Marcelo Augusto Marretto Esquisatto

Extracellular matrix composition of different regions of the knee joint cartilage in cattle

Articular cartilage covering the bone ends at the joint shows different chemical composition in different regions, depending on the mechanical and biological properties of that region. Several studies have shown a relationship between the chemical composition of the cartilage and biomechanical forces. In the present study we analysed five different knee joints divided into the following regions: F1-medial and lateral border of the patellar surface, F2-patellar surface of the femur, F3-medial and lateral condyles, P-articular surface of the patella and T-medial and lateral condyle of the tibia. The main glycosaminoglycan (GAG) present in these regions was chondroitin sulfate. Analysis of total GAG after digestion of the tissue with papain showed that in F2 and F3 there was a larger quantity of GAG/mg tissue, probably due to the dynamic character of the biomechanical forces in these regions. No significant differences were found for the extract and D1 fractions of the different regions. Analysis of the D4 fraction showed that the protein content was higher in the F3 and P regions than in their opposite T and F2 regions. The differences among the five regions may be a result of the non-uniform presence of biomechanical forces supported by these regions. It is important to consider that the intensity and direction of stress in different parts of a tissue may influence the composition of the extracellular matrix.

Changes in the connective tissue sheath of Wistar rat nerve with aging.

The alterations due to aging in the peripheral nerves can affect the physiology of these structures. Thus, the purpose of the present study was to describe the activity of the MMP-2 and MMP-9, as well as the structure and composition of the extracellular matrix of the rat sciatic nerve during maturation and aging. Our results have shown that the extracellular matrix of the sciatic nerve of 30-, 180- and 730-day-old Wistar rats present ultrastructural, morphometrical and biochemical changes during aging. The perineurium was the structure most affected by age, as evidenced by a decrease in thickness and in collagen fibril content. Cytochemical analysis detected proteoglycans in the basal membrane of Schwann cells and around perineural cells, as well as on the collagen fibrils of the perineurium and endoneurium at all ages. Biochemical analyses showed that the quantity of non-collagenous proteins was higher in 730-day-old animals compared to other ages, while the uronic acid content was higher in 30-day-old animals. Morphometrical analysis detected greater numbers of myelinated fibers and increased myelin thickness in 180-day-old animals. Zymography analysis detected greater amounts and activity of MMP-2 and MMP-9 in 180- and 730-day-old animals compared to younger rats. In conclusion, our results showed changes in the structural organization and composition of extracellular matrix of the sciatic nerve during aging, such as increase in the non-collagenous protein content and higher MMP-2 and MMP-9 activity, decrease in uronic acid concentration and in collagen fibril content in the perineurium, as well as degeneration of nerve fibers.

Differing energy densities with laser 670 nm 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-β1 (Transforming Growth Factor β1) 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.

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.

Biochemical and morphological alterations in the Achilles tendon of mdx mice

Dystrophin‐deficient muscles have repeated cycles of necrosis and regeneration, being susceptible to injury induced by muscle contractions. Some studies have demonstrated that tendons are also affected in mdx mice, based especially on the changes in biomechanical properties arising from the respective linked muscles. However, most studies have focused only on alterations in the myotendinous junction. Thus, the purpose of this work was to study biochemical and morphological alterations in the Achilles tendons of 60‐day‐old mdx mice. Hydroxyproline quantification, showed higher collagen concentration in the mdx mice as compared with the control. No difference between the tendons of both groups was found in the noncollagenous proteins dosage, and in the amount of collagen type III detected in the western blotting analysis. The zymography for gelatinases detection showed higher amounts of metaloproteinase‐2 (active isoform) and of metalloproteinase‐9 (latent isoform) in the mdx mice. Measurements of birefringence, using polarization microscopy, showed higher molecular organization of the collagen fibers in the tendons of mdx mice in comparison to the control group, with presence of larger areas of crimp. Ponceau SS‐stained tendon sections showed stronger staining of the extracellular matrix in the mdx groups. Toluidine blue‐stained sections showed more intense basophilia in tendons of the control group. In morphometry, a higher number of inflammatory cells was detected in the epitendon of mdx group. In conclusion, the Achilles tendon of 60‐day‐old mdx mice presents higher collagen concentration and organization of the collagen fibers, enhanced metalloproteinase‐2 activity, as well as prominent presence of inflammatory cells and lesser proteoglycans. Microsc. Res. Tech. 78:85–93, 2015.

Structure and composition of arytenoid cartilage of the bullfrog (Lithobates catesbeianus) during maturation and aging

The aging process induces progressive and irreversible changes in the structural and functional organization of animals. The objective of this study was to evaluate the effects of aging on the structure and composition of the extracellular matrix of the arytenoid cartilage found in the larynx of male bullfrogs (Lithobates catesbeianus) kept in captivity for commercial purposes. Animals at 7, 180 and 1080 days post-metamorphosis (n=10/age) were euthanized and the cartilage was removed and processed for structural and biochemical analysis. For the structural analyses, cartilage sections were stained with picrosirius, toluidine blue, Weigerts resorcin-fuchsin and Von Kossa stain. The sections were also submitted to immunohistochemistry for detection of collagen types I and II. Other samples were processed for the ultrastructural and cytochemical analysis of proteoglycans. Histological sections were used to chondrocyte count. The number of positive stainings for proteoglycans was quantified by ultrastructural analysis. For quantification and analysis of glycosaminoglycans were used the dimethyl methylene blue and agarose gel electrophoresis methods. The chloramine T method was used for hydroxyproline quantification. At 7 days, basophilia was observed in the pericellular and territorial matrix, which decreased in the latter over the period studied. Collagen fibers were arranged perpendicular to the major axis of the cartilaginous plate and were thicker in older animals. Few calcification areas were observed at the periphery of the cartilage specimens in 1080-day-old animals. Type II collagen was present throughout the stroma at the different ages. Elastic fibers were found in the stroma and perichondrium and increased with age in the two regions. Proteoglycan staining significantly increased from 7 to 180 days and reduced at 1080 days. The amount of total glycosaminoglycans was higher in 180-day-old animals compared to the other ages, with marked presence of chondroitin- and dermatan-sulfate especially in this age. The content of hydroxyproline, which infers the total collagen concentration, was higher in 1080-day-old animals compared to the other ages. The results demonstrated the elastic nature of the arytenoid cartilage of L. catesbeianus and the occurrence of age-related changes in the structural organization and composition of the extracellular matrix. These changes may contribute to alter the function of the larynx in the animal during aging.

Casearia sylvestris Improved Cutaneous Burn Repair in Diabetic Rats

Objectives: The burn repair associated with diabetes complications showed considerable changes in the healing process and the development of alternative therapies to favor the repair is important. This study investigated the efficacy of the Casearia sylvestris in the burn repair in diabetic and non-diabetic rats. Methods: The animals were divided into four groups (n = 20): (C) non-diabetics treated with carbopol gel; (G) non-diabetic treated with C. sylvestris extract in carbopol gel; (DM-C) diabetics treated with carbopol gel; (DM-G) diabetics treated with C. sylvestris extract in carbopol gel. Burn was induced using a metal plate (2 cm diameter/120 °C/20 sec). Samples were collected on the 3rd, 7th, 14th and 21st days after the injury for histomorphometric analysis (inflammatory infiltrate, fibroblasts, blood vessels and collagen), MPO (Myeloperoxidase) and NAG (N-Acetilglicosaminidase) dosage, hydroxyproline, glycosaminoglycans, TGF-β1, VEGF, collagen I and III. Key findings: C. sylvestris decreased the inflammatory process during the experimental period in diabetic and non-diabetic animals which has been demonstrated by MPO, NAG quantification, and TGF-β1 expression. C. sylvestris also increased blood vessels and decreased VEGF expression during the studied period in non-diabetic animals. Fibroplasia and collagenase were increased in all experimental periods in the DM-G group by means of de hydroxyproline quantification, collagen I, collagen III and glycosaminoglycans. Conclusions: C. sylvestris application can modulate favorably the time of inflammation and collagenous process in the repair of burn injuries in diabetic rats.

Electromagnetic Stimulation Combined with Aloe vera Increases Collagen Reorganization in Burn Repair

Objectives: Burns are shown as a clinical problem for their severity and multiple complications due to the time required to heal. Therapies that improve their healing are of great importance, especially for being minimally invasive, of low cost and best performance, all related to the speed and quality of healing. This study investigated the effects of the magnetic electro stimulator Haihuá CD9 isolated or in association with Aloe vera in rats skin burns. Methods: Experimental groups (n = 30/group) were: (C) Carbopol gel; (F) A. vera/Carbopol gel; (H) Haihuá+Carbopol gel; (H+F) Haihuá+A. vera/Carbopol gel. Samples were collected on the 7th, 14th, and 21st experimental days for structural and morphometric analysis, hydroxyproline and glycosaminoglycans quantification, zymography for MMP-2 and MMP-9 and Western Blotting for TGF-β1, VEGF, Collagen I and III. Key findings: The expression of TGF-β1 in H+F was increased on the 7th day and of MMP-9 on the 7th and 14th days. The expression of VEGF increased in the first experimental periods and decreased in the last for the treated groups. There was an increase in the fibroblasts and birefringent collagen fibers in groups treated with Haihuá isolated or in association with A. vera in all periods. The quantification of collagen I increased, while collagen III decreased in H+F. The higher amount of GAGs and MMP-2 active isoform was detected in H and H+F during all periods. Conclusions: Considering the results of the present study, electromagnetic stimulation in association with the A. vera extract promoted an increase in the number of fibroblasts, GAGs content, MMP-2 activity, the deposition and organization of collagen fibers, favoring the repair of injuries to second degree burns, and may also present therapeutic potential in this injury type.

Electrical stimulation: Complementary therapy to improve the performance of grafts in bone defects?: Electrical Stimulation: Complementary Therapy

The limitations of bone reconstruction techniques have stimulated the tissue engineering for the repair of large bone defects using osteoconductive materials and osteoinductive agents. This study evaluated the effects of low intensity electric current on the inorganic bovine graft in calvaria defects. Bone defects were performed with piezoelectric system in the calvaria of Wistar rats divided into four groups (n = 24): (C) without grafting and without electrical stimulation; (E) with grafting; (MC) without grafting and submitted to electrical stimulation; (MC + E) with grafting and submitted to electrical stimulation. Inflammatory, angiogenic and osteogenic events during bone repair at the 10th, 30th, 60th, and 90th days were considered. Several inflammatory markers demonstrated the efficacy of grafting in reducing inflammation, particularly when subjected to electrical stimulation. Angiogenesis and collagen organization were more evident by electrical stimulation application on the grafts. Moreover, the osteogenic cell differentiation process indicated that the application of microcurrent on grafting modulated the homeostasis of bone remodeling. It is concluded that microcurrent favored the performance of grafts in calvarial rat model. Low-intensity electrical current might improve the osteoconductive property of grafting in bone defects. Therefore, electrical current becomes an option as complementary therapy in clinical trials involving bone surgeries and injuries.

Injured Achilles Tendons Treated with Adipose-Derived Stem Cells Transplantation and GDF-5

Tendon injuries represent a clinical challenge in regenerative medicine because their natural repair process is complex and inefficient. The high incidence of tendon injuries is frequently associated with sports practice, aging, tendinopathies, hypertension, diabetes mellitus, and the use of corticosteroids. The growing interest of scientists in using adipose-derived mesenchymal stem cells (ADMSC) in repair processes seems to be mostly due to their paracrine and immunomodulatory effects in stimulating specific cellular events. ADMSC activity can be influenced by GDF-5, which has been successfully used to drive tenogenic differentiation of ADMSC in vitro. Thus, we hypothesized that the application of ADMSC in isolation or in association with GDF-5 could improve Achilles tendon repair through the regulation of important remodeling genes expression. Lewis rats had tendons distributed in four groups: Transected (T), transected and treated with ADMSC (ASC) or GDF-5 (GDF5), or with both (ASC+GDF5). In the characterization of cells before application, ADMSC expressed the positive surface markers, CD90 (90%) and CD105 (95%), and the negative marker, CD45 (7%). ADMSC were also differentiated in chondrocytes, osteoblast, and adipocytes. On the 14th day after the tendon injury, GFP-ADMSC were observed in the transected region of tendons in the ASC and ASC+GDF5 groups, and exhibited and/or stimulated a similar genes expression profile when compared to the in vitro assay. ADMSC up-regulated Lox, Dcn, and Tgfb1 genes expression in comparison to T and ASC+GDF5 groups, which contributed to a lower proteoglycans arrangement, and to a higher collagen fiber organization and tendon biomechanics in the ASC group. The application of ADMSC in association with GDF-5 down-regulated Dcn, Gdf5, Lox, Tgfb1, Mmp2, and Timp2 genes expression, which contributed to a lower hydroxyproline concentration, lower collagen fiber organization, and to an improvement of the rats’ gait 24 h after the injury. In conclusion, although the literature describes the benefic effect of GDF-5 for the tendon healing process, our results show that its application, isolated or associated with ADMSC, cannot improve the repair process of partial transected tendons, indicating the higher effectiveness of the application of ADMSC in injured Achilles tendons. Our results show that the application of ADMSC in injured Achilles tendons was more effective in relation to its association with GDF-5.