Andrea Aparecida de Aro

Biochemical and anisotropical properties of tendons.

Tendons are formed by dense connective tissue composed of an abundant extracellular matrix (ECM) that is constituted mainly of collagen molecules, which are organized into fibrils, fibers, fiber bundles and fascicles helicoidally arranged along the largest axis of the tendon. The biomechanical properties of tendons are directly related to the organization of the collagen molecules that aggregate to become a super-twisted cord. In addition to collagen, the ECM of tendons is composed of non-fibrillar components, such as proteoglycans and non-collagenous glycoproteins. The capacity of tendons to resist mechanical stress is directly related to the structural organization of the ECM. Collagen is a biopolymer and presents optical anisotropies, such as birefringence and linear dichroism, that are important optical properties in the characterization of the supramolecular organization of the fibers. The objective of this study was to present a review of the composition and organization of the ECM of tendons and to highlight the importance of the anisotropic optical properties in the study of alterations in the ECM.

Structural and biochemical alterations during the healing process of tendons treated with Aloe vera

AIMS The tendon is composed of highly organized collagen fibers that form a complex supramolecular structure. After lesions, the organization and composition of the tendon are not completely restored. Our purpose was to evaluate if the application of Aloe vera improves tendon healing, considering the effectiveness in the stimulus of collagen synthesis. MAIN METHODS The calcaneal tendon of male Wistar rats was partially transected with subsequent topical application of A. vera ointment at the injury. The animals were separated into groups with tendons treated with the A. vera extract for 7days and excised on the 7th, 14th and 21st days after surgery; control rats received only ointment base without plant extract. KEY FINDINGS Morphological analysis using polarization microscopy showed that the entire tendon undergoes a remodeling process, with disorganized collagen fibers by days 7 and 14 in plant-treated and non-treated groups and with a higher birefringence in tendons of the plant-treated group on the 21st day. A higher concentration of hydroxyproline was found in plant-treated tendons on days 7 and 14 compared with their controls. Western blots showed lower amounts of type I collagen in the plant-treated group on day 14 compared with the control. MMP-9 diminished 14days after lesion and the active isoform of MMP-2 increased on day 21 in plant-treated groups. SIGNIFICANCE The present study indicates a beneficial effect of A. vera in the tissue reorganization in the transected region of the tendon 21days after injury and is supported by an increase of active MMP-2.

Effects of Acute Inflammation Induced in the Rat Paw on the Deep Digital Flexor Tendon

The tendon is commonly affected by inflammation, and in such situations, the tissue undergoes a process of reorganization of the extracellular matrix to improve and regenerate the affected region. Little is known about the mechanisms that trigger inflammation in the tissues surrounding the affected area. The objective of this study was to biochemically and morphologically analyze the deep digital flexor tendon at the peak of acute inflammation in the rat paw. Wistar rats were divided into the following three groups: those that received injection of 1% carrageenan, those that received 0.9% NaCl, and those that received nothing. The deep digital flexor tendon was divided into the distal, proximal, and intermediate regions. For biochemical analysis, the tendons were treated with guanidine hydrochloride and analyzed by sodium dodecyl sulfate-polyacrilamide gel electrophoresis. Proteins, glycosaminoglycans (GAGs), and hydroxyproline were quantified, and metalloproteinases were analyzed. The GAGs were analyzed by agarose gel electrophoresis. Tissue sections were stained with hematoxylin–eosin, toluidine blue, and Ponceau SS. The content of proteins and GAGs was smaller in the group receiving the application of carrageenan. The concentration of hydroxyproline in the two tendon regions that respond to tension forces was higher in the inflammation group. The metalloproteinase-9 was detected in the distal region, and a thicker epitenon with cellular infiltrate was observed in the groups with inflamed paws. Meanwhile, a better organization of collagen bundles was observed in the two tension regions of that same group. Our results show that although the tendon was not directly inflamed, changes in the surrounding structural and biochemical parameters were observed.

Arrabidaea chica extract improves gait recovery and changes collagen content during healing of the Achilles tendon

INTRODUCTION Tendon lesions are still a serious clinical problem. The leaves of the Bignoniaceae Arrabidaea chica (Humb. & Bonpl.) B. Verlot. (syn. Bignonia chica (Bonpl.)) have been used in traditional medicine and described in the literature for its healing properties. However, no study has shown the effects of A. chica during tendon healing. The aim of this study was to investigate the healing properties of the A. chica leaves extract on tendons after partial transection. METHODS A partial transection in the tension region of the Achilles tendon of rats was performed with subsequent posterior topical application of A. chica extract (2.13g/mL in 0.85% saline solution) at the site of the injury. The animals (n=154) were separated into 7 groups: N - rats with tendons without transection; S7, S14 and S21 - rats with tendons treated with topical applications of saline for 7 days and sacrificed on the 7th, 14th and 21st days after surgery, respectively; A7, A14 and A21 - rats with tendons treated with topical applications of the plant extract. The transected regions of the tendons were analyzed through biochemical, morphological and functional analyses. To evaluate the type and concentration of collagen, Western blotting for collagen types I and III was performed, and the hydroxyproline concentration was determined. The participation of metalloproteinases (MMP)-2 and -9 during tendon remodelling was investigated through zymography. Gait recovery was analyzed using the catwalk system. The organization of the extracellular matrix and morphometry were detected in sections stained with haematoxylin-eosin. RESULTS The application of A. chica extract in the region of tendon injury led to an increase in the amount of hydroxyproline (mg/g tissue) on the 7th (91.5±18.9) and 21st (95.8±11.9) days after the tendon lesion relative to the control groups treated with saline (S7: 75.2±7.2; and S21: 71.9±7.9). There were decreases in collagen types I and III (as determined by densitometry) in the groups treated with the plant extract 7 days after injury (type I: 103.9±15.9; type III: 206.3±8.1) compared to the saline-treated groups (type I: 165.2±31.1; type III: 338.6±48.8). The plant extract stimulated the synthesis of MMP-2 on the 21st day after the lesion and decreased the amount of latent and active isoforms of MMP-9 on the 14th day. Analysis by the catwalk system (max contact intensity) showed that the A. chica extract improved the gait of rats on the 7th day of the healing process when compared to the saline group. CONCLUSIONS The use of A. chica extract during the healing process of the tendon leads to an increase in collagen content and improved gait recovery. Further studies will be performed to analyze the effect of this plant extract on the organization of the collagen bundles of tendons after lesions and to study its probable anti-inflammatory effect.

Structural and Biochemical Analysis of the Effect of Immobilization Followed by Stretching on the Calcaneal Tendon of Rats

Little is known about the stretching effects on the biochemical and morphological features of tendons submitted to a long period of immobilization. Our purpose was to evaluate the response of rat tendons to stretching procedures after immobilization. The animals were separated into five experimental groups: GI—control of immobilized and euthanized animals; GII—immobilized and euthanized animals; GIII—control of immobilized animals and afterward stretched or allowed free cage activity; GIV—immobilized and stretched animals; and GV—immobilized and allowed free cage activity. Analysis in SDS-PAGE showed no remarkable differences among the groups, but a prominent collagen band was observed in GV, as compared to GIV and the control group, both in the compression and tension regions. Hydroxyproline content was highest in the compression region of GII. No differences among the groups were observed in the tension region. In regard to the concentration of noncollagenous proteins, differences were detected only in the tension region, where larger concentrations were found in the GII. When GII and GIV were compared, highest values were found in the GII. A more abundant presence of sulfated glycosaminoglycans, especially chondroitin sulfate, was detected in GIV, at the compression region of tendons. The presence of dermatan sulfate was outstanding in the compression and tension regions of the GII and GV groups. In the Ponceau SS stained sections, analyzed under polarization microscopy, GII exhibited the highest disorganization of the collagen bundles, partially recovered after stretching or with only remobilization. Our results indicate that a revision in the stretching procedures, in terms of duration and periodicity of the sessions, could benefit the efficiency of the stretching in cases of previous immobilization of tendons.

Effect of the Arrabidaea chica extract on collagen fiber organization during healing of partially transected tendon

AIMS After undergoing lesions, tendons have disorganized collagen fibers compared to undamaged tendons. Arrabidaea chica leaves have the aglycones carajurin and carajurone, components of the antocyanins, with a strong pharmacological potential due to their healing properties. Thus, the aim of this study was to investigate the effect of topical application of A. chica extract during tendon healing. MAIN METHODS The calcaneal tendon of Wistar rats was partially transected with subsequent treatment with A. chica extract (2.13 g/mL) followed by excision on the 7th, 14th and 21st days. Control rats received only saline treatment. KEY FINDINGS Transmission electron microscopy analysis showed the presence of a large amount of small segments of collagen fibrils in the transected region of the tendons on the 7th day in both the control and plant-treated groups. Considering the organization of the collagen fibers, higher values of birefringence were observed under polarization microscopy in the tendons of the plant-treated group on the 14th day compared to the control group. A larger quantity of dermatan sulfate was also detected after plant treatment in the same period. However, lesser dermatan and chondroitin sulfate were detected in the plant-treated group than in the control group on the 21st day. No differences were found in the values of birefringence between these groups. Intense metachromasy was observed in both transected groups on the 21st day. SIGNIFICANCE In conclusion, the use of A. chica extract improves collagen organization and increases the quantity of dermatan sulfate on the 14th day of the tendon healing.

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.

Effect of Calendula Officinalis Cream on Achilles Tendon Healing

In recent years, the scientific community has undertaken research on plant extracts, searching for compounds with pharmacological activities that can be used in diverse fields of medicine. Calendula officinalis L. is known to have antioxidant, anti‐inflammatory, antibacterial, and wound healing properties when used to treat skin burns. Therefore, the purpose of this study was to analyze the effects of C. officinalis on the initial phase of Achilles tendon healing. Wistar rats were separated in three groups: Calendula (Cal)—rats with a transected tendon were treated with topical applications of C. officinalis cream and then euthanized 7 days after injury; Control (C)—rats were treated with only vehicle after transection; and Normal (N)—rats without tenotomy. Higher concentrations of hydroxyproline (an indicator of total collagen) and non‐collagenous proteins were observed in the Cal group in relation to the C group. Zymography showed no difference in the amount of the isoforms of metalloproteinase‐2 and of metalloproteinase‐9, between C and Cal groups. Polarization microscopy images analysis showed that the Cal group presented a slightly higher birefringence compared with the C group. In sections of tendons stained with toluidine blue, the transected groups presented higher metachromasy as compared with the N group. Immunocytochemistry analysis for chondroitin‐6‐sulfate showed no difference between the C and Cal groups. In conclusion, the topical application of C. officinalis after tendon transection increases the concentrations of collagen and non‐collagenous proteins, as well as the collagen organization in the initial phase of healing. Anat Rec, 298:428–435, 2015.

Inflammatory process induced by carrageenan in adjacent tissue triggers the acute inflammation in deep digital flexor tendon of rats.

Tendinopathy is a pathology found mainly in the rotator cuff, patellar, Achilles and flexor tendons. Tendinopathy is a significant impediment to performance in athletes and in workers in the labor market. Some studies have indicated that inflammation in adjacent tissues may affect the rotator cuff and Achilles tendon. In this study alterations were verified in the extracellular matrix (ECM) of the deep digital flexor tendon after two periods (12 and 24 hr) of induction inflammation in rat paw. Wistar rats were divided into three groups: those that received injection of 1% carrageenan; those that received 0.9% NaCl; and those that received no application. The tendon was divided into distal (d), proximal (p), and intermediate (i) regions. Biochemical analyses were performed and included non‐collagenous proteins (NCP), glycosaminoglycans (GAGs), hydroxyproline (HoPro) and metalloproteinases 2 and 9. Tissue sections were stained with toluidine blue, hematoxylin‐eosin, and Ponceau SS and observed under polarization microscopy. Remarkable results were detected that included the presence of MMP‐9, degradation of NCP and GAG and the presence of cellular infiltrate closer to digits in d region. The different concentrations of HoPro, as well as alterations in the organization of the collagen fibers showed the collagenous matrix undergoing some alterations. The results indicated that the induced inflammation in rat paw exhibited characteristics similar to the typical acute inflammatory process observed in tendons. Anat Rec, 2013.

Analysis of the Deep Digital Flexor Tendon in Rats Submitted to Stretching after Immobilization

Few studies have analyzed the effect of stretching after immobilization on the structural and biochemical properties of tendons. Here, the effect of stretching and immobilization on the proximal (p), intermediate (i), and distal (d) regions of the deep digital flexor tendon in rats was analyzed. The d region was subjected to compression and tension forces, the i region was subjected to compressive forces and the p region received tension forces. Rats were separated into five groups: GI—control for GII; GII—immobilized rats; GIII—control for GIV and GV groups; GIV—immobilized and stretched rats; and GV—immobilized rats which were allowed free cage activity. GII showed a higher molecular organization in the d and p regions as detected by measuring optical retardation, a lower concentration of hydroxyproline in the i region and a significant decrease in noncollagenous proteins found in the three regions of the tendon. Regarding the glycosaminoglycans, diminishing dermatan sulfate and the absence of chondroitin sulfate in the i region were observed in GII when compared to GI. However, in the same region of GIV, higher concentrations of chondroitin and dermatan sulfate were observed along with a strong metachromasy. An increase in hydroxyproline content in the i region and a higher molecular organization in the d and p regions were observed in GIV. Apparently, the active isoforms of metalloproteinase-2 also increased after stretching in all regions. These results suggest that stretching after immobilization contributed to the increase in molecular organization and to the synthesis of extracellular matrix components.