Arnica montana L. associated with microcurrent accelerates the dermis reorganisation of skin lesions.

The aim of this study was to test the effect of electrical stimulation in association with topical Arnica montana gel on organisational changes in the dermis during tissue repair. An experimental rat incisional skin lesion was used for the study. This involved making an incisional lesion on the dorsum of the animals using a scalpel. Ninety-six animals were used divided into the following groups: control (C), microcurrent (MC); topical treatment with Arnica montana gel (ARN); the ARN + microcurrent (ARN + MC). Treatments were administered daily, and injured tissue samples were collected and processed on Days 2, 6 and 10 for dermis analyses. Myeloperoxidase levels were greater in control than in treatment groups on Days 2 and 6. F4/80 expression was similar among all treatment groups and greater than that in control on Day 2. On Day 6, the expression of vascular endothelial growth factor was higher in the MC group than that in other groups, whereas transforming growth factor-ß expression increased in the MC and ARN + MC groups on Day 10. The expression of matrix metalloproteinase-2 was higher in the ARN + MC group when compared with other groups on Day 10. Expression levels of collagen I were increased in the ARN and ARN + MC groups when compared with control and MC groups on Day 6, while expression of collagen III was enhanced in MC, ARN, and ARN + MC groups when compared with the control. The protocol combining microcurrent with topical application of ARN reduces the inflammatory process, increases myofibroblasts proliferation and decreases the presence of macrophages in the dermis during skin repair in rats.

Electrical stimulation therapy and rotary jet-spinning scaffold to treat bone defects.

The combination of electrical stimulation (ES) and bone tissue engineering (BTE) has been successful in treatments of bone regeneration. This study evaluated the effects of ES combined with PCL + ß-TCP 5% scaffolds obtained by rotary jet spinning (RJS) in the regeneration of bone defects in the calvaria of Wistar rats. We used 120 animals with induced bone defects divided into 4 groups (n = 30): (C) without treatment; (S) with PCL+ ß-TCP 5% scaffold; (ES) treated with ES (10 µA/5 min); (ES + S) with PCL + ß-TCP 5% scaffold. The ES occurred twice a week during the entire experimental period. Cell viability (in vitro: Days 3 and 7) and histomorphometric, histochemical, and immunohistochemical (in vivo; Days 30, 60, and 90) analysis were performed. In vitro, ES + S increased cell viability after Day 7 of incubation. In vivo, it was observed modulation of inflammatory cells in ES therapy, which also promoted blood vessels proliferation, and increase of collagen. Moreover, ES therapy played a role in osteogenesis by decreasing ligand kappa B nuclear factor-TNFSF11 (RANKL), increasing alkaline phosphatase (ALP), and decreasing the tartarate-resistant acid phosphatase. The combination of ES with RJS scaffolds may be a promising strategy for bone defects regeneration, since the therapy controlled inflammation, favored blood vessels proliferation, and osteogenesis, which are important processes in bone remodeling.

Acmella oleracea extract increases collagen content and organization in partially transected tendons.

Acmella oleracea contains spilanthol as the main active compound, which possesses analgesic and anti-inflammatory effects that can favor tendon reorganization. To analyze the effect of A. oleracea on the content and organization of collagen in injured tendons, the calcaneal tendon of male Lewis rats was partially transected and treated at the site of injury with a topical application of 20% A. oleracea ointment (AO group) or with the ointment base without the plant extract (B group). The animals were euthanized 21 days after partial transection. Higher collagen concentration was observed in the AO group than in the B group, and morphological analysis using polarization microscopy showed higher birefringence in the AO group than in the B group, indicating higher collagen organization. No difference was observed in the number of fibroblasts, blood vessels, proteoglycan distribution, and maximum load between the B and AO groups. In conclusion, topical application of 20% A. oleracea ointment increased the molecular organization and content of collagen, thus indicating a potential application in tendon repair. Studies on the later phases of the tendon healing process are necessary to demonstrate the possible biomechanical changes after the application of A. oleracea ointment.

Influence of microcurrent on the modulation of remodelling genes in a wound healing assay.

The literature has shown the beneficial effects of microcurrent (MC) therapy on tissue repair. We investigated if the application of MC at 10 µA/90 s could modulate the expression of remodeling genes transforming growth factor beta (Tgfb), connective tissue growth factor (Ctgf), insulin-like growth factor 1 (Igf1), tenascin C (Tnc), Fibronectin (Fn1), Scleraxis (Scx), Fibromodulin (Fmod) and tenomodulin in NIH/3T3 fibroblasts in a wound healing assay. The cell migration was analyzed between days 0 and 4 in both fibroblasts (F) and fibroblasts + MC (F+MC) groups. On the 4th day, cell viability and gene expression were also analyzed after daily MC application. Higher expression of Ctgf and lower expression of Tnc and Fmod, respectively, were observed in the F+MC group in relation to F group (p < 0.05), and no difference was observed between the groups for the genes Tgfb, Fn1 and Scx. In cell migration, a higher number of cells in the scratch region was observed in group F+MC (p < 0.05) compared to group F on the 4th day, and the cell viability assay showed no difference between the groups. In conclusion, MC therapy at an intensity/time of 10 µA/90 s with 4 daily applications did not affect cell viability, stimulated fibroblasts migration with the involvement of Ctgf, and reduced the Tnc and Fmod expression.

Acupuncture and moxibustion stimulate fibroblast proliferation and neoangiogenesis during tissue repair of experimental excisional injuries in adult female Wistar rats.

OBJECTIVE: To investigate the effects of acupuncture and moxibustion on the repair of excisional skin injuries on the back of adult female Wistar rats. METHODS: 90 animals were divided into three groups: C, control; A, acupuncture treatment (needled at traditional acupuncture points BL13, BL17 and ST36); M, moxibustion treatment (overlying same traditional acupuncture points). They were euthanased on days 7, 14 and 21 after injury for removal and preparation of tissue for analysis. RESULTS: The treated groups (A and M) showed no changes regarding the structural analysis relative to the control (C) group. The total number of fibroblast cells in the A and M groups were significantly higher than those in the C group on days 14 and 21. The number of granulocytes was significantly less in the A and M groups compared with the C group on days 14 and 21. The total number of newly formed vessels increased on day 21 and was significantly higher in the A and M groups. The amount of birefringent collagen fibre detected on day 21 was significantly higher in the C group. The amount of glycosaminoglycan and hydroxyproline was similar between the groups. The amount of collagen I did not differ between the groups in any period, despite the increased amount detected over time. The amount of type III collagen did not differ between the groups but the detected amount decreased over the course of the experiment. The amount of transforming growth factor ß1 (TGF-ß1) and vascular endothelial growth factor (VEGF) in the A and M rats was similar but inferior to C rats across all experimental periods. CONCLUSIONS: Acupuncture and moxibustion stimulated fibroblast proliferation and neoangiogenesis, and extended the period of collagen fibre reorganisation in the repair of excisional injuries in adult female rats.

Effects of low-level laser therapy on the organization of articular cartilage in an experimental microcrystalline arthritis model.

The aim of this study was to evaluate the effects of low-level laser therapy using the gallium arsenide laser (λ = 830 nm) on the articular cartilage (AC) organization from knee joint in an experimental model of microcrystalline arthritis in adult male Wistar rats. Seventy-two animals were divided into three groups: A (control), B (induced arthritis), and C (induced arthritis + laser therapy). The arthritis was induced in the right knee using 2 mg of Na4P2O7 in 0.5 mL of saline solution. The treatments were daily applied in the patellar region of the right knee after 48 h of induction. On the 7th, 14th, and 21st days of treatment, the animals were euthanized and their right knees were removed and processed for structural and biochemical analysis of the AC. The chondrocytes positively labeled for the TUNEL reaction were lower in C than in B on the 14th and 21st days. The content of glycosaminoglycans and hydroxyproline in A and C was higher than B on the 21st day. The amount of tibial TNF-α in B and C was lower than in A. The amount of tibial BMP-7 in B and C was higher than in A. The femoral MMP-13 was lower in B and C than for A. The tibial TGF-ß for C was higher than the others. The femoral ADAMT-S4 content of A and C presented similar and inferior data to B on the 21st day. The AsGa-830 nm therapy preserved the content of glycosaminoglycans, reduced the cellular changes and the inflammatory process compared to the untreated group.

Differing energy densities with laser 670nm InGaP controls inflammation and collagen reorganization in burns.

PURPOSE: This study compared different energy densities of laser on second degrees burns in rats aiming to determine the most effective dosimetry in stimulation of the healing process. METHODS: Burns were induced in the dorsal skin of 54 animals divided into three groups (n: 18): 1-without treatment; 2-irradiated lesions by the Indium Gallium Phosphide (InGaP) 670nm (4.93J/cm2) laser; 3-irradiated lesions by the InGaP-670nm (9.86J/cm2) laser. Samples were collected on the 2, 10 and 18 days after injury for structural, morphometry, biochemical analysis and Western blotting. RESULTS: The energy densities examined were effective in significantly increasing the total number of fibroblasts and blood vessels and reduce the number of inflammatory cells particularly in irradiated lesions with 9.86J/cm2. This same energy density significantly increased the amount of GAGs (Glycosaminoglycans), decreased the TGF-ß1 (Transforming Growth Factor ß1) and increased the VEGF (Vascular and Endothelial Growth Factor) during the experimental period. This energy density also significantly increased the Collagen type I and decreased Collagen type III and the active isoform of metalloproteinase 9 (MMP-9). CONCLUSIONS: The energy density of 9.86J/cm2 was more effective in promoting cellular responses related to neoangiogenesis, decreasing inflammation and collagen fibers reorganization.

Low-level laser therapy stimulates tissue repair and reduces the extracellular matrix degradation in rats with induced arthritis in the temporomandibular joint.

The objective of this study was to characterize morphological and biochemistry action of low-level laser therapy (LLLT) on induced arthritis in the temporomandibular joint (TMJ) of rats. Twenty-four male Wistar rats were randomly divided into groups with 12 animals each: (AG) group with arthritis induced in the left TMJ and (LG) group with arthritis induced in the left TMJ and treated with LLLT (830 nm, 30 mW, 3 J/cm(2)). Right TMJs in the AG group were used as noninjected control group (CG). Arthritis was induced by intra-articular injection of 50 µl Complete Freund's Adjuvant (CFA) and LLLT began 1 week after arthritis induction. Histopathological analysis was performed using sections stained with hematoxylin-eosin, Toluidine Blue, and picrosirius. Biochemical analysis was determined by the total concentration of sulfated glycosaminoglycans (GAGs) and evaluation of matrix metalloproteinases (MMP-2 and MMP-9). Statistical analysis was performed using paired and unpaired t tests, with p < 0.05. Compared to AG, LG had minor histopathological changes in the TMJ, smaller thickness of the articular disc in the anterior (p < 0.0001), middle (p < 0.0001) and posterior regions (p < 0.0001), high birefringence of collagen fibers in the anterior (p < 0.0001), middle (p < 0.0001) and posterior regions (p < 0.0001) on the articular disc, and statistically lower activity of MMP-2 latent (p < 0.0001), MMP-2 active (P = 0.02), MMP-9 latent (p < 0.0001), and MMP-9 active (p < 0.0001). These results suggest that LLLT can increase the remodeling and enhancing tissue repair in TMJ with induced arthritis.

Effects of microcurrent therapy on excisional elastic cartilage defects in young rats.

The effects of microcurrent application on the elastic cartilage defects in the outer ear of young animals were analyzed. Sixty male Wistar rats were divided into a control (CG) and a treated group (TG). An excisional lesion was created in the right outer ear of each animal. Daily treatment was started after 24h and consisted of the application of a low-intensity (20µA) continuous electrical current to the site of injury for 5min. The animals were euthanized after 7, 14 and 28 days of injury and the samples were submitted to analyses. In CG, areas of newly formed cartilage and intense basophilia were seen at 28 days, while in TG the same observations were made already at 14 days. The percentage of birefringent collagen fibers was higher in CG at 28 days. The number of connective tissue cells and granulocytes was significantly higher in TG. Ultrastructural analysis revealed the presence of chondrocytes in TG at 14 days, while these cells were observed in CG only at 28 days. Cuprolinic blue staining and the amount of glycosaminoglycans were significantly higher in TG at 14 days and 28 days. The amount of hydroxyproline was significantly higher in TG at all time points studied. The active isoform of MMP-2 was higher activity in TG at 14 days. Immunoblotting for type II collagen and decorin was positive in both groups and at all time points. The treatment stimulated the proliferation and differentiation of connective tissue cells, the deposition of glycosaminoglycans and collagen, and the structural reorganization of these elements during elastic cartilage repair.

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

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.

Electroacupuncture increases the concentration and organization of collagen in a tendon healing model in rats.

The aim of this study was to investigate the effect of electroacupuncture (EA) on the composition and organization of the extracellular matrix of the rat Achilles tendon after a partial transection during the proliferative phase of healing. Wistar rats were divided into three groups: rats that were not tenotomized (G1), tenotomized rats (G2), and rats that were tenotomized and submitted to EA (G3). EA was applied 15 days after injury at the ST36 and BL57 acupoints for 20 min, three times per week on alternate days for a total of six sessions. Biochemical analyses were performed using non-collagenous proteins, glycosaminoglycans, and hydroxyproline quantifications. An analysis of metalloproteinase-2 was carried out by zymography. The general organization of the extracellular matrix and the metachromasy of the tendons were analyzed under light microscopy. The organization of the bundles of collagen fibers was analyzed by birefringence analysis. The results showed that EA did not alter the concentration of non-collagenous proteins or glycosaminoglycans or the enzymatic activity of metalloproteinase-2 in the transected tendons. However, the concentration of hydroxyproline was significantly increased when these tendons were treated by EA. The analysis of birefringence showed a higher organization of collagen fibers in the group treated by EA. These results indicate, for the first time, that EA may offer therapeutic benefits for the treatment of tendon injuries by increasing the concentration of collagen and by inducing a better molecular organization of the collagen fibers, which may improve the mechanical strength of the tendon after injury.