Ultraviolet radiation-induced degradation of dermal extracellular matrix and protection by green tea catechins: a randomized controlled trial.

BACKGROUND: Loss and remodelling of the dermal extracellular matrix (ECM) are key features of photodamaged human skin. Green tea catechins (GTCs) have been explored for their anti-inflammatory and chemopreventive properties, but data on the impact of GTCs on ultraviolet radiation (UVR)-induced changes to the dermal ECM are lacking. AIM: To investigate the effect of an inflammatory dose of solar-simulated UVR on human dermal ECM and potential for protection by GTCs in a double-blind randomized controlled trial. METHODS: In total, 50 healthy white (Fitzpatrick skin type I-II) adults aged 18-65 years were randomized to a combination of GTCs 540 mg plus vitamin C 50 mg or to placebo twice daily for 12 weeks. The impact of solar-simulated UVR at 3 × minimal erythema dose on the dermal collagen and elastic fibre networks was assessed by histology and immunohistochemistry in all participants at baseline. The impact of GTC supplementation on UVR-induced effects was compared between the groups post-supplementation. RESULTS: The area of papillary dermis covered by collagen and elastic fibres was significantly lower (P < 0.001) in UVR-exposed skin than in unexposed skin. Significantly lower levels of fibrillin-rich microfibrils (P = 0.02), fibulin-2 (P < 0.001) and fibulin-5 (P < 0.001) were seen in UVR-exposed than unexposed skin, while procollagen-1 deposition was significantly higher in UVR-exposed skin (P = 0.01). Following GTC supplementation, the UVR-induced change in fibulin-5 was abrogated in the active group but not the placebo group, with no difference between the two groups for other components. CONCLUSIONS: Acute UVR induced significant changes in the human dermal collagen and elastic fibre networks, whereas oral GTCs conferred specific UVR protection to fibulin-5. Future studies could explore the impact of GTCs on the effects of repeated suberythemal UVR exposure of human skin.

Effects of photobiomodulation therapy on the extracellular matrix of human dental pulp cell sheets.

Photobiomodulation therapy (PBMT) and the cell sheet (CS) technology improve processes relevant to tissue regeneration. The aim of this study was to investigate the effects of different PBMT parameters on the architecture (histology), protein composition (Western blotting and immunohistochemistry) and ultrastructure [scanning electron microscopy (SEM) and transmission electron microscopy (TEM)] of the extracellular matrix (ECM) synthesized by CSs composed by human dental pulp stem cells (hDPSCs). METHODS: Thawed cells were recharacterized by the expression profile of the surface molecules of mesenchymal stem cells (MSCs) using flow cytometry. Clonogenic medium supplemented with vitamin C (20 µg/ml) was used for obtaining the CSs. PBMT was performed with continuous-wave diode laser (660 nm, 20 mW, 0.028cm2, 0.71 W/cm2) in punctual and contact mode. The CSs were allocated in 3 experimental groups: Control: no further treatment; PBMT1 [4 s, 3 J/cm2 (lower energy density), 0.08 J/point] and PBMT2 [7 s, 5 J/cm2 (higher energy density), 0.14 J/point]. Statistical comparisons were performed (p ≤ .05). RESULTS: The cells presented the classical immunoprofile of MSCs. Type I and type III collagens and fibronectin were present in the ECM of the CSs. PBMT1 induced higher amount of fibronectin. The overall ultrastructure of the CSs in the PBMT1 was epithelial-like, whereas the PBMT2 leads to CSs with fusiform cells arranged in bundles. TEM identified a more mature ECM and signs of apoptosis and necrosis in the PBMT2 group. CONCLUSION: PBMT influence the composition and ultrastructure of the ECM of CSs of hDPSCs. Thus, PBMT, specifically when applied in the lower energy density, could be of importance in the determination of the mechanical quality of CSs, which may favor cell therapy by improving the CS transplantation approach.

Photobiomodulation of extracellular matrix enzymes in human nucleus pulposus cells as a potential treatment for intervertebral disk degeneration.

Intervertebral disc (IVD) degeneration is associated with imbalances between catabolic and anabolic responses, regulated by extracellular matrix (ECM)-modifying enzymes such as matrix metalloproteinases (MMPs) and their endogenous tissue inhibitors of metalloproteinases (TIMPs). Potential contributing factors, such as interleukin (IL)-1ß and tumor necrosis factor (TNF)-α, derived from infiltrated, activated macrophages within IVD tissues, can trigger abnormal production of ECM-modifying enzymes and progression of IVD degeneration. Novel therapies for regulating ECM-modifying enzymes can prevent or ameliorate IVD degeneration. Photobiomodulation (PBM), known to regulate wound repair, exhibits regenerative potential by modulating biological molecules. This study examined the effects of PBM, administered at various wavelengths (630, 525, and 465 nm) and energy densities (16, 32, and 64 J/cm2), on the production of ECM-modifying enzymes in replicated degenerative IVD. Our results showed that PBM selectively inhibited the production of ECM-modifying enzymes in a dose- and wavelength-dependent manner, suggesting that it could be a novel tool for treating symptomatic IVD degeneration.

Perilla frutescens leaves extract ameliorates ultraviolet radiation-induced extracellular matrix damage in human dermal fibroblasts and hairless mice skin.

ETHNOPHARMACOLOGICAL RELEVANCE: Perilla frutescens (L.) Britt. (Lamiaceae) is a traditional herb that is consumed in East Asian countries as a traditional medicine. This traditional herb has been documented for centuries to treat various diseases such as depression, allergies, inflammation and asthma. However, the effect of Perilla frutescens on skin has not been characterized well. AIM OF THE STUDY: The present study aimed to investigate the effect of Perilla frutescens leaves extract (PLE) on ultraviolet radiation-induced extracellular matrix damage in human dermal fibroblasts and hairless mice skin. MATERIALS AND METHODS: Human dermal fibroblasts and Skh-1 hairless mice were irradiated with UV and treated with PLE. Protein and mRNA levels of various target molecules were analyzed by western blotting and quantitative RT-PCR, respectively. Histological changes of mouse skin were analyzed by H&E staining. To elucidate underlying mechanism of PLE, activator protein-1 (AP-1) DNA binding assay and the measurement of reactive oxygen species (ROS) were performed. RESULTS: PLE significantly inhibited basal and UV-induced MMP-1 and MMP-3 expression dose-dependently, and also decreased UV-induced phosphorylation of extracellular signal-regulated kinases and c-Jun N-terminal kinases. This inhibitory effects of PLE on MMP-1 and MMP-3 were mediated by reduction of ROS generation and AP-1 DNA binding activity induced by UV. Furthermore, PLE promoted type I procollagen production irrespective of UV irradiation. In the UV-irradiated animal model, PLE significantly reduced epidermal skin thickness and MMP-13 expression induced by UV. CONCLUSION: Our results demonstrate that PLE has the protective effect against UV-induced dermal matrix damage. Therefore, we suggest that PLE can be a potential agent for prevention of skin aging.

Oral administration of Aloe vera gel powder prevents UVB-induced decrease in skin elasticity via suppression of overexpression of MMPs in hairless mice.

This study reports the effects of oral Aloe vera gel powder (AVGP) containing Aloe sterols on skin elasticity and the extracellular matrix in ultraviolet B (UVB)-irradiated hairless mice. Ten-week-old hairless mice were fed diets containing 0.3% AVGP for 8 weeks and irradiated UVB for 6 weeks. Mice treated with AVGP showed significant prevention of the UVB-induced decrease in skin elasticity. To investigate the mechanism underlying this suppression of skin elasticity loss, we measured the expression of matrix metalloproteinase (MMP)-2, -9, and -13. AVGP prevented both the UVB-induced increases in MMPs expressions. Moreover, we investigated hyaluronic acid (HA) content of mice dorsal skin and gene expression of HA synthase-2 (Has2). In the results, AVGP oral administration prevented UVB-induced decreasing in skin HA content and Has2 expression and attenuates the UVB-induced decrease in serum adiponectin, which promotes Has2 expression. These results suggested that AVGP has the ability to prevent the skin photoaging.

Effect of low-level laser therapy on metalloproteinase MMP-2 and MMP-9 production and percentage of collagen types I and III in a papain cartilage injury model.

Osteoarthritis (OA) resulting from injury or disease is associated with increased levels of several matrix metalloproteinases (MMPs), which degrade all components of the complex extracellular matrix in the cartilage. The objective of this study is to investigate the effect of low-level laser therapy (LLLT) on papain-induced joint damage in rats by histopathology and analysis of metalloproteinase 2 and 9 production. Sixty male Wistar rats were randomly distributed into four groups of 15 animals: (1) non-injury negative control, (2) injury positive control, (3) treated with LLLT at 50 mW, and (4) treated with LLLT at 100 mW. OA was induced in animals using papain (4 % solution) followed by treatment with LLLT. After 7, 14, and 21 days, the animals were euthanized. The articular lavage was collected and centrifuged; then, the supernatant was stored prior to protein analysis by western blot. The material was stained with hematoxylin and eosin for histopathological analysis, and Picrosirius Red was used to estimate the percentage of collagen fibers. To determine normal distribution, ANOVA and Tukey's post hoc test were used for comparison between and within each group at each time period. All data are expressed as mean and standard deviation values, with the null hypothesis considered as p < 0.05. Both laser groups (50 and 100 mW) were effective in tissue repair, decreasing collagen type III expression and increasing type I expression in all experimental periods; however, LLLT at 50 mW reduced metalloproteinase 9 more than at 100 mW in 21 days. LLLT at 50 mW was more efficient in the modulation of matrix MMPs and tissue repair.

Epilobium angustifolium extract demonstrates multiple effects on dermal fibroblasts in vitro and skin photo-protection in vivo.

Stress-induced fibroblast senescence is thought to contribute to skin aging. Ultraviolet light (UV) radiation is the most potent environmental risk factor in these processes. An Epilobium angustifolium (EA) extract was evaluated for its capacity to reverse the senescent response of normal human dermal fibroblasts (NHDF) in vitro and to exhibit skin photo-protection in vivo. The HPLC-UV-MS analysis of the EA preparation identified three major polyphenol groups: tannins (oenothein B), phenolic acids (gallic and chlorogenic acids) and flavonoids. EA extract increased the cell viability of senescent NHDF induced by serum deprivation. It diminished connective tissue growth factor and fibronectin gene expressions in senescent NHDF. Down-regulation of the UV-induced release of both matrix metalloproteinase-1 and -3 and the tissue inhibitor of matrix metalloproteinases-1 and -2, and also down-regulation of the gene expression of hyaluronidase 2 were observed in repeatedly UV-irradiated NHDF after EA extract treatment. Interestingly, EA extract diminished the down-regulation of sirtuin 1 dampened by UV-irradiation. The application of EA extract using a sub-irritating dose protected skin against UV-induced erythema formation in vivo. In summary, EA extract diminished stress-induced effects on NHDF, particularly on connective tissue growth factor, fibronectin and matrix metalloproteinases. These results collectively suggest that EA extract may possess anti-aging properties and that the EA polyphenols might account for these benefits.

Glutamine supplementation prevents collagen expression damage in healthy urinary bladder caused by radiotherapy.

OBJECTIVE: Patients who have had pelvic radiotherapy as part of their cancer therapy may develop subsequent urinary bladder effects such as hyperactive bladder, incontinence, and dysuria. Therefore, the goal of this study was to evaluate whether glutamine supplementation could prevent collagen expression damage in healthy urinary bladder caused by radiotherapy. METHODS: Fifteen adult Wistar rats were separated into a control group that received food and water ad libitum (C group), an irradiated group that received a single pelvic radiation dose of 1164 cGy (I group), and an irradiated group supplemented with l-glutamine every day during the entire experimental period (0.65 g/kg of body weight; I+G group). All animals were sacrificed 15 d after irradiation. The extracellular matrix and muscle were quantified by a morphometric method. Picro Sirius Red was used to visualize the different collagen types. Reverse transcription-polymerase chain reaction and immunohistochemistry were used to determine collagen type I and III expressions. RESULTS: The extracellular matrix (C group 36.84±4.37, I group 31.64±5.00, I+G group 35.53±2.60, P=0.0001), muscle (C group 36.43±6.15, I group 29.39±7.08, I+G group 31.38±3.14, P=0.0001), and gene expressions of collagen type I (C group 1.067±0.31, I group 0.579±0.17, I+G group 1.816±0.66, P=0.0009) and type III (C group 0.99±0.28, I group 0.54±0.13, I+G group 1.07±0.28, P=0.0080) were decreased in the I group. Apart from muscle, glutamine supplementation prevented these alterations. Immunohistochemistry and Picro Sirius Red showed similar results. CONCLUSION: Supplementation with l-glutamine seems to prevent bladder wall damage in relation to extracellular matrix volumetric density and collagen expression. These results suggest that glutamine supplementation could be efficient in protecting healthy tissues from the adverse effects of radiotherapy.

A novel explanation for the healing effect of the Er:YAG laser during skin rejuvenation.

The popularity of cutaneous laser resurfacing has soared in recent years. Ablative laser skin rejuvenation with carbon dioxide (CO2) and erbium:yttrium-aluminum-garnet (Er:YAG) lasers has been popularized and their side effects individually reported. It has been suggested that initial collagen contraction and thermal damage modulate wound healing. Progress in laser technology permits precise tissue removal and minimal thermal damage. However, mechanisms for cosmetic improvement have not yet been completely determined. In the present short communication, we would like to suggest a possible mechanism for the healing effects exerted by the Er:YAG laser.

Pulsed dye laser-induced inflammatory response and extracellular matrix turnover in rat vocal folds and vocal fold fibroblasts.

BACKGROUND AND OBJECTIVES: Disruption of the vocal fold extracellular matrix (ECM) can induce a profound and refractory dysphonia. Pulsed dye laser (PDL) irradiation has shown early promise as a treatment modality for disordered ECM in patients with chronic vocal fold scar; however, there are limited data addressing the mechanism by which this laser energy might induce cellular and extracellular changes in vocal fold tissues. In this study, we examined the inflammatory and ECM modulating effects of PDL irradiation on normal vocal fold tissues and cultured vocal fold fibroblasts (VFFs). STUDY DESIGN/MATERIALS AND METHODS: We evaluated the effects of 585 nm PDL irradiation on inflammatory cytokine and collagen/collagenase gene transcription in normal rat vocal folds in vivo (3-168 hours following delivery of approximately 39.46 J/cm(2) fluence) and VFFs in vitro (3-72 hours following delivery of 4.82 or 9.64 J/cm(2) fluence). We also examined morphological vocal fold tissue changes 3 hours, 1 week, and 1 month post-irradiation. RESULTS: PDL irradiation altered inflammatory cytokine and procollagen/collagenase expression at the transcript level, both in vitro and in vivo. Additionally, PDL irradiation induced an inflammatory repair process in vivo that was completed by 1 month with preservation of normal tissue morphology. CONCLUSIONS: PDL irradiation can modulate ECM turnover in phenotypically normal vocal folds. Additional work is required to determine if these findings extend to disordered ECM, such as is seen in vocal fold scar. Lasers Surg. Med. 41:585-594, 2009. (c) 2009 Wiley-Liss, Inc.

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

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.

Possible roles of mast cell-derived chymase for skin rejuvenation.

The relationships between mast cell-derived chymase, angiotensin II, and extracellular matrix production in the skin after intense pulsed light (IPL) were clarified in hamsters. Dorsal areas of the hamsters were irradiated once or twice a week by IPL. The index of extracellular matrix production in the skin was defined as the depth stained with Azan-Mallory stain from the epidermis to the dermis at the point of maximum thickness. The index had significantly increased 7 days after IPL irradiation in sections treated once or twice with IPL compared with that of untreated control sections. The numbers of mast cells, chymase-positive cells, and angiotensin II-positive cells had also significantly increased in IPL-irradiated areas. Significant increases in chymase and angiotensin II activities were observed in the extracts obtained from IPL-irradiated skin. Mast cell-derived chymase may be involved via angiotensin II formation in the dermal extracellular matrix production that occurs after IPL irradiation.

Effects of low-power red laser on dentine-pulp interface after cavity preparation. An ultrastructural study.

OBJECTIVE: Studies on the influence of low-power red laser on the repair of dental structures are very scarce. This study investigated the effects of the laser therapy on the ultrastructure of the dentine-pulp interface after conservative class I cavity preparation. DESIGN: Two female volunteers with 8 premolars indicated for extraction for orthodontic reasons were recruited. Class I cavities were prepared and the teeth were randomly divided into two groups. The first group received treatment with a GaA1As laser, lambda=660nm, power of 30mW and energy dose of 2J/cm(2), directly and perpendicularly into the cavity in a single visit. After the irradiation, the cavities were filled with composite resin. The second group received the same treatment, except by the laser therapy. RESULTS: Twenty-eight days post-preparation, the teeth were extracted and processed for transmission electron microscopy analysis. Two sound teeth, without cavity preparation, were also studied. The irradiated group presented odontoblast process in higher contact with the extracellular matrix and the collagen fibrils appeared more aggregated and organised than those of control group. These results were also observed in the healthy teeth. CONCLUSION: These findings suggest that laser irradiation accelerates the recovery of the dental structures involved in the cavity preparation at the predentine region.

The effect of irradiation and hyperbaric oxygenation (HBO) on extracellular matrix of the condylar cartilage after mandibular distraction osteogenesis in the rabbit.

The effects of irradiation and hyperbaric oxygenation (HBO) on the extracellular matrix of condylar cartilage after mandibular distraction were evaluated. Unilateral distraction was performed on 19 rabbits. Five study groups were included: control, low- and high-dose irradiation, and low- and high-dose irradiation groups with HBO. Additionally, four temporomandibular joints (TMJ) were used as control material. The high-dose irradiated animals were given in the TMJ 22.4 Gy/4 fractions irradiation (equivalent to 50 Gy/25 fractions). Low-dose irradiation group received a 2.2 Gy dosage. Two groups were also given preoperatively HBO 18 x 2.5ATA x 90 min. After a two-week distraction period (14 mm lengthening) and four-week consolidation period the TMJs were removed. Proteoglycan (PG) distribution of the extracellular matrix was evaluated using safranin O staining and collagen I and II using immunohistochemistry. The organization of fibrillar network was studied by polarized light microscopy. On the operated side of the control group and on the unoperated side in all, except for high-dose irradiated group, PG distribution and fibrillar network were normal appearing. In the irradiated groups, with or without HBO, the cartilaginous layer was partially or totally devoid of PG and the network structure was severely damaged. In conclusion, irradiation in conjunction with the pressure applied by distraction causes severe damage to extracellular matrix of condylar cartilage.

Therapeutic effects of electromagnetic fields in the stimulation of connective tissue repair.

The therapeutic effects of electric and magnetic fields have been studied largely for their promotion of connective tissue repair. The most widely studied application concerns bone repair and deals with acceleration of the healing of fresh fractures, delayed and non-unions, incorporation of bone grafts, osteoporosis, and osteonecrosis. More recently the effects of these fields upon the repair of cartilage and soft fibrous tissues have been described. In all these experimental systems and clinical applications an acceleration of extracellular matrix synthesis and tissue healing has been observed. A degree of specificity, in terms of the parameters of applied energy and biological response, is hypothesized.