Low-level laser irradiation modulates matrix metalloproteinase activity and gene expression in porcine aortic smooth muscle cells

BACKGROUND AND OBJECTIVES: The vascular extracellular matrix is maintained by a dynamic balance between matrix synthesis and degradation. This equilibrium is disrupted in arterial pathologies such as abdominal aortic aneurysm. Low-level laser irradiation (LLLI) promotes wound healing. However, its effect on smooth muscle cells (SMCs), a central player in these responses, has not been established. The current study was designed to determine the effects of LLLI on arterial SMC proliferation, inflammatory markers, and matrix proteins. STUDY DESIGN/MATERIALS AND METHODS: Porcine primary aortic SMCs were irradiated with a 780 nm laser diode (1 and 2 J/cm(2)). Trypan blue exclusion assay, immunofluorescent staining for collagen I and III, Sircol assay, gelatin zymography, and RT-PCR were used to monitor proliferation; collagen trihelix formation; collagen synthesis; matrix metalloproteinase-2 (MMP-2) activity, and gene expression of MMP-1, MMP-2, tissue inhibitor of MMP-1 (TIMP-1), TIMP-2, and IL-1-beta, respectively. RESULTS: LLLI-increased SMC proliferation by 16 and 22% (1 and 2 J/cm(2), respectively) compared to non-irradiated cells (P<0.01 and P<0.0005). Immediately after LLLI, trihelices of collagen I and III appeared as perinuclear fluorescent rings. Collagen synthesis was increased twofold (2 days after LLLI: 14.3+/-3.5 microg, non-irradiated control: 6.6+/-0.7 microg, and TGF-beta stimulated control: 7.1+/-1.2 microg, P<0.05), MMP-2 activity after LLLI was augmented (over non-irradiated control) by 66+/-18% (2 J/cm(2); P<0.05), and MMP-1 gene expression upregulated. However, TIMP-2 was upregulated, and MMP-2 gene expression downregulated. IL-1-beta gene expression was reduced. CONCLUSIONS: LLLI stimulates SMC proliferation, stimulates collagen synthesis, modulates the equilibrium between regulatory matrix remodeling enzymes, and inhibits pro-inflammatory IL-1-beta gene expression. These findings may be of therapeutic relevance for arterial diseases such as aneurysm where SMC depletion, weakened extracellular matrix, and an increase in pro-inflammatory markers are major pathologic components.     

Effects of the 1,320-nm Nd:YAG laser on transepidermal water loss, histological changes, and collagen remodeling in skin

The goal of the study is to investigate the effects of the 1,320-nm Nd:YAG laser on skin barrier function and dermal remodeling. Forty-eight female mice were irradiated with the 1,320-nm laser four times at an interval of 1 week (350 μs, 10 mm, single pass 10% overlap). Three doses were used: 18, 22, and 26 J/cm². Transepidermal water loss (TEWL) was measured at day 0, 1, 7, 21, 30, and 60 after treatments. Skin biopsies were taken to observe the changes in dermal structures and quantify the increases in the number of fibroblasts and collagen content. The dose of 22 J/cm² induced the lowest ratio of TEWL (0.58±0.06, p<0.05), highest degree of fibroblast proliferation (45.68±5.70, p<0.05), and the most increase in type I collagen fibers (49.8%, p<0.05) and hydroxyproline content (85.7%, p<0.05). The dose of 26 J/cm² caused marked inflammatory infiltration and the most increase in type III collagen fibers. The increase of type I collagen fibers was greater than type III collagen fibers for all three doses (p<0.05). Our results demonstrated that 22 J/cm² was most effective in enhancing skin barrier function and stimulating new collagen synthesis. Dermal remodeling after the 1,320-nm laser was mainly through the synthesis and deposition of collagen type I. Inflammatory reactions were in favor of the formation of type III collagen.     

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/cm² continuous); G4, injured + LLLT (4 J/cm², 20 Hz); G5, injured; G6, injured + LLLT (4 J/cm² continuous); and G7, injured + LLLT (4 J/cm², 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.     

Effect of low-level laser therapy on healing of tenotomized Achilles tendon in streptozotocin-induced diabetic rats

Diabetes mellitus (DM) is associated with musculoskeletal damage. Investigations have indicated that healing of the surgically tenotomized Achilles tendon was considerably augmented following low-level laser therapy (LLLT) in non-diabetic, healthy animals. The aim of the present study was to evaluate the effect of LLLT on the Achilles tendon healing in streptozotocin-induced diabetic (STZ-D) rats via a biomechanical evaluating method. Thirty-three rats were divided into non-diabetic (n?=?18) and diabetic (n?=?15) groups. DM was induced in the rats by injections of STZ. The right Achilles tendons of all rats were tenotomized 1 month after STZ injections. The two experimental groups (n?=?6 for each group) of non-diabetic rats were irradiated with a helium–neon (He–Ne) laser at 2.9 and 11.5 J/cm² for ten consecutive days. The two experimental groups of diabetic rats (n?=?5 for each group) were irradiated with a He–Ne laser at 2.9 and 4.3 J/cm² for ten consecutive days. The tendons were submitted to a tensiometric test. Significant improvements in the maximum stress (MS) values (Newton per square millimeter) were found following LLLT at 2.9 J/cm² in both the non-diabetic (p?=?0.031) and diabetic (p?=?0.019) experimental groups when compared with their control groups. LLLT at 2.9 J/cm² to the tenotomized Achilles tendons in the non-diabetic and diabetic rats significantly increased the strength and MS of repairing Achilles tendons in our study.     

The effects of low-level laser irradiation on differentiation and proliferation of human bone marrow mesenchymal stem cells into neurons and osteoblasts—an in vitro study

Bone marrow-derived mesenchymal stem cells (BMSCs) are promising for use in regenerative medicine. Several studies have shown that low-level laser irradiation (LLLI) could affect the differentiation and proliferation of MSCs. The aim of this study was to examine the influence of LLLI at different energy densities on BMSCs differentiation into neuron and osteoblast. Human BMSCs were cultured and induced to differentiate to either neuron or osteoblast in the absence or presence of LLLI. Gallium aluminum arsenide (GaAlAs) laser irradiation (810 nm) was applied at days 1, 3, and 5 of differentiation process at energy densities of 3 or 6 J/cm² for BMSCs being induced to neurons, and 2 or 4 J/cm² for BMSCs being induced to osteoblasts. BMSCs proliferation was evaluated by MTT assay on the seventh day of differentiation. BMSCs differentiation to neurons was assessed by immunocytochemical analysis of neuron-specific enolase on the seventh day of differentiation. BMSCs differentiation to osteoblast was tested on the second, fifth, seventh, and tenth day of differentiation via analysis of alkaline phosphatase (ALP) activity. LLLI promoted BMSCs proliferation significantly at all energy densities except for 6 J/cm² in comparison to control groups on the seventh day of differentiation. LLLI at energy densities of 3 and 6 J/cm² dramatically facilitated the differentiation of BMSCs into neurons (p < 0.001). Also, ALP activity was significantly enhanced in irradiated BMSCs differentiated to osteoblast on the second, fifth, seventh, and tenth day of differentiation (p < 0.001 except for the second day). Using LLLI at 810 nm wavelength enhances BMSCs differentiation into neuron and osteoblast in the range of 2–6 J/cm², and at the same time increases BMSCs proliferation (except for 6 J/cm²). The effect of LLLI on differentiation and proliferation of BMSCs is dose-dependent. Considering these findings, LLLI could improve current in vitro methods of differentiating BMSCs prior to transplantation.     

Photobiomodulation does not influence maturation and mildly improves functional healing of mouse achilles tendons

Tendon rupture can occur at any age and is commonly treated nonoperatively, yet can result in persisting symptoms. Thus, a need exists to improve nonoperative treatments of injured tendons. Photobiomodulation (PBM) therapy has shown promise in the clinic and is hypothesized to stimulate mitochondrial-related metabolism and improve healing. However, the effect of PBM therapy on mitochondrial function during tendon maturation and healing are unknown, and its effect on tendon structure and function remain unclear. In this study, near-infrared light (980:810 nm blend, 2.5 J/cm²) was applied at low (30 mW/cm²) or high (300 mW/cm²) irradiance to unilateral Achilles tendons of CD-1 mice during postnatal growth (maturation) as well as adult mice with bilateral Achilles tenotomy (healing). The chronic effect of PBM therapy on tendon structure and function was determined using histology and mechanics, and the acute effect of PBM therapy on mitochondrial-related gene expression was assessed. During maturation and healing, collagen alignment, cell number, and nuclear shape were unaffected by chronic PBM therapy. We found a sex-dependent effect of PBM therapy during healing on mechanical outcomes (eg, increased stiffness and Young's modulus for PBM-treated females, and increased strain at ultimate stress for PBM-treated males). Mitochondria-related gene expression was marginally influenced by PBM therapy for both maturation and healing studies. This study was the first to implement PBM therapy during both growth and healing of the murine tendon. PBM therapy resulted in marginal and sex-dependent effects on the murine tendon. Clinical significance: PBM may be beneficial for tendon healing because functional remodeling improves without adverse effects.     

Joint immobilization reduces the expression of sensory neuropeptide receptors and impairs healing after tendon rupture in a rat model

Healing after mobilization versus immobilization was assessed in a model of rat Achilles tendon rupture, by RT‐PCR at 8 and 17 days and by histological analyses at 14 and 28 days postrupture. The expression of mRNA for extracellular matrix (ECM) molecules (collagen type I and type III, versican, decorin, and biglycan), and the subjective histological maturation of the healing area were analyzed. Effects of immobilization on healing were related to changes in the peripheral expression of substance P (NK₁)‐ and calcitonin gene‐related peptide (CRLR and RAMP‐1)‐ receptors. At 8 days postinjury, mRNA levels for ECM molecules were equal in both groups. However, by day 17, the ECM mRNA expression in the mobilized group had increased up to ～14× that of the immobilized group, which were comparable to intact tendon values. Histological analysis confirmed a higher regenerating activity in the mobilized group, with an increased amount of blood vessels, fibroblasts, and new collagen. The expression of sensory neuropeptide receptors in the mobilized group exhibited a significant increase from 8 to 17 days postinjury similar to the increased ECM mRNA expression, whereas the immobilized group at 17 days exhibited levels comparable to the intact tendon values. Therefore, immobilization postrupture appears to hamper tendon healing, a process which may prove to be directly linked to a downregulated peripheral sensitivity to sensory neuropeptide stimulation. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:274–280, 2009     

Low-level light-emitting diode therapy increases mRNA expressions of IL-10 and type I and III collagens on Achilles tendinitis in rats

The present study investigated the effects of low-level light-emitting diode (LED) therapy (880?±?10 nm) on interleukin (IL)-10 and type I and III collagen in an experimental model of Achilles tendinitis. Thirty male Wistar rats were separated into six groups (n?=?5), three groups in the experimental period of 7 days, control group, tendinitis-induced group, and LED therapy group, and three groups in the experimental period of 14 days, tendinitis group, LED therapy group, and LED group with the therapy starting at the 7th day after tendinitis induction (LEDT delay). Tendinitis was induced in the right Achilles tendon using an intratendinous injection of 100 μL of collagenase. The LED parameters were: optical power of 22 mW, spot area size of 0.5 cm², and irradiation time of 170 s, corresponding to 7.5 J/cm² of energy density. The therapy was initiated 12 h after the tendinitis induction, with a 48-h interval between irradiations. The IL-10 and type I and III collagen mRNA expression were evaluated by real-time polymerase chain reaction at the 7th and 14th days after tendinitis induction. The results showed that LED irradiation increased IL-10 (p?<?0.001) in treated group on 7-day experimental period and increased type I and III collagen mRNA expression in both treated groups of 7- and 14-day experimental periods (p?<?0.05), except by type I collagen mRNA expression in LEDT delay group. LED (880 nm) was effective in increasing mRNA expression of IL-10 and type I and III collagen. Therefore, LED therapy may have potentially therapeutic effects on Achilles tendon injuries.     

Effects of the combination of low-level laser irradiation and recombinant human bone morphogenetic protein-2 in bone repair

Low-level laser irradiation (LLLI) and recombinant human bone morphogenetic protein type 2 (rhBMP-2) have been used to stimulate bone formation. LLLI stimulates proliferation of osteoblast precursor cells and cell differentiation and rhBMP-2 recruits osteoprogenitor cells to the bone healing area. This in vivo study evaluated the effects of LLLI and rhBMP-2 on the bone healing process in rats. Critical bone defects were created in the parietal bone in 42 animals, and the animals were divided into six treatment groups: (1) laser, (2) 7?μg of rhBMP-2, (3) laser and 7?μg of rhBMP-2, (4) 7?μg of rhBMP-2/monoolein gel, (5) laser and 7?μg rhBMP-2/monoolein gel, and (6) critical bone defect controls. A gallium-aluminum-arsenide diode laser was used (wavelength 780?nm, output power 60?mW, beam area 0.04?cm(2), irradiation time 80?s, energy density 120?J/cm(2), irradiance 1.5?W/cm(2)). After 15?days, the calvarial tissues were removed for histomorphometric analysis. Group 3 defects showed higher amounts of newly formed bone (37.89%) than the defects of all the other groups (P?相似文献   

Is cultured tendon fibroblast a good model to study tendon healing?

Cultured tendon fibroblasts (CTFs) from intact explants are widely used to study tendon healing in vitro. The significance of these findings may rely on similarities between CTFs and healing tendon fibroblasts in situ. Our purpose was to compare CTFs with fibroblasts cultured from healing tendons. We cultured CTFs from intact and healing tendons at day 7 and day 14 postinjury in a rat model of patellar donor site injury. The mRNA expression of COL1A1, COL3A1, decorin, and biglycan, with or without supplementation of 1 ng/mL TGF‐β1, was compared by quantitative real‐time RT‐PCR. The expression of proliferation cell nuclear antigen (PCNA) and α‐smooth muscle actin (α‐SMA) was determined by immunostain. COL3A1 and decorin mRNA in CTFs was lower as compared to day 7 healing fibroblasts, but its biglycan mRNA level was higher than day 14 healing fibroblasts. TGF‐β1 increased COL1A1 and decorin mRNA in CTFs, but decreased the mRNA of all four genes in day 7 healing tendon fibroblasts. CTFs exhibited lower PCNA immunopositivity as compared to day 7 and day 14 healing fibroblasts, but a higher α‐SMA immunopositivity than cultured day 14 healing fibroblasts. These findings showed that CTFs did not resemble healing tendon cells with respect to major cellular activities related to tendon healing. Thus, fibroblasts from healing tendon may be a more appropriate model for studying cellular activities in tendon healing. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:374–383, 2008     

Compression therapy promotes proliferative repair during rat Achilles tendon immobilization

Achilles tendon ruptures are treated with an initial period of immobilization, which obstructs the healing process partly by a reduction of blood circulation. Intermittent pneumatic compression (IPC) has been proposed to enhance tendon repair by stimulation of blood flow. We hypothesized that daily IPC treatment can counteract the deficits caused by 2 weeks of immobilization post tendon rupture. Forty‐eight Sprague‐Dawley SD) rats, all subjected to blunt Achilles tendon transection, were divided in three equal groups. Group A was allowed free cage activity, whereas groups B–C were immobilized at the operated hindleg. Group C received daily IPC treatment. Two weeks postrupture the rats were euthanatized and the tendons analyzed with tensile testing and histological assessments of collagen organization and collagen III‐LI occurrence. Immobilization significantly reduced maximum force, energy uptake, stiffness, tendon length, transverse area, stress, organized collagen diameter and collagen III‐LI occurrence by respectively 80, 75, 77, 22, 47, 65, 49, and 83% compared to free mobilization. IPC treatment improved maximum force 65%, energy 168%, organized collagen diameter 50%, tendon length 25%, and collagen III‐LI occurrence 150% compared to immobilization only. The results confirm that immobilization impairs healing after tendon rupture and furthermore demonstrate that IPC‐treatment can enhance proliferative tendon repair by counteracting biomechanical and morphological deficits caused by immobilization. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:852–858, 2010     

Comparison of the effects of laser, ultrasound, and combined laser + ultrasound treatments in experimental tendon healing

BACKGROUND AND OBJECTIVE: Therapeutic ultrasound (US) and laser (L) treatments accelerate and facilitate wound healing, and also have beneficial effects on tendon healing. This randomized control study was designed to evaluate the effects of low-intensity US and low-level laser therapy (LLLT) on tendon healing in rats. STUDY DESIGN/MATERIALS AND METHODS: Eighty-four healthy male Swiss-Albino rats were divided into three groups consisting of 28 rats, the left Achilles tendons were used as treatment and the right Achilles tendons as controls. The right and left Achilles tendons of rats were traumatized longitudinally. The treatment was started on postinjury day one. We applied the treatment protocols including low-intensity US treatment in Group I (US Group), Sham US in Group II (SUS Group), LLLT in Group III (L Group), Sham L in Group IV (SL Group), US and LLLT in Group V (US + L Group), and Sham US and Sham L in Group VI (SUS + SL Group). The US treatment was applied with a power of 0.5 W/cm2, a frequency of 1 MHz, continuously, 5 minutes daily. A low-level Ga-As laser was applied with a 904 nm wavelength, 6 mW average power, 1 J/ cm2 dosage, 16 Hz frequency, for 1 minute duration, continuously. In the control groups, the similar procedures as in the corresponding treatment groups were applied with no current (Sham method). The treatment duration was planned for 9 days (sessions) in all groups, except the rats used for biochemical evaluation on the 4th day of treatment, which were treated for 4 days. We measured the levels of the tissue hydroxyproline for biochemical evaluation on the 4th, 10th, and 21st days following the beginning of treatment and the tendon breaking strength on the 21st day following the beginning of treatment for biomechanical evaluation. Seven rats in each group were killed on the 4th, 10th, and 21st days for biochemical evaluation and on the 21st day for biomechanical evaluation. RESULTS: The hydroxyproline levels were found to be significantly increased in the treatment groups on the 10th and 21st days compared to their control groups (P < 0.05). In comparison of the treatment groups on the 4th, 10th, and 21st days of the treatment, the levels of tissue hydroxyproline were found to be more increased in combined US+L Group compared with US Group and L Group, but the difference was not significant (P > 0.05). In comparison of the tendon breaking strengths, it was found as significantly increased in the treatment groups compared with their control groups (P < 0.05), although there was no significant difference between the treatment groups. CONCLUSIONS: Although US, L, and combined US + L treatments increased tendon healing biochemically and biomechanically more than the control groups, no statistically significant difference was found between them. Also we did not find significantly more cumulative positive effects of combined treatment. As a result, both of these physical modalities can be used successfully in the treatment of tendon healing.     

Unfavorable effect of knee immobilization on Achilles tendon healing in rabbits

This study was undertaken to assess the effect of knee immobilization on the treatment of Achilles tendon rupture. After their Achilles tendons were severed, rabbits were divided into 2 groups. In Group A, only the ankle joint was immobilized. In Group B, both the knee and ankle joints were immobilized. At 4 weeks after surgery, both the ultimate tensile force and stiffness of the severed tendons were significantly greater in Group A than in Group B. In Group A, dense collagen fibers were seen in the repaired tendons, and the bundles of collagen fibers were parallel to one another along the axis of the tendons. In contrast, in Group B, dilated veins and capillaries were seen in the repaired tendons, and the proliferation of connective tissue containing collagen fibers was severely reduced around these veins and capillaries and was in general irregular and uneven. These results suggest that knee immobilization retards the healing of a ruptured Achilles tendon without suture, due to congestion and tension deprivation produced by keeping the tendon static.     

Unfavorable effect of knee immobilization on Achilles tendon healing in rabbits

This study was undertaken to assess the effect of knee immobilization on the treatment of Achilles tendon rupture. After their Achilles tendons were severed, rabbits were divided into 2 groups. In Group A, only the ankle joint was immobilized. In Group B, both the knee and ankle joints were immobilized. At 4 weeks after surgery, both the ultimate tensile force and stiffness of the severed tendons were significantly greater in Group A than in Group B. In Group A, dense collagen fibers were seen in the repaired tendons, and the bundles of collagen fibers were parallel to one another along the axis of the tendons. In contrast, in Group B, dilated veins and capillaries were seen in the repaired tendons, and the proliferation of connective tissue containing collagen fibers was severely reduced around these veins and capillaries and was in general irregular and uneven. These results suggest that knee immobilization retards the healing of a ruptured Achilles tendon without suture, due to congestion and tension deprivation produced by keeping the tendon static.     

Unfavorable effect of knee immobilization on Achilles tendon healing in rabbits

This study was undertaken to assess the effect of knee immobilization on the treatment of Achilles tendon rupture. After their Achilles tendons were severed, rabbits were divided into 2 groups. In Group A, only the ankle joint was immobilized. In Group B, both the knee and ankle joints were immobilized. At 4 weeks after surgery, both the ultimate tensile force and stiffness of the severed tendons were significantly greater in Group A than in Group B. In Group A, dense collagen fibers were seen in the repaired tendons, and the bundles of collagen fibers were parallel to one another along the axis of the tendons. In contrast, in Group B, dilated veins and capillaries were seen in the repaired tendons, and the proliferation of connective tissue containing collagen fibers was severely reduced around these veins and capillaries and was in general irregular and uneven. These results suggest that knee immobilization retards the healing of a ruptured Achilles tendon without suture, due to congestion and tension deprivation produced by keeping the tendon static.     

Effects of different setting of diode laser on the mRNA expression of growth factors and type I collagen of human gingival fibroblasts

The aim of this study was to analyze the influence of non-surgical applications of diode laser (940 nm) on the cell proliferation and mRNA expressions of type I collagen and growth factors in human gingival fibroblasts (GF). Gingival fibroblasts were isolated from human gingival connective tissue of systemically healthy individuals. Cells were treated with different laser parameters as follows; (1) Infected pocket setting (power: 2 W, pulse interval: 1 ms, pulse length: 1 ms, 20 s/cm²); (2) Perio-pocket setting (power: 1.5 W, pulse interval: 20 ms, pulse length: 20 ms, 20 s/cm²); and (3) Biostimulation setting (power: 0.3 W in continuous wave, 20 s/cm²). Proliferation of GF was evaluated after different laser applications using a real-time cell analyzer. Total RNA was isolated on day 2 and cDNA synthesis was performed. Type I collagen, insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF) and transforming growth factor-beta (TGF-β) mRNA expressions were determined with quantitative RT-PCR. In a proliferation experiment, no significant differences were observed in the different laser applications when compared to the control group. Statistically significant increases in IGF, VEGF, and TGF-β mRNA expressions were noted in the laser groups when compared to the untreated control group (p < 0.05). A significant increase in collagen type I mRNA expression was noted in only biostimulation set-up of diode laser (p < 0.05). The results of this study demonstrate that non-surgical laser applications modulate behavior of gingival fibroblasts inducing growth factors mRNA expressions and these applications can be used to improve periodontal wound healing.     

Murine tendon function is adversely affected by aggrecan accumulation due to the knockout of ADAMTS5

The present study examined the effect of ADAMTS5 (TS5) knockout on the properties of murine flexor digitorum longus (FDL) and Achilles tendons. FDL and Achilles tendons were analyzed using biomechanical testing, histology, and immunohistochemistry; further characterization of FDL tendons was conducted using transmission electron microscopy (collagen fibril ultrastructure), SDS-PAGE (collagen content and type), fluorescence-assisted carbohydrate electrophoresis for chondroitin sulfate and hyaluronan, and Western blotting for aggrecan, versican, and decorin abundance and distribution. FDL tendons of TS5(-/-) mice showed a 33% larger cross-sectional area, increased collagen fibril area fraction, and decreased material properties relative to those of wild type mice. In TS5(-/-) mice, aggrecan accumulated in the pericellular matrix of tendon fibroblasts. In Achilles tendons, cross-sectional area, stress relaxation, and structural properties were similar in TS5(-/-) and wild type mice; however, the TS5(-/-) tendons exhibited a higher tensile modulus and a weakened enthesis. These results demonstrate that TS5 deficiency disturbs normal tendon collagen organization and alters biomechanical properties. Hence, the role of ADAMTS5 in tendon is to remove pericellular and interfibrillar aggrecan to maintain the molecular architecture responsible for normal tissue function.     

Biostimulatory effect of low-level laser therapy on keratinocytes in vitro

Epithelial cells play an important role in reparative events. Therefore, therapies that can stimulate the proliferation and metabolism of these cells could accelerate the healing process. To evaluate the effects of low-level laser therapy (LLLT), human keratinocytes were irradiated with an InGaAsP diode laser prototype (LASERTable; 780?±?3 nm; 40 mW) using 0.5, 1.5, 3, 5, and 7 J/cm² energy doses. Irradiations were done every 24 h totaling three applications. Evaluation of cell metabolism (MTT assay) showed that LLLT with all energy doses promoted an increase of cell metabolism, being more effective for 0.5, 1.5, and 3 J/cm². The highest cell counts (Trypan blue assay) were observed with 0.5, 3, and 5 J/cm². No statistically significant difference for total protein (TP) production was observed and cell morphology analysis by scanning electron microscopy revealed that LLLT did not promote morphological alterations on the keratinocytes. Real-time polymerase chain reaction (qPCR) revealed that LLLT also promoted an increase of type I collagen (Col-I) and vascular endothelial growth factor (VEGF) gene expression, especially for 1.5 J/cm², but no change on fibroblast growth factor-2 (FGF-2) expression was observed. LLLT at energy doses ranging from 0.5 to 3 J/cm² promoted the most significant biostimulatory effects on cultured keratinocytes.     

A novel minimally invasive surgery combined with early exercise therapy promoting tendon regeneration in the treatment of spontaneous Achilles tendon rupture

ObjectiveAcute closed spontaneous Achilles tendon rupture often occurs in elderly individuals and is usually accompanied with many complications. Conventional surgical approaches to remove the tendon lesions and enthesophytes are highly traumatic and cause complications. In this study, a previously established minimally invasive surgical approach was modified and combined with a Kazakh exercise therapy to reduce trauma, improve wound healing, and promote tendon regeneration in the management of acute closed spontaneous Achilles tendon rupture.MethodsFifty-two patients with acute closed spontaneous Achilles tendon rupture were randomly classified into 2 groups. Group A included 23 patients that were treated with the novel approach. Group B included 29 patients that were treated with a continuous medial oblique surgical approach. Follow-up examinations were performed at post-operative weeks 12 and 24, and year 2. Outcomes were assessed by Achilles tendon rupture score (ATRS), a heel-rise endurance test, and ultrasonographic and multislice spiral computerized tomography.ResultsMean ATRS in Group A was 68.6 and 86.0 at post-operative week 12 and 24, respectively, significantly higher than that in Group B (55.9 and 72.0, respectively). Recovery of patients in Group A was significantly better compared to Group B (p < 0.01), allowing them to participate in early rehabilitating kinesiotherapy. Patients in Group A rarely experienced complications after surgery, such as infection and Achilles tendon exposure, while in Group B, the wound healing was slower, the inside flaps were prone to necrosis and infection, and Achilles tendon exposure occurred in 10% of patients.ConclusionsThe novel minimally invasive surgery is more advantageous in the treatment of acute closed spontaneous Achilles tendon rupture over previous approaches by promoting wound healing and tendon regeneration.     

Low-level laser irradiation induces in vitro proliferation of stem cells from human exfoliated deciduous teeth

The aim of this study was to evaluate the effect of low-level laser irradiation (LLLI) on the proliferation and viability of stem cells from human exfoliated deciduous teeth (SHED). Cells were irradiated or not (control) with an InGaAlP laser diode (660 nm, 30 mW, continuous action mode) using two different energy densities (0.5 J/cm²—16 s; 1.0 J/cm²—33 s). Irradiation was performed at 0 and 48 h, with the laser probe fixed at a distance of 0.5 cm from the cells. Cell proliferation was analyzed at 0, 24, 48, and 72 h by the Trypan blue exclusion method and MTT assay. Cell cycle and Ki67 expression were analyzed by flow cytometry. Apoptosis-related events were evaluated by expression of annexin V/PI and nuclear morphological changes by staining with DAPI. Differences between groups at each time were analyzed by the Kruskal–Wallis and Mann–Whitney tests, adopting a level of significance of 5% (p < 0.05). The results showed that an energy density of 1.0 J/cm² promoted an increase in cell proliferation at 48 and 72 h compared to the control and 0.5 J/cm² groups. Cell cycle analysis revealed a predominance of cells in the S and G2/M phases in the irradiated groups. This finding was confirmed by the increased expression of Ki67. Low positive staining for annexin V and PI was observed in all groups, and no nuclear changes were detected, indicating that cell viability was not affected by the energy densities tested. It can be concluded that the LLLI parameters used (660 nm, 30 mW, 1.0 J/cm²) promote the proliferation of SHEDs and the maintenance of cell viability.