Influência de inibidores de metaloproteinases na degradação da camada híbrida / Influence of metalloproteinase inhibitors on hybrid layer degradation

Objetivo: realizar uma revisão de literatura acerca da eficácia de utilização da clorexidina (CHX) e de outros tipos de inibidores de metaloproteinases (MMPs) na resistência de união da camada híbrida. Métodos: a busca bibliográfica foi realizada na base de dados PubMed, nos meses de novembro e dezembro de 2018. A pesquisa ocorreu em três fases, com os descritores previamente selecionados. Foram incluídas publicações dos últimos 10 anos no formato de pesquisas científicas realizadas in vitro ou in vivo. Após análise, obedecendo aos critérios de inclusão e exclusão, foram incluídos sete estudos na presente revisão. Resultados/Revisão de literatura: na interface adesiva, os estudos mostram que as MMPs são ativadas durante a etapa de ataque ácido realizada nos protocolos de aplicação de sistemas adesivos, podendo ser ativada tanto por procedimentos adesivos com condicionamento ácido prévio como por sistemas adesivos autocondicionantes. Além da CHX, outras substâncias foram pesquisadas e se mostraram eficazes na inibição de MMPs. Considerações finais: por meio da inibição da atividade das MMPs, é possível obter uma maior durabilidade da interface adesiva e uma menor degradação hidrolítica do colágeno presente na camada híbrida. (AU)

Objective: to perform a literature review on the efficacy of chlorhexidine (CHX) and other types of metalloproteinase inhibitors (MMPs) on hybrid layer bond strength. Methods: the bibliographic search was performed in PubMed, in the months of november and december of 2018. The research was carried out in three phases with the previously selected descriptors. Publications have been included in the last 10 years in the form of scientific research conducted in vitro or in vivo. After analysis, following the inclusion and exclusion criteria, 7 studies were included in the present review. Results / Literature review: in the adhesive interface, the studies show that the MMPs are activated during the acid attack stage carried out in the application protocols of adhesive systems, and can be activated either by adhesive procedures with prior acid conditioning or self-etching adhesive systems. In addition to CHX, other substances were investigated and shown to be effective in inhibiting MMPs. Final considerations: through the inhibition of the MMPs activity it is possible to obtain a greater durability of the adhesive interface and lower hydrolytic degradation of the collagen present in the hybrid layer. (AU)

Os efeitos das Metaloproteinases da matriz extracelular - MMPS e clorexidina no mecanismo de adesão dentária / The effects of metaloproteinases of the extracellular matrix - MMPs and chlorexidine in the dental adhesion mechanism

Introdução: A adesão da resina composta à dentina ocorre pela formação da camada híbrida. Assim, sua degradação ocasiona a perda da resistência de união na interface resina/dentina, influenciando na longevidade da restauração. Após o condicionamento ácido e aplicação do sistema adesivo na dentina desmineralizada, fibras colágenas não envolvidas por sistema adesivo ficam desprotegidas e suscetíveis ao ataque das metaloproteinases (MMPs). Objetivos: Esta revisão buscou esclarecer o efeito das MMPs na degradação da camada híbrida e os efeitos da clorexidina no processo de adesão. Materiais e métodos: Foi realizada uma revisão da literatura por meio de uma busca bibliográfica nas bases de dados Pubmed/ Medline, Scielo e Google Acadêmico, utilizados estudos publicados nos anos de 2005 a 2018. Foi realizada a busca pelos seguintes descritores: Dentistry, MMPs, Chlorhexidine. Resultados: Estas enzimas, presentes na própria dentina, são reativadas pelo ácido fosfórico ou pelos monômeros ácidos dos adesivos autocondicionantes e iniciam a degradação. A aplicação da clorexidina (CHX) na dentina, após o condicionamento ácido, impede ou retarda a degradação das fibras de colágeno da camada híbrida. Conclusão: Concluiu-se que a ligação adesiva à dentina diminui com o passar dos anos devido à ação das MMPs que degradam o colágeno não infiltrado por monômeros adesivos na parte mais profunda da camada híbrida. Além disso, a clorexidina como inibidor terapêutico em sistemas adesivos convencionais é capaz de inibir as MMPs e assim a ligação adesiva à dentina pode ser mantida estável por um período de tempo mais longo.

Introduction: The adhesion of the composite resin to the dentin occurs by the formation of the hybrid layer. Thus, its degradation causes loss of union resistance on interface resin / dentin interface, directly influencing the longevity of the restoration. After the acid etching and the application of the adhesive system into demineralized dentin, collagen fibers not involved by adhesive system get unprotected and susceptibles to attack by metalloproteinases (MMPs). The enzymes, present in the dentin itself, are rehabilitated by phosphoric acid or by the acids monomers of the self-etching adhesives initiating degradation. The application of chlorhexidine (CHX) in the dentin, after acid conditioning, prevents or slows down the degradation of the collagen fibers of the hybrid layer. This literature review sought to clarify the effect of MMPs on the degradation of the hybrid layer and the effects of chlorhexidine on the adhesion process. It was concluded that the adhesive bonding to dentin decreases with the passage of years due in part to the action of MMPs, which degrade collagen not infiltrated by adhesive monomers in the deepest part of the hybrid layer. In addition, the use of chlorhexidine as a therapeutic inhibitor in conventional adhesive systems is capable of inhibiting the MMPs and thus the adhesive bonding to the dentin can be kept stable for a longer period of time.

Extracellular matrix remodeling and TGF-ß1/Smad signaling in diabetic colon mucosa.

Diabetes is associated with metabolic and functional alterations in the gut. Using an experimental model of streptozotocin (STZ)-induced diabetes in rodents, we analyzed the extracellular matrix (ECM) and TGF-ß/Smad signaling in the colon mucosa. Male rats were divided into normal control, diabetic and insulin treated diabetic groups during 4 and 9 weeks. Sirius red staining showed marked increase in the extracellular matrix deposition in diabetic mucosa. High levels of fibrillar collagen (I and III) and fibronectin mRNAs were also detected with an imbalance between MMPs/TIMPs activities. Moreover, an increased mesenchymal cell proliferation together with an enhanced expression of myofibroblasts markers vimentin and α-SMA were observed. TGF-ß/Smad signaling-related genes were determined using RT-PCR, Western blotting, and immunohistochemistry. Diabetic rats showed a significant up-regulation of TGF-ß1, TGF-ß receptors and the effectors p-Smad2/3 in the mucosa compared with control rats. Insulin treatment attenuated the stimulating effect of diabetes on colon ECM deposition and TGF-ß/Smad signaling. In conclusion, the overall results showed a deregulation of the TGFß1 pathway associated with the appearance of myofibroblasts and the accumulation of ECM in the mucosa of diabetic colon. These data provide the first in vivo evidence that TGF-ß1/Smad is a key component of intestinal tissue remodeling in diabetes.

Quantitative analysis of collagen fibrillar structure in canine uterus exposed to acetate of medroxyprogesterone using second harmonic generation microscopy / Análisis cuantitativo de la estructura de fibras colágenas en útero canino expuesto a acetato de medroxiprogesterona utilizando generación microscópica de un segundo harmónico

Collagen is the most abundant structural protein found in organs and is responsible for providing tissues with structure and function. In order to investigate in canine uteri the potential effect of medroxyprogesterone acetate (MPA) on the changes in collagen deposition were grouped as nulliparous (n=11), multiparous (n=11) and treated with MPA (n=11; nulliparous with two treatments; 5 mg/kg; i.m.). The amount of collagen was studied in the fold and basal regions of the endometrium and myometrium using second harmonic generation with a two-photon spectral confocal microscope, quantified using ImageJ software with a color segmentation plugin, was expressed as fraction area (%) and analyzed by ANOVA (p<0,05). No differences were observed between groups in the fold (p=0,3995) or base (p=0,7392) of the endometrium and myometrium (p=0,1781). In conclusion, our data demonstrate that two doses of MPA (5 mg/kg; i.m.) do not affect the total collagen deposition in canine uteri undergoing contraceptive treatment.

El colágeno es la proteína estructural más abundante presente en órganos y es responsable de proporcionar la sostén y función a los tejidos. Para investigar en caninos el efecto potencial del acetato de medroxiprogesterona (MPA) sobre cambios en el depósito de colágeno en útero, éstos fueron agrupados como nulíparos (n = 11), multíparos (n = 11) y tratados con MPA (n = 11, nulíparos con dos tratamientos 5 mg/kg, im). El colágeno fue evaluado en el pliegue y regiones basales del endometrio y en miometrio utilizando la Generación de un Segundo Harmónico con un microscopio confocal espectral y dos fotones y cuantificado utilizando el software ImageJ a partir de la segmentación de colores. Los resultados fueron expresados y analizados como fracción de área (%; ANOVA; p<0,05). No se observaron diferencias entre los grupos en el pliegue (p = 0,3995) y base (p=0,7392) del endometrio y tampoco en miometrio (p=0,1781). En conclusión, nuestra evidencia demuestra que dos dosis de MPA (5 mg/kg, i.m.) no afectan el depósito total de colágeno en úteros caninos expuestos a tratamiento anticonceptivo.

Effects of glycosylated (2S,4R)-hydroxyproline on the stability and assembly of collagen triple helices.

Functionalized collagen-mimetic peptides (CMPs) have been widely used in the preparation of collagen-related biomaterials. Among the reported results, the induced noncovalent interactions between the implanted functional groups or moieties were frequently the key elements to promote the self-assembly of small CMPs. In this work, we designed and synthesized a series of glycosylated CMPs in which 4-O-[ß-D-galactopyranosyl]-(2S,4R)-4-hydroxyproline (Hyp(Gal)) was incorporated to explore the effects of glycosylation on the stability and assembly of collagen triple helices. Circular dichroism measurements showed that glycosylation of hydroxyproline slightly destabilized the collagen triple helices, but did not reduce their refolding rate. Compared to non-glycosylated CMPs, the incorporation of Hyp(Gal) speeded up the assembly of CMPs, indicating that this modification could assist the self-assembly of CMPs into higher-order structures, such as fibrils. O-Galactosylation of hydroxyproline imposes contrary effects on the triple helix stability and the self-assembly rate of collagen triple helices, exhibiting a piece of important and useful information for designing collagen-related biomaterials. Our finding also suggests that instead of stabilizing the triple helical conformation of CMPs, installing additional forces between CMPs could be a crucial factor to promote the assembly of CMPs into large-scale constructs.

Sorafenib prevents liver fibrosis in a non-alcoholic steatohepatitis (NASH) rodent model.

Liver fibrosis occurring as an outcome of non-alcoholic steatohepatitis (NASH) can precede the development of cirrhosis. We investigated the effects of sorafenib in preventing liver fibrosis in a rodent model of NASH. Adult Sprague-Dawley rats were fed a choline-deficient high-fat diet and exposed to diethylnitrosamine for 6 weeks. The NASH group (n=10) received vehicle and the sorafenib group (n=10) received 2.5 mg·kg(-1)·day(-1) by gavage. A control group (n=4) received only standard diet and vehicle. Following treatment, animals were sacrificed and liver tissue was collected for histologic examination, mRNA isolation, and analysis of mitochondrial function. Genes related to fibrosis (MMP9, TIMP1, TIMP2), oxidative stress (HSP60, HSP90, GST), and mitochondrial biogenesis (PGC1α) were evaluated by real-time quantitative polymerase chain reaction (RT-qPCR). Liver mitochondrial oxidation activity was measured by a polarographic method, and cytokines by enzyme-linked immunosorbent assay (ELISA). Sorafenib treatment restored mitochondrial function and reduced collagen deposition by nearly 63% compared to the NASH group. Sorafenib upregulated PGC1α and MMP9 and reduced TIMP1 and TIMP2 mRNA and IL-6 and IL-10 protein expression. There were no differences in HSP60, HSP90 and GST expression. Sorafenib modulated PGC1α expression, improved mitochondrial respiration and prevented collagen deposition. It may, therefore, be useful in the treatment of liver fibrosis in NASH.

Quantitative imaging of enzymatic vitreolysis-induced fiber remodeling.

PURPOSE: Collagen fiber remodeling in the vitreous body has been implicated in cases of vitreomacular traction, macular hole, and retinal detachment, and also may occur during pharmacologic vitreolysis. The purpose of this study was to evaluate quantitative polarized light imaging (QPLI) as a tool for studying fiber organization in the vitreous and near the vitreoretinal interface in control and enzymatically perturbed conditions. METHODS: Fiber alignment was measured in anterior-posterior sections of bovine and porcine vitreous. Additional tests were performed on bovine lenses and nasal-temporal vitreous sections. Effects of proteoglycan degradation on collagen fiber alignment using trypsin and plasmin were assessed at the microstructural level using electron microscopy and at the global level using QPLI. RESULTS: Control vitreous showed fiber organization patterns consistent with the literature across multiple-length scales, including the global anterior-posterior coursing of vitreous fibers, as well as local fibers parallel to the equatorial vitreoretinal interface and transverse to the posterior interface. Proteoglycan digestion with trypsin or plasmin significantly increased fiber alignment throughout the vitreous (P < 0.01). The largest changes (3×) occurred in the posterior vitreous where fibers are aligned transverse to the posterior vitreoretinal interface (P < 0.01). CONCLUSIONS: Proteoglycan loss due to enzymatic vitreolysis differentially increases fiber alignment at locations where tractions are most common. We hypothesize that a similar mechanism leads to retinal complications during age-related vitreous degeneration. Structural changes to the entire vitreous body (as opposed to the vitreoretinal interface alone) should be evaluated during preclinical testing of pharmacological vitreolysis candidates.

Proanthocyanidins rapidly stabilize the demineralized dentin layer.

While proanthocyanidins (PA) are effective in improving collagen's resistance to collagenolytic degradation, the direct incorporation of PA into an adhesive system is detrimental to the light-curing thereof. Conversely, the use of PA as a primer could circumvent this issue, but little is known about the efficacy of PA in stabilizing collagen when applied in a clinically relevant manner. This study investigated the pre- and post-digestion morphology of an acid-etched dentin collagen layer that underwent PA treatment for time periods on a scale of seconds. The null hypothesis, that there is no difference between the PA-treated and untreated control group, had to be rejected, since it was revealed that the untreated control could not survive 1 hr of exogenous collagenase digestion, while the PA-treated collagen could sustain at least 16 hrs of digestion with no perceptible changes in collagen structure. In addition, the stabilizing effect of the gold-standard cross-linker glutaraldehyde at comparable experimental conditions was found to be almost non-existent within the 5, 15, or 30 sec of cross-linking permitted. Therefore, PA have been proven to be extraordinarily efficient in stabilizing demineralized dentin collagen against enzymatic challenges in a clinically relevant setting, likely due to the non-covalent nature of their interaction with collagen molecules.

Single-arm study for the characterization of human tissue response to injectable poly-L-lactic acid.

BACKGROUND: Injectable poly-L-lactic acid (PLLA) is a synthetic polymer indicated for the correction of facial wrinkles and folds. Animal studies have shown that implantation of PLLA stimulates collagen synthesis; human studies have been limited. OBJECTIVE: To investigate human tissue response to injectable PLLA. METHODS AND MATERIALS: In this exploratory single-arm, open-label study, 14 healthy subjects were administered injectable PLLA; punch biopsies at 3, 6, and 12 months were analyzed for qualitative and quantitative changes from baseline in collagen types I and III and assessed for inflammatory responses. RESULTS: Quantitative and qualitative increases were observed for collagen types I and III at 3 and 6 months and were statistically significant for collagen type I at 3 and 6 months. Post hoc analyses at 12 months showed nominal collagen increases but were hindered by technical difficulties. The degree of inflammatory response was similar to baseline at 3, 6, and 12 months; all subjects were found to have no or mild inflammation after baseline. Adverse events were mild and among those reported previously. CONCLUSION: Results of this study in humans found statistically significant stimulation of collagen type I with no or mild inflammatory response after administration of injectable PLLA.

A possible mechanism of basic fibroblast growth factor-promoted scarless wound healing: the induction of myofibroblast apoptosis.

Although recent clinical reports have indicated that recombinant basic fibroblast growth factor (bFGF) promotes scarless wound healing, the mechanism remains unclear. The present study was carried out to elucidate the mechanisms. The protein levels of cellular α-smooth muscle actin increased at 2-4 days after TGFß treatment alone and at 4 to 6 days after a costimulation of bFGF and TGFß. A spontaneous contraction of stressed myofibroblast-collagen matrix was cancelled by bFGF, which was restored under the presence of C3 exotransferase or Y27632. bFGF stimulation of myofibroblasts as well as fibroblasts elicited a transient Rac and Rho activation. bFGF promoted apoptosis of the myofibroblasts but not of the fibroblasts, even in the presence of two different inhibitors, either LY294002 or an Akt inhibitor. The present study suggests that the phosphatidylinositol-3-kinase to Akt as well as the Rho to Rho kinase signaling pathway is involved in bFGF-promoted myofibroblast apoptosis, and bFGF can promote the scarless wound healing upon the induction of apoptosis of myofibroblasts, but not fibroblasts.

Dextrose prolotherapy and corticosteroid injection into rat Achilles tendon.

PURPOSE: To assess the mechanical behavior and the histology of collagen fibers after prolotherapy with 12.5% dextrose into rat Achilles tendons and to compare with those of corticosteroid treatment. METHODS: Out of 60 adult female Wistar rats (70 tendons), 15 received 12.5% dextrose (group I); 15 were treated with corticosteroid injection (group II); and 15 were given 0.9% saline injection (group III), all into the right Achilles tendon, whereas 13 animals received no injections (group IV). Three doses of each substance (groups I, II, and III) were given at a 5-day interval. Collagen fiber color was quantitatively assessed in three samples from each group and in five samples from the control group using picrosirius red staining under polarized and nonpolarized light. Twelve tendons from each group treated with the test substance and 20 tendons from the control group were submitted to the tensile strength test. RESULTS: There was no statistical difference across the groups with respect to maximum load at failure (n.s.) and absorbed energy (n.s.). With respect to tendon rupture, there was no difference between the myotendinous and the tendinous regions (n.s.). However, hematoxylin-eosin staining revealed statistical significance in lymphocytic inflammatory infiltrate (P = 0.008) and in parallel fiber orientation (P = 0.003) when comparing groups to the control group, without significance for either neovascularization (n.s.) or the presence of fibroblasts (n.s.). Likewise, there was no significant difference between the percentage of mature (n.s.) and immature (n.s.) fibers. CONCLUSIONS: Dextrose was not deleterious to the tendinous tissue, as it did not change the mechanical and histological properties of Achilles tendons in rats. The data obtained in this study may help clinicians in their daily work as they suggest that injections of 12.5% dextrose caused no harm to the tendons, although the clinical importance in humans still needs to be defined.

Exposed collagen in aged resin-dentin bonds produced on sound and caries-affected dentin in the presence of chlorhexidine.

PURPOSE: To evaluate the effect of chlorhexidine on the presence of collagen in aged resin-dentin bonds produced on sound and caries-affected dentin. MATERIALS AND METHODS: Flat dentin surfaces were obtained from 16 sound molars, from which 8 were microbiologically processed for induction of caries. Single Bond 2 was applied to both sound and caries-affected substrates. In half of the teeth assigned for 6-month storage in water, the phosphoric acid demineralized dentin was impregnated with 2% chlorhexidine before the application of the adhesive. Specimens (2 x 2 x 5 mm) were produced and stored in water for 24 h, or 6 months in either water or mineral oil. The specimens were subjected to histological processing and sections were stained with Goldner's Trichrome. The thickness of the zone of exposed collagen was measured by optical microscopy and the data were subjected to two-way ANOVA and Tukey's test (α = 0.05). RESULTS: There was no statistically significant difference (p > 0.05) between sound and caries-affected dentin regardless of the storage condition. For both substrates, significantly greater collagen exposure was observed after 6 months in water. Chlorhexidine-treated groups resulted in similar collagen exposure to that of the control and 6 months in water groups (p > 0.05), while no increase of the exposed collagen zone was observed after mineral oil storage. CONCLUSION: Aging in water resulted in degradation of the resin-dentin bond, as demonstrated by the increase of the zone of exposed collagen. However, the degradation of the exposed collagen was decelerated in the presence of chlorhexidine.

Degradation of human collagen isoforms by Clostridium collagenase and the effects of degradation products on cell migration.

Clostridium collagenase has been widely used in biomedical research to dissociate tissues and isolate cells; and, since 1965, as a therapeutic drug for the removal of necrotic wound tissues. Previous studies found that purified collagenase-treated extracellular matrix stimulated cellular response to injury and increased cell proliferation and migration. This article presents an in vitro study investigating the digestive ability of Clostridium collagenase on human collagen types I, III, IV, V and VI. Our results showed that Clostridium collagenase displays proteolytic power to digest all these types of human collagen, except type VI. The degradation products derived were tested in cell migration assays using human keratinocytes (gold surface migration assay) and fibroblasts (chemotaxis cell migration assay). Clostridium collagenase itself and the degradation products of type I and III collagens showed an increase in keratinocyte and fibroblast migration, type IV-induced fibroblast migration only, and the remainder showed no effects compared with the control. The data indicate that Clostridium collagenase can effectively digest collagen isoforms that are present in necrotic wound tissues and suggest that collagenase treatment provides several mechanisms to enhance cell migration: collagenase itself and collagen degradation products.

Polarized light microscopic analysis of bone formation after inhibition of cyclooxygenase 1 and 2.

Potassium diclofenac is a potent nonsteroidal anti-inflammatory drug (NSAID) and COX isoforms (COX-1 and COX-2) inhibitor. Quantitative analysis of birefringence with polarized light microscopy is a useful method to investigate the macromolecular orientation and organization of collagen fibers in connective tissues. The aim of this research was to analyze the collagen structure and maturation in bone formed after potassium diclofenac administration, during first molar orthodontic movement. Sixty Wistar rats were divided in two equal groups (N = 30): control (C) and potassium diclofenac (PD). The animals in Group C received 0.9% saline solution and the PD group received potassium diclofenac Cataflam (5 mg/kg). Animals were sacrificed 3, 7, or 14 days after a NiTi unilateral closed-coil spring was stretched between the upper right first molar and the incisors. The first molar area was fixed, decalcified, and histologically processed using picrosirius pigment. The collagen birefringence of bone turnover was analyzed by phase retardation. Two-way ANOVA and Tukey's test showed that optical retardation was influenced by time and treatment. There was increase in the collagen organization over time. On the third day, the C group showed better collagen organization than the PD group. Potassium diclofenac interfered in collagen maturation, reducing fibril organization in the initial phase of orthodontic movement.

Comparative study of clinicofunctional staging of oral submucous fibrosis with qualitative analysis of collagen fibers under polarizing microscopy.

OBJECTIVE: The aim of the study was to assess the severity of the disease in oral submucous fibrosis (OSF), correlate the clinical, functional staging with histopathological staging, and analyze collagen distribution in different stages of OSF using the picrosirius red stain under polarizing microscopy. MATERIALS AND METHODS: The study included randomly incorporated 50 subjects, of whom 40 were patients with OSF, and 10 were in the control group. Clinical, functional staging in OSF cases was done depending upon definite criteria. A histopathological study was conducted using the hematoxylin and eosin stain and picrosirius red stain. Collagen fibers were analyzed for thickness and polarizing colors. Furthermore, clinical, functional, and histopathological stages were compared. STATISTICAL ANALYSIS: Descriptive data which included mean, SD, and percentages were calculated for each group. Categorical data were analyzed by the chi-square test. Multiple group comparisons were made by one-way ANOVA followed by Student's t-test for pairwise comparisons. For all tests, a P-value of 0.05 or less was considered for statistical significance. RESULTS: As the severity of the disease increased, clinically, there was definite progression in subjective and objective symptoms. Polarized microscopic, examination revealed, there was a gradual decrease in the green-greenish yellow color of the fibers and a shift to orange red-red color with increase in severity of the disease. Thereby, it appeared that the tight packing of collagen fibers in OSF progressively increased as the disease progressed from early to advanced stages. We observed that the comparison of functional staging with histopathological staging was a more reliable indicator of the severity of the disease. CONCLUSION: In the present study, we observed that mouth opening was restricted with advancing stages of OSF. The investigation also points to the importance of assessing the cases of OSF, especially with regard to functional and histological staging in planning the treatment.

Sugar (sucrose) and fatty acid compounds with triglycerides on the treatment of wounds: experimental study in rats.

BACKGROUND: In the last 10 years, the use of essential fatty acids (EFA) compounds for the treatment of wounds has increased in Brazil, while there has been reducing indication for the use of sugar. OBJECTIVE: The aim of this study was to evaluate the response to saline, sugar and EFA on induced wounds in rats. METHODS: A wound of 400 mm(2) was produced on the back of each Wistar rat. The rats were divided into three groups, each being treated with either saline, sugar or EFA. All the animals received a closed dressing on the wounds, changed daily. Measures were taken in four moments, and the values of wound area reduction by healing, cellular inflammatory response, collagen ordering and types I and III collagen density were assessed. RESULTS: The wound healing was equal in all the three groups, but sugar promoted an inflammatory response modulation between the 7th and 14th days. On the 20th post-operative day, there were no differences between the three treated groups concerning types I and III collagen. CONCLUSIONS: The wounds healed in the three groups. The sugar group promoted effective cellular inflammatory response modulation. There were no differences between all the treated groups regarding types I and III collagen at the end of this study.

Açúcar (sacarose) e triglicerídeos de cadeia média com ácidos graxos essenciais no tratamento de feridas cutâneas: estudo experimental em ratos / Sugar (sucrose) and fatty acid compounds with triglycerides on the treatment of wounds: experimental study in rats

FUNDAMENTOS: Na última década, as indicações de uso tópico de compostos com ácidos graxos essenciais (AGE-TG) para o tratamento de feridas aumentaram no Brasil, e houve declínio das indicações do açúcar. OBJETIVOS: Estudar o efeito da aplicação de solução fisiológica de cloreto de sódio a 0,9 por cento, de açúcar e de AGE-TG sobre feridas experimentalmente induzidas em ratos. MÉTODOS: Foi induzida uma ferida de 400 mm2 no dorso de cada rato Wistar, constituindo três grupos tratados separadamente com solução fisiológica de cloreto de sódio a 0,9 por cento, açúcar e AGE-TG. Todos os animais receberam curativo oclusivo sobre a lesão, trocado a cada 24 horas. As aferições realizadas em quatro momentos consistiram na determinação do percentual de redução das áreas das feridas, da reação inflamatória celular, da ordenação do colágeno e da densidade de colágeno dos tipos I e III nas cicatrizes. RESULTADOS: A cicatrização ocorreu igualmente nos grupos estudados, mas o açúcar modulou positivamente a reação inflamatória entre o 7º e 14º dias. No 20º dia, não houve diferenças na quantidade de colágeno dos tipos I e III entre os grupos tratados. CONCLUSÕES: As feridas cicatrizaram nos três grupos. O grupo açúcar apresentou uma modulação positiva da resposta inflamatória celular. Não houve diferenças na quantidade de colágeno dos tipos I e III ao final do experimento nos grupos tratados.

BACKGROUND: In the last 10 years, the use of essential fatty acids (EFA) compounds for the treatment of wounds has increased in Brazil, while there has been reducing indication for the use of sugar. OBJECTIVE: The aim of this study was to evaluate the response to saline, sugar and EFA on induced wounds in rats. METHODS: A wound of 400 mm2 was produced on the back of each Wistar rat. The rats were divided into three groups, each being treated with either saline, sugar or EFA. All the animals received a closed dressing on the wounds, changed daily. Measures were taken in four moments, and the values of wound area reduction by healing, cellular inflammatory response, collagen ordering and types I and III collagen density were assessed. RESULTS: The wound healing was equal in all the three groups, but sugar promoted an inflammatory response modulation between the 7th and 14th days. On the 20th post-operative day, there were no differences between the three treated groups concerning types I and III collagen. CONCLUSIONS: The wounds healed in the three groups. The sugar group promoted effective cellular inflammatory response modulation. There were no differences between all the treated groups regarding types I and III collagen at the end of this study.

Role of collagen fibers in acupuncture analgesia therapy on rats.

Acupuncture, a traditional Chinese therapeutic technique, has been put into practice for more than 4000 years and widely used for pain management since 1958. However, what is the mechanism underlying the acupuncture for analgesia effects by stimulation of acupoints, what substances receive the original mechanical acupuncture signals from the acupoints, or what transforms these signals into effective biological signals are not well understood. In this work, the role of collagen fibers at acupoints during acupuncture analgesia on rats was investigated. When the structure of the collagen fibers at Zusanli (ST36) was destroyed by injection of type I collagenase, the needle force caused by the acupuncture declined and the analgesic effects of rotation or lift-thrusting manipulations was attenuated accompanying the restraint of the degranulation ratios of mast cells. We propose that collagen fibers play an important role in acupuncture-induced analgesia, and they participate in signal transmission and transform processes.

Fasudil attenuates myocardial fibrosis in association with inhibition of monocyte/macrophage infiltration in the heart of DOCA/salt hypertensive rats.

OBJECTIVE: To determine the effects of fasudil, a Rho-kinase inhibitor, on mineralocorticoid-induced myocardial remodeling, we investigated whether fasudil would suppress myocardial fibrosis and inflammation in deoxycorticosterone-acetate (DOCA)/salt hypertensive rats. METHODS: Sprague-Dawley rats treated with DOCA combined with 1% NaCl and 0.2% KCl in the drinking water after receiving left nephrectomy were given fasudil (10 mg/kg/day; n = 20) or vehicle (n = 20). Systolic blood pressure (SBP) was measured biweekly. Myocardial monocyte/macrophage infiltration and myocardial fibrosis were determined histologically. Expressions of mRNA of procollagen I (PI), procollagen III (PIII), monocyte chemoattractant protein (MCP)-1, interleukin (IL)-6, type-1 plasminogen activator inhibitor (PAI-1), transforming growth factor (TGF)-beta1, and c-fos were determined. RESULTS: SBP was significantly increased on day 14 after treatment with DOCA/salt. Extent of interstitial and perivascular fibrosis was significantly increased on day 28. Expressions of mRNA of PI, PIII, MCP-1, IL-6, PAI-1, TGF-beta1, and c-fos were significantly increased on day 14. Although SBP did not differ between the fasudil and vehicle groups, extent of monocyte/macrophage infiltration and fibrosis was attenuated in the fasudil group. Expressions of mRNA of these factors except TGF-beta1 were also attenuated. CONCLUSION: Fasudil attenuates myocardial fibrosis possibly via suppression of monocyte/macrophage infiltration of the heart in DOCA/salt hypertensive rats.

Extracellular matrix containing mutated fibrillin-1 (Fbn1) down regulates Col1a1, Col1a2, Col3a1, Col5a1, and Col5a2 mRNA levels in Tsk/+ and Tsk/Tsk embryonic fibroblasts.

It is known that the extracellular matrix (ECM) is able to signal to cells and thereby direct or modulate the transcription of certain mRNAs. This signaling plays an important role in tumor invasion and metastasis, wound healing, remodeling of the ECM and cell differentiation. There are several mechanisms whereby the ECM signals cells to change their metabolism: (1) receptor molecules binding to specific domains in the ECM, (2) direct phagocytosis of the ECM molecules or domains into the cell, (3) structural changes of the ECM domains. We report the effect of an ECM containing either mutant or normal Fbn1 on the transcription levels of several collagen mRNAs. Tsk/Tsk, Tsk/+ and +/+ mouse embryonic fibroblast cell lines were used. Tsk/Tsk cells produce only mutated fibrillin-1 which arises from mRNA containing an in-frame duplication of exons 17-40. To test the effect of the ECM containing mutant Fbn1, cells of the Tsk/Tsk, Tsk/+ and the wild-type (+/+) genotype were each grown on an ECM produced by either Tsk/Tsk, Tsk/+ cells or by wild-type cells (+/+). The embryonic cells were genotyped by Northern analyses for Fbn1 and grown to confluence. The cultures were then harvested and the cells removed, leaving the matrix in the flasks. Matrices produced from Tsk/Tsk, Tsk/+ and from +/+ cells were reseeded with Tsk/Tsk cells, Tsk/+ cells or +/+ cells. The cells were plated at a confluent concentration and incubated on the matrices for 48 h, after which total RNA was harvested and cDNA generated. Real-time PCR using cDNA or Northern analyses using RNA were performed for Fbn1 and Types I, III and V collagens. The PCR and Northern results were normalized using beta-actin and GAPDH, respectively. The Northern analyses showed that the steady state levels of mRNA for Col1a1 were depressed in both Tsk/Tsk and +/+ cells when grown on the matrix produced by Tsk/Tsk cells. Real-time PCR was then performed with primers specific for Col1a2, Col3a1, Col5a1 and Col5a2. The results showed that cells with the Tsk/Tsk, Tsk/+, and +/+ genotype all had lower steady-state levels of the above 4 collagen mRNAs when grown on the matrix produced by homozygous Tsk/Tsk cells or the matrix produced by heterozygous Tsk/+ cells compared with those grown on a matrix produced by +/+ cells. We hypothesize that the mutated Fbn1 molecules with many additional EGF-calcium binding regions and TGF-beta binding domains may (1) change the homeostasis of the ECM by binding additional growth factors and/or (2) present a radically different ECM 3-dimensional architecture. Either or both of these changes could signal the cell to produce less collagen.