Low-Level Laser Irradiation Precondition for Cardiac Regenerative Therapy.
- 1State Key Laboratory of Cardiovascular Disease and Key laboratory of Cardiac Regenerative Medicine, Fuwai Hospital, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College , Beijing, China .
The purpose of this article was to review the molecular mechanisms of low-level laser irradiation (LLLI) preconditioning for heart cell therapy.
Stem cell transplantation appears to offer a better alternative to cardiac regenerative therapy. Previous studies have confirmed that the application of LLLI plays a positive role in regulating stem cell proliferation and in remodeling the hostile milieu of infarcted myocardium. Greater understanding of LLLI’s underlying mechanisms would be helpful in translating cell transplantation therapy into the clinic.
Studies investigating LLLI preconditioning for cardiac regenerative therapy published up to 2015 were retrieved from library sources and Pubmed databases.
LLLI preconditioning stimulates proliferation and differentiation of stem cells through activation of cell proliferation signaling pathways and alteration of microRNA expression. It also could stimulate paracrine secretion of stem cells and alter cardiac cytokine expression in infarcted myocardium.
LLLI preconditioning provides a promising approach to maximize the efficacy of cardiac cell-based therapy. Although many studies have reported possible molecular mechanisms involved in LLLI preconditioning, the exact mechanisms are still not clearly understood.
Photobiomodulation of Dental Derived Mesenchymal Stem Cells: A Systematic Review.
- 11 Department of Restorative Dentistry, School of Dentistry, University of Sao Paulo , Sao Paulo, SP, Brazil .
- 22 Department of Dentistry, School of Dentistry, Faculdades Metropolitanas Unidas , Sao Paulo, SP, Brazil .
- 33 School of Dentistry, Federal University of Para , Belem, PA, Brazil .
- 44 Department of Biodentistry, School of Dentistry, Ibirapuera University , Sao Paulo, SP, Brazil .
This study aimed to conduct a systematic review of the literature published from 2000 to August 2015, to investigate the effect of photobiomodulation (PBM) therapy on dentoalveolar-derived mesenchymal stem cells (ddMSCs), assessing whether a clear conclusion can be reached from the data presented.
Systematic reviews provide the best evidence on the effectiveness of a procedure and permit investigation of factors that may influence the performance of a method. To the best of our knowledge, no previous systematic review has evaluated the effects of PBM only on ddMSCs.
The search was conducted in PubMed /MEDLINE®, Scopus and Web of Science databases, and reported according to the Preferred Reporting Items for Systematic Reviews and Metaanalyses (PRISMA Statement). Original research articles investigating the effects of PBM therapy on ddMSCs, published from 2000 to August 2015, were retrieved and used for this review according to the following eligibility criteria: evaluating PBM therapy, assessing stem cells of dentoalveolar origin, published in English, dealing with cells characterized as stem cells, and using light that did not need external chromophores.
From the initial 3467 potentially relevant articles identified, 6 were excluded because they were duplicates, and 3453 were considered ineligible based on the inclusion criteria. Therefore, eight articles remained, and these were fully analyzed in order to closely check exclusion criteria items. Only one of them was excluded because the cultured cells studied were not characterized as stem cells. Finally, seven articles served as the basis for this systematic review.
PBM therapy has no deleterious effects on ddMSCs. Although no other clear conclusion was obtained because of the scarce number of publications, the results of these studies are pointing to an important tendency of PBM therapy to improve ddMSCs’ viability and proliferation.
Combined effects of low-level laser therapy and human bone marrow mesenchymal stem cell conditioned medium on viability of human dermal fibroblasts cultured in a high-glucose medium.
- 1Department of Biology and Anatomical Sciences, School of Medicine, Shahid Beheshti University of Medical Sciences, 1985717443, 19395/4719, Tehran, Iran.
- 2Department of Biology and Anatomical Sciences, School of Medicine, Shahid Beheshti University of Medical Sciences, 1985717443, 19395/4719, Tehran, Iran. firstname.lastname@example.org.
- 3Urogenital Stem Cell Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran. email@example.com.
- 4Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.
- 5Department of Immunology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
- 6Department of Biology and Anatomical Sciences, School of Medicine, Shahid Beheshti University of Medical Sciences, 1985717443, 19395/4719, Tehran, Iran. firstname.lastname@example.org.
Low-level laser therapy (LLLT) exhibited biostimulatory effects on fibroblasts viability. Secretomes can be administered to culture mediums by using bone marrow mesenchymal stem cells conditioned medium (BM-MSCs CM). This study investigated the combined effects of LLLT and human bone marrow mesenchymal stem cell conditioned medium (hBM-MSCs CM) on the cellular viability of human dermal fibroblasts (HDFs), which was cultured in a high-glucose (HG) concentration medium. The HDFs were cultured either in a concentration of physiologic (normal) glucose (NG; 5.5 mM/l) or in HG media (15 mM/l) for 4 days. LLLT was performed with a continuous-wave helium-neon laser (632.8 nm, power density of 0.00185 W/cm2 and energy densities of 0.5, 1, and 2 J/cm2). About 10 % of hBM-MSCs CM was added to the HG HDF culture medium. The viability of HDFs was evaluated using dimethylthiazol-diphenyltetrazolium bromide (MTT) assay. A significantly higher cell viability was observed when laser of either 0.5 or 1 J/cm2 was used to treat HG HDFs, compared to the control groups. The cellular viability of HG-treated HDFs was significantly lower compared to the LLLT?+?HG HDFs, hBM-MSCs CM-treated HG HDFs, and LLLT?+?hBM-MSCs CM-treated HG HDFs. In conclusion, hBM-MSCs CM or LLLT alone increased the survival of HG HDFs cells. However, the combination of hBM-MSCs CM and LLLT improved these results in comparison to the conditioned medium.
11 Postgraduate Department, Cruzeiro do Sul University São Paulo , SP, Brasil .
22 UMR-7365, Faculté de Médecine, CNRS-Université de Lorraine , Vandoeuvre-lés-Nancy, France .
33 Biochemistry and Biophysics Laboratory of Institute Butantan São Paulo , SP, Brasil .
44 Center for Research and Innovation in Laser , São Paulo, SP, Brasil .
55 Institute for Physiotherapy, Bergen University College , Bergen Norway.
66 Phisiotherapy Research Group, Department of Global and Public Health, University of Bergen , Bergen Norway.
77 Biomedicine Engineering, Mogi das Cruzes University , Mogi das Cruzes, SP, Brasil.
88 Department of Medical, Oral and Biotechnological Sciences, “G. d’Annunzio” University , Chieti, Italy .
The objective of this study was to evaluate the effect of laser irradiation on dog bone marrow stem cells.
Low doses of low-level red laser positively affect the viability of mesenchymal stem cells, and also increase proliferation.
Low-level laser (wavelength, 660?nm; power output, 50?mW), was applied to dog bone marrow stem cell cultures (DBMSC). The energy densities delivered varied from 1 to 12J/cm2. The effect of the laser irradiation was evaluated on cell proliferation measured with the MTT colorimetric test, cell cycle phase, and on lipidic peroxidation (free radical production).
The results indicate that laser irradiation to DBMSC did not change the morphology of the cells, but significantly increased their viability and the number of cells at the G2/M phase with 6, 10, and 12?J/cm2. On the other hand, malonaldehyde production was significantly enhanced with 8?J/cm2.
The parameters used to irradiate DBMSC increased significantly proliferation without producing high levels of reactive oxygen species (ROS).
Effect of low-level laser irradiation on proliferation and viability of human dental pulp stem cells.
Zaccara IM1, Ginani F, Mota-Filho HG, Henriques ÁC, Barboza CA.
1Postgraduate Program in Dentistry, Federal University of Rio Grande do Norte, Natal, RN, Brazil, email@example.com.
A positive effect of low-level laser irradiation (LLLI) on the proliferation of some cell types has been observed, but little is known about its effect on dental pulp stem cells (DPSCs). The aim of this study was to identify the lowest energy density able to promote the proliferation of DPSCs and to maintain cell viability. Human DPSCs were isolated from two healthy third molars. In the third passage, the cells were irradiated or not (control) with an InGaAlP diode laser at 0 and 48 h using two different energy densities (0.5 and 1.0 J/cm²). Cell proliferation and viability and mitochondrial activity were evaluated at intervals of 24, 48, 72, and 96 h after the first laser application. Apoptosis- and cell cycle-related events were analyzed by flow cytometry. The group irradiated with an energy density of 1.0 J/cm² exhibited an increase of cell proliferation, with a statistically significant difference (p?<?0.05) compared to the control group at 72 and 96 h. No significant changes in cell viability were observed throughout the experiment. The distribution of cells in the cell cycle phases was consistent with proliferating cells in all three groups. We concluded that LLLI, particularly a dose of 1.0 J/cm², contributed to the growth of DPSCs and maintenance of its viability. This fact indicates this therapy to be an important future tool for tissue engineering and regenerative medicine involving stem cells.
Low Reactive Level Laser Therapy for Mesenchymal Stromal Cells Therapies.
Low reactive level laser therapy (LLLT) is mainly focused on the activation of intracellular or extracellular chromophore and the initiation of cellular signaling by using low power lasers. Over the past forty years, it was realized that the laser therapy had the potential to improve wound healing and reduce pain and inflammation. In recent years, the term LLLT has become widely recognized in the field of regenerative medicine. In this review, we will describe the mechanisms of action of LLLT at a cellular level and introduce the application to mesenchymal stem cells and mesenchymal stromal cells (MSCs) therapies. Finally, our recent research results that LLLT enhanced the MSCs differentiation to osteoblast will also be described.
Effect of Low-Level Laser Therapy on Human Adipose-Derived Stem Cells: In Vitro and In Vivo Studies.
Low-level laser therapy (LLLT) continues to receive much attention in many clinical fields. Also, LLLT has been used to enhance the proliferation of various cell lines, including stem cells. This study investigated the effect of LLLT on human adipose-derived stem cells (ADSCs) through in vitro and in vivo studies.
Low-level laser irradiation of cultured ADSCs was performed using a 830 nm Ga-Al-As (gallium-aluminum-arsenide) laser. Then, proliferation of ADSCs was quantified by a cell counting kit-8. In the in vivo study, irradiated ADSCs or non-irradiated ADSCs were transplanted, and then, low-level laser irradiation of each rat was performed as per the protocol. Cell viability was quantified by immunofluorescent staining using the human mitochondria antibody.
In the in vitro study, the laser-irradiated groups showed an increase in absorbance compared to the control group. Also, in the in vivo study, there was a significant increase in the number of human ADSCs in the laser-irradiated groups compared to the control group (p < 0.001).
Our study showed that LLLT could enhance the proliferation and viability of ADSCs. The ADSCs enhanced by LLLT could be applied in various clinical fields. With the use of LLLT, the proliferation and viability of various cells can be enhanced, besides ADSCs.
Enhancement of Ischemic Wound Healing by Spheroid Grafting of Human Adipose-Derived Stem Cells Treated with Low-Level Light Irradiation.
We investigated whether low-level light irradiation prior to transplantation of adipose-derived stromal cell (ASC) spheroids in an animal skin wound model stimulated angiogenesis and tissue regeneration to improve functional recovery of skin tissue. The spheroid, composed of hASCs, was irradiated with low-level light and expressed angiogenic factors, including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (FGF), and hepatocyte growth factor (HGF). Immunochemical staining analysis revealed that the spheroid of the hASCs was CD31+, KDR+, and CD34+. On the other hand, monolayer-cultured hASCs were negative for these markers. PBS, human adipose tissue-derived stromal cells, and the ASC spheroid were transplanted into a wound bed in athymic mice to evaluate the therapeutic effects of the ASC spheroid in vivo. The ASC spheroid transplanted into the wound bed differentiated into endothelial cells and remained differentiated. The density of vascular formations increased as a result of the angiogenic factors released by the wound bed and enhanced tissue regeneration at the lesion site. These results indicate that the transplantation of the ASC spheroid significantly improved functional recovery relative to both ASC transplantation and PBS treatment. These findings suggest that transplantation of an ASC spheroid treated with low-level light may be an effective form of stem cell therapy for treatment of a wound bed.
Photomed Laser Surg. 2015 Feb 18. [Epub ahead of print]
Photomodulation of Proliferation and Differentiation of Stem Cells By the Visible and Infrared Light.
Objective: The aim of this article is to review experimental studies of visible and infrared light irradiation of human and animal stem cells (SCs) in vitro and in vivo to assess photobiomodulation effects on their proliferation and differentiation. Background data: The clinical application of light irradiation remains controversial, primarily because of the complexity of the rational choice of irradiation parameters. In laboratories, the theoretical justification underlying the choice of irradiation parameters also remains a challenge.
Methods: A systematic review was completed of original research articles that investigated the effects of light irradiation on human and animal SCs in vitro and in vivo (to June 2014). Relevant articles were sourced from PubMed and MEDLINE®. The search terms were laser (light) therapy (irradiation), stem cells, and phototherapy, stem cells.
Results: The analysis revealed the importance of cell type when choosing the cell irradiation parameters. The influence of wavelength on the SC proliferation rate seemed to be nonsignificant. The high values of increased proliferation or differentiation were obtained using high power density, low energy density, and short exposure time. SC exposure to light without inducers did not lead to their differentiation. The maximum differentiation was achieved using irradiation parameters different from the ones needed to achieve the maximum proliferation of the same cells.
Conclusions: Increased power density and reduced energy density were needed to increase the SC response. Based on the analysis, we have presented a graph of the cell response to generalized photostimulus, and introduced the concepts of “photostress” and “photoshock” to describe the stages of this response.
Lasers Med Sci. 2015 Jan 8. [Epub ahead of print]
Vascular regeneration effect of adipose-derived stem cells with light-emitting diode phototherapy in ischemic tissue.
The objective of this study was to investigate the effects on the vascular regeneration of adipose-derived stem cells (ASCs) by using red light-emitting diode (LED) irradiation in ischemic hind limbs. Low-level light therapy (LLLT) has been shown to enhance proliferation and cytokine secretion of a number of cells. ASCs are an attractive cell source for vascular tissue engineering. This approach is hindered because transplanted ASCs decline rapidly in the recipient tissue. Ischemic hind limbs were treated with LLLT from an LED array (660 nm) at an irradiance of 50 mW/cm2 and a radiant exposure of 30 J/cm2. LLLT, ASC transplantation, and ASC transplantation with LLLT (ASC + LLLT) were applied to ischemic limbs, and cell survival and differentiation, and secretion of vascular endothelial growth factor and basic fibroblast growth factor of the ASCs were evaluated by immunostaining and Western blot analyses. Vascular regeneration was assessed by immunostaining and hematoxylin and eosin staining. In the ASC + LLLT group, the survival of ASCs was increased due to the decreased apoptosis of ASCs. The secretion of growth factors was stimulated in this group compared with ASCs alone. The ASC + LLLT group displayed improved treatment efficacy including neovascularization and tissue regeneration compared with ASCs alone. In particular, quantitative analysis of laser Doppler blood perfusion image ratio showed that blood perfusion was enhanced significantly (p?<?0.05) by ASC + LLLT treatment. These data suggest that LLLT is an effective biostimulator of ASCs in vascular regeneration, which enhances the survival of ASCs and stimulates the secretion of growth factors in ischemic limbs.
Einstein (Sao Paulo) 2014 Mar;12(1):75-81.
Low-level laser irradiation induces in vitro proliferation of mesenchymal stem cells.
- 1Universidade Federal do Rio Grande do Norte, Natal, RN, Brasil.
- 2Universidade Federal de Pernambuco, Recife, PE, Brasil.
Objective : To evaluate the effect of low-level laser irradiation on the proliferation and possible nuclear morphological changes of mouse mesenchymal stem cells.
Methods : Mesenchymal stem cells derived from bone marrow and adipose tissue were submitted to two applications (T0 and T48 hours) of low-level laser irradiation (660nm; doses of 0.5 and 1.0J/cm2). The trypan blue assay was used to evaluate cell viability, and growth curves were used to analyze proliferation at zero, 24, 48, and 72 hours. Nuclear alterations were evaluated by staining with DAPI (4′-6-diamidino-2-phenylindole) at 72 hours.
Results : Bone marrow-derived mesenchymal stem cells responded to laser therapy in a dose-dependent manner. Higher cell growth was observed when the cells were irradiated with a dose of 1.0J/cm2, especially after 24 hours (p<0.01). Adipose-derived mesenchymal stem cells responded better to a dose of 1.0J/cm2, but higher cell proliferation was observed after 48 hours (p<0.05) and 72 hours (p<0.01). Neither nuclear alterations nor a significant change in cell viability was detected in the studied groups.
Conclusion : Low-level laser irradiation stimulated the proliferation of mouse mesenchymal stem cells without causing nuclear alterations. The biostimulation of mesenchymal stem cells using laser therapy might be an important tool for regenerative therapy and tissue engineering.
Evid Based Complement Alternat Med. 2013; 2013: 594906.
Low-Level Laser Stimulation on Adipose-Tissue-Derived Stem Cell Treatments for Focal Cerebral Ischemia in Rats
Copyright © 2013 Chiung-Chyi Shen et al.
This study investigated the effects of large-area irradiation from a low-level laser on the proliferation and differentiation of i-ADSCs in neuronal cells. MTT assays indicated no significant difference between the amount of cells with (LS+) and without (LS?) laser treatment (P > 0.05). However, immunofluorescent staining and western blot analysis results indicated a significant increase in the neural stem-cell marker, nestin, following exposure to low-level laser irradiation (P < 0.05). Furthermore, stem cell implantation was applied to treat rats suffering from stroke. At 28 days posttreatment, the motor functions of the rats treated using i-ADSCs (LS+) did not differ greatly from those in the sham group and HE-stained brain tissue samples exhibited near-complete recovery with nearly no brain tissue damage. However, the motor functions of the rats treated using i-ADSCs (LS?) remained somewhat dysfunctional and tissue displayed necrotic scarring and voids. The western blot analysis also revealed significant expression of oligo-2 in the rats treated using i-ADSCs (LS+) as well as in the sham group (P < 0.05). The results demonstrated that low-level laser irradiation exerts a positive effect on the differentiation of i-ADSCs and can be employed to treat rats suffering from ischemic stroke to regain motor functions.
Stroke has become a common disease and has been shown to be associated with the consumption of high amounts of oil and salt. These dietary habits cause blood vessels to narrow and become prone to occlusion, which can lead to stroke. Strokes can be broadly classified into 2 categories: ischemic and hemorrhagic strokes. Differing treatment methods are used according to stroke type and lesion location. Therefore, at stroke onset, computed tomography or magnetic resonance imaging is often used for diagnosis to provide physicians with a basis of treatment . The treatment methods used for the 2 types of stroke are different. (1) Ischemic stroke is caused by thrombosis or blood clots in the brain vessels, which prevent blood flow into the brain tissues. Anticoagulants or antiplatelet medications must be administered to patients as soon as possible. (2) Hemorrhagic stroke is caused by the rupture or hemorrhaging of brain vessels caused by peripheral brain trauma (subarachnoid hemorrhage). The majority of cases of hemorrhagic stroke are caused by brain trauma (e.g., car and workplace accidents). Treating these patients often requires neurosurgical interventions . Ischemic strokes constitute the majority of stroke cases. When patients suffer from ischemic stroke, the brain tissues necrotize gradually because of the lack of nutrients if anticoagulants or antiplatelet medications are not administered within 3h of the stroke. After necrosis occurs, necrotic brain tissue cannot regenerate or recover its functions even if blood-vessel reperfusion occurs . In recent years, stem cell treatment has become an innovative therapy for brain tissues that cannot regenerate.
Body fat extraction (e.g., abdomen, thigh, or buttock fat) has been used for body shaping procedures in the past, and the extracted fat was discarded as medical waste. Presently, medical experts have confirmed that adipose tissues contain large amounts of mesenchymal stem cells that exhibit in vitro proliferation and multiple differentiation ability, characteristics that facilitate the repair and regeneration of damaged tissues or organs . In addition, adipose-derived stem cells (ADSCs) possess several characteristics: (1) they can be easily harvested in abundant quantities without invasiveness, (2) they can proliferate in in vitro cultures, and (3) they can be applied to a wide range of body tissue types because these cells migrate to lesion sites automatically to repair damage. Studies have shown that ADSCs can differentiate into many different cell types, such as adipose, bone, cartilage, smooth muscle, cardiac muscle, endothelial, blood, liver, and even neuronal cells . Because of these characteristics, ADSCs will likely be one of the major sources of autologous stem cells in the future.
The effectiveness of laser therapy on biological bodies has been confirmed, and laser therapy has been successfully applied in new technological applications such as microsurgery. Lasers can be classified as high or low power, depending on the energy levels used. High-power lasers involve using high energy levels to provoke blood coagulation, stop bleeding, cut tissues, and even damage cells, whereas low-level lasers involve using electromagnetic or photochemical processes to achieve therapeutic effects . A low-level laser is defined as a laser with extremely low power and energy too low to destroy the molecular bonding capacity (e.g., hydrogen bonds and van der Waals forces) of tissues. Therefore, low-level lasers do not cause molecular structural change, protein denaturation, or cell death. Irradiation from low-level lasers on tissue does not cause an obvious temperature increase at the treatment site (less than 0.1 to 0.5°C); thus, any physiological responses of the tissues are produced by the stimulation of the laser itself, and this mechanism is described by the theory of laser biostimulation . In addition, by using an appropriate energy level, low-level lasers are primarily used to stimulate biological cells to induce or strengthen physiological responses for facilitating local blood circulation, regulating cell functions, promoting immunological functions, and facilitating cell metabolism and proliferation. Using lasers to generate these physiological changes enables treatment goals such as anti-inflammation and wound-healing promotion to be met [8, 9]. Many studies have shown that low-level laser irradiation exerts beneficial biological effects on bone, neuronal, and skin healing [10–12]. However, the type, wavelength, power, and energy level of lasers used in previous studies have varied, and various effects have been observed in different cells when differing levels of laser energy were applied. Previous studies have shown that using a low-level laser with an 820–830nm wavelength can reduce neural damage, facilitate neuronal healing, and accelerate neural recovery after an osteotomy [13, 14]. Using a low-level laser with a 660nm wavelength has been demonstrated to exert healing effects on musculoskeletal injuries and inflammation . In addition, many studies have indicated that a low-level laser with a 660nm wavelength can effectively promote neural regeneration and accelerate the reinnervation of muscle fibers to promote the recovery of motor functions [16–18]. Recent evidence has suggested that protein aggregates such as ?-amyloid- (A?) associated neurotoxicity and dendrite atrophy might be a consequence of brain-derived neurotrophic-factor (BDNF) deficiency. Meng et al. observed that the upregulation of BDNF caused by using low-level laser therapy (LLLT) to activate the extracellular signal-regulated kinase (ERK)/cAMP response element-binding protein (CREB) pathway can ameliorate A?-induced neuron loss and dendritic atrophy, thus identifying a novel pathway through which LLLT protects against A?-induced neurotoxicity .
Currently, most low-level laser therapies in practical clinical applications emphasize treatment courses covering an extensive tissue area in a relatively short period. Multichannel-laser hair treatment, which is currently available for the physical treatment of alopecia, is one such example. Therefore, we used a large-area LLLT that differs from the irradiation methods previously reported in the literature (such as single-spot low-energy laser exposure or the scanning method) to increase the local area of exposure . In this study, stem cells were extracted from adipose tissues, and neural-stem-cell- (NSC-) differentiating agents were used to culture these ADSCs to transform them into induced adipose-derived stem cells (i-ADSCs), which were used as experimental cells. First, we investigated the effects of large-area irradiation from a low-level laser on the proliferation and differentiation of i-ADSCs into neuronal cells. We then investigated whether i-ADSCs treated with laser irradiation and injected via an intravenous route could integrate and survive in various locations in rat brains. We investigated if this treatment could improve the neurological dysfunctions caused by ischemic brain damage in rats and if rats could produce BDNF, using this treatment. Studies using intravenous injection to transplant i-ADSC for the treatment of ischemic stroke with protocols similar to that used in the current study are rare. In this study, we further combined the biostimulation theory from large-area LLLT with transplantation of i-ADSCs to induce neural differentiation to investigate the effectiveness of ischemic stroke treatment. In a previous study , we established a transient ischemia-reperfusion stroke rodent model by using right-sided middle cerebral artery occlusion (MCAO) to simulate acute clinical insults. The effectiveness of the treatment was assessed by comparing HE-staining and western blot analysis, as well as the evaluation of motor skill indices by using the rotarod and grip-strength tests. Our protocol has the potential to be developed for application in the clinical treatment of patients with ischemic stroke.
2. Materials and Methods
2.1. Isolation and Culture of ADSCs
A flowchart illustrating the experimental design of the study is shown in Figure 1. Eight-week-old male Sprague-Dawley (SD) rats were used for isolating rat ADSCs. The ADSCs were harvested from the rats’ subcutaneous anterior abdominal wall. Inguinal fat pads were excised, washed sequentially in serial dilutions of betadine, and finely minced as tissues in phosphate-buffered saline (PBS). The tissues were digested with 0.3% of Type I collagenase (Sigma) at 37°C for 60min. The digested tissue/cell suspension was filtered through a 100-mesh filter to remove the debris, and the filtrate was centrifuged at 1000rpm for 10min. The cellular pellet was resuspended using DMEM/F12 (10% FBS, 1% P/S) and then cultured for 24h at 37°C in 5% CO2. Unattached cells and debris were then removed and the adherent cells were cultured using fresh medium. The cells were cultured to 80% confluency before being released with 0.05% trypsin and then subcultured.
In this study, i-ADSCs obtained from culturing ADSCs by adding NSC-differentiating agents were used as experimental cells. ADSCs within 3–5 passages were detached and induced using NSC media supplementation. ADSCs were resuspended in a serum-free DMEM/F12 medium supplemented with an N2 supplement (Sigma), 20ng/mL of epidermal growth factor (Gibco, NY, USA), and fibroblast growth factor (Gibco, NY, USA).
2.3. Setup of a Low-Level Laser Application Method
The probe of the laser irradiation device was fixed vertically on a clean, open experimental bench. The distance between the probe and the cell culture dish was 30cm. Laser irradiation was applied in a 25°C environment by using an AlGaInP-diode laser (Konftec Co., Taipei, Taiwan) with a wavelength of 660nm at an output power of 50mW and frequency of 50Hz. In the control group, the cells that did not receive laser irradiation treatment, i-ADSCs (LS?) (n = 10), were compared with the experimental cells, i-ADSCs (LS+) (n = 10), which were subjected to a laser irradiation treatment of 10min. The cells in the i-ADSCs (LS?) group were cultured for 7 days, whereas the cells in the i-ADSCs (LS+) group were treated using low-level lasers on the following day for 10min and then cultured for 6 days. The cells receiving laser irradiation were collected at various times for analysis according to the purposes of the experimental protocols. After the completion of the cultures, an optical microscope was used for observing cell morphology.
2.4. MTT Assay
The principle of the MTT (3-[4,5-dimethylthiazol-2-y1]-2,4-diphenyltetrazolium bromide) assay is that the mitochondria of living cells can transform the yellow chemical substance MTT tetrazolium into the purple non-water-soluble substance MTT formazan through the effect of succinate dehydrogenase. DMSO can be used to dissolve the purple-colored products. No such response occurs in dead cells. An optical absorbance of 570nm was measured using an enzyme immunoassay analyzer. A higher absorbance value indicates a larger amount of cells. In this study, ADSCs neuronal predifferentiation was first distributed in a 96-well plate with approximately 10000 cells per well. The cells in the i-ADSCs (LS?) group were then cultured in a 37°C environment and a 5% CO2 environment for 5 days and 7 days, respectively. The cells in the i-ADSCs (LS+) group were cultured in a 37°C environment and a 5% CO2 environment for 4 days and 6 days, respectively. After the completion of the cultures, the medium was removed by several rinses with PBS. An MTT solution of 100?L was added to each well of a 96-well plate in the dark (1mL of MTT reagent was added to 9mL of phenol-red-free, serum-free medium) and incubated in a 37°C, 5% CO2 environment for 2h. The MTT solution was then removed and the cells were dissolved using DMSO. An optical absorbance of 570nm was measured using an enzyme immunoassay analyzer to compare the values between the different groups.
2.5. Immunocytochemistry of i-ADSCs
After 7 days in culture, the subcultured neurospheres were washed using 0.1M PBS 3 times and fixed with 4% paraformaldehyde for 1h. Following the fixation, the cells were permeated with 0.1% of Triton X-100 for 10min and then blocked with 5% nonfat milk for 30min. The phenotypic expression of these neurospheres was examined by implementing immunocytochemical staining accompanied by antibodies against glial fibrillary acidic proteins (GFAPs) for astrocytes, mouse monoclonal antinestins for NSCs, and doublecortin (DCX), which has recently been used as a marker for neurogenesis. Briefly, the fixed cells were washed 3 times in cold PBS. After washing with PBS, the aforementioned primary antibodies were added and the slides were maintained at room temperature overnight. In the following day, the primary antibodies were removed by washing 3 times with PBS and the secondary antibodies were added before incubating the cells for 1h. After washing off the secondary antibodies, the cells were incubated with tertiary antibodies tagged with peroxidase-antiperoxidase for 1h. The tertiary antibodies were washed off using PBS. The cells were incubated with DAPI (Sigma, St. Louis, MO, USA) diluted with the cell culture medium for 10min. Finally, the cells were mounted with 90% glycerol and examined using fluorescent microscopy (Olympus IX-71, Inc., Trenton, NJ, USA).
2.6. Animals and Induction of the MCAO Model
In a previous study , we established a transient ischemia-reperfusion stroke rodent model, using right-sided middle cerebral artery occlusion (MCAO) to simulate acute clinical insults. All of the experimental procedures were approved by the Institutional Animal Care and Use Committee of Taichung Veteran General Hospital, Taiwan. Thirty-two adult male SD rats were randomly allocated to 3 groups: the i-ADSCs (LS?) therapy group (n = 12), the i-ADSCs (LS+) therapy group (n = 12), and a sham group (n = 8). The rats in all 3 groups were euthanized on the 28th day after MCAO was performed. For MCAO procedures, anesthesia was induced using 4% isoflurane (Baxter, USA) and maintained using 2% isoflurane. A midline cervical incision was made to isolate the right bilateral common carotid artery. A 25mm-long 3-0 nylon surgical thread was then inserted into the right carotid bifurcation. In this study, 2 rounds were used to provide a more complete blockage of blood flow in the artery. When the blunted distal end met resistance, the proximal end of the thread was tightened at the carotid bifurcation. The right common, internal, and external carotid arteries were carefully separated from the adjacent vagal nerve, and the distal portions of the external and common carotid arteries were ligated. A small incision was subsequently made at the proximal portion of the external carotid artery, and a 3-0 nylon monofilament suture was gently inserted (approximately 18mm) into the internal carotid artery. After 60min of MCAO, the nylon surgical thread was removed to allow complete reperfusion of the ischemic area. During ischemia, rectal temperature was monitored and maintained at approximately 37°C by using a heating pad and an overhead lamp. The anesthetized rats intravenously received i-ADSCs at a concentration of 2 × 107mL?1 via their femoral veins. The rats in the sham group underwent the same surgical procedures except that the right-sided middle cerebral artery was not occluded.
2.7. Rotarod Test
An accelerating rotarod test was performed for each rat before and on the 7th, 14th, 21st, and 28th day after cerebral ischemia-reperfusion was induced. Before the ischemia-reperfusion experiment was conducted, the animals were subjected to 3 training sessions per day for 3 days on the accelerating rotarod to obtain stable duration on the rotarod spindle. The diameter of the rotarod spindle was 7cm. The surface of the rotarod spindle was made of knurled Perspex to provide an adequate grip, which prevented animals from slipping off the spindle. The speed of the spindle was increased from 4 to 40rpm over a period of 5min and the duration that the animal stayed on the device was recorded. The rats that were capable of staying on the rotarod longer than 150s after 3 training sessions were selected for the experiments. On the testing days, the animals were tested twice, and the longest durations on the rotarod were recorded.
2.8. Grip Strength Test
Each rat was supported in a horizontal position approximately parallel to a grip bar (Model DPS-5R: range 0–5 kgf, Japan). The researcher set the rat’s forepaws on the grip bar and pulled the animal horizontally away from the bar by the base of its tail until the rat released its grip. The pulling motion was smooth and continuous. The researcher supported the rat by the abdomen when the grip was released. The reading on the strain gauge remained constant at the point of maximal value, which was recorded as the measure of forepaw grip strength. The researcher supported the rat body by both the chest and the base of the tail at an angle of ?45° down the tail. The rat was facing away from the grip bar. The rat was encouraged to grasp the bar by moving its hind paw to the bar. When the rat grasped the bar with both hind paws, establishing a “full” grip, the upper body of the rat was lowered so that the rat was in a nearly horizontal position. The rat was pulled horizontally by the base of the tail until it released its grip and was supported as previously described. The reading on the strain gauge remained constant at the point of maximal value (force was measured in grams), which was recorded as the measurement of forepaw grip strength. Three values were obtained in succession, and the median value was used as the daily score. The data were expressed as the percentage of the baseline (preischemic) value.
2.9. Hematoxylin-Eosin Staining of the Cerebellum
The SD rats were anesthetized using 10% chloral hydrate (4?L/kg), administered intraperitoneally, and were euthanized on the 28th day after the MCAO operation and sham treatment. For each rat, the left cerebellum was rapidly removed and postfixed in formalin for 24h. The postfixed tissues were embedded in paraffin wax and 6-?m-thick serial coronal sections were obtained and mounted on poly-L-lysine-coated glass slices. To assess the histological changes in the MCAO and sham groups, the paraffin-embedded left cerebellum sections were stained using hematoxylin-eosin (HE), according to standard protocol before the assay was performed.
2.10. Western Blot Analysis
Proteins were extracted from the rat brains by using a cold lysis buffer (10mM of tetra sodium pyrophosphate, 20mM of Hepes, 1% Triton X-100, 100mM NaCl, 2?g/mL of aprotinin, 2?g/mL of leupeptin, and 100?g/mL of phenylmethylsulfonyl fluoride). The protein concentrations from tissue extracts or ADSC-conditioned medium were determined using the Bradford protein assay. Equal amounts of protein were placed in a 2× sample buffer (0.125M Tris-HCl, pH 6.8, 2% glycerol, 0.2mg/mL of bromophenol blue dye, 2% SDS, and 10% ?-mercaptoethanol) and electrophoresed through 10% SDS-polyacrylamide gel. The proteins were then transferred onto a nitrocellulose membrane by using electroblotting. The membranes were blocked for 1h at room temperature in a Tris-buffered saline with Tween-20 (TBST) and 5% nonfat milk. The primary antibodies (1:1000) with appropriate dilutions were incubated for 1h at room temperature in TBST and 5% nonfat milk. The blots were then washed and incubated with a peroxidase-conjugated secondary antibody (1:2000) for 1h in TBST. The chemiluminescent substrate for the secondary antibody was developed using the ECL detection system (Amersham, UK). The blots were exposed to film for 3–5min and then developed.
2.11. Statistical Analysis
The data were expressed as the mean value ± standard error of the mean. The statistical significance of the differences between the groups was determined using a one-way analysis of variance followed by Tukey’s test. An alpha level of less than 0.05 (P < 0.05) was considered statistically significant.
3.1. Effects of the Low-Level Laser on Cell Morphology
The ADSCs were passaged 3–5 times after the initial plating of the primary culture. Rat ADSCs appeared to be a monolayer of large and flat cells (Figure 2(a)). Many cells in the i-ADSCs (LS?) and i-ADSCs (LS+) groups induced a neuronal phenotype and exhibited, among one another, bipolar and multipolar elongations of neuronally induced cell-forming networks. The results show that the stem cells in both i-ADSCs (LS?) and i-ADSCs (LS+) groups developed tentacles, indicating that ADSCs were facilitating the induction of differentiation into neuronal cells. Comparative optical micrographs revealed that some attached cells exhibited a spread-out shape with a spindle-like and fibroblastic phenotype in the i-ADSCs (LS?) group (Figure 2(b)). However, most of i-ADSCs-expressing neurites extended radially, connecting like bridges with those from adjacent cells in the i-ADSCs (LS+) group (Figure 2(c)).
3.2. Effects of the Low-Level Laser on Cell Proliferation and Differentiation
In this study, MTT assays were performed on Day 5 and Day 7 to evaluate the effects of large-area low-level laser irradiation on the facilitation of cell proliferation. After analyzing the optical absorbance values, the results showed that, on Day 5, cell activity was slightly higher in the i-ADSCs (LS+) group compared with that in the i-ADSCs (LS?) group. However, the difference was not statistically significant (P > 0.05). On Day 7, the cell amounts in both the i-ADSCs (LS+) and i-ADSCs (LS?) groups were larger than the amounts on Day 5. However, the cell proliferation rates were similar on Day 5 without major differences (P > 0.05) (Figure 3).
In this study, immunofluorescent staining and western blots were used to evaluate the effects of large-area low-level laser irradiation on the facilitation of cell differentiation. Immunofluorescent staining was performed for the NSC marker, nestin, glial cell marker, GFAP antibody, and neuronal precursor-cell-marker protein, DCX. After the staining was completed, fluorescent microscopy was used to observe the amount of fluorescence expression of each antibody. The results showed that the fluorescence expression of the nestin was higher in the cells in the i-ADSCs (LS+) group than that in the cells in the i-ADSCs (LS?) group. These results indicated that ADSC differentiation into neuronal cells was facilitated after large-area low-level laser irradiation (Figure 4(a)). For GFAP, no difference was observed in the amount of fluorescence expression of GFAP between the i-ADSCs (LS?) and i-ADSCs (LS+) groups (Figure 4(b)). Furthermore, for DCX, no difference was observed between the 2 groups because the cells in both groups still exhibited stem cell morphology (Figure 4(c)).
Western blot is used to quantify the expression of marker proteins. Therefore, western blot analysis was used to compare the amount of nestin expression between the i-ADSCs (LS?) and i-ADSCs (LS+) groups in this study. The results showed that cells in the i-ADSCs (LS+) group exhibited a substantially higher nestin expression compared with the cells in the i-ADSCs (LS?) group (P < 0.05) (Figure 5). Regarding the results of GFAP and DCX, no difference was observed in the fluorescence expressions of the i-ADSCs (LS?) and i-ADSCs (LS+) groups. Therefore, western blot analyses were not shown for GFAP and DCX. This result is consistent with the findings obtained using immunofluorescent staining.
3.3. Evaluation of Behavior Recovery after Stroke in the Animals
In this study, treadmill and forepaw-grip tests were used to evaluate motor function recovery after stem-cell transplantation treatment in rats with ischemic stroke. The treadmill test was performed on Day 7 after stem-cell transplantation was performed on the rats with stroke. The rats from either the i-ADSCs (LS+) group or the i-ADSCs (LS?) group were unable to run as quickly as the rats in the sham group. From Day 14, the rats in the i-ADSCs (LS+) group gradually recovered the ability to run. By contrast, it was observed that the rats in the i-ADSCs (LS?) group recovered slightly; however, the degree of recovery was lower than that in the i-ADSCs (LS+) group. On Day 21 after stem-cell transplantation, the recovery of running function was still more satisfactory in the i-ADSCs (LS+) group than in the i-ADSCs (LS?) group. On Day 28, the motor function of the rats in the i-ADSCs (LS+) group was approaching the level of the rats in the sham group, whereas the performance of the rats in the i-ADSCs (LS?) group was still considerably weaker than that of the sham group (Figure 6).
Grip-strength tests were performed on Day 7 after stem-cell transplantation. The findings were similar to the treadmill test results; the grip behavior of the rats in both the i-ADSCs (LS+) group and the i-ADSCs (LS?) group was worse than that of the rats in the sham group. On Day 14, the grip strength of the rats in the i-ADSCs (LS+) group was considerably recovered. By contrast, although the rats in the i-ADSCs (LS?) group showed some progress, the improvement was minimal. On Day 21, the rats in the i-ADSCs (LS+) group continued to recover, whereas it was observed that the animals in the i-ADSCs (LS?) group were not recovering as quickly. On Day 28, grip strength recovery in the i-ADSCs (LS+) group approached that of the sham group, whereas grip strength in the i-ADSCs (LS?) group was still lower (Figure 7).
3.4. Repair of Brain Tissues after Treatment in Animals
After euthanizing the rats, the brain specimens were treated with paraffin and then sliced. The HE-immunostaining method was used to observe the repair of brain tissues after stem-cell transplantation was performed. An upright microscope (10x) was used to macroscopically observe the brain tissue, and the results showed that brain tissue was completely repaired in the rats in the i-ADSCs (LS+) group with nearly no necrotic brain tissue. By contrast, obvious necrotic scars were observed at the ischemic sites of the brain tissue from the rats in the i-ADSCs (LS?) group (Figure 8(a)). Observed under a microscope and magnified 200 times, the results showed that the brain tissue from the rats in the i-ADSCs (LS+) group was as dense as normal brain tissues, whereas numerous cavities were observed in the brain tissue from rats in the i-ADSCs (LS?) group (Figure 8(b)).
Oligodendrocytes, which are glial cells found in normal brains, form myelin in the central nervous system. Oligodendrocytes substantially decrease after brain cells are damaged, leading to myelin collapse and the loss of neural-signal conduction. Therefore, a western blot was used to analyze the amount of expression of the oligodendrocyte cell protein, oligo-2, to confirm the repair of brain tissues after stem-cell transplantation was performed in rats with stroke. The results showed that the amount of oligo-2 protein response in the brain tissues of the i-ADSCs (LS+) group was as high as that in the sham group. By contrast, the oligo-2 protein response in the i-ADSCs (LS?) group was substantially lower (P < 0.05). These results indicated that stem-cell transplantation treatments can repair brain tissues damaged by ischemia (Figure 9).
Stem cells possess the ability to proliferate, regenerate, differentiate, and secrete cytokines. Previous studies have proven that stem-cell therapy substantially improves the damage caused by stroke. Stem cells are derived from various sites. Most previous studies have used bone marrow mesenchymal stem cells; however, we used adipose stem cells in the present study, which are easily accessible, and abundant and exhibit high differentiation and proliferation activity. Moreover, adipose stem cells do not trigger strong immune reactions (resulting in low exclusion) and rarely form teratomas. In the literature, it has been demonstrated that adipose stem cells are essential adult stem cells that differentiate into various mesoderm tissues similarly to bone marrow mesenchymal stem cells [21–23]. Furthermore, adipose stem cells are more easily accessible than mesenchymal stem cells; therefore, adipose stem cells can be used as a substitute for bone marrow mesenchymal stem cells for repairing damaged tissues in the future. Adipose stem cells are generally more practical than bone marrow mesenchymal stem cells for use in research [23–26].
In this study, we used large-area low-level laser irradiation to induce ADSCs to differentiate into neuronal cells. The MTT assay analysis showed that the cell activity of the i-ADSCs increased on Days 5 and 7 of culture after large-area low-level laser irradiation [27, 28]. However, although cell activity increased on both Days 5 and 7, the activity was also increased for the group that did not receive laser irradiation treatment. No significant difference was observed between the groups with or without laser treatment (P > 0.05). Previous studies have shown that, depending on irradiation parameters, various types of cell respond differently to laser irradiation . Although the mechanism underlying this phenomenon remains obscure, several hypotheses have been proposed to explain the mechanism of laser action . We speculate that the lack of significant effects might be attributable to the short duration of low-level laser irradiation, which might not provide sufficient energy to the ADSCs for them to facilitate proliferation. Alternatively, using low-level lasers on the ADSCs might not sufficiently enhance cell proliferation. Therefore, future studies should investigate the appropriate duration of low-level laser irradiation and determine how much energy is required to accelerate cell proliferation [31,32].
To understand if using large-area low-level lasers exerted a positive effect on cell differentiation, we used immunofluorescent staining and western blot analysis to evaluate whether these lasers were capable of accelerating the induction of cell differentiation . The immunofluorescent staining results showed that the nestin level in the group with i-ADSCs treated using large-area low-level laser irradiation increased substantially compared with that of the group that did not receive laser treatment. This result indicated that large-area low-level laser can accelerate the differentiation of ADSCs into neuronal cells [34, 35]. LLLT has been demonstrated to regulate neuronal function both in vitro and in vivo. Previous studies have reported that laser treatments accelerated nerve cell sprouting and cell migration, which begin within 24h of seeding. During the first week of cultivation, irradiated cultures contain a high number of neurons exhibiting large perikaria and branched neuronal fibers, which interconnect to form networks . The possible mechanism of LLLT at the cellular level has been attributed to the acceleration of electron transfer reactions, resulting in the increase of reactive oxygen species and Ca2+as versatile second messengers . Previous studies have shown that applying LLLT could influence cellular processes by altering DNA synthesis and protein expression , biomodulating cytoskeletal organization , and stimulating cellular proliferation . Such properties suggest that LLLT, or interventions with similar neurobiological effects, can be used to treat neurodegeneration, a phenomenon that underlies debilitating clinical conditions.
However, no obvious differences were observed for GFAP and DCX in the test results. This can be explained by the differentiation agent used in this study, which exerts its effects primarily by inducing ADSCs to differentiate into NSCs. Therefore, although the ADSCs were treated using a low-level laser, they still did not differentiate into neural glia cells. The DCX protein can only be discovered after neurons have been formed from NSCs, which demonstrates that DCX is a late-stage protein that cannot be expressed when cells still exhibit the morphology of NSCs. Therefore, the amount of DCX expression is not affected by low-level laser exposure. Western blot analysis was used to determine the amount of nestin expression after i-ADSCs were exposed to large-area low-level laser irradiation and culture for 7 days. The results showed that the amount of nestin was higher with laser treatment. This is similar to the findings from the immunofluorescent-staining method, indicating that laser irradiation can accelerate the differentiation of ADSCs into neuronal cells. These results indicated that ADSCs can be induced to differentiate into neuronal cells after treatment by large-area laser irradiation for 10min. Future studies should establish the precise duration of large-area LLLT required to achieve improved results.
In this study, treadmill and forepaws grip tests were used to evaluate motor function recovery after stem cell transplantation treatments in rats with stroke. The treadmill test results showed that the running function was weaker for the rats in the sham group on Day 7 after treatment. We speculate that this might have been because the brain-tissue lesion area from ischemia was too large; therefore, the transplanted stem cells did not have sufficient time to noticeably repair brain tissue. Therefore, the motor functions of the rats remained impaired on Day 7. When tested on Day 14, recoveries in motor functions were observed in both the i-ADSCs (LS?) and i-ADSCs (LS+) groups, with superior recovery in the i-ADSCs (LS+) group, indicating that the damaged brain tissue was repaired. On Day 21, the recovery of the rats’ running function was more satisfactory in the i-ADSCs (LS+) group than in the i-ADSCs (LS?) group, indicating that the brain tissue repair capability was superior to that in the i-ADSCs (LS+) group. When tested on Day 28, the running function of the rats in the i-ADSCs (LS+) group was close to that of the rats in the sham group. However, the motor function of the i-ADSCs (LS?) group remained impaired, indicating that hind-paw motor function recovery was accelerated after i-ADSCs (LS+) treatment.
The grip test results were similar to the treadmill test results. On Day 7 after stem-cell transplantation treatment of the rats with stroke, grip strength was low for both the i-ADSCs (LS+) group and i-ADSCs (LS?) groups because the damaged brain tissues were just about to be repaired; therefore, the forepaws of the rats remained weak. When tested on Day 14, no major improvement was observed in the grip strength of the rats in the i-ADSCs (LS?) group. By contrast, the grip strength improved considerably in the i-ADSCs (LS+) group, indicating that the brain tissue repair capability was more satisfactory in the i-ADSCs (LS+) group than in the i-ADSCs (LS?) group. On Days 21 and 28, the tests showed that grip strength recovery was more satisfactory in the i-ADSCs (LS+) group than in the i-ADSCs (LS?) group. On Day 28, the grip strength in the i-ADSCs (LS+) group recovered to a level close to that of the sham group, indicating that, after i-ADSCs (LS+) treatment, the damaged brain tissues of the rats with stroke were repaired quickly, enabling the recovery of forepaw-grip strength. Based on these results, we concluded that damaged brain tissues can be repaired faster and motor function can be recovered efficiently in rats with stroke after i-ADSCs (LS+) treatment.
The i-ADSCs could differentiate into neuronal cells after transplantation into the brain. As a result, they moved and repaired damaged cerebral tissue selectively and improved cerebral functions by enhancing angiogenesis, renewal of neurons, and proliferation of nerve cells [40, 41]. In this study, we used the HE-immunostaining method and western blot analysis to evaluate brain tissue repair on Day 28 after stem cell transplantation treatment was performed in rats with stroke. The results of immunostaining were observed using a microscope. The structures were magnified 200 times, which showed that the brain tissue in the stroke lesion was dense and similar to that of normal brain tissue in the i-ADSCs (LS+) group. By contrast, numerous cavities were observed in the ischemic lesions of the brain tissues of the rats in the i-ADSCs (LS?) group. These results indicated that necrotizing brain tissue after ischemia was quickly repaired when i-ADSCs (LS+) was used to treat the rats with stroke. We speculate that using i-ADSCs (LS+) treatment can accelerate the induction of ADSCs to differentiate into neuronal cells. Therefore, although the same number of stem cells was transplanted for treatment, a greater number of NSCs were observed in the i-ADSCs (LS+) group than in the i-ADSCs (LS?) group. Furthermore, because NSCs can protect damaged brain tissues from continuous deterioration, they can also help brain tissues to repair. Therefore, transplanting a greater amount of NSCs would likely assist in repairing brain tissues more effectively.
Regarding the results of the western blot analysis, the oligo-2 amounts after stem-cell transplantation treatment were analyzed. Because oligodendrocytes form myelin in the central nervous system, myelin levels collapse when oligodendrocytes die. Nerve conduction is delayed or interrupted after the death of oligodendrocytes, leading to limb disabilities. Therefore, the evaluation of oligo-2 could be a crucial reference in assessing the degree of brain tissue recovery after stem-cell transplantation treatments in rats with stroke. In this study, using western blot analysis showed that the amount of expression of oligo-2 in brain tissues treated with i-ADSCs (LS+) was similar to that in normal brain tissues. By contrast, brain tissues treated with i-ADSCs (LS?) exhibited a lower oligo-2 expression, indicating that using i-ADSCs (LS+) treatment in rats with stroke can repair myelin in the central nervous system, leading to the recovery of neural-signal conduction and motor function. Based on these experimental results, we concluded that using i-ADSCs (LS+) treatment in rats with stroke cannot only accelerate the repair of damaged brain tissues for the partial recovery of motor functions, but also enable the central nervous system to recover the velocity of neural-signal conduction. These results confirm that the transplantation of i-ADSCs (LS+) can accelerate repairs in rats with ischemic stroke because i-ADSCs (LS+) can more efficiently differentiate into NSCs.
In this study, we used i-ADSCs treated with large-area low-level laser irradiation to evaluate the effects of a low-level laser on cell proliferation and differentiation. The results showed that although a low-level laser cannot facilitate cell proliferation, it can accelerate the induction of ADSCs differentiating into NSCs. In this study, we successfully created large-area cell and tissue damage in rat brains by using an embolic stroke animal model. Stem-cell transplantation with either i-ADSCs (LS+) or i-ADSCs (LS?) was performed to evaluate the degree of repair after stroke in the animals. Because large-area low-level laser irradiation can accelerate the differentiation of ADSCs into NSCs, and NSCs can protect damaged brain tissues to prevent continuous deterioration from damage and to help with repair, the motor function recovery was thus superior in the rats treated using i-ADSCs (LS+) compared with that in the rats treated using i-ADSCs (LS?). From the brain tissue slices from each group of rats, we discovered that i-ADSCs (LS+) treatment more effectively repaired necrotizing brain tissues after ischemia in rat brains. Furthermore, the western blot analysis also showed that the amount of oligo-2 increased in i-ADSCs (LS+)-treated rats with stroke, confirming the repair of myelin in cerebral neurons to further assist in the recovery of neural-signal conduction in the central nervous system.
Therefore, in the present study we demonstrated that using large-area low-level lasers exerts positive effects on inducing ADSCs differentiation, and it effectively treated ischemic stroke in rats, regarding motor function recovery. In future studies, the effects of large-area low-level laser irradiation time and the appropriate dosage for the proliferation and differentiation of ADSCs should be evaluated. If the optimal irradiation time and dosage for ADSC proliferation and differentiation can be discovered in animal experiments similar to those in this study, we believe that superior experimental results can be obtained. Furthermore, if primate or canine experimental animals can be used to conduct the experimental protocols described herein, the concerns associated with individual animal differences and errors associated with motor function assessments can be minimized to obtain more reliable experimental data. Therefore, the findings of this study contribute to the development of cell therapy, which can benefit patients with stroke.
Conflict of Interests
There is no conflict of interests.
This work was supported by grants from the Taichung Veterans General Hospital and Central Taiwan University of Science and Technology (Grant. TCVGH-CTUST 1027701) and the National Science Council (Grant. NSC 102-2314-B-075A-019-MY2), Taiwan.
Stem Cells Dev. 2012 Sep 1;21(13):2508-19. doi: 10.1089/scd.2011.0695. Epub 2012 Apr 20.
MicroRNA-193 pro-proliferation effects for bone mesenchymal stem cells after low-level laser irradiation treatment through inhibitor of growth family, member 5.
State Key Laboratory of Cardiovascular Medicine, Fuwai Hospital and Cardiovascular Institute, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, China.
The enhanced proliferation of mesenchymal stem cells (MSCs) can be helpful for the clinical translation of cell therapy. Low-level laser irradiation (LLLI) has been demonstrated as regulating MSC proliferation. MicroRNAs (miRNAs) are involved in various pathophysiologic processes in stem cells, but the role of miRNAs in the LLLI-based promotion of MSC proliferation remains unclear. We found that the proliferation level and cell cycle-associated genes in MSCs were increased after LLLI treatment in a time-dependent manner. Microarray assays revealed subsets of miRNAs to be differentially regulated, and these dynamic changes were confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) after LLLI. miR-193 was the most highly up-regulated miRNA, and the change in it was related with the proliferation level. Gain-loss function experiments demonstrated that miR-193 could regulate the proliferation of MSCs, including human’s and rat’s, but could not affect the apoptosis and differentiation level. Blockade of miR-193 repressed the MSC proliferation induced by LLLI. By qRT-PCR, we found that miR-193, in particular, regulated cyclin-dependent kinase 2 (CDK2) expression. Bioinformatic analyses and luciferase reporter assays revealed that inhibitor of growth family, member 5 (ING5) could be the best target of miR-193 to functionally regulate proliferation and CDK2 activity, and the mRNA and protein level of ING5 was regulated by miR-193. Furthermore, the ING5 inhibited by small interfering RNA (siRNA) could up-regulate the proliferation of MSCs and the expression of CDK2. Taken together, these results strongly suggest that miR-193 plays a critical part in MSC proliferation in response to LLLI stimulation, which is potentially amenable to therapeutic manipulation for clinical application.
Stem Cell Rev. 2011 Nov;7(4):869-82.
Influence of low intensity laser irradiation on isolated human adipose derived stem cells over 72 hours and their differentiation potential into smooth muscle cells using retinoic acid.
Laser Research Centre, Faculty of Health Sciences, University of Johannesburg, P.O. Box 17011, Doornfontein 2028, South Africa.
Human adipose derived stem cells (hADSCs), with their impressive differentiation potential, may be used in autologous cell therapy or grafting to replace damaged tissues. Low intensity laser irradiation (LILI) has been shown to influence the behaviour of various cells, including stem cells.
This study aimed to investigate the effect of LILI on hADSCs 24, 48 or 72 h post-irradiation and their differentiation potential into smooth muscle cells (SMCs).
hADSCs were exposed to a 636 nm diode laser at a fluence of 5 J/cm(2). hADSCs were differentiated into SMCs using retinoic acid (RA). Morphology was assessed by inverted light and differential interference contrast (DIC) microscopy. Proliferation and viability of hADSCs was assessed by optical density (OD), Trypan blue staining and adenosine triphosphate (ATP) luminescence. Expression of stem cell markers, ?1-integrin and Thy-1, and SMC markers, smooth muscle alpha actin (SM-?a), desmin, smooth muscle myosin heavy chain (SM-MHC) and smoothelin, was assessed by immunofluorescent staining and real-time reverse transcriptase polymerase chain reaction (RT-PCR).
Morphologically, hADSCs did not show any differences and there was an increase in viability and proliferation post-irradiation. Immunofluorescent staining showed expression of ?1-integrin and Thy-1 72 h post-irradiation. RT-PCR results showed a down regulation of Thy-1 48 h post-irradiation. Differentiated SMCs were confirmed by morphology and expression of SMC markers.
LILI at a wavelength of 636 nm and a fluence of 5 J/cm(2) does not induce differentiation of isolated hADSCs over a 72 h period, and increases cellular viability and proliferation. hADSCs can be differentiated into SMCs within 14 days using RA.
Lasers Med Sci. 2011 Sep 29. [Epub ahead of print]
Effects of low-level laser irradiation on mesenchymal stem cell proliferation: a microarray analysis.
Department of Surgery, Cardiovascular Institute & Fuwai Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, 167 Beilishi Road, Beijing, 100037, China.
Increased proliferation after low-level laser irradiation (LLLI) has been well demonstrated in many cell types including mesenchymal stem cells (MSCs), but the exact molecular mechanisms involved remain poorly understood. The aim of this study was to investigate the change in mRNA expression in rat MSCs after LLLI and to reveal the associated molecular mechanisms. MSCs were exposed to a diode laser (635 nm) as the irradiated group. Cells undergoing the same procedure without LLLI served as the control group. Proliferation was evaluated using the MTS assay. Differences in the gene expression profiles between irradiated and control MSCs at 4 days after LLLI were analyzed using a cDNA microarray. Gene ontology and pathway analysis were used to find the key regulating genes followed by real-time PCR to validate seven representative genes from the microarray assays. This procedure identified 119 differentially expressed genes. Real-time PCR confirmed that the expression levels of v-akt murine thymoma viral oncogene homolog 1 (Akt1), the cyclin D1 gene (Ccnd1) and the phosphatidylinositol 3-kinase, catalytic alpha polypeptide gene (Pik3ca) were upregulated after LLLI, whereas those of protein tyrosine phosphatase non-receptor type 6 (Ptpn6) and serine/threonine kinase 17b (Stk17b) were downregulated. cDNA microarray analysis revealed that after LLLI the expression levels of various genes involved in cell proliferation, apoptosis and the cell cycle were affected. Five genes, including Akt1, Ptpn6, Stk17b, Ccnd1 and Pik3ca, were confirmed and the PI3K/Akt/mTOR/eIF4E pathway was identified as possibly playing an important role in mediating the effects of LLLI on the proliferation of MSCs.
Lasers Med Sci. 2011 May 20. [Epub ahead of print]
The effects of low-level laser irradiation on differentiation and proliferation of human bone marrow mesenchymal stem cells into neurons and osteoblasts-an in vitro study.
Department of Hematology, Faculty of Medical Science, Tarbiat Modares University, Tehran, Iran, firstname.lastname@example.org.
Bone marrow-derived mesenchymal stem cells (BMSCs) are promising for use in regenerative medicine. Several studies have shown that low-level laser irradiation (LLLI) could affect the differentiation and proliferation of MSCs. The aim of this study was to examine the influence of LLLI at different energy densities on BMSCs differentiation into neuron and osteoblast. Human BMSCs were cultured and induced to differentiate to either neuron or osteoblast in the absence or presence of LLLI. Gallium aluminum arsenide (GaAlAs) laser irradiation (810 nm) was applied at days 1, 3, and 5 of differentiation process at energy densities of 3 or 6 J/cm(2) for BMSCs being induced to neurons, and 2 or 4 J/cm(2) for BMSCs being induced to osteoblasts. BMSCs proliferation was evaluated by MTT assay on the seventh day of differentiation. BMSCs differentiation to neurons was assessed by immunocytochemical analysis of neuron-specific enolase on the seventh day of differentiation. BMSCs differentiation to osteoblast was tested on the second, fifth, seventh, and tenth day of differentiation via analysis of alkaline phosphatase (ALP) activity. LLLI promoted BMSCs proliferation significantly at all energy densities except for 6 J/cm(2) in comparison to control groups on the seventh day of differentiation. LLLI at energy densities of 3 and 6 J/cm(2) dramatically facilitated the differentiation of BMSCs into neurons (p? nm wavelength enhances BMSCs differentiation into neuron and osteoblast in the range of 2-6 J/cm(2), and at the same time increases BMSCs proliferation (except for 6 J/cm(2)). The effect of LLLI on differentiation and proliferation of BMSCs is dose-dependent. Considering these findings, LLLI could improve current in vitro methods of differentiating BMSCs prior to transplantation.
Lasers Med Sci. 2011 Jan 28. [Epub ahead of print]
Low-level laser therapy: a useful technique for enhancing the proliferation of various cultured cells.
Alghamdi KM, Kumar A, Moussa NA.
Department of Dermatology, Vitiligo Research Chair, College of Medicine, King Saud University, PO Box 240997, Riyadh, 11322, Saudi Arabia, email@example.com.
The aim of this work is to review the available literature on the details of low-level laser therapy (LLLT) use for the enhancement of the proliferation of various cultured cell lines including stem cells. A cell culture is one of the most useful techniques in science, particularly in the production of viral vaccines and hybrid cell lines. However, the growth rate of some of the much-needed mammalian cells is slow. LLLT can enhance the proliferation rate of various cell lines. Literature review from 1923 to 2010. By investigating the outcome of LLLT on cell cultures, many articles report that it produces higher rates of ATP, RNA, and DNA synthesis in stem cells and other cell lines. Thus, LLLT improves the proliferation of the cells without causing any cytotoxic effects. Mainly, helium neon and gallium-aluminum-arsenide (Ga-Al-As) lasers are used for LLLT on cultured cells. The results of LLLT also vary according to the applied energy density and wavelengths to which the target cells are subjected. This review suggests that an energy density value of 0.5 to 4.0 J/cm(2) and a visible spectrum ranging from 600 to 700 nm of LLLT are very helpful in enhancing the proliferation rate of various cell lines. With the appropriate use of LLLT, the proliferation rate of cultured cells, including stem cells, can be increased, which would be very useful in tissue engineering and regenerative medicine.
Photomed Laser Surg. 2010 Aug;28 Suppl 1:S157-65.
Red-light light-emitting diode irradiation increases the proliferation and osteogenic differentiation of rat bone marrow mesenchymal stem cells.
Li WT, Leu YC, Wu JL.
Department of Biomedical Engineering, Chung Yuan Christian University, Chung-Li, Taiwan, Republic of China. firstname.lastname@example.org
OBJECTIVE: The objective of this study was to investigate the effects on the proliferation and osteogenic differentiation of rat mesenchymal stem cells (MSCs) by using red-light light-emitting diode (LED) irradiation.
BACKGROUND DATA: Low-level light irradiation (LLLI) has been shown to enhance proliferation and cytokine secretion of a number of cells. MSCs are capable of regenerating various mesenchymal tissues and are essential in supporting the growth and differentiation of hematopoietic stem cells within the bone marrow.
MATERIALS AND METHODS: Rat bone marrow MSCs were treated with single or multiple doses of LLLI from an LED array (630 nm) at the irradiances of 5 and 15 mW/cm(2), and radiant exposures of 2 and 4 J/cm(2). The proliferation, clonogenic potential, and osteogenic differentiation of MSCs were evaluated after illumination.
RESULTS: The growth of MSCs was enhanced by red-light LLLI, and the effect became more obvious at low cell density. A single dose of LLLI led only to a short-term increase in MSCs proliferation. A maximal increase in cell proliferation was observed with multiple exposures of LLLI at 15 mW/cm(2) and 4 J/cm(2). The number of colony-forming unit fibroblasts increased when cells were illuminated under the optimal parameter. During osteogenesis, significant increases (p < 0.01) in both alkaline phosphatase and osteocalcin expressions were found in the MSCs that received light irradiation.
CONCLUSION: Our data demonstrated that MSCs proliferation was enhanced by multiple exposures to LLLI from 630-nm LEDs, and cell growth depended on the plating density. Furthermore, multiple dose of LLLI could enhance the osteogenic potential of rat MSCs.
J Transl Med. 2010 Feb 16;8(1):16. [Epub ahead of print]
Lasers, stem cells, and COPD.
Lin F, Josephs SF, Alexandrescu DT, Ramos F, Bogin V, Gammill V, Dasanu CA, De Necochea-Campion R, Patel AN, Carrier E, Koos DR.
ABSTRACT: The medical use of low level laser (LLL) irradiation has been occurring for decades, primarily in the area of tissue healing and inflammatory conditions. Despite little mechanistic knowledge, the concept of a non-invasive, non-thermal intervention that has the potential to modulate regenerative processes is worthy of attention when searching for novel methods of augmenting stem cell-based therapies. Here we discuss the use of LLL irradiation as a photoceutical for enhancing production of stem cell growth/chemoattractant factors, stimulation of angiogenesis, and directly augmenting proliferation of stem cells. The combination of LLL together with allogeneic and autologous stem cells, as well as post-mobilization directing of stem cells will be discussed.
Photomed Laser Surg. 2009 Apr;27(2):227-33.
Implantation of low-level laser irradiated mesenchymal stem cells into the infarcted rat heart is associated with reduction in infarct size and enhanced angiogenesis.
Tuby H, Maltz L, Oron U.
Department of Zoology, The George S. Wise Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv, Israel.
OBJECTIVE: The aim of the present study was to evaluate the possible beneficial effects of implantation of laser-irradiated mesenchymal stem cells (MSCs) into the infarcted rat heart.
BACKGROUND DATA: It was demonstrated that low-level laser therapy (LLLT) upregulates cytoprotective factors in ischemic tissues.
MATERIALS AND METHODS: MSCs were isolated from rat bone marrow and grown in culture. The cells were laser irradiated with a Ga-Al-As laser (810 nm wavelength), labeled with 5-bromo-2’deoxyuridine (BrdU), and then implanted into infarcted rat hearts. Non-irradiated cells were similarly labeled and acted as controls. Hearts were excised 3 wk later and cells were stained for BrdU and c-kit immunoreactivity.
RESULTS: Infarcted hearts that were implanted with laser-treated cells showed a significant reduction of 53% in infarct size compared to hearts that were implanted with non-laser-treated cells. The hearts implanted with laser-treated cells prior to implantation demonstrated a 5- and 6.3-fold significant increase in cell density that positively immunoreacted to BrdU and c-kit, respectively, as compared to hearts implanted with non-laser-treated cells. A significantly 1.4- and 2-fold higher level of angiogenesis and vascular endothelial growth factor, respectively, were observed in infarcted hearts that were implanted with laser-treated cells compared to non-laser-treated implanted cells.
CONCLUSION: The findings of the present study provide the first evidence that LLLT can significantly increase survival and/or proliferation of MSCs post-implantation into the ischemic/infarcted heart, followed by a marked reduction of scarring and enhanced angiogenesis. The mechanisms associated with this phenomenon remain to be elucidated in further studies.
Lasers Surg Med. 2008 Dec;40(10):726-33.
In vitro effects of low-level laser irradiation for bone marrow mesenchymal stem cells: proliferation, growth factors secretion and myogenic differentiation.
Hou JF, Zhang H, Yuan X, Li J, Wei YJ, Hu SS.
Department of Surgery, Cardiovascular Institute and Fu Wai Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, People’s Republic of China.
BACKGROUND AND OBJECTIVES: Bone marrow derived mesenchymal stem cells (BMSCs) have shown to be an appealing source for cell therapy and tissue engineering. Previous studies have confirmed that the application of low-level laser irradiation (LLLI) could affect the cellular process. However, little is known about the effects of LLLI on BMSCs. The aim of this study was designed to investigate the influence of LLLI at different energy densities on BMSCs proliferation, secretion and myogenic differentiation.
STUDY DESIGN/MATERIALS AND METHODS: BMSCs were harvested from rat fresh bone marrow and exposed to a 635 nm diode laser (60 mW; 0, 0.5, 1.0, 2.0, or 5.0 J/cm(2)). The lactate dehydrogenase (LDH) release was used to assess the cytotoxicity of LLLI at different energy densities. Cell proliferation was evaluated by using 3-(4, 5-dimethylithiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) and 5-bromo-2′-deoxyuridine (BrdU) assay. Production of vascular endothelial growth factor (VEGF) and nerve growth factor (NGF) were measured by enzyme-linked immunosorbent assay (ELISA). Myogenic differentiation, induced by 5-azacytidine (5-aza), was assessed by using immunocytochemical staining for the expression of sarcomeric alpha-actin and desmin.
RESULTS: Cytotoxicity assay showed no significant difference between the non-irradiated group and irradiated groups. LLLI significantly stimulated BMSCs proliferation and 0.5 J/cm(2) was found to be an optimal energy density. VEGF and NGF were identified and LLLI at 5.0 J/cm(2) significantly stimulated the secretion. After 5-aza induction, myogenic differentiation was observed in all groups and LLLI at 5.0 J/cm(2) dramatically facilitated the differentiation.
CONCLUSIONS: LLLI stimulates proliferation, increases growth factors secretion and facilitates myogenic differentiation of BMSCs. Therefore, LLLI may provide a novel approach for the preconditioning of BMSCs in vitro prior to transplantation.
Lasers Surg Med. 2008 Aug;40(6):433-8.
Stem cell proliferation under low intensity laser irradiation: a preliminary study.
Eduardo Fde P, Bueno DF, de Freitas PM, Marques MM, Passos-Bueno MR, Eduardo Cde P, Zatz M.
Hospital Israelita Albert Einstein, Unit of Bone Marrow Transplantation, São Paulo 05651-901, SP, Brazil.
BACKGROUND AND OBJECTIVES: Phototherapy with low intensity laser irradiation has shown to be effective in promoting the proliferation of different cells. The aim of this in vitro study was to evaluate the potential effect of laser phototherapy (660 nm) on human dental pulp stem cell (hDPSC) proliferation.
STUDY DESIGN/MATERIALS AND METHODS: The hDPSC cell strain was used. Cells cultured under nutritional deficit (10% FBS) were either irradiated or not (control) using two different power settings (20 mW/6 seconds to 40 mW/3 seconds), with an InGaAIP diode laser. The cell growth was indirectly assessed by measuring the cell mitochondrial activity through the MTT reduction-based cytotoxicity assay.
RESULTS: The group irradiated with the 20 mW setting presented significantly higher MTT activity at 72 hours than the other two groups (negative control–10% FBS–and lased 40 mW with 3 seconds exposure time). After 24 hours of the first irradiation, cultures grown under nutritional deficit (10% FBS) and irradiated presented significantly higher viable cells than the non-irradiated cultures grown under the same nutritional conditions.
CONCLUSIONS: Under the conditions of this study it was possible to conclude that the cell strain hDPSC responds positively to laser phototherapy by improving the cell growth when cultured under nutritional deficit conditions. Thus, the association of laser phototherapy and hDPSC cells could be of importance for future tissue engineering and regenerative medicine. Moreover, it opens the possibility of using laser phototherapy for improving the cell growth of other types of stem cells.
Lasers Surg Med. 2008 Jan;40(1):38-45.
Primary myogenic cells see the light: improved survival of transplanted myogenic cells following low energy laser irradiation.
Shefer G, Ben-Dov N, Halevy O, Oron U.
Department of Cell & Development Biology, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv 69978, Israel. email@example.com
BACKGROUND AND OBJECTIVES: There is a substantial need for finding new avenues to promote muscle recovery when acute skeletal muscle loss extends beyond the natural capacity of the muscle to recover. Maintenance and regeneration of skeletal muscles depend mainly on resident stem cells known as satellite cells. Nevertheless, there are situations in which a significant loss of muscle tissue exhausts the satellite cell pool. For such cases, cell therapy and tissue engineering are becoming promising alternatives. Thus far, attempts to supplement damaged host muscles with donor satellite cells by means of myoblast transplantation therapy were mostly unsuccessful due to massive and rapid loss of donor cells within few hours after transplantation. This study aims at following the effects of low-energy-laser irradiation on the fate of implanted myoblasts.
STUDY DESIGN: Primary myogenic cells, harvested from male rat skeletal muscles, were irradiated with low energy laser, seeded on a biodegradable scaffold and expanded in vitro. The scaffold containing cells was transplanted into partially excised muscles of host female rats. Donor cells were identified in the host muscle tissue, using Y-chromosome in situ hybridization.
RESULTS: In this study, we show that laser irradiated donor primary myogenic cells not only survive, but also fuse with host myoblasts to form a host-donor syncytium.
CONCLUSIONS: Our data show that the use of low energy laser irradiation (LELI), a non-surgical tool, is a promising means to enhance both the survival and functionality of transplanted primary myogenic cells.
Lasers Med Sci. 2007 Aug 23; [Epub ahead of print]
The effect of low level laser irradiation on adult human adipose derived stem cells.
Mvula B, Mathope T, Moore T, Abrahamse H.
Laser Research Group, Faculty of Health Sciences, University of Johannesburg, P. O. Box 17011, Doornfontein, Johannesburg, South Africa, 2028, firstname.lastname@example.org.
This study investigated the effect of low level laser irradiation on primary cultures of adult human adipose derived stem cells (ADSC) using a 635-nm diode laser, at 5 J/cm(2) with a power output of 50.2 mW and a power density of 5.5 mW/cm(2). Cellular morphology did not appear to change after irradiation. Using the trypan blue exclusion test, the cellular viability of irradiated cells increased by 1% at 24 h and 1.6% at 48 h but was not statistically significant. However, the increase of cellular viability as measured by ATP luminescence was statistically significant at 48 h (p < 0.05). Proliferation of irradiated cells, measured by optical density, resulted in statistically significant increases in values compared to nonirradiated cells (p < 0.05) at both time points. Western blot analysis and immunocytochemical labeling indicated an increase in the expression of stem cell marker beta1-integrin after irradiation. These results indicate that 5 J/cm(2) of laser irradiation can positively affect human adipose stem cells by increasing cellular viability, proliferation, and expression of beta1-integrin.
Lasers Surg Med. 2007 Apr;39(4):373-8.
Low-level laser irradiation (LLLI) promotes proliferation of mesenchymal and cardiac stem cells in culture.
Tuby H, Maltz L, Oron U.
Department of Zoology, The George S. Wise Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv 69978, Israel.
BACKGROUND AND OBJECTIVES: Low-level laser irradiation (LLLI) was found to promote the proliferation of various types of cells in vitro. Stem cells in general are of significance for implantation in regenerative medicine. The aim of the present study was to investigate the effect of LLLI on the proliferation of mesenchymal stem cells (MSCs) and cardiac stem cells (CSCs).
STUDY DESIGN/MATERIALS AND METHODS: Isolation of MSCs and CSCs was performed. The cells were cultured and laser irradiation was applied at energy densities of 1 and 3 J/cm2.
RESULTS: The number of MSCs and CSCs up to 2 and 4 weeks respectively, post-LLLI demonstrated a significant increase in the laser-treated cultures as compared to the control.
CONCLUSION: The present study clearly demonstrates the ability of LLLI to promote proliferation of MSCs and CSCs in vitro. These results may have an important impact on regenerative medicine.
Conf Proc IEEE Eng Med Biol Soc. 2007;2007:5830-33.
Effects of low level red-light irradiation on the proliferation of mesenchymal stem cells derived from rat bone marrow.
Li WT, Leu YC.
Department of Biomedical Engineering, Chung-Yuan Christian University, Chung-Li, 32023 Taiwan, ROC. email@example.com
Mesenchymal stem cells (MSCs) are capable of regenerating various mesenchymal tissues and are essential in supporting the growth and differentiation of hematopoietic stem cells within the bone marrow microenvironment in vivo. To achieve clinically meaningful numbers of cells, many approaches have been used to maintain the differentiation potentialities and expand enough cells for clinical treatments. Previously, we have reported that low level light irradiation (LLLI) using 630 nm light emitting diodes (LEDs) could enhance replicative and colony formation potentials of MSCs derived from human bone marrow. The purpose was to study the effect on the proliferation of MSCs derived from the rat bone marrow by red light LLLI (630 nm) under different parameters of irradiation. The irradiance used was 5, 10 or 15 mW/cm2, and the radiant exposure was 2 or 4 J/cm2. Rat MSCs were irradiated at room temperature with single and multiple exposures. The results showed that the proliferation of MSCs plated at the low density (100 cells/well) and high density (1000 cells/well) was enhanced by multiple exposures of red-light LED treatment. The rate of proliferation of MSCs plated at the high density was not as high as those plated at the low density. The optimal parameter for LLLI was at irradiance of 15 mW/cm2, and radiant exposure of 4 J/cm2. The effect on the proliferation of cells by single dose irradiation was temporary. Multiple stimuli may be necessary for the enhancement of cell growth.
Lasers Med Sci. 2005 Dec;20(3-4):138-46. Epub 2005 Nov 16.
Low level laser irradiation stimulates osteogenic phenotype of mesenchymal stem cells seeded on a three-dimensional biomatrix.
Abramovitch-Gottlib L, Gross T, Naveh D, Geresh S, Rosenwaks S, Bar I, Vago R.
Department of Biotechnology Engineering, Ben-Gurion University of the Negev, Beer-Sheva, 84105, Israel. firstname.lastname@example.org
Mesenchymal stem cells (MSCs) seeded on three-dimensional (3D) coralline (Porites lutea) biomatrices were irradiated with low-level laser irradiation (LLLI). The consequent phenotype modulation and development of MSCs towards ossified tissue was studied in this combined 3D biomatrix/LLLI system and in a control group, which was similarly grown, but was not treated by LLLI. The irradiated and non irradiated MSC were tested at 1-7, 10, 14, 21, 28 days of culturing via analysis of cellular distribution on matrices (trypan blue), calcium incorporation to newly formed tissue (alizarin red), bone nodule formation (von Kossa), fat aggregates formation (oil red O), alkaline phosphatase (ALP) activity, scanning electron microscopy (SEM) and electron dispersive spectrometry (EDS). The results obtained from the irradiated samples showed enhanced tissue formation, appearance of phosphorous peaks and calcium and phosphate incorporation to newly formed tissue. Moreover, in irradiated samples ALP activity was significantly enhanced in early stages and notably reduced in late stages of culturing. These findings of cell and tissue parameters up to 28 days of culture revealed higher ossification levels in irradiated samples compared with the control group. We suggest that both the surface properties of the 3D crystalline biomatrices and the LLLI have biostimulatory effects on the conversion of MSCs into bone-forming cells and on the induction of ex-vivo ossification.
J Cell Sci. 2002 Apr 1;115(Pt 7):1461-9.
Low-energy laser irradiation promotes the survival and cell cycle entry of skeletal muscle satellite cells.
Shefer G, Partridge TA, Heslop L, Gross JG, Oron U, Halevy O.
Department of Animal Sciences, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100, Israel.
Low energy laser irradiation (LELI) has been shown to promote skeletal muscle cell activation and proliferation in primary cultures of satellite cells as well as in myogenic cell lines. Here, we have extended these studies to isolated myofibers. These constitute the minimum viable functional unit of the skeletal muscle, thus providing a close model of in vivo regeneration of muscle tissue. We show that LELI stimulates cell cycle entry and the accumulation of satellite cells around isolated single fibers grown under serum-free conditions and that these effects act synergistically with the addition of serum. Moreover, for the first time we show that LELI promotes the survival of fibers and their adjacent cells, as well as cultured myogenic cells, under serum-free conditions that normally lead to apoptosis. In both systems, expression of the anti-apoptotic protein Bcl-2 was markedly increased, whereas expression of the pro-apoptotic protein BAX was reduced. In culture, these changes were accompanied by a reduction in the expression of p53 and the cyclin-dependent kinase inhibitor p21, reflecting the small decrease in viable cells 24 hours after irradiation. These findings implicate regulation of these factors as part of the protective role of LELI against apoptosis. Taken together, our findings are of critical importance in attempts to improve muscle regeneration following injury.
Biochim Biophys Acta. 1999 Jan 11;1448(3):372-80.
Low-energy laser irradiation affects satellite cell proliferation and differentiation in vitro.
Ben-Dov N, Shefer G, Irintchev A, Wernig A, Oron U, Halevy O.
Department of Animal Sciences, Hebrew University of Jerusalem, Rehovot, Israel.
Low-energy laser (He-Ne) irradiation was found to promote skeletal muscle regeneration in vivo. In this study, its effect on the proliferation and differentiation of satellite cells in vitro was evaluated. Primary rat satellite cells were irradiated for various time periods immediately after preparation, and thymidine incorporation was determined after 2 days in culture. Laser irradiation affected thymidine incorporation in a bell-shaped manner, with a peak at 3 s of irradiation. Three seconds of irradiation caused an induction of cell-cycle regulatory proteins: cyclin D1, cyclin E and cyclin A in an established line of mouse satellite cells, pmi28, and proliferating cell nuclear antigen (PCNA) in primary rat satellite cells. The induction of cyclins by laser irradiation was compatible with their induction by serum refeeding of the cells. Laser irradiation effect on cell proliferation was dependent on the rat’s age. At 3 weeks of age, thymidine incorporation in the irradiated cells was more than twofold higher than that in the controls, while at 6 weeks of age this difference had almost disappeared. Myosin heavy chain (MHC) protein levels were twofold lower in the irradiated than in the control cells, whereas the proliferation of the irradiated cells was twofold higher. Fusion percentage was lower in the irradiated compared to non-irradiated cells. In light of these data, the promoting effect of laser irradiation on skeletal muscle regeneration in vivo may be due to its effect on the activation of early cell-cycle regulatory genes in satellite cells, leading to increased proliferation and to a delay in cell differentiation.