Angiogenesis

PLoS One. 2015; 10(6): e0122776.
Published online 2015 Jun 11. doi:  10.1371/journal.pone.0122776
PMCID: PMC4465903

Enhancement of Ischemic Wound Healing by Spheroid Grafting of Human Adipose-Derived Stem Cells Treated with Low-Level Light Irradiation

In-Su Park,1 Phil-Sang Chung,1,2 and Jin Chul Ahn1,3,4,*
Michael Hamblin, Academic Editor
1Beckman Laser Institute Korea, Dankook University, 119 Dandae-ro, Cheonan, Chungnam, 330–714, Korea
2Department of Otolaryngology-Head and Neck Surgery, College of Medicine, Dankook University, 119 Dandae-ro, Cheonan, Chungnam, 330–714, Korea
3Department of Biomedical Science, Dankook University, Cheonan, Chungnam, 330–714, Korea
4Biomedical Translational Research Institute, Dankook University, Cheonan, Chungnam, 330–714, Korea
Massachusetts General Hospital, UNITED STATES
Competing Interests: The authors have declared that no competing interests exist.

Conceived and designed the experiments: ISP PSC JCA. Performed the experiments: ISP. Analyzed the data: ISP JCA. Contributed reagents/materials/analysis tools: ISP PSC JCA. Wrote the paper: ISP.

Author information ? Article notes ? Copyright and License information ?
Received 2014 Oct 14; Accepted 2015 Feb 12.

Abstract

We investigated whether low-level light irradiation prior to transplantation of adipose-derived stromal cell (ASC) spheroids in an animal skin wound model stimulated angiogenesis and tissue regeneration to improve functional recovery of skin tissue. The spheroid, composed of hASCs, was irradiated with low-level light and expressed angiogenic factors, including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (FGF), and hepatocyte growth factor (HGF). Immunochemical staining analysis revealed that the spheroid of the hASCs was CD31+, KDR+, and CD34+. On the other hand, monolayer-cultured hASCs were negative for these markers. PBS, human adipose tissue-derived stromal cells, and the ASC spheroid were transplanted into a wound bed in athymic mice to evaluate the therapeutic effects of the ASC spheroid in vivo. The ASC spheroid transplanted into the wound bed differentiated into endothelial cells and remained differentiated. The density of vascular formations increased as a result of the angiogenic factors released by the wound bed and enhanced tissue regeneration at the lesion site. These results indicate that the transplantation of the ASC spheroid significantly improved functional recovery relative to both ASC transplantation and PBS treatment. These findings suggest that transplantation of an ASC spheroid treated with low-level light may be an effective form of stem cell therapy for treatment of a wound bed.

Introduction

Formation of new blood vessels, either by angiogenesis or by vasculogenesis, is critical for normal wound healing. Angiogenesis aids in the repair of damaged tissue by regenerating blood vessels and thus improves blood flow in chronic, disease-impaired wounds [1]. To accelerate skin regeneration, many skin tissue engineering techniques have been investigated, including the use of various scaffolds, cells, and growth factors [2]. However, only a subset of the tissue functions can be restored with existing tissue engineering techniques.

Human adipose-derived mesenchymal stem cells (hASCs), which are found in adipose tissue, provide an attractive source of cell therapy for regeneration of damaged skin because they are able to self-renew and are capable of differentiating into various cells [3, 4]. Recent clinical trials involving stem cell therapy aimed to increase vascularization to a sufficient level for wound perfusion and healing [5]. However, several studies claim that the effects of stem cell therapy are not significant in the absence of scaffolds or stimulators [6]. Recently, various scaffolds or growth factors have been studied to increase skin regeneration when using stem cells [7].

Low-level light irradiation (LLLI) has been implemented for various purposes for some time, such as to provide pain relief, to reduce inflammation, and to improve local circulation. Moreover, many studies have demonstrated that LLLI has positive biostimulatory effects on stem cells [8]. For example, LLLT can positively affect hASCs by increasing cellular viability, proliferation and migration [9, 10]; LLLI also enhances vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) secretion [8]; and Low-level light therapy (LLLT) enhanced tissue healing by stimulating angiogenesis in various animal models of ischemia [11]. Hypoxic preconditioning results have been reported in enhanced survival of human mesenchymal stem cells [12]. Since cells within a spheroid are naturally exposed to mild hypoxia, they are naturally preconditioned to an ischemic environment [13]. In ischemia models, spheroids of stem cells present improved therapeutic efficacy via enhanced cell viability and paracrine effects [14]. Hypoxia stimulates the production of growth factors, such as VEGF that induce angiogenesis and endothelial cell (EC) survival [13]. In two-dimensional cultures, growth factors secreted from cells are released and diluted into the culture supernatant, preventing cells from responding to the released factors [14].

Several experimental strategies for endothelial differentiation of stem cells have been developed, including 2D-cell culture in EC growth medium containing VEGF and FGF, 3D spheroid culture on substrates with immobilized polypeptides, and genetic modification of stem cells [12, 15, 16]. However, no reports have yet been produced discussing high-ratio EC differentiation of hASCs in 3D-cultured stem cells without growth factors and peptides.

In this study, LLLI was used to promote a hypoxic spheroid of hASCs (which we refer to as a ‘spheroid’) by weakening cell-matrix adhesion. Differentiation and secretion of FGF and VEGF growth factors were also enhanced by LLLI. hASCs can differentiate into ECs without EC growth medium containing VEGF and FGF. The vascularization and potential therapeutic efficacy of ASC spheroids treated with LLLI (L-spheroid) were evaluated by injecting spheroids into a mouse excisional wound splinting model.

Materials and Methods

Culture of ASCs

The hASCs were supplied by Cell Engineering for Origin, CEFO (Seoul, Korea) under a material transfer agreement. hASCs were isolated from the adipose tissue and were cultured in low-glucose Dulbecco’s modified Eagle’s medium F-12 (DMEM/F-12; Welgene, Daegu, Korea) supplemented with 10% fetal bovine serum (FBS, Welgene), 100 units/ml penicillin, and 100 ?g/ml streptomycin at 37°C in a 5% CO2 incubator. The hASCs between passage 5 and 8 were used for all experiments.

Spheroid formation

hASCs were split and seeded on 24 well polystyrene plate (low cell binding surface) at a density of 7.5 × 104cells/cm2,andallowedtoadhereat37°C. Within 3 days of culture, hASCs formed spheroids by Low-Level light irradiation (L-spheroids). The light source used was LED (light emitting diode; WON Technology Co., Ltd., korea) designed to fit over a standard multi-well plate (12.5 × 8.5 cm) for cell culture. The LED was had an emission wavelength peaked at 660 nm. The irradiance at the surface of the cell monolayer was measured by a power meter (Orion, Ophir Optronics Ltd., UT). To obtain the energy dose of 6 J/cm2,exposure time for LED array was 10 min under power density of 10 mW/cm2 (1 milliwatt × second = 0.001 joules) (Table 1). L-spheroid sizes were measured by counting the area of individual cell clusters by image analysis. The diameters of L-spheroids were presented as median ± SD (n = 8 per group).

Table 1

Summary table of parameters for the light irradiation.

Cells viability assay

After 3 days of culture, the cell viability of the spheroids was analyzed by using a live/dead viability cytotoxicity assay kit (Molecular probes, Carlsbad, CA). Briefly, 1 ml of HEPES-buffered saline solution (HBSS) containing 2 ?l of SYTO 10 green fluorescent nucleic acid stain solution and 2 ?l of red (ethidium homodimer-2) nucleic acid stain solution were added to plates, and these were then incubated at 37°C in a 5% CO2 incubator for 15 min. The negative control was prepared by freezing cells at -80°C for 30 min. Images were quantified by using the ImageJ software (NIH, Bethesda, MD), and the percentage of live/dead cells was scored by counting pixels in each image.

Fluorescence-activated cell sorting (FACS)

The cells were washed with phosphate buffered saline (PBS) containing 0.5% bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO) and were stained in PBS containing 1% BSA, with either isotype controls or antigen specific antibodies, for 60 min. CD34 (BD Biosciences, San Jose, CA), KDR (Beckman Coulter, Brea, CA), CD31 (Beckman Coulter), CD45 (Abcam, Cambridge, MA), CD90 (BD Biosciences), CD105 (Caltac Laboratories, Burlingham, CA), and CD29 (Millipore, Waltham, MA) human antibodies were used. The cells were washed three times with PBS containing 0.5% BSA and were resuspended in PBS for flow cytometry using an Accuri device (BD Biosciences). The isotype IgG was used as a negative control.

Human angiogenic protein analysis

To analyze the expression profiles of angiogenesis-related proteins, we used a Human Angiogenesis Array Kit (R&D Systems, Ltd., Abingdon, UK). Cell samples (5 × 106 cells) were harvested, and 150 ?g of protein were mixed with 15 ?l of biotinylated detection antibodies. After pre-treatment, the cocktail was incubated with the array overnight at 4°C on a rocking platform. Following washing to remove unbound material, streptavidin–horseradish and chemiluminescent detection reagents are added sequentially. The signals on the membrane film were detected by scanning on an image reader LAS-3000 (Kodak, Rochester, NY) and were quantified using the MultiGauge 4.0 software (Kodak). The positive signals seen on developed film were identified by placing a transparency overlay on the array image and aligning it with the two pairs of positive control spots in the corners of each array.

ELISA assay for angiogenic growth factor production

Angiogenic growth factor production in the spheroid was assayed with a commercially available ELISA kit (R&D Systems) according to the manufacturer’s protocols. The concentrations are expressed as the amount of angiogenic growth factor per 104 cells at a given time.

Immunofluorescence staining

Indirect immunofluorescence staining was performed using a standard procedure. In brief, tissues cryosectioned at a 4-?m thickness were fixed with 4% paraformaldehyde, blocked with 5% BSA/PBS (1 h, 24°C), washed twice with PBS, treated with 0.1% Triton X-100/PBS for 1 min, and washed extensively in PBS. The sections were stained with specific primary antibodies and fluorescent-conjugated secondary antibodies (Table 2) using a M.O.M kit according to the manufacturer’s instructions (Vector Laboratories, Burlingame, CA). The cells were counterstained with DAPI (4,6-diamino-2-phenylindole dihydrochloride; Vector Laboratories). Mouse IgG (Dako, Carpinteria, CA) and rabbit IgG (Dako) antibodies was used as negative controls. To detect transplanted human cells, sections were immunofluorescently stained with anti-human nuclear antigen (HNA, Millipore). The stained sections were viewed with a DXM1200F fluorescence microscope (Nikon, Tokyo, Japan). The processed images were analyzed for fluorescence intensity using the ImageJ software (NIH).

Table 2

List of antibodies for immunofluorescence staining.

Histological staining

Samples were harvested 14 days after treatment. Specimens were fixed in 10% (v/v) buffered formaldehyde, dehydrated in a graded ethanol series, and embedded in paraffin. Specimens were sliced into 4 ?m-thick sections and were stained with hematoxylin and eosin (H&E) to examine muscle degeneration and tissue inflammation. Masson’s trichrome collagen staining was performed to assess tissue fibrosisin ischemic regions. The criteria used for the histological scores of wound healing were modified from previous reports [17] and are summarized in Table 3. The histological parameters considered were reepithelialization, dermal regeneration, granulation tissue formation, and angiogenesis. Regeneration of skin appendages was assessed by counting the number of hair follicles or sebaceous glands in the wound bed.

Table 3

Histological scoring system.

Western blot analysis

Samples were solubilized in lysis buffer [20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium dodecyl sulfate (SDS), 1 mM phenylmethylsulfonyl fluoride, 1?g/ml leupeptin, and 2 ?g/ml aprotinin] for 1 h at 4°C. The lysates were then clarified by centrifugation at 15,000 g for 30 min at 4°C, were diluted in Laemmli sample buffer containing 2% SDS and 5% (v/v) 2-mercaptoethanol, and were heated for 5 min at 90°C. The proteins were separated via SDS polyacrylamide gel electrophoresis (PAGE) using 10% or 15% resolving gels followed by transfer to nitrocellulose membranes (Bio-Rad, Hercules, CA) and then probed with antibodies against HIF-1a (Novus), CD31 (Abcam), HGF (Santa cruz), VEGF (Abcam), and FGF2 (Abcam) for 1h at room temperature (Table 2). Peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG and enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ) were used as described by the manufacturer for detection. The membranes were scanned to create chemiluminescent images that were then quantified with an image analyzer (Kodak).

Preparation of the experimental animal model

The animal studies were approved by the Dankook University Animal Use and Care Committee. Five-week-old male BALB/c nude mice (20 g body weight; Narabio, Seoul, Korea) were anesthetized with ketamine (100 mg/kg). After aseptically preparing the surgical site, two full-thickness skin wounds were created on the dorsal part using an 8-mm biopsy punch. To inhibit wound contraction, a 0.5-mm thickness silicone splint was applied, as has been previously described [18]. The splint was fixed with instant adhesive and six simple, interrupted sutures around the wound with Nylon 6–0. The wounds were randomly classified into five groups: control (n = 9), LLLT (n = 9), ASCs (15 × 105 cells; hASCs group, n = 9), spheroid (10 masses; spheroids group, n = 9), and spheroid + LLLT (10 masses; spheroids + LLLT group, n = 9). In the ASCs, spheroid, and spheroid + LLLT groups, 15 × 105 ASCs in 100 ?l of PBS were transplanted intradermally at four injection sites on the border between the wound and the normal skin. The control group received a PBS injection of PBS (PBS group, n = 9). The physiological status of the wound was followed up for up to 2 weeks after treatment. Tegaderm (3M Health Care, MN, USA) was used for wound protection, and an equivalent number of cells were injected in both conditions.

Low-level light therapy in skin

Light emitting diode (LED; WON Technology, Daejeon, Korea) was applied for 10 min daily from day 1 to 20. The distance from the LED to the skin flap was 8 cm. This LED model exhibited an irradiated wavelength of 660 nm and power density of 50 mW/cm2. The fluence of each flap site was 30 J/cm2 (1 milliwatt × second = 0.001 joules) (Table 1).

Gross evaluation of the wound area

The wounds were photographed using a digital camera at 3, 7, and 14 days after surgery, and the wound area was measured by tracing the wound margin and then performing the calculation using the Image J image analysis program (NIH, MD, USA). The wound area was analyzed by calculating the percentage of the current wound with respect to the original wound area. The wound was considered to be completely closed when the wound area was grossly equal to zero.

Statistical analyses

All quantitative results were obtained from triplicate samples. Data were expressed as a mean ± SD, and the statistical analyses were carried out using two-sample t tests to compared two groups of samples and a One-way Analysis of Variance (ANOVA) for the three groups. A value of p < 0.05 was considered to be statistically significant.

Results

Characterization of hASCs

hASCs obtained from human adipose tissue were expanded in vitro. The cells were positive for human MSC markers CD29 (?1 integrin), CD90 (Thy-1) and CD105 (endoglin). However, the cells were found to be negative for human endothelial cell markers CD34, CD31, and KDR (VEGF receptor) through immunofluorescent staining and flow cytometry analyses (S1A and S1B Fig). These results indicated that the expanded cells included a large population of hASCs and were not contaminated with endothelial cells (EC).

Effect of LLLI on the migration and survival of hASCs

A wound scratch test showed that the migration of LLLI-treated hASCs markedly improved relative to that of control cells cultured without LLLI after 24 hours (S2 Fig). There were statistically significant differences between the experimental and the control groups (p < 0.05). To verify the cell viability of the L-spheroid, a live/dead assay with of fluorescent dyes was carried out (S3 Fig). Non-viable cells were stained red, and viable cells were stained green. Apoptosis induced by a lack of cell-matrix interaction (anoikis) was prevented in hASCs cultured as L-spheroids.

Production of Angiogenic Factors by hASCs in L-spheroids hASCs

hASCs were cultured on non-tissue culture-treated 24-well plates in the presence of FBS and formed a floating spheroid after irradiation with low-level light (Fig 1A) 3 days after seeding. The diameter of most L-spheroids ranged from 1.2 to 1.5 mm (Fig 1B). L-spheroid cultures showed a dramatic increase in the expression of hypoxia-induced survival factors, such as hypoxia-inducible factor (HIF)-1?, relative to cells in a monolayer culture (Fig 1C). Therefore, L-spheroid hASCs seemed to be more adaptable and more resistant to hypoxia compared to hASCs in monolayer cultures. HIF-1? is known to upregulate the expression of angiogenic growth factors [19, 20], and L-spheroid hASCs showed considerable expression of angiogenic growth factors, i.e., hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), and fibroblast growth factor 2 (FGF2). The expression of angiogenic growth factors in L-spheroid hASCs was much greater than that of hASCs cultured in a monolayer culture (Fig 1D and 1E).

Fig 1

Enhanced expression of hypoxia-induced survival factors and angiogenic growth factors in hASC L-spheroids.

Endothelial Cell Differentiation of hASC L-spheroid hASCs

The endothelial phenotype of the L-spheroid cells was also evaluated via immunofluorescent staining for a variety of endothelial cell surface markers. First, the surface markers of hASCs expanded in DMEM-F12/FBS were examined. The cells expressed CD29 (?1 integrin), CD90 (Thy-1), and CD105 (endoglin), as well as MSC surface antigens, but not CD34, CD31, or KDR (S2B and S2C Fig). This indicated that the cells used in the study included a large population of hASCs without endothelial lineage cell contamination. Immunofluorescence staining also revealed that L-spheroids expressed a variety of EC surface markers, including CD34, CD31, and KDR (VEGF receptor) (Fig 1B). The cell population of the L-spheroids was further characterized via flow cytometry. In a monolayer culture with LLLI, EC markers (CD34, CD31, and KDR) were detected in less than 1% of cells. Conversely, L-spheroids were composed of a population of cells positive for CD34, CD31, and KDR (Fig 2B).

Fig 2

Endothelial phenotyping of L-spheroids.

Survival of ASCs in the wound bed

After 14 days, fluorescence microscopy was used to identify caspase 3-positive cells and HNA-positive cells throughout the wound bed to determine whether locally transplanted ASCs were incorporated into the healing wound. In the L-spheroid and L-spheroid + LLLT groups, ASCs were observed in the regenerated skin tissue (Fig 3A). The L-spheroid + LLLT group exhibited significantly increased numbers of HNA-positive cells (ASC group: 11%; L-spheroid group: 46%; L-spheroid + LLLT group: 51% per DAPI-positive cells) (Fig 3B) and decreased proportions of caspase 3-positive ASCs (ASC group: 36%; L-spheroid group: 12%; L-spheroid + LLLT group: 9% per HNA-positive cells) (Fig 3C). The HNA+ cell per DAPI+ cell ratio of the L-spheroid + LLLT group was 4.6 times higher than that of the hASC group.

Fig 3

Survival of transplanted hASCs in the wound bed.

Enhanced secretion of growth factors from grafted hASCs in the wound bed

Transplantation of hASCs into the wound bed enhanced the paracrine secretion of angiogenic growth factors. Double immunofluorescent staining of HNA and human angiogenic growth factors bFGF, VEGF, and HGF indicated the presence of secretion from transplanted hASCs in the ASC or spheroid group (Fig 4A). Secreted human growth factors were mainly distributed in the vicinity of transplanted hASCs (HNA-positive cells), and as compared to the ASC group, more growth factor-positive ASCs were observed in the L-spheroid + LLLT group (Fig 4A). A Western blot assay showed that significantly higher levels of VEGF, bFGF, and HGF were secreted by the L-spheroid and L-spheroid + LLLT groups than by the control group, and greater amounts of growth factors were observed in the L-spheroid + LLLT group than in the ASC group (Fig 4B). However, no significant difference was observed between the ASC-treated tissues and the control tissues, indicating that the L-spheroid groups were more effective than the ASC group at increasing transplanted cell retention and angiogenic growth factor expression.

Fig 4

Enhanced secretion of angiogenic growth factors from hASCs in the wound bed.

Angiogenic efficacy in the wound bed

Many of the CD31+ cells in the L-spheroid + LLLT group were double stained for smooth muscle actin (SMA). ECs and perivascular cells differentiated from injected human cells were detected via ?SMA and human CD31 antibodies, respectively (Fig 5A and 5B). A Western blot assay presented significantly higher levels of CD31 secreted by the L-spheroid and L-spheroid + LLLT groups than by the control group (Fig 5C and 5D) and greater amounts of growth factors in the L-spheroid + LLLT group than in the ASC group (Fig 4D). However, there was no significant difference between the ASC-treated tissues and the control tissues. These findings suggest the greater effectiveness of the L-spheroid + LLLT treatment for angiogenesis in the wound bed.

Fig 5

Endothelial cell and smooth muscle cell differentiation of transplanted cells.

Differentiation of ASCs into epithelial cells

To determine whether the L-spheroid ASCs could contribute to the epidermal structure, immunohistochemistry for pan-cytokeratin was performed at 14 days (Fig 6). Some cytokeratin-positive ASCs were found in the epidermis or the sebaceous glands in the spheroid and L-spheroid + LLLT group.

Fig 6

Differentiation of ASCs into epithelial cells.

Wound closure and dermal reaction

An excisional wound splinting model was prepared, and the silicon splints remained tightly adherent to the skin and restricted wound contraction during the experimental period (Fig 7A). At 7 and 14 days after the surgery, the L-spheroid and L-spheroid + LLLT groups exhibited significantly smaller wound areas than did the other groups. At 7 days, the L-spheroid + LLLT group showed a significantly smaller wound area than the ASCs and the L-spheroid groups did. No significant difference was observed between control and LLLT group at any time (Fig 7B and 7C). At 14 days, all of the wounds of the L-spheroid and L-spheroid + LLLT groups achieved complete closure, but not all of the wounds of the control and LLLT groups had completely closed. The histological observation showed that skin regeneration was much greater in the L-spheroid and L-spheroid + LLLT groups compared to the control group. Our data indicated that the L-spheroid enhanced re-epithelialization and granulation at 14 days (Fig 7D). Furthermore, the L-spheroid groups appeared to have an increased number of skin appendages (Fig 7F and 7G). The L-spheroid groups displayed significantly increased numbers of hair follicles and sebaceous glands 14 days (Fig 7D–7G).

Fig 7

Evaluation of the wound closure.

Discussion

The formation of spheroids is affected by the cell-matrix adhesion strength [21]. Moreover, LLLI can promote the migration of hASCs [9, 22, 23]. In this study, within 3 days of culture on non–tissue culture–treated 24-well plates, hASCs formed spheroids as a result of low-level light irradiation (L-spheroids) (Fig 1A). Notably, an increased HIF-1? expression and the consequent induction of protein for VEGF and FGF occurred at a fluence of 660 nm. It had been previously reported that hypoxia mediates the angiogenic switch in agglomerates of tumor cells larger than 200 ?m in diameter [12]. To confirm that a hypoxic environment developed in the spheroid cultures of the hASCs, we detected protein expression of HIF-1? (Fig 1B). HIF-1? expression is primarily induced by hypoxia, but its induction can also be mediated by growth factors and cytokines [9, 22, 23]. This protein is stabilized at low oxygen tensions while at higher oxygen tensions it is rapidly degraded by oxygen-dependent prolyl hydroxylase enzymes. HIF-1? regulates the cellular response to physiological and pathological hypoxia by activating genes that are important to cellular adaptation and survival pathways under hypoxic conditions [12, 24, 25]. In our experimental model, we observed that the non-irradiated group expresses HIF-1? in response to the hypoxic environment formed in the hASC spheroid. In the irradiated groups, 660-nm light alone is able to increase HIF-1? expression in this model regardless of the fluence used (Fig 1B). In this case, induction does not depend on oxygen tension and involves the activation of a different regulatory mechanism, possibly mediated by mitogen-activated protein kinase and the phosphatidylinositol 3-kinase/Akt signaling pathway [26]. Oxidative stress can also increase the expression of this transcription factor in the spheroid. Numerous studies have reported that hypoxia induces the production of growth factors correlated with endothelial cell growth and function [27]. In practice, hASCs can differentiate in vitro into functional endothelial cells in the presence of angiogenic factors such as 50 ng/mlVEGF [28]. Therefore, it is expected that the endothelial differentiation of hASCs in the spheroid might be up-regulated by angiogenic factors, such as VEGF (Fig 1C and 1D). In two-dimensional cultures, growth factors secreted from the cells are released and diluted in the culture supernatant, preventing cells from responding to the released factors. Conversely, in L-spheroid cultures, if growth factors are secreted from stem cells after 3D cell aggregation, the factors might be stored in the cell spheroid and may then stimulate endothelial differentiation of the stem cells (Fig 2).

Clinical studies of stem cell transplantation have raised several questions concerning cell therapy. The density and complexity of vascular networks formed by the synergistic dual cell system were reported to be was many times greater than those observed with ASC- containing and EC- or SMC-containing implants [29]. In addition, gel-assisted subcutaneous injection of VEGF- and bFGF-expressing ECs formed mature vasculature, whereas those expressing VEGF did not form mature vasculature. In this study, L-spheroid ASCs accelerated wound closure with an increased level of re-epithelialization, neovascularization, and regeneration of skin appendages. This is likely to be due to the enhanced survival of L-spheroid hASCs along with increased paracrine secretion (Fig 3). Our data revealed the presence of an increased number of ASCs and a decreased percentage of caspase 3-positive ASCs at 14 days in the L-spheroid group relative to the ASCs group. These data suggest that LLLI enhanced the survival of the spheroid ASCs by inhibiting apoptosis. In addition, VEGF, bFGF, and HGF positive-ASCs were detected in the wound bed (Fig 4). VEGF is the most effective and specific growth factor that regulates angiogenesis [30]; bFGF is an important growth factor in wound healing because it affects the migration and proliferation of fibroblasts, angiogenesis, and matrix deposition [31]; and HGF is another potent proangiogenic factor that induces migration and proliferation and inhibits apoptotic cell death of ECs [32]. The L-spheroid complex appeared to promote vasculogenesis through a synergistic effect in the wound bed (Fig 5). It is possible that LLLI enhances cellular responses in terms of gene expression, secretion of growth factors, and cell proliferation through an increase in the mitochondrial membrane potential and the ATP and cAMP levels [26]. In addition to the sebaceous glands, some cytokeratin positive-ASCs were observed in the regenerated epidermis (Fig 6). Several recent studies that reported ASCs to enhance wound repair as a result of differentiation and of their paracrine effects are consistent with the results of our study. For example, Smith et al (2010) reported that MSCs migrate into the wound area [33] and differentiate into keratinocytes, endothelial cells, sweat glands, sebaceous glands, and hair follicles [17, 34]. Additionally, Wu et al (2007) have shown that MSCs secrete paracrine factors, such as VEGF, bFGF, epidermal growth factor, keratinocyte growth factor, insulin-like growth factor, and hepatocyte growth factor, and stimulate the deposition of extracellular matrix [17, 35]. In this study, the L-spheroid groups were found to exhibit rapid wound closure and a higher histological score relative to the ASCs group (Fig 7). In skin bioengineering, the ultimate goal is to rapidly produce a construct that offers complete restoration of functional skin, ideally involving the regeneration of all skin appendages and layers [2]. Interestingly, our results showed that the L-spheroid groups present significantly increased numbers of sebaceous glands than the ASCs group. These results suggest that L-spheroid ASCs enhanced not only survival, but also the functionality of the transplanted ASCs in the wound bed.

Conclusions

hASC L-spheroids transplantation accelerates tissue regeneration through the differentiation of ECs and through growth factor secretion. We emphasize the significance of the application of a 3D spheroid culture of stem cells with LLLI to achieve a high-ratio of EC differentiation of hASCs and to enhance treatment efficiency of L-spheroid transplantation relative to single-cell transplantation in the wound bed. These results may provide more effective therapeutic methods to treat delayed skin regeneration.

 

Supporting Information

S1 Fig

Immunofluorescence staining and flow cytometry analyses of hASCs.

hASCs (passage 4) were stained with CD29, CD90 and CD105 for mesenchymal stem cell identification, with KDR, CD31 and CD34 for endothelial lineage cell identification, and SMA for smooth muscle cell identification. Scale bar: 200 ?m (B) Flow cytometry analysis; hASCs cultured for 1 days were stained for CD29, CD90, CD105, CD45, CD31, CD34 and KDR expression and analyzed by flow cytometry.

(TIF)

S2 Fig

Migration of BMSCs by wound scratch test.

LLLI treated hASCs scratch wound at 24 h (*, p < 0.05, compared with LLLT group, t-test, n = 3 in each group).

(TIF)

S3 Fig

Fluorescence microscopic image of Live/Dead stain on day 1.

The middle-section was 500 ?m from the L-spheroid surface. Live cells were stained by calcein AM (green), and dead ones were stained with ethidium homodimer (red). Scale bar: 500 ?m.

(TIF)

Funding Statement

This study was supported by a grant of the Ministry of Science, ICT and Future Planning grant funded by the Korea government (2012K1A4A3053142, NRF-2014R1A1A1038199). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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J Dent (Tehran). 2014 May; 11(3): 319–327.
Published online 2014 May 31.
PMCID: PMC4290760

Effect of Low-Level Laser on Healing of Temporomandibular Joint Osteoarthritis in Rats

Ali Peimani1 and Farimah Sardary2
1Assistant Professor, Department of Oral Surgery, Dental School of Rafsanjan University, Rafsanjan, Iran
2Assistant Professor, Department of Oral Medicine, Dental School of Rafsanjan University, Rafsanjan, Iran
Corresponding author: F. Sardary, Department of Oral Medicine, Dental School of Rafsanjan University, Rafsanjan, Iran, moc.oohay@hamiraf_iradrasrd
Author information ? Article notes ? Copyright and License information ?
Received 2013 Dec 28; Accepted 2014 Mar 23.

Abstract

Objective:

Temporomandibular disorders (TMD) are clinical conditions characterized by pain and sounds of the temporomandibular joint (TMJ). This study was designed to assess the effect of low-level laser therapy (LLLT) on healing of osteoarthritis in rats with TMD.

Materials and Methods:

Thirty-two male Wistar rats (250–200 g) were housed in standard plastic cages. After injection of Complete Freund’s adjuvant into the TMJ, rats were randomly divided into two groups of 16 (case and control) and anesthetized; then osteoarthritis was induced via intraarticular injection of 50 µl of Complete Freund’s adjuvant; into the bilateral TMJs. In the case group, LLLT was done transcutaneously for 10 minutes daily, starting the day after the confirmation of osteoarthritis. Exposure was performed for 10 minutes at the right side of the TMJ with 880 nm low-level laser with 100 mW power and a probe diameter of 0.8 mm. Control rats were not treated with laser.

Results:

After three days of treatment the grade of cartilage defects, number of inflammatory cells, angiogenesis, number of cell layers and arthritis in rats in the case group were not significantly different compared with controls (P>0.05). After seven days, the grade of cartilage defects, number of inflammatory cells, number of cell layers, and arthritis in the case group improved compared to controls (P<0.05); angiogenesis in both groups was similar.

Conclusion:

Treatment of TMD with LLLT after 7 days of irradiation with a wavelength of 880 nm was associated with a greater improvement compared to the control group.

Keywords: TMJ disorders, laser therapy, osteoarthritis

INTRODUCTION

Temporomandibular disorders are a varied set of clinical conditions characterized by pain in the temporomandibular joint (TMJ) and/or the masticatory muscles. In the body, the TMJ is a synovial, bilateral joint with unique morphology and function, and a stress-sensitive cartilage that is subject to extensive tissue remodeling [13]. Associations between developments of osteoarthritis-like, degenerative changes of articular cartilage and common dysfunction of TMJ have been reported previously. Commonly, progressive and more degenerative processes, with unknown cause in most cases, occur after osteoarthritic changes in the TMJ during life [48].

Pain relief and functional recovery can be achieved by inhibiting the factors causing cartilage deformity as much as possible, since they result in cartilage loss and articular deformity. Use of non-steroidal anti-inflammatory drugs (NSAIDs), synovectomy, steroids and immunosuppressants as treatment methods for artificially induced osteoarthritis have been studied [913]. Development of drugs or treatment methods that are not harmful seems necessary because of the complications and side effects of most of the existing methods [14]. Low-level laser therapy (LLLT) is a treatment approach with a wide range of applications and with biomodulative and analgesic purposes. In several studies, LLLT was used for the treatment of soft tissue injuries, rheumatoid arthritis, musculoskeletal pain and dental problems. Though controversy was observed in its efficacy, positive clinical results have been reported [1417]. Some studies evaluated the use of LLLT for treatment of atherosclerosis, non-healing ulcers, and various degenerative conditions [1820]. Also, augmentation of heat shock proteins and pathophysiological improvement of arthritic cartilage resulted in an osteoarthritis model for treatment with LLL [21]. Dental and periodontal treatment applications of LLLT have been the subject of many in vivo and in vitro studies; and due to its ability to expedite the healing process, it has been used after gingivoplasty and gingivectomy [2223]. LLLT for temporomandibular disorders, in spite of the common treatment modes available, has proven capable of relieving pain in minutes after administration, bringing about a significant improvement for the patient [24]. Analgesic effects reported by most authors in the literature were the main reason for the use of LLLT for TMD [2527].

Nonetheless, it has been shown that often laser therapy can be used in lieu of anti-inflammatory medication; thus, preventing side effects [26]. However, LLLT is not the definitive treatment for temporomandibular disorders, regardless of all benefits of laser treatment.

Because of ethical reasons much of the research cannot be done on humans and studies in animal models are used for this purpose. Rats may be used as convenient animals for experimental studies for treatment of TMD, due to the similarity of the TMJ of rats and humans. Therefore, our study was designed to assess the effect of LLLT on healing of osteoarthritis in rats with TMD.

MATERIALS AND METHODS

Thirty-two male Wistar rats (200–250 g) were housed in standard plastic cages with food and water available ad libitum. Ethical review of the animal procedures was obtained from the Institutional Animal Care and Use Committee of Rafsanjan University of Medicine, and all experiments were designed to minimize animal suffering and to use the minimum number of animals required to achieve a valid statistical evaluation. The rats were anesthetized intraperitoneally with a ketamine and xylazine mixture (Figure 1-a). Osteoarthritis was induced with an intraarticular injection of 50 µl of Complete Freund’s adjuvant (CFA), (oil/saline at a ratio of 1:1) into the bilateral TMJs using a 30-gauge needle and 1-mL syringe (Figure 1-b). After injection of CFA into the TMJ, using random-maker software “Random Allocation”, the rats were randomly divided into two groups of 16 (case and control). In the case group, LLLT was done transcutaneously for 10 minutes every day, using a LLL AZOR-2k (Azor Medical Equipment, Moscow, Russia), starting the day after the confirmation of osteoarthritis.

Fig 1.

Osteoarthritis induced by an intraarticular injection of 50 µl of complete Freund’s adjuvant, into the bilateral TMJs in rats

Exposure was performed for 10 minutes at the right side of TMJ with 880 nm low-level laser with 100 mW power and a probe diameter of 0.8 mm (Figure 1-c). In the control group, rats were not treated with laser, and the same procedure was performed, but the probe was turned off. Each rat was kept in one cage and was able to rotate 360 degrees to obtain food and water freely.

The heads of the rats were dissected from euthanized rats on day three (8 rats in the case and 8 rats in the control groups), and on day seven (8 rats in the case and 8 rats in the control groups). The separated heads were fixed in 10% formalin and were then carefully oriented in the paraffin blocks (Figure 1-d). The TMJ was removed and fixed in 4% paraformaldehyde and demineralized in 15% EDTA.

The specimens were dehydrated in graded concentrations of alcohol and xylene, embedded in paraffin, and cut serially into 4 µm sagittal sections.

Next, they were stained with hematoxylineosin. An observer, blinded to the experimental design, in consultation with two pathologists, evaluated histopathological alterations of the joints after standardization of the measurements.

The presence of angiogenesis, grade of cartilage defects, the number of cell layers, the number of inflammatory cells, and arthritis were assessed (Figure 3). Arthritis in rats was confirmed using clinical signs and based on swelling and redness.

Fig 3.

Photomicrographs of the histopathological analysis of TMJ. A: control group, B: case group (H&E staining 100×)

All statistical analyses were performed using SPSS software (version 20; SPSS Inc., Chicago, IL).

The data were presented and statistically significant differences among the groups were compared using the Mann-Whitney U test. P-values less than 0.05 were considered to indicate statistical significance.

RESULTS

Figure 2 shows the algorithm of the study, number of rats, treatment, follow-up and analyses.

Fig 2.

Flowchart of the study

Results of the comparison of the frequencies, grade of cartilage defects, number of inflammatory cells, number of cell layers, arthritis and angiogenesis between the case and the control groups after three days of treatment are shown in Table 1.

Table 1.

Comparison of the frequencies of studied variables between the case and control groups three days after the intervention

As shown, the grade of cartilage defect in all rats in the case group was irregular and superficial erosion, and deep defects in the cartilage were more than in the control group, but differences were not statistically significant (P>0.05).

Inflammatory cells and angiogenesis between the groups were similar (P>0.05).

Half the rats in the control group showed one cell layer; while most rats in the case group showed more than one cell layer; also, arthritis in most rats in the case group was average but in the control group was severe. Differences in the number of cell layers and arthritis between the case and control groups were not statistically significant (P>0.05).

Table 2 shows the the results seven days after the intervention assessing the grade of cartilage defect, number of inflammatory cells, number of cell layers, arthritis and angiogenesis between the case and the control groups. The grade of cartilage, number of inflammatory cells, number of cell layers, and arthritis in the case group improved compared to the controls (P<0.05); but angiogenesis was similar between the case and the control groups after seven days (P=0.05).

Table 2.

Comparison of the frequencies of studied variables between the case and control groups seven days after the intervention

Arthritis in all rats in the case group on day 3 was severe or average but on day seven, arthritis in half of these rats improved while in the control group slight arthritis progressed to severe arthritis. These changes between groups were statistically significant (P=0.005). Also, the grade of cartilage defect, number of inflammatory cells and number of cell layers in the case group improved after increasing the time of treatment but in controls exhibited no change after increasing the treatment time.

DISCUSSION

The data of histological analysis in this study suggest that no improvement was observed in the case group after 3 days of irradiation around the TMJ compared to the controls with regard to the grade of cartilage defect, number of inflammatory cells, number of cell layers, arthritis and angiogenesis. With an increase in the laser irradiation days from 3 days to 7 days, statistically significant improvements in the grade of cartilage defect, number of inflammatory cells and number of cell layers were seen.

Based on our findings, increase in the laser irradiation sessions can be useful for treatment of osteoarthritis in rats with TMD.

The stimulatory effects of 630 nm low level laser irradiation on bone formation in the condylar region during mandibular advancement in rabbits were assessed by Abtahi et al, [28]. They showed that after 3 weeks of irradiation around TMJ, a significant increase in newly formed bone was observed.

Other studies reported an over-arching clinical rationale for use of LLL in conditions such as arthritis. Shen and colleagues assessed the efficacy and safety of 650 nm laser irradiation in 40 patients with knee osteoarthritis which were randomly allocated to an active laser group or to a placebo laser group (20 per group). They showed the advantages of laser treatment in these patients [29]. Also, Ekim et al. [30] evaluated the efficacy of LLLT in patients with rheumatoid arthritis with carpal tunnel syndrome and showed that laser therapy seemed to be effective for pain and hand function and suggested that LLLT may be used as a good alternative for treatment of patients with rheumatoid arthritis and carpal tunnel syndrome [33].

These studies concluded that this treatment is associated with anti-inflammatory effects. In agreement with previous in vitro and in vivo studies, our findings showed that laser irradiation can be useful for treatment of TMD.

Cho et al. [35] reported the induction of osteoarthropathy into both knees of 25 normal rabbits and demonstrated that edema and heat sensation significantly decreased and no inflammatory cells were observed histologically in the groups treated with LLLT, as compared to the control groups. They reported that after 2 weeks, no significant treatment effect was seen, but significant improvement was observed after 4 weeks of treatment. They believed that LLLT should be continued for at least 3 weeks in these patients. Brosseau et al. [36] reported that LLLT is effective for rheumatoid arthritis. Amano et al. [37] reported that LLLT is effective for rheumatoid arthritis due to a direct photochemical effect. Our study differed from the studies mentioned above, as we induced non-inflammatory osteoarthritis. However, unlike our study Brosseau et al. [36] reported that LLLT is not effective in osteoarthritis.

The wavelength and the type of irradiation as well as the time of exposure are key factors for the efficacy of laser therapy, and we think that time of exposure, use of just one wavelength and a narrow spectrum of therapeutic doses should be noted as limitations of this study. Therefore, we suggested further studies in order to examine the efficacy of LLL irradiation in various conditions and time periods.

CONCLUSION

Treatment of TMD with LLLT after 7 days of irradiation with a wavelength of 880 nm was associated with a greater improvement compared to the control group.

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Lasers Med Sci.  2012 Aug 25. [Epub ahead of print]

Laser and LED phototherapies on angiogenesis.

de Sousa AP, Paraguassú GM, Silveira NT, de Souza J, Cangussú MC, Dos Santos JN, Pinheiro AL.

Source

Center of Biophotonics, School of Dentistry, Federal University of Bahia, 62 Araújo Pinho Ave, Canela, Salvador, BA, CEP 40140-110, Brazil, paula_canti@hotmail.com.

Abstract

Angiogenesis is a key process for wound healing. There are few reports of LED phototherapy on angiogenesis, mainly in vivo. The aim of the present investigation was to evaluate histologically the angiogenesis on dorsal cutaneous wounds treated with laser (660 and 790 nm) or LEDs (700, 530, and 460 nm) in a rodent model. Twenty-four young adult male Wistar rats weighting between 200 and 250 g were used on the present study. Under general anesthesia, one excisional wound was created on the dorsum of each animal that were then randomly distributed into six groups with four animals each: G0-control; G1-laser 660 nm (60 mW, 2 mm, 10 J/cm(2)); G2-laser 790 nm (50 mW, 2 mm, 10 J/cm(2)); G3-LED 700±20 nm (15 mW, 16 mm, 10 J/cm(2)); G4-LED 530±20 nm (8 mW, 16 mm, 10 J/cm(2)); G5-LED 460±20 nm (22 mW, 16 mm, 10 J/cm(2)). Irradiation started immediately after surgery and was repeated every other day for 7 days. Animal death occurred at the eighth day after surgery. The specimens were removed, routinely processed to wax, cut and stained with HE. Angiogenesis was scored by blood vessel counting in the wounded area. Quantitative results showed that green LED (530±20 nm), red LED (700±20 nm), 790 nm laser and 660 nm laser caused significant increased angiogenesis when compared to the control group. It is concluded that both laser and LED light are capable of stimulating angiogenesis in vivo on cutaneous wounds and that coherence was not decisive on the outcome of the treatment.

J Transl Med. 2010 Feb 16;8:16.

Lasers, stem cells, and COPD.

 

Lin F, Josephs SF, Alexandrescu DT, Ramos F, Bogin V, Gammill V, Dasanu CA, De Necochea-Campion R, Patel AN, Carrier E, Koos DR.

Entest BioMedical, San Diego, CA, USA.

Abstract

The medical use of low level laser (LLL) irradiation has been occurring for decades, primarily in the area of tissue healing and inflammatory conditions. Despite little mechanistic knowledge, the concept of a non-invasive, non-thermal intervention that has the potential to modulate regenerative processes is worthy of attention when searching for novel methods of augmenting stem cell-based therapies. Here we discuss the use of LLL irradiation as a “photoceutical” for enhancing production of stem cell growth/chemoattractant factors, stimulation of angiogenesis, and directly augmenting proliferation of stem cells. The combination of LLL together with allogeneic and autologous stem cells, as well as post-mobilization directing of stem cells will be discussed.

Photomed Laser Surg. 2009 Apr;27(2):227-33.

Implantation of low-level laser irradiated mesenchymal stem cells into the infarcted rat heart is associated with reduction in infarct size and enhanced angiogenesis.

Tuby H, Maltz L, Oron U.

Department of Zoology, The George S. Wise Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv, Israel.

OBJECTIVE: The aim of the present study was to evaluate the possible beneficial effects of implantation of laser-irradiated mesenchymal stem cells (MSCs) into the infarcted rat heart. BACKGROUND DATA: It was demonstrated that low-level laser therapy (LLLT) upregulates cytoprotective factors in ischemic tissues. MATERIALS AND METHODS: MSCs were isolated from rat bone marrow and grown in culture. The cells were laser irradiated with a Ga-Al-As laser (810 nm wavelength), labeled with 5-bromo-2’deoxyuridine (BrdU), and then implanted into infarcted rat hearts. Non-irradiated cells were similarly labeled and acted as controls. Hearts were excised 3 wk later and cells were stained for BrdU and c-kit immunoreactivity. RESULTS: Infarcted hearts that were implanted with laser-treated cells showed a significant reduction of 53% in infarct size compared to hearts that were implanted with non-laser-treated cells. The hearts implanted with laser-treated cells prior to implantation demonstrated a 5- and 6.3-fold significant increase in cell density that positively immunoreacted to BrdU and c-kit, respectively, as compared to hearts implanted with non-laser-treated cells. A significantly 1.4- and 2-fold higher level of angiogenesis and vascular endothelial growth factor, respectively, were observed in infarcted hearts that were implanted with laser-treated cells compared to non-laser-treated implanted cells. CONCLUSION: The findings of the present study provide the first evidence that LLLT can significantly increase survival and/or proliferation of MSCs post-implantation into the ischemic/infarcted heart, followed by a marked reduction of scarring and enhanced angiogenesis. The mechanisms associated with this phenomenon remain to be elucidated in further studies.

Micron. 2009 Jun;40(4):413-8. Epub 2009 Feb 13.

Ultrastructural analysis of the low level laser therapy effects on the lesioned anterior tibial muscle in the gerbil.

Iyomasa DM, Garavelo I, Iyomasa MM, Watanabe IS, Issa JP.

Department of Physiotherapy, São Paulo State University, Presidente Prudente, SP, Brazil.

Low level laser therapy (LLLT) is known for its positive results but studies on the biological and biomodulator characteristics of the effects produced in the skeletal muscle are still lacking. In this study the effects of two laser dosages, 5 or 10 J/cm(2), on the lesioned tibial muscle were compared. Gerbils previously lesioned by 100 g load impact were divided into three groups: GI (n=5) controls, lesion non-irradiated; GII (n=5), lesion irradiated with 5 J/cm(2) and GIII (n=5), lesion irradiated with 10 J/cm(2), and treated for 7 consecutive days with a laser He-Ne (lambda=633 nm). After intracardiac perfusion, the muscles were dissected and reduced to small fragments, post-fixed in 1% osmium tetroxide, dehydrated in increasing alcohol concentrations, treated with propylene oxide and embedded in Spurr resin at 60 degrees C. Ultrafine cuts examined on a transmission electron microscope (Jeol 1010) revealed in the control GI group a large number of altered muscle fibers with degenerating mitochondria, intercellular substance containing degenerating cell fragments and budding blood capillaries with underdeveloped endothelial cells. However, groups GII and GIII showed muscle fibers with few altered myofibrils, regularly contoured mitochondria, ample intermembrane spaces and dilated mitochondrial crests. The clean intercellular substance showed numerous collagen fibers and capillaries with multiple abluminal processes, intraluminal protrusions and several pinocytic vesicles in endothelial cells. It was concluded that laser dosages of 5 or 10 J/cm(2) delivered by laser He-Ne (lambda=633 nm) during 7 consecutive days increase mitochondrial activity in muscular fibers, activate fibroblasts and macrophages and stimulate angiogenesis, thus suggesting effectivity of laser therapy under these experimental conditions.

Lasers Surg Med. 2006 Aug;38(7):682-8.

Modulations of VEGF and iNOS in the rat heart by low level laser therapy are associated with cardioprotection and enhanced angiogenesis.

Tuby H, Maltz L, Oron U.

Department of Zoology, The George S. Wise Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv, 69978, Israel.

Abstract

BACKGROUND AND OBJECTIVES: It has been shown previously that low-level laser therapy (LLLT) significantly reduces infarct size following induction of myocardial infarction in rats and dogs. The aim of the present study was to investigate the effect of LLLT on the expression of vascular endothelial growth factor (VEGF) and inducible nitric oxide synthase (iNOS). STUDY DESIGN AND

MATERIAL AND METHODS: Myocardial infarction was induced by occlusion of the left descending artery in 87 rats. LLLT was applied to intact and post-infarction. VEGF, iNOS, and angiogenesis were determined.

RESULTS: Both the laser-irradiated rat hearts post-infarction and intact hearts demonstrated a significant increase in VEGF and iNOS expression compared to non-laser-irradiated hearts. LLLT also caused a significant elevation in angiogenesis.

CONCLUSIONS: It is concluded that VEGF and iNOS expression in the infarcted rat heart is markedly upregulated by LLLT and is associated with enhanced angiogenesis and cardioprotection.

Photomed Laser Surg. 2005 Oct;23(5):470-5.

Effect of In-Ga-Al-P diode laser irradiation on angiogenesis in partial ruptures of Achilles tendon in rats.

Salate AC, Barbosa G, Gaspar P, Koeke PU, Parizotto NA, Benze BG, Foschiani D.

Department of Physiotherapy, Federal University of Sao Carlos, Sao Paulo, Brazil.

OBJECTIVE: This study was conducted to analyze the effect of different irradiances of low-level laser therapy (LLLT) on angiogenesis after partial rupture of Achilles tendon of rats. BACKGROUND DATA: METHODS: Ninety-six animals were divided into three groups subject to treatment during 3, 5, and 7 days post-lesion. Thirty-two animals were used in each group. The groups were further divided into four subgroups with eight animals in each, receiving In-Ga-Al-P laser (660 nm) treatment at (1) mean output of 10 mW, (2) 40 mW during 10 sec, (3) a sham subgroup, and (4) a non-treatment subgroup. Each animal was subjected to a lesion of the Achilles tendon by dropping a 186-g weight from a 20-cm height over the tendon. Treatment was initiated 6 h post-injury for all the groups. Blood vessels were colored with India ink injection and were examined in a video microscope. RESULTS: Laser exposure promoted an increase in blood vessel count when compared to controls. The 40-mW group showed early neovascularization, with the greatest number of microvessels after three laser applications. The 10-mW subgroup showed angiogenesis activity around the same time as the sham laser group did, but the net number of vessels was significantly higher in the former than in the controls. After seven irradiations, the subgroup receiving 40 mW experienced a drop in microvessel number, but it was still higher than in the control groups. CONCLUSIONS: LLLT of different intensities seems to promote neovascularization in damaged Achilles tendons of rats after partial rupture compared to controls.

Histol Histopathol. 2004 Jan;19(1):43-8.

The effects of low laser irradiation on angiogenesis in injured rat tibiae.

Garavello I, Baranauskas V, da Cruz-Höfling MA.

Department of Semiconductors Instruments, Institute of Biology, State University of Campinas (UNICAMP), Campinas (SP), Brazil.

Abstract

The influence of He-Ne laser radiation on the formation of new blood vessels in the bone marrow compartment of a regenerating area of the mid-cortical diaphysis of the tibiae of young adult rats was studied. A small hole was surgically made with a dentistry burr in the tibia and the injured area received a daily laser therapy over 7 or 14 days transcutaneously starting 24 h from surgery. Incident energy density dosages of 31.5 and 94.5 Jcm(-2) were applied during the period of the tibia wound healing investigated. Light microscopic examination of histological sections of the injured area and quantification of the newly-formed blood vessels were undertaken. Low-level energy treatment accelerated the deposition of bone matrix and histological characteristics compatible with an active recovery of the injured tissue. He-Ne laser therapy significantly increased the number of blood vessels after 7 days irradiation at an energy density of 94.5 Jcm(-2), but significantly decreased the number of vessels in the 14-day irradiated tibiae, independent of the dosage. These effects were attributed to laser treatment, since no significant increase in blood vessel number was detected between 8 and 15 non-irradiated control tibiae. Molecular mechanisms involved in low-level laser therapy of angiogenesis in post-traumatic bone regeneration needs further investigation.

Antioxid Redox Signal. 2002 Oct;4(5):785-90.

 

Promotion of angiogenesis by low energy laser irradiation.

Mirsky N, Krispel Y, Shoshany Y, Maltz L, Oron U.

The Faculty of Science, Haifa University at Oranim, Tivoon 36006, Israel.

The effect of low energy laser (He-Ne) irradiation (LELI) on the process of angiogenesis in the infarcted rat heart and in the chick chorioallantoic membrane (CAM), as well as the proliferation of endothelial cells in tissue culture, was investigated. Formation of new blood vessels in the infarcted rat heart was monitored by counting proliferating endothelial cells in blood vessels. In the CAM model, defined areas were laser-irradiated or nonirradiated and blood vessel density was recorded in each site in the CAM at various time intervals. Laser irradiation caused a 3.1-fold significant increase in newly formed blood vessels 6 days post infarction, as compared with nonirradiated rats. In the CAM model, a slight inhibition of angiogenesis up to 2 days post irradiation and a significant enhancement of angiogenesis in the laser-irradiated foci as compared with control nonirradiated spots were evident. The LELI caused a 1.8-fold significant increase in the rate of proliferation in endothelial cells in culture over nonirradiated cells. It is concluded that LELI can promote the proliferation of endothelial cells in culture, which may partially explain the augmentation of angiogenesis in the CAM model and in the infarcted heart. These results may have clinical significance by offering therapeutic options to ameliorate angiogenesis in ischemic conditions.

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