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Effect of autologous mesenchymal stem cells induced by low level laser therapy on cardiogenesis in the infarcted area following myocardial infarction in rats

Hana Tuby1, Tali Yaakobi1, Lidya Maltz1, Yaakov Delarea2, Orit Sagi-Assif2, Uri Oron1* - (Publication)
This study showed rats that were give a heart attach and then treated with the laser on their shins saw a 55% reduction in infarct size in the heart showing that the stem cells migrated released from the bone migrated to the heart.
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 1Department of Zoology, The George S. Wise Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv, Israel

2Department of Cell Biology and Immunology, The George S. Wise Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv, Israel

Email: *oronu@post.tau.ac.il

Received 27 May 2013; revised 29 June 2013; accepted 16 July 2013

ABSTRACT

In this study, we investigated the hypothesis that photo- biostimulation by low-energy laser therapy (LLLT) applied to the bone marrow (BM) of myocardial in- farcted rats may attenuate the scarring processes that follow myocardial infarction (MI). Wistar rats under- went experimental MI. LLLT (Ga-Al-As diode laser) was applied to the BM of the exposed tibia at differ- ent time intervals post-MI (4 hrs, 48 hrs and 5 days). Sham-operated infarcted rats served as control. In- farct size was significantly reduced (55%) in the la- ser-treated rats as compared to the control non-treat- ed rats, at 2 weeks post-MI. A significant 3-fold in- crease was observed in the density of desmin immu- nopositive stained cells 14 days post-MI in the infarc- ted area of the laser-treated rats as compared to the non-laser-treated controls. The electron microscopy from the control infarcted rat hearts revealed a typi- cal interphase area between the intact myocardium and the infarcted area, with conspicuous fibroblasts with collagen deposition dispersed among them. In rats that were laser treated (to BM), the interphase zone demonstrated cells with different intracellular struc- tures. There was also a significant increase in the per- centage of c-kit positive cells and macrophages in the circulating blood of the laser treated rats as compar- ed to control non treated ones. In the majority of the cells clusters of myofibrils anchored to well-developed Z-lines and structures resembling the morphological characteristics of mature intact cardiomyocytes were evident. In conclusion, LLLT to the BM of rats post- MI induces cardiogenesis mainly at the borders of the infarcted area in the heart.

Keywords: Low-Level Laser Therapy; Myocardial Infarction; Macrophage; Desmin; Ultrastructure; c-Kit Positive Cells

1. INTRODUCTION

Regenerative capacity and mitotic activity in the heart are confined mainly to the lower vertebrates [1]. Amputation of ~20% of the zebrafish’s ventricular myocardium re- sulted in full regeneration without scarring [2]. In am- phibians, heart injury was associated with increased cell proliferation of myocytes and enhanced regeneration [3]. The adult mammalian heart was traditionally considered to be a post-mitotic organ with terminally differentiated cardiac myocytes. However, this dogma has recently been challenged by several studies and reviews [4-8]. These studies have suggested that cardiac myocytes are replaced throughout the lifespan even in the human heart, and that myocytes can regenerate from resident cardiac progenitor cells (CPC) as well as from bone marrow (BM). Studies in human infarcted hearts have shown evidence of cytoki- nesis of cells in the heart and evidence of cardiac stem cells that are activated in response to ischemic injury. This growth response is attenuated in chronic heart fail- ure [9]. Some studies have reported that cardiac myocyt- es can be derived from BM; specifically, side population precursor cells following induction of myocardial infarc- tion (MI) by left anterior descending artery (LAD) liga- tion [10-12]. Contradicting these findings, other laborato- ries using genetic markers have reported that lineage ne- gative, c-kit+ BM cells did not differentiate into cardio- myocytes [13]. It was also suggested that BM-derived stem cells may stimulate the small population of stem cells in the ischemic heart to proliferate and differentiate to enhance cardiac repair post-MI [14]. In a recent study transient regenerative potential in the mouse heart was demonstrated during the neonatal period [15].

Low-level laser therapy (LLLT) has been found to modulate various biological processes [16,17], such as increasing mitochondrial respiration and ATP synthesis [18], facilitating wound healing and promoting the proc- ess of skeletal muscle regeneration and angiogenesis [19- 21]. In an experimental model of the infarcted heart in rats and dogs, it was demonstrated that LLLT application directly to the infarcted area in the heart at optimal power parameters significantly reduced scar tissue formation [22-24]. This phenomenon was partially attributed to a significant elevation in ATP content, heat shock proteins, vascular endothelial growth factor (VEGF), inducible ni- tric oxide (NO) synthase, and angiogenesis in the ischemic zone of the laser-irradiated rats, as compared to non- irradiated rats [25].

The effect of photobiostimulation on stem cells or pro- genitor cells has not been extensively studied. LLLT ap- plication to normal human neural progenitor cells signi- ficantly increases ATP production in these cells [26]. LLLT delivery to MSCs and cardiac stem cells in vitro caused a significant enhancement in their proliferation rate [27,28]. LLLT has also been shown to increase the proliferation rate of adipose-derived stem cells in vitro [29]. Recently, we demonstrated that LLLT application to autologous BM could induce mesenchymal stem cells (MSCs) in the BM to proliferate and cause their recruit- ment and specific homing in on the infarcted rat heart and not on other organs [30,31]. The laser treatment to the BM also caused a marked and statistically significant reduction of 79% in the scarring and ventricular dilata- tion followed MI as compared to infarcted non-laser- treated rats. The aim of the present study was to investi- gate the possibility that induction of stem cells in the BM of rats by LLLT could also affect cardiogenesis in the in- farcted rat heart.

2. MATERIALS AND METHODS

2.1. Experimental Procedures

A total of 21 Wistar male rats, weighing 200 - 250 gr, that underwent ligation of the LAD artery to induce MI, were used as described by us previously [23]. All the ex- perimental procedures were approved by the animal care committee of Tel-Aviv University. Briefly, rats were anes- thetized with Avertin (1 ml/100 g body weight I.P.) and the lungs were ventilated. Thoractomy was performed by invasion of the intercostals muscles between the 5th and 6th rib to expose the heart. The LAD artery was occluded 2 mm from the origin with 5-0 polypropylene thread (Ethicon Inc., Cincinnati, OH). Following LAD artery occlusion the chest muscles and skin were sutured and the rats were ventilated until they woke up. The infarcted rats were divided randomly into two groups. In one group LLLT was applied directly to the BM 4 hrs, 48 hrs and 5 days post-MI (see below). The second group was non-laser-treated (the rat’s bone was exposed for the same duration as the laser-treated group but the laser was not turned on). Food and water were supplied ad libitum. Rats were sacrificed 14 days post-MI.

2.2. Laser Application

After induction of MI rats were randomly assigned to a laser-treated or control non-laser-treated group. A diode (Ga-Al-As) laser, wavelength 804 nm with a tunable po- wer output of maximum of 400 mW (Lasotronic Inc., Zug, Switzerland) for application to the BM was used. The laser device was equipped with a metal-backed glass fiber optic (1.5 mm diameter). An infrared viewer (Laso- tronic Inc. Zug, Switzerland) and infrared-sensitive de-tecting card (Newport, Inc., Irvine, CA) were used to de- termine the infrared irradiation area. Laser application was done by a 10 mm longitudinal cut in the skin above the medial aspect, and further delicate cleaning of the bone surface was carried out. The tip of the fiber optic (1.5 mm diameter) was placed perpendicularly to the center of the exposed medial aspect of the tibia and power den- sity of 10 mW/cm2 was applied to the BM. The laser was applied for a duration of 100 sec (energy density 1.0 J/cm2). Left or right exposed tibias were chosen at random for LLLT application. In sham-operated infarcted rats that served as control the tibias were exposed and the fi- ber optic was placed as described above but the laser beam was not turned on.

2.3. Histology and Electron Microscopy

A defined cross-section sample (2 mm thick) from the central part of the infarcted area was taken from all hearts for histology. Eight micron paraffin sections were pre- pared from the tissue samples of each heart. Infarct size was determined using Masson’s trichrome staining as described by us previously [23]. Three observers, blinded to control or laser-treated rats, analyzed infarct size. Six microscopic slides from the infarcted area of each heart were chosen at random for determination of infarct size. Infarct size was expressed as the percentage of the total infarcted area relative to the total area of the left ventri- cle (LV) in each section, using image analysis software Sigma Scan Pro (Sigma, St. Louis, MO).

For electron microscopy three tissue samples from each of the control and laser-irradiated rat hearts were taken from the interphase zone between the infarcted and non-infarcted tissue by macroscopic examination. Fixa- tion was performed in 3.5% glutaraldehyde in 0.1 M ca- codylate buffer for 24 hrs followed by embedment in Epon-812. Semi-thin sections (1 micron) were prepared in order to localize the interphase zone. Thin sections were then prepared and stained with uranyl acetate and lead citrate followed by examination with a Jeol electron microscope.

2.4. Immunohistochemistry

The total number of cells immunostained for desmin (bone marrow cells or newly formed) in the infarcted area were determined using a desmin kit (Zytomed Laboratory, Ber- lin, Germany). The procedure was performed at room temperature with anti-mouse (dilution 1:25 - 1:50) primary antibody for 60 min. Following washing, slides were in- cubated with HRP secondary antibody for mouse for 30 min followed by DAB Chromogen system (Covance Inc., Dedham). Slides were rinsed again in wash buffer, stain- ed in Hematoxylin for nuclei detection, mounted and viewed using a Zeiss microscope equipped with a camera and video screen. The total number of desmin immuno- stained cells within the infarcted area was counted and their density expressed as the percentage of the total area of the infarct using SigmaPro software.

2.5. Flow Cytometry Analysis

Blood samples were taken 2 and 7 days post-IR injury for fluorescence-activated cell sorting (FACS) analysis. 100 μl of blood were mixed with different antibodies: anti-mouse CD117 (c-kit) PE (eBioscience San Diego, USA) and rat IgG2b isotype control PE (eBioscience San Diego, USA) and anti-rat macrophage marker PE (eBio- science San Diego, USA) and mouse IgG2a K isotype control PE (eBioscience San Diego, USA), were used for the FACS analysis according to the manufacturer’s guide- lines. Forty five min post incubation of the whole fresh blood with the relevant antibodies, 2 ml of Fix/Lyse so- lution (eBioscience, San Diego, USA) was added. After mixture the suspended cells were left for 60 min in the dark at room temperature. Centrifugation was performed for 10 min, supernatant was removed and washing of the pellet was performed with 2 ml of Flow Cytometry Stain- ing Buffer Solution (eBioscience, San Diego, California, USA). After another centrifugation for 10 minutes the supernatant was decanted. The pellet containing mono- nucleated cells was resuspended in 200 μl of flow stain buffer for FACS analysis.

2.6. Statistical Analysis

The SigmaStat 2.0 (Sigma, St. Luis, USA) software was used for statistical analysis. Tests were performed first for normality distribution, followed by parametric (stu- dent’s t-test) test.

3. RESULTS

Application of LLLT to the infarcted heart caused a sig- nificant (p = 0.049) reduction of 55% in infarct size as compared to control. The present of macrophages and c- kit positive cells in the blood was determined by FACS analysis (Figure 1). It was found that at 5 days post MI there was a statistical significant 2-fold higher concentra- tion of macrophages and significant 1.4-fold higher c-kit positive cells (mesenchymal cells) in the laser treated rats as compared to the infarcted non laser treated rats. Des- min immunostaining of histological sections of the in- farcted zone from laser-treated rats demonstrated a higher density of positively stained cells than in the non laser-treated ones (Figures 2-4). In the interphase zone, cells extending from the myocardium towards the in 

Figure 1. Percent (out of total mononucleated cells) of macro- phages and c-kit positive cells in blood of control and laser treated rats (to the bone marrow) 5 days post MI as revealed by FACS analysis. The results are mean ± S.E.M of 15 rats at each group. Statistical significance *p < 0.05; **p < 0.01.

Figure 2. Representative desmin immunostained light micro- graphs of the infarcted zone of non-laser-treated rats (a, c) and laser-treated rats (to the bone marrow at 4 and 48 hrs and 5 days) (b, d) taken 2 weeks post-MI. Note that the zone in the control non-laser-treated rats contains mainly collageneous mate- rial with a few desmin immunopositive cells in the infarcted area (a, c); while in the laser-treated rats the zone displays posi- tive desmin staining in extended outgrowths (arrow) from the myocardium (MC) in (b), and in the cytoplasm of many cells in the infarcted area in (d). IF, Infarcted area. Bar = 50 μm.

farcted area showed higher immunostaining for desmin in the laser-treated rat hearts as compared to the control non-treated ones (Figure 2). The cell density of desmin immune-positive cells was also determined quantitatively in histological sections of both the infarcted laser-treated rats and infarcted non-laser-treated rats. The cell density was significantly (p < 0.01) 3-fold higher in the infarcted area of the laser-treated rats as compared to the non-la- ser-treated controls (Figure 4).

The electron micrographs of all samples taken from the control non-laser-treated infarcted rat hearts revealed a typical interphase area between intact and infarcted heart (Figure 5(a)). Adjacent to the non-ischemic intact myocardium there were conspicuous fibroblasts with col- lagen deposition dispersed among them (Figure 5(a)). In all samples taken from the laser-irradiated hearts the in- terphase zone between intact and infarcted area demon- strated different characteristics to those of the non-laser- treated infarcted rat hearts. Cells with newly-formed or- ganized contractile myofilaments dispersed in the cyto- plasm were detected in groups of several cells (Figure 5(b)). In these cells numerous mitochondria, clusters of ribosomes, and conspicuous clusters of contractile pro- teins were evident in the cytoplasm (Figures 6-8). Some cells contained dispersed contractile myofilaments in the cytoplasm that were still in an early stage of organization (Figure 6). The organization of newly-formed contractile myofilaments in the cytoplasm was observed in various

Figure 3. Representative desmin immunostained light micro- graphs of the interphase of the infarcted zone of laser-treated rats. Note that desmin positively stained cross-sections of myo- fibers (arrows) intermingled in the infarcted zone in (a). In (b) immunopositively stained cross-sections of myofibers (arrow) are visible in the infarcted area (IF). In (c) newly-formed car- diomyocytes (NC) are seen, with the desmin immunostaining mainly confined to the Z-line. Bar = 50 μm.

Figure 4. Density of desmin positively stained area (relative to total area) in the infarcted areas of control (non-laser-treated) and laser-treated (to the bone marrow) rats at 14 days post-MI. Results are mean+ S.E.M from 6 - 8 rats in each group. **p < 0.01.

Figure 5. Electron micrographs of typical interphase zone be- tween myocardium and infarcted area of control non-laser- treated (a) and laser-treated (b) to bone marrow rats. Note intact myocardium (MY) and adjacent fibroblast (FB) in the infarcted area surrounded by collagen (CL) deposition in (a). In (b) sev- eral newly-formed cardiomyocytes (marked with asterix) with conspicuous well-organized myofilaments (MF) in their cyto- plasm are evident adjacent to blood capillaries (CA). EN, En- dothelial cell.

degrees of maturation in those cells. In some cells the myofilaments were dispersed in the cytoplasm and in others they were organized in clusters anchored to well- developed Z-lines (Figure 7(a)). In certain cells the myo- filaments were organized parallel to the longitudinal di- rection of the cells, resembling the morphological char- acteristics of mature intact cardiomyocytes (Figure 7(b)). Some of the cells were also seen in a process of forma- tion of typical intercalated disc between them (Figure 9).

4. DISCUSSION AND CONCLUSION

The most significant outcome of this study was the ap- pearance of newly-formed cardiomyocytes following laser treatment to the BM, as indicated by light and electron microscopy. There was a 3-fold increase in the density of

Figure 6. Electron micrographs of most probably newly-formed cardiomyocytes at an early stage of organization of contractile myofilaments. Note myofilaments (MF) in the cytoplasm. M, Mitochondrion. Bar = 1 μm.

Figure 7. Electron micrographs of most probably newly-formed cardiomyocytes with early (a) and late (b) stages of the organi- zation of the contractile myofilaments in the cytoplasm. Note contractile myofilaments that are dispersed (DMF) in the cyto- plasm with a few organized in clusters anchored to Z-lines (Z) in (a). In (b) myofilaments (MF) are organized in parallel to the longitudinal axis of the cardiomyocyte, resembling their orga- nization in mature cardiomyocyte. N, Nucleus. Bar = 1 μm.

desmin immunostained cells in the infarcted rat hearts that had been laser treated. Desmin is a protein found in the cytoplasm of developing myocytes and cardiomyo- cytes [32]. The significantly higher occurrence of des- min-positive cells in the infarcted area of the laser- treated hearts may indicate the synthesis of new contrac- tile proteins in the developing new cardiomyocytes, re- sembling the process that takes place during embryonic development. The ultrastructural features of the cells in the interphase between the intact myocardium and the

Figure 8. Electron micrographs of typical interphase zone be- tween myocardium and infarcted area of laser-treated infarcted rat heart. Note numerous mitochondria (M) in the cytoplasm of the cardiomyocytes in (a) and (b). Also note organized contrac- tile myofilament with well-developed Z-lines (Z), some dis- persed myofilaments and clusters of ribosomes (R). Bar = 1 μm.

Figure 9. Electron micrographs of typical intercalated disk formation in the interphase region of the infarcted heart of la- ser-treated rats. Formation of intercalated disks (ID) between cells (marked with asterix) is evident. Note that the most proba- bly newly-formed cardiomyocytes contain clusters of myofila- ments (MF) in the cytoplasm that are conspicuous in their obli- que or cross-sections (arrows). Bar = 1 μm.

infarcted myocardium of the laser-treated rats, as shown in this study, clearly resemble the characteristics of car- diomyocytes during embryonic development of the heart [33]. Furthermore, the clusters of ribosomes and the nu- merous clusters of mitochondria in the cytoplasm of these cells may characterize cells that are active in the synthe- sis of proteins. It was previously demonstrated that direct LLLT to the infarcted hearts of rats, dogs and pigs caus- ed a significant reduction of scarring post-MI [23,24]. It was suggested that part of this reduction could be ex- plained by the regenerative response that takes place in the interphase zone [24].

The results of the present study indicate that the LLLT

applied to autologous BM attenuates the concentration of macrophages and MSC in the circulating blood. We have previously shown that LLLT application to the BM of infarcted rats caused a 2 fold enhancement in the rate of proliferation of MSC in the BM [30]. Those cells that most probably leave the BM to the circulating blood in- deed show a significant elevation of their concentration (as reveled by the FACS analysis in the present paper) at 5 days post MI. Consequently these cells probably home in on the infarcted heart, and even migrate specifically to the infarcted area [30]. These cells may induce cardiac stem cells to differentiate to newly-formed cardiomyo- cytes, as suggested previously by Hatzistergos et al. [14]. Indeed, it was found that endogenous c-kit+ cardiac stem cells were increased by 20-fold in the rat infarcted heart compared to control, following transcardial injection of BM-derived MSCs [14]. Such induction may be enabled due to paracrine secretion of various growth factors by the laser-stimulated MSC that originated from the BM. The possibility that paracrine secretion occurs in im- planted stem cells during cell therapy to the heart post- MI has been suggested previously [34]. Another mecha- nism that may take place after homing of stem cells to the infarcted heart of the laser-stimulated rats is that these cells continue to proliferate in the appropriate mi-lieu of the interphase zone in the infarcted heart and then differentiate to cardiomyocytes [30].

Another possible mechanism that maybe associates with the reduction of infarct size is the significant increase in the concentration of macrophages in the circulation fol- lowing LLLT to the BM as revealed from the FACS analysis in the present study. These findings corroborate with studies indicating that macrophages activity in the infarcted area at early stages post MI cause reduction of scarring post MI [35,36]. Thus, it could be postulated that more macrophages that will eventually home in the infarcted area from the circulating blood in the laser treated rats will also contribute to the reduction of scar- ring.

Although the findings of the present study do not in- dicate the extent of regenerative capacity of the rat in- farcted heart post-laser-irradiation, they do reveal a shift from practically no cardiomyocytes in the tissue samples taken from the non-laser-treated hearts, to the presence of newly-formed cardiomyocytes in all the electron mi- croscope sections taken from the hearts of rats that are laser-treated to the BM.

In conclusion, to the best of our knowledge, this is the first study to demonstrate the appearance of newly-form- ed cardiomyocytes in the infarcted area following LLLT to autologous BM in the infarcted rat heart. The mecha- nisms associated with this phenomenon remain to be elu- cidated in further studies.

5. ACKNOWLEDGEMENTS

This study was partially supported by the Elizabeth and Nicholas Shle- zak Super-center for Cardiac Research and Medical Engineering. The authors wish to acknowledge N. Paz for editing the manuscript and V. Wexler for helping with preparation of the figures.

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Original Source: http://www.scirp.org/journal/jbise

Influence of Low Level Laser Radiation on Migration of Stem Cells

Levon Gasparyan, Grigory Brill, Anu Makela - (Publication)
View Resource

 Abstract: The long term effects of low level laser therapy can involve treatment mechanisms connected with activation of stem cells.

In the current study migration of stem cells was tested under the influence of laser light alone as well as in case of combined influence of light and stromal cell-derived factor-1α (SDF-1α). This cytokine plays a role in lymphocyte trafficking, hematopoietic progenitor cell and stem cell homing.

To investigate the light influence on stem cells, we analyzed factor-dependent cell-Patersen (FDCP)-mix multipotent progenitor cells.

Migration of the stem cell line was tested using Transwell system (Corning, NY) under influence of red diode laser (λ=659.6 nm, 19.5 mW) or infrared diode laser (λ=958 nm, 36 mW) during 15 min at continuous wave, as well as in case of applying 150 ng/ml SDF-1α.

Group 1 cells were a group of control, group 2 cells received only red light irradiation, while group 3 cells had IR light irradiation. Group 4 cells were treated with 150 ng/ml SDF-1α. Group 5 cells were irradiated with red laser light in addition to 150 ng/ml SDF-1α, and group 6 cells by IR light and 150 ng/ml SDF-1α.

The count of migrated cells was 1496,5±409 (100%) in case of control. Red and IR laser light increased migration activity of stem cells up to 1892±283 (126%) and 2255,5±510 (151%) accordingly. Influence of SDF-1α was more significant, than effects of light irradiation alone 3365,5±489 (225%). Combined effects of light irradiation and SDF-1α were significantly stronger 5813±1199 (388%) for SDF-1α and red laser light, and 6391,5±540 (427%) for SDF-1α and IR laser light irradiation.

Preliminary study results showed that laser light irradiation can activate stem cell migration in vitro. The results are more reliable in the case of combined application of light and SDF-1α. These results are giving ground to consider that stem cell reactions to light irradiation can be one of the factors of light therapy.

Key words: low level laser irradiation, low level laser therapy, stem cells, SDF-1, stromal cell-derived factor-1

INTRODUCTION

More than 30 years ago first reports about biological effects of low doses of laser light were presented. Currently low level laser therapy (LLLT) is successfully applied in the treatment of numerous diseases and pathological conditions. LLLT exhibits positive effects for the treatment of disorders, having in common failure of blood supply with development of acute or chronic tissue hypoxia, different level of destruction of tissues, following decreased regenerative abilities of tissues and organs, defects in immune system, and altered cell metabolism. At the same time some important mechanisms of influence of laser light on the body are still far to be fully understood [1 - 8].

Recent studies discovered important role of bone marrow hematopoetic stem cell (HSCs) for naturally occurred recovery and regeneration processes, following tissue hypoxia and injury. The three clinically important steps in this natural process are mobilization of stem cells from the bone marrow, homing of these cells to the site of injury, and differentiation of the stem cell into a functional cell of the injured tissue [9]. Different methods of stem cell therapy, the treatment method, based on mobilization and transplantation of stem cells, proves to be effective method of therapy for different disorders.

We proposed a hypothesis that wide range of positive effects following laser therapy can be connected to increased activity of stem cells in damaged tissues. To test that, we examined in vitro the influence of laser light on migration of stem cells in absence and in presence of stromal cell-derived factor-1 (SDF-1), a potent chemoattractor for lymphocytes, monocytes, HSCs, which plays a critical role in the stem cell migration towards areas of tissue injury and hypoxia.

MATERIALS AND METHODS

To investigate the light influence on stem cells, we analyzed factor-dependent cell-Patersen (FDCP)-mix multipotent progenitor cells. The FDCP-mix stem cell line was maintained in ISCOVE’S medium supplemented with 20% horse serum and penicillin/streptomycin in the presence of 20 ng/ml IL-3. The cells were supplied with fresh medium each 5 days. Migration of the stem cell line was tested using Transwell system (Corning, NY). The cells were washed with PBS once and re-suspended in the medium containing 0.1% BSA (2x106/ml). Then, 600 μl of the mixture was irradiated by red diode laser (λ=659.6 nm, 19.5 mW) or infrared diode laser (λ=958 nm, 36 mW) during 15 min at continuous wave. Next, 100 μl of the mixture (2x105 cells) was seeded into upper chambers of the Transwell system, and the filters were placed into the wells containing 600? μl of the medium with or without 150 ng/ml SDF-1α. The plate was incubated for 4 h (37°C, 5% CO2, humidified atmosphere), after which the cells were collected and counted by a FACS sorter (Beckton Dickinson) during 1 min. All samples were performed in duplicate.

Group 1 cells are control group, group 2 cells received only red light irradiation, while group 3 cells – only IR light irradiation. Group 4 cells were treated with 150 ng/ml SDF-1α. Group 5 cells were irradiated with red laser light in addition to 150 ng/ml SDF-1α, and group 6 cells – IR light and 150 ng/ml SDF-1α.

RESULTS

Small amount of stem cells can migrate without SDF-1α or laser light influence. The count of migrated cells in control group was 1496,5±409 (Fig). This amount was considered as 100%. Red and IR laser light at the above mentioned dosage and methods of irradiation increased migration activity of stem cells up to 1892±283 (126%) and 2255,5±510 (151%) accordingly. Influence of SDF-1α was more noticeable, than effects of red or IR laser light irradiation alone - 3365,5±489 (225%). It is important to stress attention on the finding, that rate of stem cell migration towards the filter and SDF-1α containing medium was much higher after laser irradiation of cells - 5813±1199 (388%) for red laser light, and 6391,5±540 (427%) for IR laser light irradiation.

DISCUSSION

The main scientific result of this study is the fact, that red and infrared laser light irradiation can activate migration of stem cells in vitro. Moreover, red and IR laser radiation can up-regulate the rate of stem cell migration towards higher SDF-1α gradient.

How to explain the direct effects of mobility of stem cells in vitro under red and IR laser light irradiation, and use this fact for better understanding the wide range of therapeutic effects of laser therapy?

Modern medical science has accepted that every pathologic condition or disease should be treated according to its clinical stage and symptoms, considering its pathogenesis and etiology. Similar treatment methods can be applied only for the treatment of different diseases, having common pathogenesis.

Not very many examples of successful application of the similar or close therapy method for the treatment of different pathologies are known in modern medicine. Steroid hormone therapy is one of such cases.

Another illustration of successful application of the similar treatment techniques for treatment of different disorders is stem cell therapy, a novel treatment method, which is still under development. Growing data suggests, that transplanted stem cell can successfully and for long period of time improve heart myocardial contractility and other heart functions after myocardial infarction, can support neoangiogenesis in areas of tissue infarction and damage, can replace several cell types in tissues, including β-cells in diabetes models, neurons, cardiomyocytes, hematopoetic cells of different lineages and so on, as well as be useful in the treatment of atherosclerosis [9].

The main principle of stem cell therapy is the idea of replacement of damaged and dead cells in injured tissues and organs with new healthy ones. It is known, that severe stress, tissue hypoxia and damage mobilizes some hematopoetic stem cells (HSCs) from bone marrow to peripheral bloodstream. After that HSCs can migrate towards hypoxic tissues and reach them. Finally they can start to proliferate to the cells types, typical for that damaged tissues. HSCs in the tissues are also able to produce several cytokines, chemokines, growthfactors, improve survival of damaged cells and limit apoptosis. As a result of some tissue regeneration, improvement in the function of a damaged organ can be achieved. Similar and even stronger regeneration and treatment effects can be displayed after transplantation of fetal or adult HSCs to recipient [10-12].

Low laser light irradiation is one other example of application of the same factor for the treatment of number of disorders, which, at first glance, have nothing or very little in common in their pathogenesis. Laser light can accelerate wound and burn healing, improve condition of patients after myocardial infarction and stroke, can support hematopoiesis of bone marrow after X-ray radiation or during cancer chemotherapy, can help for the treatment of diabetic angiopathy and neuropathy, as well as reduce atherosclerotic plaque formation. In cellular and tissue level LLLT exhibits positive effects for the treatment of disorders, having in common failure of blood supply with development of acute or chronic tissue hypoxia, different level of destruction of tissues, following with decreased regenerative abilities of cells, as well as altered cell metabolism [6, 7, 13, 14].

One can see that the therapeutic applications of LLLT and stem cell therapy are very close. So, earlier we proposed the hypotheses that one of the mechanisms of light therapy includes acceleration of tissue repair due to better mobilization of stem cells to the spot of injury after laser light irradiation [15]. That process should include several phases, including activation of stem cell migration towards area of tissue damage and hypoxia.

Stem cells are being investigated for their potential use in regenerative medicine. Stem cells share the following two defining characteristics: the capacity to differentiate into a spectrum of different cell types and the capacity to renew themselves [16]. The biological principle that underlies stem cell therapy is tissue-directed differentiation. For example, adult stem cells isolated from liver tissue and re-injected into liver become hepatocytes, whereas the same cells injected into myocardium become myocytes. [17] Stem cells have been engrafted into a broad spectrum of tissues, including regenerating bone, neural tissue, dystrophic skeletal muscle, and injured skeletal muscle. [18]. Myocardial regeneration is perhaps the most widely studied and debated example of stem cell plasticity. The most promising results have been obtained after transplantation and mobilization of bone marrow cells to the area of infarction.

The three clinically important steps in this natural process are mobilization of stem cells from the bone marrow, homing of these cells to the site of injury, and differentiation of the stem cell into a functional cell of the injured tissue [19].

Stem cell repair of cardiac and vascular tissue is a naturally occurring process after injury [20, 21] Circulating CD34+ mononuclear cell counts and plasma levels of endothelial growth factor are significantly increased in patients with acute myocardial infarction, peaking on day 7 after onset [22]. Due to limitations of the naturally occurring repair process after myocardium infarction and other injuries or pathologies several stem cell transplantation strategies were proposed and tested.

At present, however, enthusiasm for the therapeutic potential of strategies of stem cell transplantation is limited by certain practical considerations. For example, the number of stem cells, required for injection for the treatment of myocardial infarction, can be harvested approximately from 6 l of donor blood [23].

Other important limitation for autologous bone marrow stem/progenitor cell mobilization is a recent finding, that circulating endothelial progenitor cells in patients with coronary heart disease are impaired with respect to number and functional activity. Moreover, Heeschen et al [24] reported that regeneration and functional ability of bone marrow-derived mononuclear cells (BM-MNCs) in patients with chronic ischemic cardiomyopathy (ICMP) are also limited. In spite of the fact that, the number of BM-MNCs isolated from bone marrow aspirates of 18 patients with ICMP and 8 healthy subjects s did not differ, the colony-forming capacity of BM-MNCs from patients with ICMP was significantly lower compared with BM-MNCs from healthy controls. Likewise, the migratory response to SDF-1 and vascular endothelial growth factor (VEGF) was significantly reduced in BM-MNCs derived from patients with ICMP compared with BM-MNCs from healthy controls. The reduced neovascularization capacity in vivo of BM-MNCs derived from patients with ICMP closely correlated with the in vitro assessment of SDF-1-induced migration and colony-forming capacity.

The need for development of new methods for mobilization, as well as for homing of stem cells to the site of injury is therefore evident.

Several growth factors, chemokines and cytokines are involved in the regulation of stem cell mobilization, homing and differentiation. Stromal cell-derived factor-1 (SDF-1) is one of them. SDF-1 is a chemokine playing an important role in the trafficking of hematopoietic stem cells. SDF-1 is expressed on stromal cells of various tissues. CXCR4 is the only known receptor for SDF-1 [25]. SDF-1/CXCR4 interaction is reported to play an important physiological role during embryogenesis in hematopoiesis, vascular development, cardiogenesis, and cerebellar development [26-28].

Recently, several investigators have reported that CD34+ cells, classically considered to be hematopoietic stem cells, expressed CXCR4, and that SDF-1 could induce CD34+ cell migration in vitro [29]. Accordingly, SDF-1 is considered as one of the key regulators of hematopoietic stem cell trafficking between the peripheral circulation and bone marrow. SDF-1 has also been shown to effect CD34+ cell proliferation and mobilization and to induce angiogenesis in vivo [30 -32].

Hattori et al [31] reported that plasma elevation of SDF-1 induced mobilization of mature and immature hematopoietic progenitors and stem cells, including endothelial progenitor cells (EPCs). However, application of granulocyte colony-stimulating factor (G-CSF) for stem cell mobilization is widely accepted nowadays.

Yamaguchi et al [23] studied the effects of SDF-1 on migration and accumulation of EPCs. SDF-1 induced EPCs migration in a dose dependent manner in vitro. The magnitude of migration was similar to that induced by VEGF. Authors also reported that locally (in hind-limb ischemic muscle of experimental animals) administered SDF-1 could augment the local accumulation of transplanted EPCs from peripheral blood, thereby resulting in enhanced neovascularization. As a result, cell transplantation not only improved neovascularization but also reduced adverse biological consequences such as limb necrosis and auto-amputation in the mouse ischemic hind-limb model. These studies also disclosed that systemic EPCs transplantation improved myocardial neovascularization and cardiac function corresponding to reduced left ventricular scarring. Authors concluded that, at least under the experimental conditions used in the study, the effect of SDF-1 on neovascularization appears to result primarily from its ability to enhance the recruitment and incorporation of transplanted EPCs.

Damas at al. [33] reported that SDF-1α, at least in high concentrations, may mediate anti-inflammatory and matrix-stabilizing effects in unstable angina. These effects may promote plaque stabilization, and therapeutic intervention that enhances SDF-1 α activity could potentially be beneficial in acute coronary syndromes. Authors demonstrated significantly altered SDF-1/CXCR4 expression in patients with angina, with particularly marked changes in those with unstable disease, with low SDF-1 levels in plasma and altered expression of its corresponding receptor on peripheral blood mononuclear cells (PBMC). In contrast to the raised plasma levels of inflammatory chemokines in patients with angina plasma levels of SDF-1 and the surface expression of its corresponding receptor (CXCR4) on PBMC appear to be down-regulated in these patients. Thus, although persistent inflammation may involve up-regulation of inflammatory chemokines, recent studies suggest that inflammatory cytokines (eg, TNF-α and IL-1) may decrease the expression of SDF-1 and CXCR4.

Future progress of stem therapy techniques probably will include development of incubation methods for enhancement stem cell mobility and homing ability, as well as for faster proliferation into desire tissue cells. Increasing migration abilities will help to achieve better and faster results.

The ability of laser light to activate migration and mobility of different cells is well known. It was noticed, that irradiation of sperm cells in vitro can increase their mobility and fertility [34]. Moreover, this effect is more pronounced in case of damaged cells with low mobility rate. This gives a ground to assume that laser light irradiation in certain dosage and condition can improve functional abilities of cells. Future experiments are required to ascertain if stem cells respond to the laser light the same way.

The main finding on this study is that red and IR laser light can stimulate stem cell migration in vitro, and especially increase migration towards SDF-1α gradient. Stem cell ability to migrate towards tissues with higher SDF-1 concentration is one of the key mechanisms of stem cell homing. These results are giving ground to speculate that activation of stem cell migration can be one of the mechanisms of low level laser therapy. Taking into consideration that the combined of SDF-1 and laser irradiation had the strongest effect on stem cell homing, it would be reasonable to assume that this combination could be used in not only increasing the activity of stem cells but also in determining the main area of stem cell mobilization and homing. The current study did not aim to study the mechanisms of increased migration ability, which will be study in the future. But it is possible to suggest following explanation: laser irradiation can change the metabolism of stem cells, increase ATP production and so increase the migration, as well as up-regulate CXCR4 receptor expression or syntheses de novo. More studies are required to test if the laser light irradiation in vivo is able to make homing of transplanted stem cells to the area of damage more efficient, to check the influence of laser light on the mobilization rate of stem cells from bone marrow, to investigate if laser light can enhance functional abilities of stem cells. These studies would be desirable for better understanding of the mechanisms of laser therapy and for development of more effective methods of stem cell therapy.

References

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2. Karu T. The Science of Low Power Laser Therapy, Gordon & Breach, London, 1998.

3. Baxter G.D. Therapeutic Lasers: Theory and Practice, Churchill Livingstone, London, 1994.

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10. Orlic D., Hill J., Arai A. Stem Cells for Myocardial Regeneration Circulation Research. 2002;91:1092.

11. Hodgson D., Behfar A., Zingman L.V., Kane G.C., Perez-Terzic C., Alekseev A.E., Puceat M., and Terzic A. Stable benefit of embryonic stem cell therapy in myocardial infarction. Am J Physiol Heart Circ Physiol, August 1, 2004; 287(2): H471 - H479.

12. Ozbaran M., Omay S. B., Nalbantgil S., Kultursay H., Kumanlioglu K., Nart D., and Pektok E. Autologous peripheral stem cell transplantation in patients with congestive heart failure due to ischemic heart disease. Eur. J. Cardiothorac. Surg., March 1, 2004; 25(3): 342 - 350.

13. Brill A.G., Shenkman B., Brill G.E. et al. Blood irradiation by He-Ne laser induces a decrease in platelet responses to physiological agonists and an increase in platelet cyclic GMP. Platelets. 2000. Vol. 11. P. 87-93.

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15. Gasparyan L.V. Stem cells and therapeutic effect of light irradiation (in Russian). Collection of abstracts of the 10th International Conference of Quantum Medicine, Moscow, 2003, pp. 43-44.

16. Graf T. Differentiation plasticity of hematopoietic cells. Blood. 2002;99:3089–3101.

17. Malouf NN, Coleman WB, Girsham JW, et al. Adult-derived stem cells from the liver become myocytes in the heart in vivo. Am J Pathol. 2001;158:1929–1935.

18. Donovan PJ, Gearhart J. The end of the beginning for pluripotent stem cells. Nature. 2001;414:92–97.

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20. Beltrami AP, Urbanek K, Kajstura J, et al. Evidence that human cardiac myocytes divide after myocardial infarction. N Engl J Med. 2001;344:1750–1757.

21. Gill M, Dias S, Hattori K, et al. Vascular trauma induces rapid but transient mobilization of VEGFR2(+)AC133(+) endothelial precursor cells. Circ Res. 2001;88:167–174.

22. Shintani S, Murohara T, Ikeda H, et al. Mobilization of endothelial progenitor cells in patients with acute myocardial infarction. Circulation. 2001;103:2776–2779.

23. Yamaguchi J, Kusano K, Masuo O, at al. Stromal Cell–Derived Factor-1 Effects on Ex Vivo Expanded Endothelial Progenitor Cell Recruitment for Ischemic Neovascularization. Circulation. 2003;107: 1322–1328.

24. Heeschen C, Lehmann R, Honold J, Assmus B, Aicher A, Walter DH, Martin H, Zeiher AM, Dimmeler S. Profoundly reduced neovascularization capacity of bone marrow mononuclear cells derived from patients with chronic ischemic heart disease. Circulation. 2004;109(13):1615-22.

25. Bleul CC, Farzan M, Choe H, et al. The lymphocyte chemoattractant SDF-1 is a ligand for LESTR/fusin and blocks HIV-1 entry. Nature. 1996;382:829–833.

26. Nagasawa T, Hirota S, Tachibana K, et al. Defects of B-cell lymphopoiesis and bone-marrow myelopoiesis in mice lacking the CXC chemokine PBSF/SDF-1. Nature. 1996;382:635–638.

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Lasers, stem cells, and COPD

Feng Lin†, Steven F Josephs†, Doru T Alexandrescu†, Famela Ramos, Vladimir Bogin, Vincent Gammill, Constantin A Dasanu, Rosalia De Necochea-Campion, Amit N Patel, Ewa Carrier and David R Koos - 2010 (Publication)
This well annotated paper looks at the biphasic response to try to find the appropriate dosage for several applications including COPD.
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Lasers, stem cells, and COPD

  • Feng Lin,
  • Steven F Josephs,
  • Doru T Alexandrescu,
  • Famela Ramos,
  • Vladimir Bogin,
  • Vincent Gammill,
  • Constantin A Dasanu,
  • Rosalia De Necochea-Campion,
  • Amit N Patel,
  • Ewa Carrier and
  • David R KoosEmail author
Contributed equally
Journal of Translational Medicine20108:16

https://doi.org/10.1186/1479-5876-8-16

Received: 7 January 2010

Accepted: 16 February 2010

Published: 16 February 2010

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.

Introduction (Personal Perspective)

We came upon the field of low level laser (LLL) therapy by accident. One of our advisors read a press release about a company using this novel technology of specific light wavelengths to treat stroke. Given the possible role of stem cells in post-stroke regeneration, we decided to cautiously investigate. As a background, it should be said that our scientific team has been focusing on the area of cord blood banking and manufacturing of disposables for processing of adipose stem cells for the past 3 years. Our board has been interested in strategically refocusing the company from services-oriented into a more research-focused model. An unbiased exploration into the various degenerative conditions that may be addressed by our existing know-how led us to explore the condition of chronic obstructive pulmonary disease (COPD), an umbrella term covering chronic bronchitis and emphysema, which is the 4th largest cause of death in the United States. As a means of increasing our probability of success in treatment of this condition, the decision was made to develop an adjuvant therapy that would augment stem cell activity. The field of LLL therapy attracted us because it appeared to be relatively unexplored scientific territory for which large amounts of clinical experience exist. Unfortunately, it was difficult to obtain the cohesive "state-of-the-art" description of the molecular/cellular mechanisms of this therapy in reviews that we have searched. Therefore we sought in this mini-review to discuss what we believe to be relevant to investigators attracted by the concept of "regenerative photoceuticals". Before presenting our synthesis of the field, we will begin by describing our rationale for approaching COPD with the autologous stem cell based approaches we are developing.

COPD as an Indication for Stem Cell Therapy

COPD possesses several features making it ideal for stem cell based interventions: a) the quality of life and lack of progress demands the ethical exploration of novel approaches. For example, bone marrow stem cells have been used in over a thousand cardiac patients with some indication of efficacy [1, 2]. Adipose-based stem cell therapies have been successfully used in thousands of race-horses and companion animals without adverse effects [3], as well as numerous clinical trials are ongoing and published human data reports no adverse effects (reviewed in ref [4]). Unfortunately, evaluation of stem cell therapy in COPD has lagged behind other areas of regenerative investigation; b) the underlying cause of COPD appears to be inflammatory and/or immunologically mediated. The destruction of alveolar tissue is associated with T cell reactivity [5, 6], pathological pulmonary macrophage activation [7], and auto-antibody production [8]. Mesenchymal stem cells have been demonstrated to potently suppress autoreactive T cells [9, 10], inhibit macrophage activation [11], and autoantibody responses [12]. Additionally, mesenchymal stem cells can be purified in high concentrations from adipose stromal vascular tissue together with high concentrations of T regulatory cells [4], which in animal models are approximately 100 more potent than peripheral T cells at secreting cytokines therapeutic for COPD such as IL-10 [13, 14]. Additionally, use of adipose derived cells has yielded promising clinical results in autoimmune conditions such as multiple sclerosis [4]; and c) Pulmonary stem cells capable of regenerating damaged parenchymal tissue have been reported [15]. Administration of mesenchymal stem cells into neonatal oxygen-damaged lungs, which results in COPD-like alveoli dysplasia, has been demonstrated to yield improvements in two recent publications [16, 17].

Based on the above rationale for stem cell-based COPD treatments, we began our exploration into this area by performing several preliminary experiments and filing patents covering combination uses of stem cells with various pharmacologically available antiinflammatories, as well as methods of immune modulation. These have served as the basis for two of our pipeline candidates, ENT-111, and ENT-894. As a commercially-oriented organization, we needed to develop a therapeutic candidate that not only has a great potential for efficacy, but also can be easily implemented as part of the standard of care. Our search led us to the area of low level laser (LLL) therapy. From our initial perception as neophytes to this field, the area of LLL therapy has been somewhat of a medical mystery. A pubmed search for "low level laser therapy" yields more than 1700 results, yet before stumbling across this concept, none of us, or our advisors, have ever heard of this area of medicine.

On face value, this field appeared to be somewhat of a panacea: clinical trials claiming efficacy for conditions ranging from alcoholism [18], to sinusitis [19], to ischemic heart disease [20]. Further confusing was that many of the studies used different types of LLL-generating devices, with different parameters, in different model systems, making comparison of data almost impossible. Despite this initial impression, the possibility that a simple, non-invasive methodology could exist that augments regenerative potential in a tissue-focused manner became very enticing to us. Specific uses envisioned, for which intellectual property was filed included using light to concentrate stem cells to an area of need, to modulate effects of stem cells once they are in that specific area, or even to use light together with other agents to modulate endogenous stem cells.

The purpose of the current manuscript is to overview some of the previous work performed in this area that was of great interest to our ongoing work in regenerative medicine. We believe that greater integration of the area of LLL with current advancements in molecular and cellular biology will accelerate medical progress. Unfortunately, in our impression to date, this has been a very slow process.

What is Low Level Laser Irradiation?

Lasers (Light amplification by stimulated emission of radiation) are devices that typically generate electromagnetic radiation which is relatively uniform in wavelength, phase, and polarization, originally described by Theodore Maiman in 1960 in the form of a ruby laser [21]. These properties have allowed for numerous medical applications including uses in surgery, activation of photodynamic agents, and various ablative therapies in cosmetics that are based on heat/tissue destruction generated by the laser beam [22, 23, 24]. These applications of lasers are considered "high energy" because of their intensity, which ranges from about 10-100 Watts. The subject of the current paper will be another type of laser approach called low level lasers (LLL) that elicits effects through non-thermal means. This area of investigation started with the work of Mester et al who in 1967 reported non-thermal effects of lasers on mouse hair growth [25]. In a subsequent study [26], the same group reported acceleration of wound healing and improvement in regenerative ability of muscle fibers post wounding using a 1 J/cm2 ruby laser. Since those early days, numerous in vitro and in vivo studies have been reported demonstrating a wide variety of therapeutic effects involving LLL, a selected sample of which will be discussed below. In order to narrow our focus of discussion, it is important to first begin by establishing the current definition of LLL therapy. According to Posten et al [27], there are several parameters of importance: a) Power output of laser being 10-3 to 10-1 Watts; b) Wavelength in the range of 300-10,600 nm; c) Pulse rate from 0, meaning continuous to 5000 Hertz (cycles per second); d) intensity of 10-2-10 W/cm(2) and dose of 0.01 to 100 J/cm2. Most common methods of administering LLL radiation include lasers such as ruby (694 nm), Ar (488 and 514 nm), He-Ne (632.8 nm), Krypton (521, 530, 568, and 647 nm), Ga-Al-As (805 or 650 nm), and Ga-As (904 nm). Perhaps one of the most distinguishing features of LLL therapy as compared to other photoceutical modalities is that effects are mediated not through induction of thermal effects but rather through a process that is still not clearly defined called "photobiostimulation". It appears that this effect of LLL is not depend on coherence, and therefore allows for use of non-laser light generating devices such as inexpensive Light Emitting Diode (LED) technology [28].

To date several mechanisms of biological action have been proposed, although none are clearly established. These include augmentation of cellular ATP levels [29], manipulation of inducible nitric oxide synthase (iNOS) activity [30, 31], suppression of inflammatory cytokines such as TNF-alpha, IL-1beta, IL-6 and IL-8 [32, 33, 34, 35, 36], upregulation of growth factor production such as PDGF, IGF-1, NGF and FGF-2 [36, 37, 38, 39], alteration of mitochondrial membrane potential [29, 40, 41, 42] due to chromophores found in the mitochondrial respiratory chain [43, 44] as reviewed in [45], stimulation of protein kinase C (PKC) activation [46], manipulation of NF-κB activation [47], direct bacteriotoxic effect mediated by induction of reactive oxygen species (ROS) [48], modification of extracellular matrix components [49], inhibition of apoptosis [29], stimulation of mast cell degranulation [50], and upregulation of heat shock proteins [51]. Unfortunately these effects have been demonstrated using a variety of LLL devices in non-comparable models. To add to confusion, dose-dependency seems to be confined to such a narrow range or does not seem to exist in that numerous systems therapeutic effects disappear with increased dose.

In vitro studies of LLL

In areas of potential phenomenology, it is important to begin by assessing in vitro studies reported in the literature in which reproducibility can be attained with some degree of confidence, and mechanistic dissection is simpler as compared with in vivo systems. In 1983, one of the first studies to demonstrate in vitro effects of LLL was published. The investigators used a helium neon (He-Ne) laser to generate a visible red light at 632.8 nm for treatment of porcine granulosa cells. The paper described upregulation of metabolic and hormone-producing activity of the cells when exposed for 60 seconds to pulsating low power (2.8 mW) irradiation [52]. The possibility of modulating biologically-relevant signaling proteins by LLL was further assessed in a study using an energy dose of 1.5 J/cm2 in cultured keratinocytes. Administration of He-Ne laser emitted light resulted in upregulated gene expression of IL-1 and IL-8 [53]. Production of various growth factors in vitro suggests the possibility of enhanced cellular mitogenesis and mobility as a result of LLL treatment. Using a diode-based method to generate a similar wavelength to the He-Ne laser (363 nm), Mvula et al reported in two papers that irradiation at 5 J/cm2 of adipose derived mesenchymal stem cells resulted in enhanced proliferation, viability and expression of the adhesion molecule beta-1 integrin as compared to control [54, 55]. In agreement with possible regenerative activity based on activation of stem cells, other studies have used an in vitro injury model to examine possible therapeutic effects. Migration of fibroblasts was demonstrated to be enhanced in a "wound assay" in which cell monolayers are scraped with a pipette tip and amount of time needed to restore the monolayer is used as an indicator of "healing". The cells exposed to 5 J/cm2 generated by an He-Ne laser migrated rapidly across the wound margin indicating a stimulatory or positive influence of phototherapy. Higher doses (10 and 16 J/cm2) caused a decrease in cell viability and proliferation with a significant amount of damage to the cell membrane and DNA [56]. In order to examine whether LLL may positively affect healing under non-optimal conditions that mimic clinical situations treatment of fibroblasts from diabetic animals was performed. It was demonstrated that with the He-Ne laser dosage of 5 J/cm2 fibroblasts exhibited an enhanced migration activity, however at 16 J/cm2 activity was negated and cellular damage observed [57]. Thus from these studies it appears that energy doses from 1.5 J/cm2 to 5 J/cm2 are capable of eliciting "biostimulatory effects" in vitro in the He-Ne-based laser for adherent cells that may be useful in regeneration such as fibroblasts and mesenchymal stem cells.

Studies have also been performed in vitro on immunological cells. High intensity He-Ne irradiation at 28 and 112 J/cm2 of human peripheral blood mononuclear cells, a heterogeneous population of T cells, B cells, NK cells, and monocytes has been described to induce chromatin relaxation and to augment proliferative response to the T cell mitogen phytohemaglutin [58]. In human peripheral blood mononuclear cells (PBMC), another group reported in two papers that interleukin-1 alpha (IL-1 alpha), tumor necrosis factor-alpha (TNF-alpha), interleukin-2 (IL-2), and interferon-gamma (IFN-gamma) at a protein and gene level in PBMC was increased after He-Ne irradiation at 18.9 J/cm2 and decreased with 37.8 J/cm2 [59, 60]. Stimulation of human PBMC proliferation and murine splenic lymphocytes was also reported with He-Ne LLL [61, 62]. In terms of innate immune cells, enhanced phagocytic activity of murine macrophages have been reported with energy densities ranging from 100 to 600 J/cm2, with an optimal dose of 200 J/cm2 [63]. Furthermore, LLL has been demonstrated to augment human monocyte killing mycobacterial cells at similar densities, providing a functional correlation [64].

Thus from the selected in vitro studies discussed, it appears that modulation of proliferation and soluble factor production by LLL can be reliably reproduced. However the data may be to some extent contradictory. For example, the over-arching clinical rationale for use of LLL in conditions such as sinusitis [65], arthritis [66, 67], or wound healing [68] is that treatment is associated with anti-inflammatory effects. However the in vitro studies described above suggested LLL stimulates proinflammatory agents such as TNF-alpha or IL-1 [59, 60]. This suggests the in vivo effects of LLL may be very complex, which to some extent should not be surprising. Factors affecting LLL in vivo actions would include degree of energy penetration through the tissue, the various absorption ability of cells in the various tissues, and complex chemical changes that maybe occurring in paracrine/autocrine manner. Perhaps an analogy to the possible discrepancy between LLL effects in vitro versus in vivo may be made with the medical practice of extracorporeal ozonation of blood. This practice is similar to LLL therapy given that it is used in treatment of conditions such as atherosclerosis, non-healing ulcers, and various degenerative conditions, despite no clear mechanistic understanding [69, 70, 71]. In vitro studies have demonstrated that ozone is a potent oxidant and inducer of cell apoptosis and inflammatory signaling [72, 73, 74]. In contrast, in vivo systemic changes subsequent to administration of ozone or ozonized blood in animal models and patients are quite the opposite. Numerous investigators have published enhanced anti-oxidant enzyme activity such as elevations in Mg-SOD and glutathione-peroxidase levels, as well as diminishment of inflammation-associated pathology [75, 76, 77, 78]. Regardless of the complexity of in vivo situations, the fact that reproducible, in vitro experiments, demonstrate a biological effect provided support for us that there is some basis for LLL and it is not strictly an area of phenomenology.

Animal Studies with LLL

As early as 1983, Surinchak et al reported in a rat skin incision healing model that wounds exposed He-Ne radiation of fluency 2.2 J/cm2 for 3 min twice daily for 14 days demonstrated a 55% increase in breaking strength over control rats. Interestingly, higher doses yielded poorer healing [79]. This application of laser light was performed directly on shaved skin. In a contradictory experiment, it was reported that rats irradiated for 12 days with four levels of laser light (0.0, 0.47, 0.93, and 1.73 J/cm2) a possible strengthening of wounds tension was observed at the highest levels of irradiation (1.73 J/cm2), however it did not reach significance when analyzed by resampling statistics [80]. In another wound-healing study Ghamsari et al reported accelerated healing in the cranial surface of teats in dairy cows by administration of He-Ne irradiation at 3.64 J/cm2 dose of low-level laser, using a helium-neon system with an output of 8.5 mW, continuous wave [81]. Collagen fibers in LLL groups were denser, thicker, better arranged and more continuous with existing collagen fibers than those in non-LLL groups. The mean tensile strength was significantly greater in LLL groups than in non-LLL groups [82]. In the random skin flap model, the use of He-Ne laser irradiation with 3 J/cm2 energy density immediately after the surgery and for the four subsequent days was evaluated in 4 experimental groups: Group 1 (control) sham irradiation with He-Ne laser; Group 2 irradiation by punctual contact technique on the skin flap surface; Group 3 laser irradiation surrounding the skin flap; and Group 4 laser irradiation both on the skin flap surface and around it. The percentage of necrotic area of the four groups was determined on day 7-post injury. The control group had an average necrotic area of 48.86%; the group irradiated on the skin flap surface alone had 38.67%; the group irradiated around the skin flap had 35.34%; and the group irradiated one the skin flap surface and around it had 22.61%. All experimental groups reached statistically significant values when compared to control [83]. Quite striking results were obtained in an alloxan-induced diabetes wound healing model in which a circular 4 cm2 excisional wound was created on the dorsum of the diabetic rats. Treatment with He-Ne irradiation at 4.8 J/cm2 was performed 5 days a week until the wound healed completely and compared to sham irradiated animals. The laser-treated group healed on average by the 18th day whereas, the control group healed on average by the 59th day [84].

In addition to mechanically-induced wounds, beneficial effects of LLL have been obtained in burn-wounds in which deep second-degree burn wounds were induced in rats and the effects of daily He-Ne irradiation at 1.2 and 2.4 J/cm2 were assessed in comparison to 0.2% nitrofurazone cream. The number of macrophages at day 16, and the depth of new epidermis at day 30, was significantly less in the laser treated groups in comparison with control and nitrofurazone treated groups. Additionally, infections with S. epidermidis and S. aureus were significantly reduced [85].

While numerous studies have examined dermatological applications of LLL, which may conceptually be easier to perform due to ability to topically apply light, extensive investigation has also been made in the area of orthopedic applications. Healing acceleration has been observed in regeneration of the rat mid-cortical diaphysis of the tibiae, which is a model of post-injury bone healing. A small hole was surgically made with a dentistry burr in the tibia and the injured area and LLL was administered over a 7 or 14 day course transcutaneously starting 24 h from surgery. Incident energy density dosages of 31.5 and 94.5 J/cm2 were applied during the period of the tibia wound healing. Increased angiogenesis was observed after 7 days irradiation at an energy density of 94.5 J/cm2, but significantly decreased the number of vessels in the 14-day irradiated tibiae, independent of the dosage [86]. In an osteoarthritis model treatment with He-Ne resulted in augmentation of heat shock proteins and pathohistological improvement of arthritic cartilage [87]. The possibility that a type of preconditioning response is occurring, which would involve induction of genes such as hemoxygenase-1 [88], remains to be investigated. Effects of LLL therapy on articular cartilage were confirmed by another group. The experiment consisted of 42 young Wistar rats whose hind limbs were operated on in order to immobilize the knee joint. One week after operation they were assigned to three groups; irradiance 3.9 W/cm2, 5.8 W/cm2, and sham treatment. After 6 times of treatment for another 2 weeks significantpreservation of articular cartilage stiffness with 3.9 and 5.8 W/cm2 therapy was observed [89].

Muscle regeneration by LLL was demonstrated in a rat model of disuse atrophy in which eight-week-old rats were subjected to hindlimb suspension for 2 weeks, after which they were released and recovered. During the recovery period, rats underwent daily LLL irradiation (Ga-Al-As laser; 830 nm; 60 mW; total, 180 s) to the right gastrocnemius muscle through the skin. After 2-weeks the number of capillaries and fibroblast growth factor levels exhibited significant elevation relative to those of the LLL-untreated muscles. LLL treatment induced proliferation in satellite cells as detected by BRdU [90].

Other animal studies of LLL have demonstrated effects in areas that appear unrelated such as suppression of snake venom induced muscle death [91], decreasing histamine-induced vasospasms [92], inhibition of post-injury restenosis [93], and immune stimulation by thymic irradiation [94].

Clinical Studies Using LLL

Growth factor secretion by LLL and its apparent regenerative activities have stimulated studies in radiation-induced mucositis. A 30 patient randomized trial of carcinoma patients treated by radiotherapy alone (65 Gy at a rate of 2 Gy/fraction, 5 fractions per week) without prior surgery or concomitant chemotherapy suffering from radiation-induced mucositis was performed using a He-Ne 60 mW laser. Grade 3 mucositis occured with a frequency of 35.2% in controls and at 7.6% of treated patients. Furthermore, a decrease in "severe pain" (grade 3) was observed in that 23.8% in the control group experienced this level of pain, as compared to 1.9% in the treatment group [95]. A subsequent study reported similar effects [96].

Healing ability of lasers was also observed in a study of patients with gingival flap incisions. Fifty-eight extraction patients had one of two gingival flap incisions lased with a 1.4 mW He-Ne (670 nm) at 0.34 J/cm2. Healing rates were evaluated clinically and photographically. Sixty-nine percent of the irradiated incisions healed faster than the control incisions. No significant difference in healing was noted when patients were compared by age, gender, race, and anatomic location of the incision [97]. Another study evaluating healing effects of LLL in dental practice examined 48 patients subjected to surgical removal of their lower third molars. Treated patients were administered Ga-Al-As diode generated 808 nm at a dose of 12 J. The study demonstrated that extraoral LLL is more effective than intraoral LLL, which was more effective than control for the reduction of postoperative trismus and swelling after extraction of the lower third molar [98].

Given the predominance of data supporting fibroblast proliferative ability and animal wound healing effects of LLL therapy, a clinical trial was performed on healing of ulcers. In a double-blinded fashion 23 diabetic leg ulcers from 14 patients were divided into two groups. Phototherapy was applied (<1.0 J/cm2) twice per week, using a Dynatron Solaris 705(R) LED device that concurrently emits 660 and 890 nm energies. At days 15, 30, 45, 60, 75, and 90 mean ulcer granulation and healing rates were significantly higher for the treatment group as compared to control. By day 90, 58.3% of the ulcers in the LLL treated group were fully healed and 75% achieved 90-100% healing. In the placebo group only one ulcer healed fully [68].

As previously mentioned, LLL appears to have some angiogenic activity. One of the major problems in coronary artery disease is lack of collateralization. In a 39 patient study advanced CAD, two sessions of irradiation of low-energy laser light on skin in the chest area from helium-neon B1 lasers. The time of irradiation was 15 minutes while operations were performed 6 days a week for one month. Reduction in Canadian Cardiology Society (CCS) score, increased exercise capacity and time, less frequent angina symptoms during the treadmill test, longer distance of 6-minute walk test and a trend towards less frequent 1 mm ST depression lasting 1 min during Holter recordings was noted after therapy [99].

Perhaps one of the largest clinical trials with LLL was the NEST trial performed by Photothera. In this double blind trial 660 stroke patients were recruited and randomized: 331 received LLL and 327 received sham. No prespecified test achieved significance, but a post hoc analysis of patients with a baseline National Institutes of Health Stroke Scale score of <16 showed a favorable outcome at 90 days on the primary end point (P < 0.044) [100]. Currently Photothera is in the process of repeating this trial with modified parameters.

Relevance of LLL to COPD

A therapeutic intervention in COPD would require addressing the issues of inflammation and regeneration. Although approaches such as administration of bone marrow stem cells, or fat derived cellular components have both regenerative and anti-inflammatory activity in animal models, the need to enhance their potency for clinical applications can be seen in the recent Osiris's COPD trial interim data which reported no significant improvement in pulmonary function [101]. Accordingly, we sought to develop a possible rationale for how LLL may be useful as an adjunct to autologous stem cell therapy.

Table 1 depicts some of the properties of LLL that provide a rationale for the combined use with stem cells. One of the basic properties of LLL seems to be ability to inhibit inflammation at the level of innate immune activation. Representative studies showed that LLL was capable of suppressing inflammatory genes and/or pathology after administration of lipopolysaccharide (LPS) as a stimulator of monocytes [102] and bronchial cells [34], in vitro, and leukocyte infiltration in vivo [103, 104]. Inflammation induced by other stimulators such as zymosan, carrageenan, and TNF-alpha was also inhibited by LLL [32, 105, 106]. Growth factor stimulating activity of LLL was demonstrated in both in vitro and in vivo experiments in which augmentation of FGF-2, PDGF and IGF-1 was observed [36, 37, 107]. Endogenous production of these growth factors may be useful in regeneration based on activation of endogenous pulmonary stem cells [108, 109]. Another aspect of LLL activities of relevance is ability to stimulate angiogenesis. In COPD, the constriction of blood vessels as a result of poor oxygen uptake is results in a feedback loop culminating in pulmonary hypertension. Administration of angiogenic factors has been demonstrated to be beneficial in several animal models of pulmonary pathology [110, 111]. The ability of LLL to directly induce proliferation of HUVEC cells [112], as well as to augment production of angiogenic factors such as VEGF [113], supports the possibility of creation of an environment hospitable to neoangiogenesis which is optimal for stem cell growth. In fact, a study demonstrated in vivo induction of neocapillary formation subsequent to LLL administration in a hindlimb ischemia model [114]. The critical importance of angiogenesis in stem cell mediated regeneration has previously been demonstrated in the stroke model, where the major therapeutic activity of exogenous stem cells has been attributed to angiogenic as opposed to transdifferentiation effects [115].
Table 1

Examples of LLL Properties Relevant to COPD

COPD Property

LLL Experiment

LLL Details

Ref

Inflammation

In vivo. Decreased joint inflammation in zymosan-induced arthritis

Semiconductor laser (685 nm and 830 nm) at (2.5 J/cm2)

[105]

 

In vitro. Suppression of LPS-induced bronchial inflammation and TNF-alpha.

655 nm at of 2.6 J/cm2

[34]

 

In vivo. Carrageenan-induced pleurisy had decreased leukocyte infiltration and cytokine (TNF-alpha, IL-6, and MCP)

660 nm at 2.1 J/cm2

[106]

 

In vitro. LPS stimulated Raw 264.7 monocytes had reduced gene expression of MCP-1, IL-1 and IL-6

780 nm diode laser at 2.2 J/cm2)

[102]

 

In vivo. Suppression of LPS-stimulated neutrophil influx, myeloperoxidase activity and IL-1beta in bronchoalveolar lavage fluid.

660 nm diode laser at 7.5 J/cm2

[35]

 

In vitro. Inhibition of TNF-alpha induced IL-1, IL-8 and TNF-alpha mRNA in human synoviocytes

810 nm (5 J/cm2) suppressed IL-1 and TNF, (25 J/cm2) also suppressed IL-8

[32]

 

In vivo. Reduction of TNF-alpha in diaphragm muscle after intravenous LPS injection.

4 sessions in 24 h with diode Ga-AsI-Al laser of 650 nm and a total dose of 5.2 J/cm2

[104]

 

In vivo. Inhibition of LPS induced peritonitis and neutrophil influx

3 J/cm2 and 7.5 J/cm2

[103]

Growth Factor Production

 

In vivo. Upregulation of TGF-β and PDGF in rat gingiva after incision.

He-Ne laser (632.8 nm) at a dose of 7.5 J/cm2

[36]

 

In vitro. Osteoblast-like cells were isolated from fetal rat calvariae had increased IGF-1

Ga-Al-As laser (830 nm) at (3.75 J/cm2).

[107]

 

In vitro. Upregulated production of IGF-1 and FGF-2 in human gingival fibroblasts.

685 nm, for 140 s, 2 J/cm2

[37]

Angiogenesis


Original Source: https://translational-medicine.biomedcentral.com/articles/10.1186/1479-5876-8-16

Lasers, stem cells, and COPD

Feng Lin1†, Steven F Josephs1†, Doru T Alexandrescu2†, Famela Ramos1, Vladimir Bogin3, Vincent Gammill4, Constantin A Dasanu5, Rosalia De Necochea-Campion6, Amit N Patel7, Ewa Carrier6, David R Koos1* - (Publication)
View Resource

 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, nonthermal

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.

Introduction (Personal Perspective)

We came upon the field of low level laser (LLL) therapy

by accident. One of our advisors read a press release

about a company using this novel technology of specific

light wavelengths to treat stroke. Given the possible role

of stem cells in post-stroke regeneration, we decided to

cautiously investigate. As a background, it should be

said that our scientific team has been focusing on the

area of cord blood banking and manufacturing of disposables

for processing of adipose stem cells for the past 3

years. Our board has been interested in strategically

refocusing the company from services-oriented into a

more research-focused model. An unbiased exploration

into the various degenerative conditions that may be

addressed by our existing know-how led us to explore

the condition of chronic obstructive pulmonary disease

(COPD), an umbrella term covering chronic bronchitis

and emphysema, which is the 4th largest cause of death

in the United States. As a means of increasing our probability

of success in treatment of this condition, the

decision was made to develop an adjuvant therapy that

would augment stem cell activity. The field of LLL therapy

attracted us because it appeared to be relatively

unexplored scientific territory for which large amounts

of clinical experience exist. Unfortunately, it was difficult

to obtain the cohesive “state-of-the-art” description of

the molecular/cellular mechanisms of this therapy in

reviews that we have searched. Therefore we sought in

this mini-review to discuss what we believe to be relevant

to investigators attracted by the concept of “regenerative

photoceuticals”. Before presenting our synthesis

of the field, we will begin by describing our rationale for

approaching COPD with the autologous stem cell based

approaches we are developing.

COPD as an Indication for Stem Cell Therapy

COPD possesses several features making it ideal for

stem cell based interventions: a) the quality of life and

lack of progress demands the ethical exploration of

novel approaches. For example, bone marrow stem cells

have been used in over a thousand cardiac patients with

some indication of efficacy [1,2]. Adipose-based stem

cell therapies have been successfully used in thousands

of race-horses and companion animals without adverse

effects [3], as well as numerous clinical trials are

ongoing and published human data reports no adverse

effects (reviewed in ref [4]). Unfortunately, evaluation of

stem cell therapy in COPD has lagged behind other

areas of regenerative investigation; b) the underlying

cause of COPD appears to be inflammatory and/or

immunologically mediated. The destruction of alveolar

tissue is associated with T cell reactivity [5,6], pathological

pulmonary macrophage activation [7], and auto-antibody

production [8]. Mesenchymal stem cells have been

demonstrated to potently suppress autoreactive T cells

[9,10], inhibit macrophage activation [11], and autoantibody

responses [12]. Additionally, mesenchymal stem

cells can be purified in high concentrations from adipose

stromal vascular tissue together with high concentrations of T regulatory cells [4], which in animal

models are approximately 100 more potent than peripheral

T cells at secreting cytokines therapeutic for COPD

such as IL-10 [13,14]. Additionally, use of adipose

derived cells has yielded promising clinical results in

autoimmune conditions such as multiple sclerosis [4];

and c) Pulmonary stem cells capable of regenerating

damaged parenchymal tissue have been reported [15].

Administration of mesenchymal stem cells into neonatal

oxygen-damaged lungs, which results in COPD-like

alveoli dysplasia, has been demonstrated to yield

improvements in two recent publications [16,17].

Based on the above rationale for stem cell-based

COPD treatments, we began our exploration into this

area by performing several preliminary experiments and

filing patents covering combination uses of stem cells

with various pharmacologically available antiinflammatories,

as well as methods of immune modulation. These

have served as the basis for two of our pipeline candidates,

ENT-111, and ENT-894. As a commerciallyoriented

organization, we needed to develop a therapeutic

candidate that not only has a great potential for efficacy,

but also can be easily implemented as part of the

standard of care. Our search led us to the area of low

level laser (LLL) therapy. From our initial perception as

neophytes to this field, the area of LLL therapy has been

somewhat of a medical mystery. A pubmed search for

“low level laser therapy” yields more than 1700 results,

yet before stumbling across this concept, none of us, or

our advisors, have ever heard of this area of medicine.

On face value, this field appeared to be somewhat of a

panacea: clinical trials claiming efficacy for conditions

ranging from alcoholism [18], to sinusitis [19], to

ischemic heart disease [20]. Further confusing was that

many of the studies used different types of LLL-generating

devices, with different parameters, in different model

systems, making comparison of data almost impossible.

Despite this initial impression, the possibility that a simple,

non-invasive methodology could exist that augments

regenerative potential in a tissue-focused manner

became very enticing to us. Specific uses envisioned, for

which intellectual property was filed included using light

to concentrate stem cells to an area of need, to modulate

effects of stem cells once they are in that specific

area, or even to use light together with other agents to

modulate endogenous stem cells.

The purpose of the current manuscript is to overview

some of the previous work performed in this area that was

of great interest to our ongoing work in regenerative medicine.

We believe that greater integration of the area of

LLL with current advancements in molecular and cellular

biology will accelerate medical progress. Unfortunately, in

our impression to date, this has been a very slow process.

What is Low Level Laser Irradiation?

Lasers (Light amplification by stimulated emission of

radiation) are devices that typically generate electromagnetic

radiation which is relatively uniform in wavelength,

phase, and polarization, originally described by Theodore

Maiman in 1960 in the form of a ruby laser [21]. These

properties have allowed for numerous medical applications

including uses in surgery, activation of photodynamic

agents, and various ablative therapies in cosmetics that are

based on heat/tissue destruction generated by the laser

beam [22-24]. These applications of lasers are considered

“high energy” because of their intensity, which ranges

from about 10-100 Watts. The subject of the current

paper will be another type of laser approach called low

level lasers (LLL) that elicits effects through non-thermal

means. This area of investigation started with the work of

Mester et al who in 1967 reported non-thermal effects of

lasers on mouse hair growth [25]. In a subsequent study

[26], the same group reported acceleration of wound healing

and improvement in regenerative ability of muscle

fibers post wounding using a 1 J/cm2 ruby laser. Since

those early days, numerous in vitro and in vivo studies

have been reported demonstrating a wide variety of therapeutic

effects involving LLL, a selected sample of which

will be discussed below. In order to narrow our focus of

discussion, it is important to first begin by establishing the

current definition of LLL therapy. According to Posten et

al [27], there are several parameters of importance: a)

Power output of laser being 10-3 to 10-1 Watts; b) Wavelength

in the range of 300-10,600 nm; c) Pulse rate from 0,

meaning continuous to 5000 Hertz (cycles per second); d)

intensity of 10-2-10 W/cm(2) and dose of 0.01 to 100 J/

cm2. Most common methods of administering LLL radiation

include lasers such as ruby (694 nm), Ar (488 and 514

nm), He-Ne (632.8 nm), Krypton (521, 530, 568, and 647

nm), Ga-Al-As (805 or 650 nm), and Ga-As (904 nm).

Perhaps one of the most distinguishing features of LLL

therapy as compared to other photoceutical modalities is

that effects are mediated not through induction of thermal

effects but rather through a process that is still not clearly

defined called “photobiostimulation”. It appears that this

effect of LLL is not depend on coherence, and therefore

allows for use of non-laser light generating devices such as

inexpensive Light Emitting Diode (LED) technology [28].

To date several mechanisms of biological action have

been proposed, although none are clearly established.

These include augmentation of cellular ATP levels [29],

manipulation of inducible nitric oxide synthase (iNOS)

activity [30,31], suppression of inflammatory cytokines

such as TNF-alpha, IL-1beta, IL-6 and IL-8 [32-36],

upregulation of growth factor production such as PDGF,

IGF-1, NGF and FGF-2 [36-39], alteration of mitochondrial

membrane potential [29,40-42] due to chromophores found in the mitochondrial respiratory

chain [43,44] as reviewed in [45], stimulation of protein

kinase C (PKC) activation [46], manipulation of NF-!B

activation [47], direct bacteriotoxic effect mediated by

induction of reactive oxygen species (ROS) [48], modification

of extracellular matrix components [49], inhibition

of apoptosis [29], stimulation of mast cell

degranulation [50], and upregulation of heat shock proteins

[51]. Unfortunately these effects have been demonstrated

using a variety of LLL devices in noncomparable

models. To add to confusion, dose-dependency

seems to be confined to such a narrow range or

does not seem to exist in that numerous systems therapeutic

effects disappear with increased dose.

In vitro studies of LLL

In areas of potential phenomenology, it is important to

begin by assessing in vitro studies reported in the literature

in which reproducibility can be attained with some

degree of confidence, and mechanistic dissection is simpler

as compared with in vivo systems. In 1983, one of

the first studies to demonstrate in vitro effects of LLL

was published. The investigators used a helium neon

(He-Ne) laser to generate a visible red light at 632.8 nm

for treatment of porcine granulosa cells. The paper

described upregulation of metabolic and hormone-producing

activity of the cells when exposed for 60 seconds

to pulsating low power (2.8 mW) irradiation [52]. The

possibility of modulating biologically-relevant signaling

proteins by LLL was further assessed in a study using an

energy dose of 1.5 J/cm2 in cultured keratinocytes.

Administration of He-Ne laser emitted light resulted in

upregulated gene expression of IL-1 and IL-8 [53]. Production

of various growth factors in vitro suggests the

possibility of enhanced cellular mitogenesis and mobility

as a result of LLL treatment. Using a diode-based

method to generate a similar wavelength to the He-Ne

laser (363 nm), Mvula et al reported in two papers that

irradiation at 5 J/cm2 of adipose derived mesenchymal

stem cells resulted in enhanced proliferation, viability

and expression of the adhesion molecule beta-1 integrin

as compared to control [54,55]. In agreement with possible

regenerative activity based on activation of stem

cells, other studies have used an in vitro injury model to

examine possible therapeutic effects. Migration of fibroblasts

was demonstrated to be enhanced in a “wound

assay” in which cell monolayers are scraped with a pipette

tip and amount of time needed to restore the

monolayer is used as an indicator of “healing”. The cells

exposed to 5 J/cm2 generated by an He-Ne laser

migrated rapidly across the wound margin indicating a

stimulatory or positive influence of phototherapy.

Higher doses (10 and 16 J/cm2) caused a decrease in

cell viability and proliferation with a significant amount

of damage to the cell membrane and DNA [56]. In

order to examine whether LLL may positively affect

healing under non-optimal conditions that mimic clinical

situations treatment of fibroblasts from diabetic animals

was performed. It was demonstrated that with the

He-Ne laser dosage of 5 J/cm2 fibroblasts exhibited an

enhanced migration activity, however at 16 J/cm2 activity

was negated and cellular damage observed [57]. Thus

from these studies it appears that energy doses from 1.5

J/cm2 to 5 J/cm2 are capable of eliciting “biostimulatory

effects” in vitro in the He-Ne-based laser for adherent

cells that may be useful in regeneration such as fibroblasts

and mesenchymal stem cells.

Studies have also been performed in vitro on immunological

cells. High intensity He-Ne irradiation at 28

and 112 J/cm2 of human peripheral blood mononuclear

cells, a heterogeneous population of T cells, B cells, NK

cells, and monocytes has been described to induce chromatin

relaxation and to augment proliferative response

to the T cell mitogen phytohemaglutin [58]. In human

peripheral blood mononuclear cells (PBMC), another

group reported in two papers that interleukin-1 alpha

(IL-1 alpha), tumor necrosis factor-alpha (TNF-alpha),

interleukin-2 (IL-2), and interferon-gamma (IFNgamma)

at a protein and gene level in PBMC was

increased after He-Ne irradiation at 18.9 J/cm2 and

decreased with 37.8 J/cm2 [59,60]. Stimulation of human

PBMC proliferation and murine splenic lymphocytes

was also reported with He-Ne LLL [61,62]. In terms of

innate immune cells, enhanced phagocytic activity of

murine macrophages have been reported with energy

densities ranging from 100 to 600 J/cm2, with an optimal

dose of 200 J/cm2 [63]. Furthermore, LLL has been

demonstrated to augment human monocyte killing

mycobacterial cells at similar densities, providing a functional

correlation [64].

Thus from the selected in vitro studies discussed, it

appears that modulation of proliferation and soluble factor

production by LLL can be reliably reproduced. However

the data may be to some extent contradictory. For

example, the over-arching clinical rationale for use of

LLL in conditions such as sinusitis [65], arthritis [66,67],

or wound healing [68] is that treatment is associated

with anti-inflammatory effects. However the in vitro studies

described above suggested LLL stimulates proinflammatory

agents such as TNF-alpha or IL-1 [59,60].

This suggests the in vivo effects of LLL may be very

complex, which to some extent should not be surprising.

Factors affecting LLL in vivo actions would include

degree of energy penetration through the tissue, the various

absorption ability of cells in the various tissues, and

complex chemical changes that maybe occurring in

paracrine/autocrine manner. Perhaps an analogy to the

possible discrepancy between LLL effects in vitro versus in vivo may be made with the medical practice of extracorporeal

ozonation of blood. This practice is similar to

LLL therapy given that it is used in treatment of conditions

such as atherosclerosis, non-healing ulcers, and

various degenerative conditions, despite no clear

mechanistic understanding [69-71]. In vitro studies have

demonstrated that ozone is a potent oxidant and inducer

of cell apoptosis and inflammatory signaling [72-74].

In contrast, in vivo systemic changes subsequent to

administration of ozone or ozonized blood in animal

models and patients are quite the opposite. Numerous

investigators have published enhanced anti-oxidant

enzyme activity such as elevations in Mg-SOD and glutathione-

peroxidase levels, as well as diminishment of

inflammation-associated pathology [75-78]. Regardless

of the complexity of in vivo situations, the fact that

reproducible, in vitro experiments, demonstrate a biological

effect provided support for us that there is some

basis for LLL and it is not strictly an area of

phenomenology.

Animal Studies with LLL

As early as 1983, Surinchak et al reported in a rat skin

incision healing model that wounds exposed He-Ne

radiation of fluency 2.2 J/cm2 for 3 min twice daily for

14 days demonstrated a 55% increase in breaking

strength over control rats. Interestingly, higher doses

yielded poorer healing [79]. This application of laser

light was performed directly on shaved skin. In a contradictory

experiment, it was reported that rats irradiated

for 12 days with four levels of laser light (0.0, 0.47, 0.93,

and 1.73 J/cm2) a possible strengthening of wounds tension

was observed at the highest levels of irradiation

(1.73 J/cm2), however it did not reach significance when

analyzed by resampling statistics [80]. In another

wound-healing study Ghamsari et al reported accelerated

healing in the cranial surface of teats in dairy cows

by administration of He-Ne irradiation at 3.64 J/cm2

dose of low-level laser, using a helium-neon system with

an output of 8.5 mW, continuous wave [81]. Collagen

fibers in LLL groups were denser, thicker, better

arranged and more continuous with existing collagen

fibers than those in non-LLL groups. The mean tensile

strength was significantly greater in LLL groups than in

non-LLL groups [82]. In the random skin flap model,

the use of He-Ne laser irradiation with 3 J/cm2 energy

density immediately after the surgery and for the four

subsequent days was evaluated in 4 experimental

groups: Group 1 (control) sham irradiation with He-Ne

laser; Group 2 irradiation by punctual contact technique

on the skin flap surface; Group 3 laser irradiation surrounding

the skin flap; and Group 4 laser irradiation

both on the skin flap surface and around it. The percentage

of necrotic area of the four groups was determined

on day 7-post injury. The control group had an average

necrotic area of 48.86%; the group irradiated on the skin

flap surface alone had 38.67%; the group irradiated

around the skin flap had 35.34%; and the group irradiated

one the skin flap surface and around it had

22.61%. All experimental groups reached statistically significant

values when compared to control [83]. Quite

striking results were obtained in an alloxan-induced diabetes

wound healing model in which a circular 4 cm2

excisional wound was created on the dorsum of the diabetic

rats. Treatment with He-Ne irradiation at 4.8 J/

cm2 was performed 5 days a week until the wound

healed completely and compared to sham irradiated animals.

The laser-treated group healed on average by the

18th day whereas, the control group healed on average

by the 59th day [84].

In addition to mechanically-induced wounds, beneficial

effects of LLL have been obtained in burn-wounds

in which deep second-degree burn wounds were

induced in rats and the effects of daily He-Ne irradiation

at 1.2 and 2.4 J/cm2 were assessed in comparison to

0.2% nitrofurazone cream. The number of macrophages

at day 16, and the depth of new epidermis at day 30,

was significantly less in the laser treated groups in comparison

with control and nitrofurazone treated groups.

Additionally, infections with S. epidermidis and S. aureus

were significantly reduced [85].

While numerous studies have examined dermatological

applications of LLL, which may conceptually be

easier to perform due to ability to topically apply light,

extensive investigation has also been made in the area

of orthopedic applications. Healing acceleration has

been observed in regeneration of the rat mid-cortical

diaphysis of the tibiae, which is a model of post-injury

bone healing. A small hole was surgically made with a

dentistry burr in the tibia and the injured area and LLL

was administered over a 7 or 14 day course transcutaneously

starting 24 h from surgery. Incident energy density

dosages of 31.5 and 94.5 J/cm2 were applied during

the period of the tibia wound healing. Increased angiogenesis

was observed after 7 days irradiation at an

energy density of 94.5 J/cm2, but significantly decreased

the number of vessels in the 14-day irradiated tibiae,

independent of the dosage [86]. In an osteoarthritis

model treatment with He-Ne resulted in augmentation

of heat shock proteins and pathohistological improvement

of arthritic cartilage [87]. The possibility that a

type of preconditioning response is occurring, which

would involve induction of genes such as hemoxygenase-

1 [88], remains to be investigated. Effects of LLL

therapy on articular cartilage were confirmed by another

group. The experiment consisted of 42 young Wistar

rats whose hind limbs were operated on in order to

immobilize the knee joint. One week after operation they were assigned to three groups; irradiance 3.9 W/

cm2, 5.8 W/cm2, and sham treatment. After 6 times of

treatment for another 2 weeks significantpreservation of

articular cartilage stiffness with 3.9 and 5.8 W/cm2 therapy

was observed [89].

Muscle regeneration by LLL was demonstrated in a rat

model of disuse atrophy in which eight-week-old rats

were subjected to hindlimb suspension for 2 weeks,

after which they were released and recovered. During

the recovery period, rats underwent daily LLL irradiation

(Ga-Al-As laser; 830 nm; 60 mW; total, 180 s) to

the right gastrocnemius muscle through the skin. After

2-weeks the number of capillaries and fibroblast growth

factor levels exhibited significant elevation relative to

those of the LLL-untreated muscles. LLL treatment

induced proliferation in satellite cells as detected by

BRdU [90].

Other animal studies of LLL have demonstrated

effects in areas that appear unrelated such as suppression

of snake venom induced muscle death [91],

decreasing histamine-induced vasospasms [92], inhibition

of post-injury restenosis [93], and immune stimulation

by thymic irradiation [94].

Clinical Studies Using LLL

Growth factor secretion by LLL and its apparent regenerative

activities have stimulated studies in radiationinduced

mucositis. A 30 patient randomized trial of carcinoma

patients treated by radiotherapy alone (65 Gy at

a rate of 2 Gy/fraction, 5 fractions per week) without

prior surgery or concomitant chemotherapy suffering

from radiation-induced mucositis was performed using a

He-Ne 60 mW laser. Grade 3 mucositis occured with a

frequency of 35.2% in controls and at 7.6% of treated

patients. Furthermore, a decrease in “severe pain” (grade

3) was observed in that 23.8% in the control group

experienced this level of pain, as compared to 1.9% in

the treatment group [95]. A subsequent study reported

similar effects [96].

Healing ability of lasers was also observed in a study

of patients with gingival flap incisions. Fifty-eight extraction

patients had one of two gingival flap incisions lased

with a 1.4 mW He-Ne (670 nm) at 0.34 J/cm2. Healing

rates were evaluated clinically and photographically.

Sixty-nine percent of the irradiated incisions healed faster

than the control incisions. No significant difference

in healing was noted when patients were compared by

age, gender, race, and anatomic location of the incision

[97]. Another study evaluating healing effects of LLL in

dental practice examined 48 patients subjected to surgical

removal of their lower third molars. Treated patients

were administered Ga-Al-As diode generated 808 nm at

a dose of 12 J. The study demonstrated that extraoral

LLL is more effective than intraoral LLL, which was

more effective than control for the reduction of postoperative

trismus and swelling after extraction of the

lower third molar [98].

Given the predominance of data supporting fibroblast

proliferative ability and animal wound healing effects of

LLL therapy, a clinical trial was performed on healing of

ulcers. In a double-blinded fashion 23 diabetic leg ulcers

from 14 patients were divided into two groups. Phototherapy

was applied (<1.0 J/cm2) twice per week, using a

Dynatron Solaris 705(R) LED device that concurrently

emits 660 and 890 nm energies. At days 15, 30, 45, 60,

75, and 90 mean ulcer granulation and healing rates

were significantly higher for the treatment group as

compared to control. By day 90, 58.3% of the ulcers in

the LLL treated group were fully healed and 75%

achieved 90-100% healing. In the placebo group only

one ulcer healed fully [68].

As previously mentioned, LLL appears to have some

angiogenic activity. One of the major problems in coronary

artery disease is lack of collateralization. In a 39

patient study advanced CAD, two sessions of irradiation

of low-energy laser light on skin in the chest area from

helium-neon B1 lasers. The time of irradiation was 15

minutes while operations were performed 6 days a week

for one month. Reduction in Canadian Cardiology

Society (CCS) score, increased exercise capacity and

time, less frequent angina symptoms during the treadmill

test, longer distance of 6-minute walk test and a

trend towards less frequent 1 mm ST depression lasting

1 min during Holter recordings was noted after therapy

[99].

Perhaps one of the largest clinical trials with LLL was

the NEST trial performed by Photothera. In this double

blind trial 660 stroke patients were recruited and randomized:

331 received LLL and 327 received sham. No

prespecified test achieved significance, but a post hoc

analysis of patients with a baseline National Institutes of

Health Stroke Scale score of <16 showed a favorable

outcome at 90 days on the primary end point (P <

0.044) [100]. Currently Photothera is in the process of

repeating this trial with modified parameters.

Relevance of LLL to COPD

A therapeutic intervention in COPD would require

addressing the issues of inflammation and regeneration.

Although approaches such as administration of bone marrow

stem cells, or fat derived cellular components have

both regenerative and anti-inflammatory activity in animal

models, the need to enhance their potency for clinical

applications can be seen in the recent Osiris’s COPD trial

interim data which reported no significant improvement

in pulmonary function [101]. Accordingly, we sought to

develop a possible rationale for how LLL may be useful as

an adjunct to autologous stem cell therapy.

Table 1 Examples of LLL Properties Relevant to COPD

COPD

Property

LLL Experiment LLL Details Ref

Inflammation In vivo. Decreased joint inflammation in zymosan-induced

arthritis

Semiconductor laser (685 nm and 830 nm) at (2.5 J/cm2)

In vitro. Suppression of LPS-induced bronchial inflammation and

TNF-alpha.

655 nm at of 2.6 J/cm2

In vivo. Carrageenan-induced pleurisy had decreased leukocyte

infiltration and cytokine (TNF-alpha, IL-6, and MCP)

660 nm at 2.1 J/cm2

In vitro. LPS stimulated Raw 264.7 monocytes had reduced gene

expression of MCP-1, IL-1 and IL-6

780 nm diode laser at 2.2 J/cm2)

In vivo. Suppression of LPS-stimulated neutrophil influx,

myeloperoxidase activity and IL-1beta in bronchoalveolar lavage

fluid.

660 nm diode laser at 7.5 J/cm2

In vitro. Inhibition of TNF-alpha induced IL-1, IL-8 and TNF-alpha

mRNA in human synoviocytes

810 nm (5 J/cm2) suppressed IL-1 and TNF, (25 J/cm2) also

suppressed IL-8

In vivo. Reduction of TNF-alpha in diaphragm muscle after

intravenous LPS injection.

4 sessions in 24 h with diode Ga-AsI-Al laser of 650 nm and

a total dose of 5.2 J/cm2

In vivo. Inhibition of LPS induced peritonitis and neutrophil influx 3 J/cm2 and 7.5 J/cm2

Growth Factor Production

In vivo. Upregulation of TGF-b and PDGF in rat gingiva after

incision.

He-Ne laser (632.8 nm) at a dose of 7.5 J/cm2

In vitro. Osteoblast-like cells were isolated from fetal rat calvariae

had increased IGF-1

Ga-Al-As laser (830 nm) at (3.75 J/cm2).

In vitro. Upregulated production of IGF-1 and FGF-2 in human

gingival fibroblasts.

685 nm, for 140 s, 2 J/cm2

Angiogenesis

In vivo. Increased fiber to capillary ratio in rabbits with ligated

femoral arteries.

Gallium-aluminum-arsenide (Ga-Al-As) diode laser, 904 nm

and power of 10 mW

In vitro. Stimulation of HUVEC proliferation by conditioned media

from LLL-treated T cells

820 nm at 1.2 and 3.6 J/cm2.

In vitro. 7-fold increased production of VEGF by cardiomyocytes,

1.6-fold increase by smooth muscle cells (SMC) and fibroblasts.

Supernatant of SMC had increased HUVEC-stimulating potential.

He:Ne continuous wave laser (632 nm). 0.5 J/cm2 for SMC,

2.1 J/cm2 for fibroblasts and 1.05 J/cm2 for cardiomyocytes.

In vitro. Direct stimulation of HUVEC proliferation 670 nm diode device at 2 and 8 J/cm2

Direct Stem Cell Effects

In vivo. LLL precondition significantly enhanced early cell survival

rate by 2-fold, decreased the apoptotic percentage of implanted

BMSCs in infarcted myocardium and increased the number of

newly formed capillaries.

635 nm at 0.96 J/cm2

In vitro. LLL stimulated MSC proliferation, VEGF and NGF

production, and myogenic differentiation after 5-aza induction.

635 nm diode laser at 0.5 J/cm2 for MSC proliferation, 5 J/

cm2 for VEGF and NGF production and for augmentation of

induced myogenic differentiation

In vitro. Increased proliferation of rat MSC. Red light LED 630 nm at 2 and 4 J/cm(2)

In vitro. Augmented proliferation of bone marrow and cardiac

specific stem cells.

GA-Al-As 810 nm at 1 and 3 J/cm2

In vitro/In vivo. Administration of LLL-treated MSC resulted 53%

reduction in infarct size, 5- and 6.3-fold significant increase in cell

density that positively immunoreacted to BrdU and c-kit,

respectively, and 1.4- and 2-fold higher level of angiogenesis and

vascular endothelial growth factor, respectively, when compared

to non-laser-treated implanted cells

Ga-Al-As laser (810 nm wavelength), 1 J/cm2

In vitro. Enhanced proliferation of adipose derived MSC in

presence of EGF.

636 nm diode, 5 J/cm2

Lin et al. Journal of Translational Medicine 2010, 8:16

http://www.translational-medicine.com/content/8/1/16

Table 1: Examples of LLL Properties Relevant to COPD (Continued)

In vitro. Enhanced proliferation and beta-1 integrin expression of

adipose derived MSC.

635 nm diode laser, at 5 J/cm2

Clinical. 660 stroke patients: 331 received LLL and 327 received

sham. No prespecified test achieved significance, but a post hoc

analysis of patients with a baseline National Institutes of Health

Stroke Scale score of <16 showed a favorable outcome at 90

days on the primary end point (P < 0.044).

808 nm. No density disclosed.

 

Table 1 depicts some of the properties of LLL that provide

a rationale for the combined use with stem cells. One

of the basic properties of LLL seems to be ability to inhibit

inflammation at the level of innate immune activation.

Representative studies showed that LLL was capable of

suppressing inflammatory genes and/or pathology after

administration of lipopolysaccharide (LPS) as a stimulator

of monocytes [102] and bronchial cells [34], in vitro, and

leukocyte infiltration in vivo [103,104]. Inflammation

induced by other stimulators such as zymosan, carrageenan,

and TNF-alpha was also inhibited by LLL

[32,105,106]. Growth factor stimulating activity of LLL

was demonstrated in both in vitro and in vivo experiments

in which augmentation of FGF-2, PDGF and IGF-1 was

observed [36,37,107]. Endogenous production of these

growth factors may be useful in regeneration based on

activation of endogenous pulmonary stem cells [108,109].

Another aspect of LLL activities of relevance is ability to

stimulate angiogenesis. In COPD, the constriction of

blood vessels as a result of poor oxygen uptake is results

in a feedback loop culminating in pulmonary hypertension.

Administration of angiogenic factors has been

demonstrated to be beneficial in several animal models of

pulmonary pathology [110,111]. The ability of LLL to

directly induce proliferation of HUVEC cells [112], as well

as to augment production of angiogenic factors such as

VEGF [113], supports the possibility of creation of an

environment hospitable to neoangiogenesis which is optimal

for stem cell growth. In fact, a study demonstrated in

vivo induction of neocapillary formation subsequent to

LLL administration in a hindlimb ischemia model [114].

The critical importance of angiogenesis in stem cell

mediated regeneration has previously been demonstrated

in the stroke model, where the major therapeutic activity

of exogenous stem cells has been attributed to angiogenic

as opposed to transdifferentiation effects [115].

Direct evidence of LLL stimulating stem cells has been

obtained using mesenchymal stem cells derived both

from the bone marrow and from the adipose tissue

[116,117]. Interestingly in vivo administration of LLL stimulated

MSC has resulted in 50% decrease in cardiac

infarct size [118]. Clinical translation of LLL has been

performed in the area of stroke, in which a 660 patient

trial demonstrated statistically significant effects in post

trial subset analysis [100].

Conclusions

Despite clinical use of LLL for decades, the field is still

in its infancy. As is obvious from the wide variety of

LLL sources, frequencies, and intensities used, no standard

protocols exist. The ability of LLL to induce

growth factor production, inhibition of inflammation,

stimulation of angiogenesis, and direct effects on stem

cells suggests the urgent need for combining this modality

with regenerative medicine, giving birth to the new

field of “regenerative photoceuticals”. Development of a

regenerative treatment for COPD as well as for other

degenerative diseases would be of considerable benefit.

Regarding COPD, such treatment would be life-saving/

life extending for thousands of affected individuals.

Ceasing smoking or not starting to smoke would considerably

impact this disease.

Acknowledgements

The authors thank Victoria Dardov and Matthew Gandjian for critical

discussions and input.

Author details

1Entest BioMedical, San Diego, CA, USA. 2Georgetown Dermatology,

Washington DC, USA. 3Cromos Pharma Services, Longview, WA, USA. 4Center

for the Study of Natural Oncology, Del Mar, CA, USA. 5Department of

Hematology and Medical Oncology, St Francis Hospital and Medical Center,

Hartford, CT, USA. 6Moores Cancer Center, University of California San Diego,

CA, USA. 7Department of Cardiothoracic Surgery, University of Utah, Salt

Lake City, UT, USA.

Authors’ contributions

FL, SFJ, DTA, FR, VB, VG, CAD, RDNC, ANP, EC, DRK contributed to literature

review, analysis and discussion, synthesis of concepts, writing of the

manuscript and proof-reading of the final draft.

Competing interests

David R Koos is a shareholder, as well as Chairman and CEO of Entest Bio.

Feng Lin is research director of Entest Bio. All other authors declare no

competing interest.

Received: 7 January 2010

Accepted: 16 February 2010 Published: 16 February 2010

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Original Source: http://www.translational-medicine.com/content/8/1/16


Light Therapy to Stem Cells: A New Therapeutic Approach in Regenerative Medicine – A Mini Review

Amir Oron1 and Uri Oron2* - 15 April 2015 (Publication)
View Resource

 Abstract

Regenerative capacity following injury or an ischemic event is confined to non mammalian vertebrates. Mammals have a limited capacity to restore organs

following injury to organs like the liver and skeletal muscles but practically no ability to regenerate organs like the heart or brain following an ischemic event

or injury. We tried a new approach in cell based therapy to improve regeneration in various organs following ischemic injury. Low-level laser therapy (LLLT)

which has photobiostimulating effects on cells was delivered to autologous bone marrow (BM) that is enriched with stem cells and various progenitor cells, in

order to induce the cells in the BM for the benefit of the injured /ischemic organs. In a model of induced myocardial infarction (MI) in rats laser application

to the BM caused a marked and significant decrease (79%) in infarct size (scarring) 3 weeks post-MI. It was also found that a significantly higher density of

c-kit positive cells (a marker of mesenchymal stem cells) in the myocardium of laser-treated rats relative to non-treated rat’s post-MI. The novel approach

presented in this study, of the use of stem cells for cell therapy to the infracted heart, avoids the need to isolate millions of stem cells, to grow them in vitro and

to inject them back into the patient. In the same line of rationale we tried to find whether LLLT to the BM could be beneficial also to kidney impairment after

ischemic reperfusion injury (IRI) to the rat kidney. C-kit positive cell density in kidneys post-IRI and laser-treatment was significantly (p=0.05) 2.4-fold higher

compared to the non laser treated group. Creatinine, blood urea nitrogen, and cystatin-C levels were significantly lower in the laser-treated rats as compared

to non-treated ones. The effect of LLLT delivery to BM was also tested on Alzheimer’s disease (AD) mice in their late stage of the disease. Mice were given

multiple (every 10 days) LLLT to BM from age 4 to 6 months. It was found that in the treated AD-mice neurological tests (Fear and Cognitive tests) revealed

a significantly (p<0.05) better neurological performance and cognitive capacity compared to the non-treated AD mice. Furthermore, concomitantly with the

improved neurological performance, ß-amyloid density in the hippocampal region of the brains was revealed to be significantly less in the laser-treated mice

as compared to control. In conclusion, a novel approach, of applying LLLT to autologeous BM in order to induce stem cells that are consequently recruited to the

injured/ischemic organ leading to a marked beneficial effect post-ischemic event or degenerative process is presented. This approach is novel in the respect

that it is stimulating the patient’s own abilities to initiate a regenerative response in an organ by the utilization of light. The possibility that this approach can

also be applied to other ischemic/injured organs or organs undergoing degenerative processes (i.e. neurodegenerative diseases), with consequent beneficial

effects, cannot be ruled out

CELL THERAPY FOR INJURED/ISCHEMIC ORGANS

Clinical trials have lately been implemented in a growing

abundance due to the extensive research and new approaches of

cell based therapies for the reconstruction of impaired organs.

Regenerative capacity following injury or an ischemic event

is confined to non mammalian vertebrates. In particular, fish

and primitive amphibians can regenerate organs like the heart,

brain and limbs. However, mammals have a limited capacity

to restore organs following injury to organs like the liver and

skeletal muscles but practically no ability to regenerate organs

like the heart or brain following an ischemic event or injury.

The mammalian heart, including the human heart, for example,

has a very limited capacity to regenerate following damage or

an acute ischemic event like myocardial infarction (MI). This is

due to the very low level of cardiomyocyte proliferation and the

limited number of cells expressing stem-cell marker proteins.

Stem-cell-based therapy was suggested as a potential solution

to the above situation. In recent years, cell-based therapy for

cardiac repair in particular has undergone a rapid transition

from basic science research to clinical reality [1-3]. The general

outcome of the clinical trials was that the procedures and longterm

outcome post-stem-cell implantation to the heart via the

coronary arteries are safe. However, improvement in long-term

functional performance of the heart was either not achieved or

was marginal [1-3].

There are several central issues pertaining to the use of cell

implantation in stem-cell therapy: the number of implanted stem

cells has to be high since there is massive cell death following

implantation or injection of cells into the heart or the blood

circulation. Another central issue in stem-cell implantation for

organ repair is the creation of a receptive cell environment in the

ischemic organ. Several factors (e.g. inhibition of inflammation

and apoptosis, secretion of cell growth factors etc.) are necessary

for optimal cell implantation [4]. The injected cells may have to

migrate from the circulating blood to the ischemic niche. They

can then remain active and secrete growth factors, exerting a paracrine effect on the ischemic tissue [5]. Alternatively, they

may stimulate the small population of stem cells in the ischemic

organ (such as the heart), to proliferate and differentiate so as to

enhance cardiac repair post-MI [6]. Another issue is the timing

of injection of the stem cells to the infarcted heart and effect of

MI (inflammatory phase) on the BM [7]. Photobiostimulation

of cells in the bone marrow (BM), that is enriched with various

progenitor cells, by low level laser therapy (LLLT) may suggest a

new approach that may overcome some of the above limitation.

This new approach will be discussed in the present mini review

[8-11].

LOW LEVEL LASER THERAPY FOR THE ISCHEMIC

HEART

In general LLLT has been found to modulate various biological

processes, such as increasing mitochondrial respiration and ATP

synthesis, facilitating wound healing, and promoting the process

of skeletal muscle regeneration and angiogenesis [12,13]. It

was previously shown that LLLT can enhance skeletal muscle

regeneration following partial excision in the rat hind limb

muscles when the laser was delivered directly to the injured

organ multiple times (for 2 min each time) following injury [14].

This phenomenon was even more prominent following cold

injury to the frog skeletal muscles indicating that enhancement

of regeneration by LLLT is probably a general phenomenon in

vertebrates and maybe more effective in cold blooded animals

which innately have a lower metabolic rate in their cells [13-15].

In an experimental model of the infarcted heart in rats and dogs, it

was demonstrated that LLLT (Diode –Ga-Al-As 810nm at a power

density of 5 mW/cm2 for 120sec duration of laser exposure

comprising 0.6 J/cm2), application directly to the infarcted area

in the heart at optimal power parameters significantly reduces

infarct size (scar tissue formation) [16,17]. This phenomenon

was partially attributed to a significant elevation in ATP content,

heat shock proteins, vascular endothelial growth factor (VEGF),

and angiogenesis in the ischemic zone of the laser-irradiated

rats, as compared to non-irradiated rats [16,17]. The mechanism

associated with the photobiostimulation by LLLT is not yet

clearly understood [12]. There is evidence that cytochrome c

oxidase and perhaps also plasma membranes in cells function as

photoacceptors of the photons, and thereafter a cascade of events

occur in the mitochondria, leading to effects on various processes

like ATP production, up-regulation of VEGF, etc [12].

The effect of photobiostimulation on stem cells or progenitor

cells has not been extensively studied [18-21]. It was previously

shown that laser application (Diode laser at 50mW/cm2 for

100sec, energy density 0.5 mW/cm2) to the mesenchymal stem

cells isolated from bone marrow or cardiac stem cells causes a

significant increase in their proliferation in vitro [20]. Based on

previous studies that showed an increase in cytoprotective effect

on the ischemic heart following LLLT, a new approach was taken

to apply laser irradiation to stem cells grown in culture prior

to their implantation to the infarcted heart as a cell therapy for

heart repair [21]. In that study it was demonstrated that MSCs

that were laser treated prior to their implantation to the rat

infarcted heart caused a significant reduction in infarct size

as compared to MSCs that were injected to the heart without

prior laser treatment. This phenomenon was also associated

with significant elevation of vascular endothelial growth factor

(VEGF) in the myocardium of the rats that received the lasertreated

MSCs. In a recent study [8] the possibility of recruiting

autologous stem cells stimulated by LLLT in the BM to the

infarcted heart was addressed. The rationale behind the attempt

to use LLLT to induce the “crude” BM in the bone was, and still is,

that one cannot significantly affect the complex process post-MI

or ischemic injury to the kidney with a single type of stem cell. The

native BM is known for its many types and subtypes of stem cells,

which are defined by their reactivity to various antibodies. The

BM also contains many progenitor cells (i.e. monocytes) that can

further differentiate, for example to macrophages. Macrophages

have been shown recently to have a crucial role in the scarring

process post-MI. Thus LLLT may induce concomitantly in the BM

various types of cells that will increase in number in the blood

circulation following their enhanced proliferation in the BM.

These cells will probably, eventually, and to a certain extent and

under certain circumstances, home in on the ischemic zone in the

ischemic organ (heart, kidney etc.). In this study [8] it was found

that when LLLT was applied in vivo to the BM, and MSCs were

isolated from that BM 3 and 6 weeks later and grown in vitro, they

grew at a higher rate of proliferation relative to MSCs isolated

from non-laser-treated BM. This indicated that the MSCs when

in the BM, following LLLT application in vivo can be induced to

proliferate to a higher rate than non-treated MSCs. Furthermore,

laser application (Diode laser 808nm at power density of 10mW/

cm2 for 100 sec comprising 1J/cm2 energy density) to the BM (at

about 20 min post-MI) caused a marked and significant decrease

(79%) in infarct size 3 weeks post-MI. This extent of infarct size

reduction was even more effective in reducing scarring than that

of laser application directly to the infarcted heart, as also found

in previous studies with infarcted rat and dog hearts [10]. Even

when laser was applied 4 hours post-MI to the BM of infarcted

rats, a marked and significant reduction in the infarcted area

was observed in the laser-treated rats compared to control.

We also found a significantly higher density of c-kit+ (a marker

of MSCs) cells in the myocardium of laser-treated rats relative

to non-treated rat’s post-MI. Moreover, it was demonstrated in

this study that c-kit+ cells post-laser application to the BM of MIinduced

rats, homed specifically in on the infarcted heart and

not on uninjured organs (i.e. liver, kidney) in the same rat [16].

It can be hypothesized that the increased number of c-kit+ cells

found in the myocardium came from proliferating MSCs in the

BM that had migrated to the circulating blood and homed onto

the infarcted heart. Another finding of this study was that of

the preferred homing of the recruited or endogenous c-kit+ cells

in on the infarcted area, rather than their random deposition

throughout the left ventricle in the heart. Indeed, at 3-weeks

post-MI the density of c-kit+ cells in the infarcted area was 27-

fold higher in the rats whose BM had been treated with LLLT as

compared to control rats. Similarly, Hatzistergos et al. [6] found

that endogenous c-kit+ cardiac stem cells increased by 20-fold

in the porcine infarcted heart as compared to control following

View Resource

Welcome to the laser-therapy.us research tool. This tool is a searchable collection of technical publications, books, videos and other resources about the use of lasers for photobiomodulation. This tool includes almost the entire U.S. library of medicine research papers on LLLT, videos from Youtube associated with therapy lasers and the tables of contents from laser therapy books. This allows users to search for a keyword or condition and see resources about using lasers to treat that condition. All the resources include links to the original source so we are not making any statement about the use of lasers for treating non-FDA cleared application, we are simple summarizing what others have said.  Where every possible, we have included a link to the orginal publication.

Here are some of our favorite queries:

This tool uses a broad match query so:

  • It does not correct spelling and searches only cold laser related subjects so do not use LLLT, cold or laser in the search bar
  • It works better with shorter search terms or even parts of search terms
  • It searches all the available fields so you can enter a body part, author, condition or laser brand.
  • Where ever possible, the detailed section about the resource will link to the sources.
  • This system is only for photobiomodulation or cold laser therapy research (including LLLT, laser acupuncture and high power laser therapy) only. It does NOT include photodynamic laser therapy (where the laser is used to react with a pharmaceutical), hot surgery lasers or cosmetic lasers. It does include some resources on weight loss and smoking cessation.

The results of the search are sorted based on 3 quality factors on a scale of 1 to 10 with 10 being the best score. Originally all the resources were given a 5-5-5 until they could be individually evaluated. These scores are purely opinion and are only used to simplify the rank of the results from more valuable to least valuable. This should not be considered a critique of any work. This system was created to help researchers (including ourselves) find the most usable resources for any cold laser therapy research. The resources are assigned values based on the following 3 factors:

  • Efficacy: The resource (especially research papers) should show a significant improvement in the condition being treated. Resources that show better results are given a higher quality score.
  • Detail: The source must give enough information that the results can be duplicated. If a resource lacks too many details that it cannot be recreated, it is given a lower detail score.
  • Lack of Bias: Many resources are created to try and show that one device is superior to its competition. Many manufacturers have staff that crank out biased papers on a regular basis on the hope that this will make their product look superior. If the author of the resource is paid by a manufacturer of the resource appears to be biased towards one device and not one technology, the resource has much less value.

Over the past few years of working with research, we found that a majority of the published resources are lacking in one of these three ranking factors.
The original goal of this research tool was to tie published resources to the protocols in the laser-therapy.us library. This connection allows users to trace each protocol back to a list of resources so the protocol can be researched and improved.

General Comments


POWER
When many of the first research papers were published, the most power laser available for therapy were less than 100mW and many systems had to be pulsed to keep the laser from burning out too quickly. Today, system are available that will deliver up to 60,000mW of continuous output. Because of these power limitation, many early studies were limited to extremely low dosages by today’s standards. It takes a 50mW system 17 minutes to deliver 50 joules at the surface of the skin. If this was spread over a large area of damage or was treating a deeper problem, the actual dosages were much less than 1J/cm2.  Today, we know that these dosages typically produce very little or no results.
WAVELENGTH
About 80% of the resources in this database are in the near infrared wavelength. There is also some interest in the red wavelength (600 to 660nm) . Other wavelengths like blue, purple, and green have very little scientific research behind them and have not gotten much traction in the core therapy market with the exception of some fringe consumer products.
Legal Disclaimer
This research tool is free to use but we make no claims about the accuracy of the information. It is an aggregation of existing published resources and it is up to the user to determine if the source of the resources has any value. The information provided through this web site should not be used for diagnosing or treating a health problem or disease. If you have or suspect you may have a health problem, you should consult your local health care provider.



The query result(s) can be shared using the following direct link. Anyone who clicks on this link in an email or on a web site will be shown the current results for the query.
https://www.laser-therapy.us/research/index.cfm?researchinput=stemcell