Healing Effects of Formulations with Extract of Sonchus oleraceus

Introduction: Scar is defined as a process of tissue regeneration characterized by fibroblasts and a disorganized extracellular matrix, formed mainly by collagen. Thus it is understood that regeneration capacity is a phenomenon that occurs in order to repair an injured tissue. However, science has been looking for alternatives to assist in the repair process, and thus phytotherapy has been used as a therapeutic resource in medicine, and scientifically proven its efficiency. The plant Sonchus oleraceus is known, among other names, as “serralha” or sowthistle. It is a cosmopolitan species, found in several agricultural regions infesting crops. In Brazil, it is more frequent to grow in the winter and its seeds can be kept viable for eight years in the soil. This plant can be used as a popular drug for diuretic proposals, in cases of anemia, liver diseases and in cases of treatment of vitiligo in the eastern region of the state of Minas Gerais, Brazil. The objective of this research was to evaluate two different products (ointment and balsam) composed by S. oleraceus in the healing of surgical wounds in rats (Rattus novergicus).Materials, Methods & Results: Sonchus oleraceus specimens were obtained at the beginning of May, during the autumnwinter season, in which there is already a flowering of this plant. The leaves and stem of S. oleraceus were selected, cleaned and dried under study at 40°C of circulating air for four days for complete dehydration, and then ground in a knife mill. The constituents were prepared in 80% alcohol (v / v) and subjected to ultrasonic extraction for 60 min. The hydroalcoholic extract of S. oleraceus was concentrated on a rotary evaporator until all alcohol evaporated and the aqueous extract was obtained. The extracts were produced at concentrations of 5%, 10% and 15% and sent for manipulation of the pharmaceutical formulations of ointment and salve. Forty Wistar rats were divided into eight groups for evaluation of different products (salve and ointment). The wounds were created on the back of the animals and the products were applied daily; for analysis, clinical evaluations and wound measurement were performed daily. After 15 days, a biopsy was performed for histopathological analysis and quantification of type I and III collagen fibers. Measurement of the wound in rats treated with ointment containing 15% of extract presented better tissue retraction, with complete retraction observed after 10 days of treatment. Type I collagen was predominant in all samples; however, the healing properties of the balsam containing 5% extract and ointment containing 5% and 15% extract were significantly different.Discussion: As a conclusion, Sonchus oleraceus influenced the healing process of the skin. This pharmacological action is probably directly related to the amount of flavonoids present in its composition, that is, the higher the extract concentration, the better its efficacy. However, a better efficacy in the ointment is emphasized by better adhesion to the skin. Studies have shown that the flavonoids present in most plants have relevant healing activity, stimulating the proliferation of fibroblasts and the production of collagen, acting on the inhibition of metalloproteinase, thus increasing the rate of synthesized collagen. Other researchers reported a high concentration of flavonoids (anthocyanins and tannins) that act as mediators of inflammation, stimulating the production of inflammatory cytokines and tumor necrosis factor, observed in this species

Cytotoxic profile of the hydroalcoholic extract of Sonchus oleraceus L. in NHI/3T3 Lines of Fibroblast Cells

Os fibroblastos, as principais células envolvidas na reepitelização, sofrem alterações fenotípicas muito marcantes, assumindo a função específica de síntese proteica. Os avanços na utilização de testes in vitro relativos a ensaios de previsões celulares permitiram que tais testes se tornassem o primeiro passo na análise da compatibilidade biológica de um composto, a fim de comprovar a morte e disfunção do metabolismo celular. Para a análise da citotoxicidade foi utilizada cultura celular e as linhagens celulares utilizadas foram: MDCK e NIH-3T3, medidas através do MTT. Após 60 horas de exposição de linhagens celulares normais de fibroblastos NHI/3T3, constatou-se que, de acordo com a CE50, o extrato em concentrações ≥2,057μg/mL estimula as necessidades celulares.Conclui-se que o extrato hidroalcoólico de S. oleraceus L. pode estimular o ritmo dos fibroblastos e aumentar a produção de colágeno

Histomorphometry and immunohistochemical expression of RANKL in femur and vertebra of rats with secondary osteoporosis

A osteoporose é uma doença comum em humanos, acometendo tanto mulheres quanto homens. A doença pode ter origem primária, relacionada à deficiência de hormônios sexuais ou a senilidade, ou secundária, cujo exemplo mais comum é o uso crônico de glicocorticóides. Independente da causa, a conseqüência da osteoporose é a diminuição da massa óssea, aumentando o risco de fraturas. Apesar de ser considerada uma doença sistêmica, a redução de massa óssea na osteoporose não é uniforme no esqueleto. RANKL é uma citocina capaz de ativar a função osteoclástica e sua expressão é indutível em osteoblastos e linfócitos T. A função desta citocina tem sido considerada um possível alvo terapêutico no tratamento da osteoporose. Para avaliar a relação da perda óssea trabecular e a expressão de RANKL, foram realizados testes imunoistoquímicos e histomorfométricos em fêmur e vértebras de ratos castrados e/ou tratados com glicocorticóides, 56 dias após a indução. A expressão imunoistoquímica de RANKL pôde ser verificada nos animais castrados, tanto machos quanto fêmeas, mas não no grupo castrado que também recebeu glicocorticóide. Entretanto, a diminuição da massa óssea em ambos os grupos foi similar na avaliação histomorfométrica. Isso pode ocorrer por causa do efeito inibitório que os glicocorticóides têm sobre o metabolismo. As análises histomorfométricas revelaram ainda, que a massa óssea trabecular avaliada por este método é similar em machos e fêmeas, e que a perda óssea não é uniforme entre o colo femoral, o côndilo femoral e o corpo vertebral. Aos 56 dias de indução, o quadro de perda óssea instalado tanto em machos quanto em fêmeas, para todos os fragmentos ósseos analisados, é compatível com o quadro de osteoporose.Osteoporosis is a common human disease affecting both men and women, and it can classified as (1) primary, related to sexual hormones deficiency or senility, or (2) secondary, for what the most common example is the chronic therapies with glucocorticoids. Whatever is its cause, the osteoporosis outcomes are bone loss and increased fracture risk. Although osteoporosis is considered a systemic condition, bone loss seen in osteoporosis is not homogeneous throughout the skeleton. RANKL is a cytokine able to activate the osteoclasts function and its expression is inducible in osteoblasts and T cells by a range of stimuli. Function of RANKL has been considered a possible therapeutic target for the treatment of osteoporosis. To evaluate the trabecular bone loss related to the RANKL expression, immunochemistry and histomorphometric assays were used in femur and vertebra of castrated and/or glucocorticoid-treated male and female rats, at the day 56 after induction. RANKL expression was evident only in the castrated group, both male and female, but not in the group of castrated rats that also received glucocorticoid therapy. But histomorphometric data showed that bone loss was similar in both groups. That could happen because glucocorticoid can inhibit osteoblast metabolism. Histomorphometry also reveals that trabecular bone mass in male is similar to female, and bone loss is not homogenous between distal and proximal femur and vertebra body. At the day 56 after induction, bone loss, in femur and vertebra, both male and female, was compatible to osteoporosis

Stress, aging and osteoporosis: an experimental study of bone loss, expression of proinflammatory cytokines and bone markers RANKL, RANK and OPG in rats

Envelhecimento, estresse e osteoporose são três condições que tomaram grande importância no mundo moderno como reflexo não apenas do estilo de vida das pessoas, mas também do crescente aumento da expectativa de vida das populações. Tais condições compartilham de diversos mecanismos comuns e a modulação de uma pode afetar direta e indiretamente as outras. A osteoporose é uma doença comum entre os idosos e, embora seja multifatorial, muitos processos inerentes ao envelhecimento contribuem com sua patogenia. O estresse crônico é capaz de comprometer a massa óssea e, assim como a osteoporose, é considerado um importante fator que compromete a qualidade de vida dos idosos. O presente trabalho busca avaliar o efeito de um protocolo de estresse moderado crônico sobre parâmetros de avaliação óssea em ratos segundo o sexo e a idade. Para isso, animais machos e fêmeas, com cinco ou 22-24 meses de idade foram submetidos à aplicação de estressores moderados durante 28 dias. Foram incluídos frio, calor, imobilização, inclinação da gaiola, isolamento, luz noturna e privação de água e de ração, aplicados quatro vezes cada durante o período. Animais controles de ambos os sexos, jovens e senis foram mantidos em gaiolas coletivas, com água e ração à vontade, sem qualquer outro tipo de intervenção. Os ratos foram pesados no início e ao final do período, e, após eutanásia, tiveram seus ossos e sangue coletados. Fêmur, tíbia e vértebra lombar foram analisados por densitometria óssea. Testes biomecânicos de flexão avaliaram força máxima, tenacidade e rigidez da cabeça e da diáfise do fêmur. Em análise histomorfométrica, foram obtidos os volumes trabeculares da cabeça do fêmur e do corpo vertebral e medidas do osso cortical e da cavidade medular da seção transversal do fêmur. A técnica de imunoistoquímica foi realizada para a análise da expressão de RANKL, RANK e OPG no fêmur e na vértebra. Do sangue foram avaliados por ELISA: estradiol e testosterona, IL-1β, IL-6 e TNF-α. O estresse foi capaz de diminuir o peso dos ratos, independente do sexo e da idade. Sobre os parâmetros de avaliação óssea, o protocolo também mostrou capacidade de reduzir muitas das medidas avaliadas, podendo comprometer a saúde óssea. Entretanto, o estresse afetou um maior número de variáveis entre os animais jovens que entre os senis. Esse resultado deve ser interpretado com cautela. Provavelmente o envelhecimento poderia retardar a resposta do tecido ósseo aos estímulos desencadeados pelo estresse. De maneira crônica, os efeitos poderiam ser mais severos nos idosos. Com relação aos hormônios sexuais, embora não tenham sido estatisticamente significantes, uma diminuição na concentração de testosterona nos grupos submetidos ao estresse pôde ser percebida. Para o estrógeno, houve um comportamento contrário entre o grupo de machos e o grupo de fêmeas no primeiro, o estresse aumentou a concentração do hormônio, enquanto no segundo, diminuiu. A variação individual das observações foi muito ampla, e um melhor delineamento do modelo, possivelmente envolvendo um número maior de repetições, torna-se necessário para esclarecimento desta questão. Sobre as citocinas inflamatórias, o estresse também mostrou apenas efeito indireto no caso da IL-1β, que diminuiu nos animais jovens, mas aumentou nos senis, e do TNF-α, que diminuiu nos machos e aumentou nas fêmeas. Diferenças na marcação imunoistoquímica foram percebidas apenas para RANKL em relação devido ao estresse. Provavelmente pelo fato de que as substâncias investigadas nesta última parte do trabalho sejam de expressão induzível, seu estudo pode tornar-se mais heterogêneo. Visto que o modelo proposto é inédito, alguns ajustes metodológicos fazem-se necessários. Estes dados são apresentados na forma de capítulos, e cada um contém um breve resumo em português e em inglês, além de um conjunto de palavras-chave.Aging, stress and osteoporosis are conditions that have taken great importance in modern world not only as reflection of lifestyle, but also due to the increasing life expectancy of populations. These conditions share many common mechanisms and modulating one of them may affect the others by direct and indirect means. Osteoporosis is a common disease among the elderly and, although its cause may be multifactorial, many processes associated with aging contribute to its pathogenesis. Chronic stress can impair bone mass and it can be considered an important factor that affects life quality for seniors, as does osteoporosis. This study aims to evaluate the effect of a chronic mild stress protocol on bone parameters in rats, and the role of sex and age on this process. To do that, male and female rats, 5 and 22-24 months old, were submitted to mild stressors during 28 days. We included cold, heat, immobilization, tilt cage, isolation, overnight illumination, and water and food deprivation; each stressor was applied four times during the period. Animals of both sexes, young and aged, were kept with water and feed at will, with no interventions, to serve as control. Rats were weighed at the beginning and at the end of the period. After euthanasia, rats had their bones and blood collected. Femur, tibia and lumbar vertebra were analyzed by densitometry. Biomechanical tests evaluated maximal strength, toughness and stiffness of the head and shaft of femoral head and diaphysis by bending test. Histomorphometric analysis provided trabecular bone volume from femoral head and vertebral body, and measures of cortical bone and marrow cavity from femoral diaphysis cross-section. Immunohistochemistry was performed to analyze expression of RANKL, RANK and OPG in femur and vertebra. From blood, ELISA detected testosterone, estradiol, IL-1β, IL-6 and TNF-α. Stress was able to decrease body weight, regardless of sex and age. The protocol employed also showed ability to reduce many of the parameters assessed, and that could harm bone health. However, stress affected a greater number of variables among young than aged animals. This result should be interpreted with caution.Probably aging could slow down bone response to the stimuli caused by stress. But with chronic stimuli, the effects of stress on bone metabolism could be more severe in the elderly. About sex hormones, although not statistically significant, a decrease in testosterone concentration in the groups subjected to stress could be perceived. Considering estradiol, there was an opposite behavior between the group of male and female group in the former, stress increased hormone concentration, while in the latter hormone levels were decreased. Individual variations were wide, so a better delineation of the model makes necessary, possibly involving a larger number of samples. Stress also showed only an indirect effect on IL-1β levels, which decreased in young animals but increased in the senile. TNF-α decreased in males and it increased in females after stress. Differences in RANKL expression assessed by immunohistochemistry were perceived only related to stress. Probably because of the inducible nature of those substances, their investigation may become more heterogeneous. Since the proposed model is a novel model, some methodological adjustments are made necessary. All these data are presented in chapters where summaries in Portuguese and English and a set of keywords can be found

Preconditioning Methods to Improve Mesenchymal Stromal Cell-Derived Extracellular Vesicles in Bone Regeneration—A Systematic Review

Mesenchymal stromal cells (MSCs) have long been used in research for bone regeneration, with evidence of their beneficial properties. In the segmental area of MSC-based therapies, MSC-derived extracellular vesicles (EVs) have also shown great therapeutic effects in several diseases, including bone healing. This study aimed to assess whether the conditioning of MSCs improves the therapeutic effects of their derived extracellular vesicles for bone regeneration. Electronic research was performed until February 2021 to recover the studies in the following databases: PubMed, Scopus, and Web of Science. The studies were screened based on the inclusion criteria. Relevant information was extracted, including in vitro and in vivo experiments, and the animal studies were evaluated for risk of bias by the SYRCLE tool. A total of 463 studies were retrieved, and 18 studies met the inclusion criteria (10 studies for their in vitro analysis, and 8 studies for their in vitro and in vivo analysis). The conditioning methods reported included: osteogenic medium; dimethyloxalylglycine; dexamethasone; strontium-substituted calcium silicate; hypoxia; 3D mechanical microenvironment; and the overexpression of miR-375, bone morphogenetic protein-2, and mutant hypoxia-inducible factor-1α. The conditioning methods of MSCs in the reported studies generate exosomes able to significantly promote bone regeneration. However, heterogeneity regarding cell source, conditioning method, EV isolation and concentration, and defect model was observed among the studies. The different conditioning methods reported in this review do improve the therapeutic effects of MSC-derived EVs for bone regeneration, but they still need to be addressed in larger animal models for further clinical application

Biodegradable polymer nanofiber membrane for the repair of cutaneous wounds in dogs - two case reports

The study of wound healing and its treatment is extremely important in veterinary medicine due to the high frequency of wounds and the difficulty in treating wounds by second intention. Thus, the objective of this study was to evaluate the use of a nanofiber membrane made of biodegradable polymers as a method of wound treatment in dogs. This study comprised two dogs with bite wounds. Debridement and cleaning was performed followed by the application of the membrane. In one dog, the wound was in the left proximal calcaneal region with clinical signs of infection, necrotic tissue, and muscle and the gastrocnemius tendon were exposed. The wound displayed rapid formation of granulation tissue which became excessive, so it was necessary to debride several times. However, with the suspension of the use of the membrane, formation of this tissue was not observed, and the wound evolved to epithelialization and fast contraction. In the second dog, there was a deep wound on the medial aspect of the proximal right hind limb, with clinical signs of infection, with muscle exposure. Once the membrane was placed, granulation tissue formed, and the membrane was used until the level of this tissue reached the skin. The wound underwent rapid epithelialization and contraction, without developing exuberant granulation tissue. Efficient wound repair was observed and the dogs exhibited greater comfort during application and use of the membrane. More studies should be conducted in dogs focusing on the application of this membrane until the appearance of healthy granulation tissue, as continued use seems to stimulate the formation of exuberant granulation tissue.O estudo da reparação e tratamento das feridas cutâneas possui extrema importância em medicina veterinária devido à alta frequência e dificuldade no tratamento por segunda intenção. Este estudo objetivou avaliar o uso da membrana de nanofibras de polímeros biodegradáveis, como alternativa no tratamento de feridas em cães. Foram incluídos no trabalho dois cães com feridas por mordedura. Foi realizado o debridamento e limpeza das feridas e a aplicação da membrana. Em um animal, a ferida em região proximal ao calcâneo esquerdo, apresentava sinais clínicos de infecção, com tecido necrótico, exposição da musculatura e do tendão gastrocnêmio. Foi observada formação rápida de tecido de granulação que se tornou exuberante, sendo necessário debridamento seriado. Porém, com a suspensão do uso da membrana a formação desse tecido não foi mais observada, evoluindo para epitelização e rápida contração. No outro animal, a ferida em face medial proximal do membro pélvico direito, era profunda, com sinais clínicos de infecção e musculatura exposta. A formação de tecido de granulação também apresentou-se rápida, utilizando-se a membrana apenas até que o nível deste tecido se igualasse ao da pele, evoluindo para rápida epitelização e contração sem formação de tecido de granulação exuberante. Foi observada reparação eficiente e com maior conforto para os pacientes durante aplicação e utilização da membrana. Acredita-se que mais estudos devam ser realizados focando a aplicação em cães até o aparecimento do tecido de granulação saudável, pois as observações deste estudo indicam possível estimulação do tecido de granulação exuberante com utilização da membrana

Composite synthetic hydroxyapatite 30%, in two physical states, as dermal filler

The aim of this study was to evaluate the response to the implantation of synthetic hydroxyapatite 30% (HAP-91®) in different physical states as dermal filler. Eighteen New Zealand rabbits were used, distributed randomly into two equal groups and then divided into three groups according to the postoperative period at 8, 21 and 49 days. One mL of HAP-91®, fluid and viscous, was implanted in the subcutaneous tissue, 1 cm proximal to the cranial crest of the right scapula. The thickness of the skin was measured before and after implantation and for the following 15 days. Pain sensitivity assessment was conducted, assigning the following scores: 0 - when the animal allowed the touch of the implant area and expressed no signs of pain; 1 - when the animal allowed the touch, but pain reaction occurred, like increase of the respiratory rate or attempt to escape; 2 - when the animal did not allow the touch to the implanted area. At 8, 21 and 49 days, biopsy of the implanted area was performed. No difference was observed between the thickness of the skin (p>0.05) and all animals received a score 0 for soreness. Histological analysis did not reveal any obvious inflammatory process, showing a predominance of mononuclear cells in samples of eight days and tissue organization around the biomaterial with a tendency to encapsulation. The results indicate that HAP-91®, both viscous and fluid, is biocompatible and suitable for dermal filling.Objetivou-se avaliar a resposta tecidual, após implantação de hidroxiapatita sintética 30% (HAP-91® ), em dois estados físicos, para preenchimento cutâneo. Foram utilizados 18 coelhos da raça Nova Zelândia, distribuídos aleatoriamente em dois grupos (HAP-91® fluida e viscosa) e, posteriormente, divididos em três subgrupos, de acordo com o período pós-implante, em oito, 21 e 49 dias. Em cada grupo foi implantado 1,0 mL de HAP-91® , fluida ou viscosa, no tecido subcutâneo, 1 cm cranial à crista da escápula direita. Foi realizada a mensuração da espessura cutânea, imediatamente antes e durante 15 dias após a implantação do compósito. Avaliações de sensibilidade dolorosa foram realizadas, atribuindo-se os escores: 0 - quando o animal permitiu o toque e não manifestou reação; 1 - quando permitiu o toque, mas manifestou reação de dor, como aumento da frequência respiratória ou tentativa de fuga; 2 - quando não permitiu o toque. Aos oito, 21 e 49 dias, foi realizada a biópsia da região implantada. Não foi observada diferença entre a espessura cutânea dos animais (p > 0,05) e todos os animais receberam escore 0 para sensibilidade dolorosa. Na análise histológica, não foi notado processo inflamatório evidente. Houve predomínio de células mononucleares, nas amostras de oito dias, e uma organização tecidual ao redor do biomaterial, com tendência ao encapsulamento. A HAP- 91® , tanto fluida quanto viscosa, é biocompatível e indicada para o preenchimento cutâneo

Composite synthetic hydroxyapatite 30%, in two physical states, as dermal filler

The aim of this study was to evaluate the response to the implantation of synthetic hydroxyapatite 30% (HAP-91®) in different physical states as dermal filler. Eighteen New Zealand rabbits were used, distributed randomly into two equal groups and then divided into three groups according to the postoperative period at 8, 21 and 49 days. One mL of HAP-91®, fluid and viscous, was implanted in the subcutaneous tissue, 1 cm proximal to the cranial crest of the right scapula. The thickness of the skin was measured before and after implantation and for the following 15 days. Pain sensitivity assessment was conducted, assigning the following scores: 0 - when the animal allowed the touch of the implant area and expressed no signs of pain; 1 - when the animal allowed the touch, but pain reaction occurred, like increase of the respiratory rate or attempt to escape; 2 - when the animal did not allow the touch to the implanted area. At 8, 21 and 49 days, biopsy of the implanted area was performed. No difference was observed between the thickness of the skin (p>0.05) and all animals received a score 0 for soreness. Histological analysis did not reveal any obvious inflammatory process, showing a predominance of mononuclear cells in samples of eight days and tissue organization around the biomaterial with a tendency to encapsulation. The results indicate that HAP-91®, both viscous and fluid, is biocompatible and suitable for dermal filling

Hydroxyapatite-lignin composite as a metallic implant-bone tissue osseointegration improver: experimental study in dogs

The study aimed to evaluate biocompatibility, osteoconduction and osseointegration of a pasty composite of hydroxyapatite (20%) and lignin (80%) as a promoter of metal implant and bone tissue integration. An intramedullary Schanz pin was implanted in both tibias of fifteen bitches. In the left tibia, the pin was coated with the biomaterial at the time of surgery. Marrow cavity was also filled with the biomaterial. Right limb did not receive the biomaterial, then constituting the control group. Tibias were harvested from five animals at 8, 60 and 150 days after surgery; three of them were analyzed by histological and biomechanical assessment and the two remaining tibias by X-ray diffraction. Results showed that the biomaterial is biocompatible, with osteoconductivity and osseointegration properties. Histological analysis and diffractograms showed the presence of hydroxyapatite in samples in all periods, although the presence of organic material of low crystallinity was variable. There was no statistical difference in the forces required for removal of the biocompatibility, osteoconductivity and osseointegration, it was not able to promote a better intramedullary pin anchorage