighboring cells and provide cues for stimulating neo-angiogenesis and extracellular matrix remodeling and assisting the endogenous regenerative response (Caplan, 2007; Sassoli et al., 2012). Moreover, the immunosuppressive properties of MSCs allow them to be used in autologous and allogenic contexts (Bernardo et al., 2012). The use of these cells in cell therapy requires their expansion in vitro in order to obtain an adequate amount of cells to be implanted in the injured tissue. Therefore, improvements of their proliferative potential during in vitro culture can significantly shorten cell preparation time and avoid contamination, thus contributing to the further development of cell-based tissue regeneration. In this context, we have previously shown that the treatment with platelet-derived rich plasma (PRP) may represent a promising approach to stimulate cell proliferation and influence stemness gene expression in cultured human MSCs (Formigli et al., 2012). Of interest, there is recent evidence suggesting that proliferation of various cultured stem cells, including MSCs, cardiac and pulpal dental stem cells, could also be enhanced by low-level laser irradiation (LLLI; Tuby et al., 2007; Eduardo Fde et al., 2008; de Villiers et al., 2011; AlGhamdi et al., 2012). This approach is widely applied in different branches of regenerative medicine and dentistry, where it is used to improve the tissue healing and repair processes. Indeed, LLLI has been found to stimulate cutaneous wound repair (Mendez et al., 2004; Maiya et al., 2005), cornea and nerve regeneration (Ratkay-Traub et al., 2001; Barbosa et al., 2010), to enhance bone remodeling and formation (Fa ´varo-Pı ´pi et al., 2011) and promote cartilage tissue regeneration (Kamali et al., 2007). In particular, we have recently demonstrated that diode laser irradiation is able to improve periodontal healing in patients affected by chronic periodontitis (Giannelli et al., 2012). Despite these data, the molecular mechanisms underlying the effects of laser The authors have no conflict of interest to declare. Marco Giannelli and Flaminia Chellini contributed equally to the study. Additional supporting information may be found in the online version of this article. Contract grant sponsor: General Project, Montespertoli, Italy. *Correspondence to: Lucia Formigli, Department of Human Anatomy, Histology and Forensic Medicine, University of Florence, Largo Brambilla, 3, 50134 Florence, Italy. E-mail: formigli@unifi.it Manuscript Received: 5 April 2012 Manuscript Accepted: 15 May 2012 Accepted manuscript online in Wiley Online Library (wileyonlinelibrary.com): 24 May 2012. DOI: 10.1002/jcp.24119 ORIGINAL RESEARCH ARTICLE 172 Journal of Journal of Cellular Physiology Cellular Physiology ß 2012 WILEY PERIODICALS, INC.