Collagen

Lasers Med Sci. 2014 Jul;29(4):1405-10. doi: 10.1007/s10103-014-1537-0. Epub 2014 Feb 20.

Do laser and led phototherapies influence mast cells and myofibroblasts to produce collagen?

De Castro IC1, Rocha CA, Gomes Henriques AC, Cavalcanti de Sousa AP, Lisboa MV, Sotero Dda R, Pinheiro AL, Cury PR, Santos JN.
Author information
1Center of Biophotonics, School of Dentistry, Federal University of Bahia, Av. Araújo Pinho, 62, Canela, Salvador, Bahia, CEP 40110-150, Brazil, isabeledecastro@gmail.com.
Abstract
Laser and LED phototherapies accelerate tissue repair. Mast cells induce the proliferation of fibroblasts and the development of local fibrosis. Increased numbers of myofibroblasts and mast cells are frequently found together in a normal wound repair, suggesting that mediators produced by the mast cells could play a role in the regulation of myofibroblast differentiation and function. The aim of this study was to analyze the involvement of mast cells on the synthesis of collagen and their influence on myofibroblast differentiation in the late phase of tissue repair on wounds treated with LLLT (? 660 nm, 10 J/cm(2), 40 mW, 252 s) or LED (? 630?±?10 nm, 10 J/cm(2), 115 mW, 87 s). A 1?×?1-cm surgical wound was created on the dorsum of 30 rats divided into three groups of ten animals each: control, laser, and LED. The animals of each group were irradiated and sacrificed 7 and 14 days after injury. The statistical analysis was performed using the Mann-Whitney and Spearman correlation tests. Laser light improved the collagen deposition rate along the time points (p?=?0.22), but when compared to the control groups during the periods studied, the number of mast cells decreased significantly (p???0.05). With respect to myofibroblasts, the results showed a trend to their reduction. No statistical significances were observed for LED light according to the parameters used in this study. It is concluded that the mast cell and myofibroblast population might participate in the collagen formation of irradiated wounds particularly in relation to laser phototherapy.
Lasers Med Sci.  2012 Nov 21. [Epub ahead of print]

LLLT improves tendon healing through increase of MMP activity and collagen synthesis.

Guerra FD, Vieira CP, Almeida MS, Oliveira LP, de Aro AA, Pimentel ER.

Source

Department of Anatomy, Cell Biology and Physiology and Biophysics, Institute of Biology, University of Campinas-UNICAMP, CP 6109, Campinas, São Paulo, 13083-970, Brazil, dgflavia@yahoo.com.br.

Abstract

The Achilles tendon has a high incidence of rupture, and the healing process leads to a disorganized extracellular matrix (ECM) with a high rate of injury recurrence. To evaluate the effects of different conditions of low-level laser (LLL) application on partially tenotomized tendons, adult male rats were divided into the following groups: G1, intact; G2, injured; G3, injured + LLL therapy (LLLT; 4 J/cm(2) continuous); G4, injured + LLLT (4 J/cm(2), 20 Hz); G5, injured; G6, injured + LLLT (4 J/cm(2) continuous); and G7, injured + LLLT (4 J/cm(2), 20 Hz until the 7th day and 2 kHz from 8 to 14 days). G2, G3, and G4 were euthanized 8 days after injury, and G5, G6, and G7 were euthanized on the 15th day. The quantification of hydroxyproline (HOPro) and non-collagenous protein (NCP), zymography for matrix metalloproteinase (MMP)-2 and MMP-9, and Western blotting (WB) for collagen types I and III were performed. HOPro levels showed a significant decrease in all groups (except G7) when compared with G1. The NCP level increased in all transected groups. WB for collagen type I showed an increase in G4 and G7. For collagen type III, G4 presented a higher value than G2. Zymography for MMP-2 indicated high values in G4 and G7. MMP-9 increased in both treatment groups euthanized at 8 days, especially in G4. Our results indicate that the pulsed LLLT improved the remodeling of the ECM during the healing process in tendons through activation of MMP-2 and stimulation of collagen synthesis.

Lasers Med Sci.   2011 Jul 15. [Epub ahead of print]

Phototherapy with low-level laser affects the remodeling of types I and III collagen in skeletal muscle repair.

de Souza TO, Mesquita DA, Ferrari RA, Dos Santos Pinto D Jr, Correa L, Bussadori SK, Fernandes KP, Martins MD.

Source

Rehabilitation Sciences, Universidade Nove de Julho – UNINOVE, 612, Avenida Francisco Matarazzo, São Paulo, SP, CEP: 05001-100, Brazil, thais.oricchio@terra.com.br.

Abstract

The purpose of this article was to analyze the photobiomodulator role of low-level laser therapy (LLLT) on the skeletal muscle remodeling following cryoinjury in rats, focusing the types I and III collagen proteins. Laser phototherapy has been employed to stimulate repair in different tissues. However, its role in skeletal muscle remodeling is not yet well clarified, especially its effect on the collagen component of the extracellular matrix. Fifty adult Wistar rats were divided into four groups: control, sham, cryoinjury, and laser-treated cryoinjury. Laser irradiation was performed three times a week on the injured region using the InGaAlP (indium-gallium-aluminum-phosphorous) laser (660 nm; beam spot of 0.04 cm(2), output power of 20 mW, power density of 0.5 mW/cm(2), energy density of 5 J/cm(2), 10-s exposure time, with a total energy dose of 0.2 J). Five animals were killed after short-term (days 1 and 7) and long-term (14 and 21) durations following injury. The muscles were processed and submitted to hematoxylin and eosin (H&E) and immunohistochemical staining. The histological slices were analyzed qualitatively, semi-quantitatively, and quantitatively. The data were submitted to statistical analysis using the Kruskal-Wallis test. The qualitative analysis of morphological aspects revealed that the muscle repair were very similar in cryoinjury and laser groups on days 1, 14 and 21. However, at 7 days, differences could be observed because there was a reduction in myonecrosis associated to formation of new vessels (angiogenesis) in the laser-treated group. The analysis of the distribution of types I and III collagen, on day 7, revealed a significant increase in the depositing of these proteins in the laser-treated group when compared to the cryoinjury group. InGaAlP diode laser within the power parameters and conditions tested had a biostimulatory effect at the regenerative and fibrotic phases of the skeletal muscle repairs, by promoting angiogenesis, reducing myonecrosis, and inducing types I and III collagen synthesis, following cryoinjury in rat.

J Photochem Photobiol B. 2010 Mar 8;98(3):211-5. Epub 2010 Jan 25.

Chondrogenic mRNA expression in prechondrogenic cells after blue laser irradiation.

Kushibiki T, Tajiri T, Ninomiya Y, Awazu K.

Frontier Research Center, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan. kushibiki@see.eng.osaka-u.ac.jp

Abstract

Low-level laser therapy (LLLT) has been used as a method for biostimulation. Cartilage develops through the differentiation of mesenchymal cells into chondrocytes, and differentiated chondrocytes in articular cartilage maintain cartilage homeostasis by synthesizing cartilage-specific extracellular matrix. The aim of this study is to evaluate the enhancement of chondrocyte differentiation and the expression levels of chondrogenic mRNA in prechondrogenic ATDC5 cells after laser irradiation. For chondrogenic induction, ATDC5 cells were irradiated with a blue laser (405 nm, continuous wave) at 100 mW/cm(2) for 180 s following incubation in chondrogenic differentiation medium. Differentiation after laser irradiation was quantitatively evaluated by the measurement of total collagen contents and chondrogenesis-related mRNAs. The total amount of collagen and mRNA levels of aggrecan, collagen type II, SOX-9, and DEC-1 were increased relative to those of a non-laser irradiated group after 14 days of laser irradiation. On the other hand, Ap-2alpha mRNA, a negative transcription factor of chondrogenesis, was dramatically decreased after laser irradiation. In addition, intracellular reactive oxygen species (ROS) were generated after laser irradiation. These results, for the first time, provide functional evidence that mRNA expression relating to chondrogenesis is increased, and Ap-2alpha is decreased immediately after laser irradiation. As this technique could readily be applied in situ to control the differentiation of cells at an implanted site within the body, this approach may have therapeutic potential for the restoration of damaged or diseased tissue.

J Invest Dermatol. 2009 Dec;129(12):2751-9. Epub 2009 Jul 9.

Regulation of skin collagen metabolism in vitro using a pulsed 660 nm LED light source: clinical correlation with a single-blinded study.

Barolet D, Roberge CJ, Auger FA, Boucher A, Germain L.

RoseLab Skin Optics Laboratory, 3333 Graham Blvd., Suite 206, Montreal, Quebec H3R 3L5, Canada. daniel.barolet@mcgill.ca

It has been reported that skin aging is associated with a downregulation in collagen synthesis and an elevation in matrix metalloproteinase (MMP) expression. This study investigated the potential of light-emitting diode (LED) treatments with a 660 nm sequentially pulsed illumination formula in the photobiomodulation of these molecules. Histological and biochemical changes were first evaluated in a tissue-engineered Human Reconstructed Skin (HRS) model after 11 sham or LED light treatments. LED effects were then assessed in aged/photoaged individuals in a split-face single-blinded study. Results yielded a mean percent difference between LED-treated and non-LED-treated HRS of 31% in levels of type-1 procollagen and of -18% in MMP-1. No histological changes were observed. Furthermore, profilometry quantification revealed that more than 90% of individuals showed a reduction in rhytid depth and surface roughness, and, via a blinded clinical assessment, that 87% experienced a reduction in the Fitzpatrick wrinkling severity score after 12 LED treatments. No adverse events or downtime were reported. Our study showed that LED therapy reversed collagen downregulation and MMP-1 upregulation. This could explain the improvements in skin appearance observed in LED-treated individuals. These findings suggest that LED at 660 nm is a safe and effective collagen-enhancement strategy.

Lasers Surg Med. 2009 Sep;41(7):487-91.

The effects of laser irradiation of cartilage on chondrocyte gene expression and the collagen matrix.

Holden PK, Li C, Da Costa V, Sun CH, Bryant SV, Gardiner DM, Wong BJ.

Department of Otolaryngology-Head and Neck Surgery, University of California, Irvine, California 92612, USA.

OBJECTIVES: Laser reshaping of cartilage is an emerging technology aimed at replacing conventional techniques for aesthetic and reconstructive surgery. Little is known about the mechanisms of wound healing following the photothermal heating during laser reshaping and, ultimately, how collagen remodels in the irradiated tissue. Healthy hyaline and elastic cartilage as found in the ear, nose, larynx, and trachea does not express collagen type I which is characteristic of fibro-cartilage and scar tissue. The aim of the study was to determine if collagen I and II gene expression occurs within laser irradiated rabbit septal cartilage. METHODS: Nasal septum harvested from freshly euthanized New Zealand White rabbits were irradiated with an Nd:YAG laser. After 2 weeks in culture, the laser spot and surrounding non-irradiated regions were imaged using immunofluorescence staining and evaluated using reverse transcription polymerase chain reaction (RT-PCR) to determine the presence of collagen I and II, and ascertain collagen I and II gene expression, respectively. RESULTS: All laser irradiated specimens showed a cessation in collagen II gene expression within the center of the laser spot. Collagen II was expressed in the surrounding region encircling the laser spot and within the non-irradiated periphery in all specimens. Immunohistochemistry identified only type II collagen. Neither collagen I gene expression nor immunoreactivity were identified in any specimens regardless or irradiation parameters. CONCLUSIONS: Laser irradiation of rabbit septal cartilage using dosimetry parameters similar to those used in laser reshaping does not result in the detection of either collagen I gene expression or immunoreactivity. Only collagen type II was noted after laser exposure in vitro following cell culture, which suggests that the cellular response to laser irradiation is distinct from that observed in conventional wound healing. Laser irradiation of cartilage can leave an intact collagen matrix which likely allows chondrocyte recovery on an intact scaffold.

Cell Tissue Bank. 2009 Nov;10(4):327-32. Epub 2009 Jul 11.

Effects of diode laser therapy on the acellular dermal matrix.

Soares LP, de Oliveira MG, de Almeida Reis SR.

Department of Oralmaxillofacial Surgery, PUCRS School of Dentistry, Porto Alegre, Rio Grande do Sul, Brazil. liviaps@ibest.com.br

Acellular dermal matrix (ADM) was subcutaneously implanted into calvarian skin of male Wistar rats (n = 40). Low-level laser (lambda 685 nm, 4 J/cm(2)) was locally applied in experimental group (n = 20) above the skin flap. Grafts were harvested at 1, 3, 7 and 14 days after surgery and underwent histological analyses. In treated animals, the extent of edema and the number of inflammatory cells were reduced (P < 0.05). The amount of collagen in graft treated with low-level laser were significantly higher than those of controls (P < 0.05) and were statistically more prominent on the 14th day after surgery. The mean count of fibroblasts was significantly higher in the low-laser therapy group within the 3rd day, showing a marked influx of fibroblasts into area. In conclusion, wound healing of the ADM appear to be positively affected by laser therapy.

Lasers Med Sci. 2007 Mar;22(1):1-3. Epub 2006 Nov 25.

A reasonable mechanism for visible light-induced skin rejuvenation.

Lubart R, Friedmann H, Lavie R, Longo L, Jacobi J, Baruchin O, Baruchin AM.

Department of Chemistry, Bar-Ilan University, Ramat-Gan, 52900, Israel.

In recent years, much research has been done in the field of non-ablative skin rejuvenation. This comes as a response to the continuous demand for a simple method of treating rhytides, UV exposure, and acne scars. Numerous researches involve visible light-pulsed systems (20-30 J/cm(2)). The mechanism of action is believed to be a selective heat-induced denaturalization of dermal collagen that leads to subsequent reactive synthesis (Bitter Jr., Dermatol. Surg., 26:836-843, 2000; Fitzpatrick et al., Arch. Dermatol., 132:395-402, 1996; Kauvar and Geronemus, Dermatol. Clin., 15:459-467, 1997; Negishi et al., Lasers Surg. Med., 30:298-305, 2002; Goldberg and Cutler, Lasers Surg. Med., 26:196-200, 2000; Hernandez-Perez and Ibeitt, Dermatol. Surg., 28:651-655, 2002). In this study, we suggest a different mechanism for photorejuvenation based on light-induced reactive oxygen species (ROS) formation. We irradiated collagen in vitro with a broadband of visible light (400-800 nm, 24-72 J/cm(2)) and used the spin trapping coupled with electron paramagnetic resonance spectroscopy to detect ROS. Irradiated collagen resulted in hydroxyl radicals formation. We propose, as a new concept, that visible light at the energy doses used for skin rejuvenation (20-30 J/cm(2)) produces high amounts of ROS, which destroy old collagen fibers, encouraging the formation of new ones. On the other hand, at inner depths of the skin, where the light intensity is much weaker, low amounts of ROS are formed, which are well known to stimulate fibroblast proliferation.

Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2007 Apr;21(4):382-5.

Expression of vascular cell adhesion molecule 1 in acellular dermal matrix grafting in pigs

[Article in Chinese]

Pan Y, Xu J, Chen S.

Department of Burns, Hainan Provincial Hospital, Haikou Hainan, 570311, P. R. China. pychuan@21cn.com

OBJECTIVE: To explore the expression of the vascular cell adhesion molecule 1 (VCAM-1) in the acellular dermal matrix grafting in pigs. METHODS: Experimental models were established with 15 Inbred Strain mini pigs, 6 full-thichness skin defect wounds, 6 cm x 6 cm in size, were produced on both-side backs of the each pig, and then the pigs were randomly divided into 3 groups. In Group A (n = 5, control) the thin auto-skin transplantation alone was made; in Group B (n = 5), the grafting was performed in the acellular allo-dermal matrix combined with the thin auto-skins; in Group C (n = 5), the grafting was performed in the acellular xeno-dermal matrix combined with the thin auto-skins. The areas of the wounds were measured and the survival condition of the grafted skins was observed at 3, 9, 21 and 30 days after the grafting. The histological samples were harvested from the grafting area at 3, 6, 9, 12, 21 and 30 days after the procedure. The flow cytometry was employed to analyze the changes in the VCAM-1 level in the sample at different time points after the grafting. RESULTS: In the 3 groups, the transplanted skin base was easily separated at 3 days after transplantation; the areas of the wound healing accounted for 94Y+/- 12%, 92%+/- 9% , and 91%+/- 11%, respectively, at 21 days; good wound healing was achieved at 30 days. At 9 and 12 days after transplantation, there was an evidently-increased level of the VCAM-1 expression in the tissue samples in the composite skin grafting groups. Compared with the control group, the difference was significant (P < 0.05); however, the VCAM-1 expression at 3 days was not statistically different between the composite skin grafting groups and the control group after transplantation. In contrast, the level of the VCAM-1 expression was significantly higher at 6 days in the control group than in the composite skin grafting groups (P < 0.05). The levels of the VCAM-1 expression were significantly lower at 30 days than at 3 days after transplantation in all the 3 groups (P < 0.01). CONCLUSION: The highest level of the VCAM-1 expression can be delayed in the composite skin grafting when compared with that in the thin auto-skins alone, which implies that the VCAM-1 expression may be correlated with angiogenesis and composite skin survival. The VCAM-1 expression is not different between the acellular allo-dermal matrix composite skin grafting groups and the acellular xeno-dermal matrix group.

Lasers Surg Med. 2005 Jul;37(1):89-96.

Identification of chondrocyte proliferation following laser irradiation, thermal injury, and mechanical trauma.

Wong BJ, Pandhoh N, Truong MT, Diaz S, Chao K, Hou S, Gardiner D.

Beckman Laser Institute and Medical Clinic, University of California-Irvine, 1002 Health Sciences Road East, Irvine, CA 92612, USA. bjwong@uci.edu

Abstract

BACKGROUND AND OBJECTIVE: Cartilage has a limited regenerative capacity, and there are a lack of reliable techniques and methods to stimulate growth of new tissue to treat degenerative diseases and trauma. This study focused on identifying chondrocyte cell proliferation in ex vivo cartilage tissue following heating Nd:YAG laser using whole-mount analysis and flow cytometry, and compared findings with results produced by contact, and water bath heating methods, mechanical injury, and the addition of transforming growth factor-beta (TGF-beta).

STUDY DESIGN/MATERIALS AND METHODS: Ex vivo rabbit nasal septal cartilages were either irradiated with an Nd:YAG laser (lambda = 1.32 microm, 2-16 seconds, 6 W/cm(2)), heated by immersion in a warm saline bath, heated by direct contact with a metal rod, or mechanically damaged by scoring with a scalpel or crushing. After treatment, specimens were incubated for 7 or 14 days in growth media containing 10 microM bromodeoxyuridine (BrdU). Additional specimens were cultured with both BrdU and TGF-beta. Both whole-mount BrdU-double-antibody detection techniques and flow cytometry were used to determine the presence of DNA replication as a marker of proliferation.

RESULT: An annular region of regenerating chondrocytes was identified surrounding the laser irradiation zone in whole-mount tissue specimens, and the diameter of this region increased with irradiation time. Using whole-mount analysis, no evidence of chondrocyte DNA replication was observed in tissues heated using non-laser methods, grown in TGF-beta, or mechanically traumatized. In contrast, flow cytometry identified the presence of BrdU-positive cells in the S-phase of the cell cycle (synthesis of DNA) for all protocols, indicating chondrocyte proliferation. The percentage of cells that are in S-phase increased with irradiation time.

CONCLUSION: These data provide evidence that laser irradiation, along with other thermal and mechanical treatments, causes a proliferative response in chondrocytes, and this is observed ex vivo in the absence of cellular and humoral repair mechanisms. The advantage of using optical methods to generate heat in cartilage is that microspot injuries could be created in tissue and scanned across surfaces in clinical applications.

PesquiOdontol Bras. 2003 Oct-Dec;17(4):307-13. Epub 2004 Apr 19.

The influence of low-level laser therapy on biomodulation of collagen and elastic fibers.

Pugliese LS, Medrado AP, Reis SR, Andrade Zde A.

Department of Basic Science, Foundation for the Development of Science, Salvador.

The study of low-level laser therapy upon extracellular matrix elements is important to understand the wound healing process under this agent. However, little is known about the interference of laser light in relation to collagen and elastic fibers. Cutaneous wounds were performed on the back of 72 Wistar rats and a Ga-Al-As low-level laser was punctually applied with different energy densities. The animals were killed after 24, 48, 72 hours and 5, 7 and 14 days. Tissues were stained with hematoxilin-eosin, sirius red fast green and orcein and then analyzed. It was observed that the treated group exhibited larger reduction of edema and inflammatory infiltrate. The treated animals presented a larger expression of collagen and elastic fibers, although without statistical significance (p > 0.05). Treatment with a dosage of 4 J/cm(2) exhibited more expressive results than that with 8 J/cm(2). In this study, the authors concluded that low-level laser therapy contributed to a larger expression of collagen and elastic fibers during the early phases of the wound healing process.