Stereological and Morphometric Study of Type 3 Collagen Formation in the Cutaneous Wounds of Diabetic Mice Treated with Mesenchymal Stem Cells

Introduction: Wound healing is a progressive, essential and complex physiological process that occurs as a restorative response after a tissue injury. It involves three phases: inflammation, proliferation and maturation. Exogenous, endogenous and pathological factors may interfere in the cicatricial process in humans and animals by altering the balance between the synthesis, degradation and remodelling of collagen and elastic fibres. Diabetes mellitus is a progressive metabolic disease that alters elastogenesis and collagenesis and induces delays in the healing process. Scientific evidence suggests that mesenchymal stem cells modulate the cicatricial response. Thus the objective of this work was to perform stereological and morphometric analysis to determine the formation of dermal fibres in cutaneous fragments of a murine model of diabetes mellitus.Materials, Methods & Results: Histological sections were obtained from the cutaneous wounds of diabetic mice. The cutaneous wounds were previously treated with autogenous mesenchymal stem cells, physiological solution or polyurethane membrane. The histological sections were subsequently processed and stained for type 1 and 3 collagen fibres and elastic fibres using Picrosirius Red and Weigert staining, respectively. Histological sections stained with Picrosirius Red presented three types of birefringence under polarised light microscopy that corresponded to red colours for type 1 collagen and green and yellow colours for type 3 collagen. Weigert staining presented three colours for histological structures under white light microscopy that corresponded to black colours for elastic fibres, variations in colour from pink to purple for other structures and dermal attachments. The elastic fibres, represented by a black colour, presented in a heterogeneous form and were either identified as thin, punctiform or rectangular fibres or as elastic agglomerates. A greater volume of elastic fibres was observed in the superficial dermis than in the deep dermis, arranged irregularly. These fibres were organised longitudinally to the dermo-epidermal junction and surrounding the blood vessels and hair follicles. The images obtained were evaluated using the Cavalieri principle of stereology to obtain quantitative data in three-dimensions (3D), represented by the volume of the dermal fibres, and by the colour segmentation method. The K-means clustering plug-in in Image J® was used to quantify the area of the dermal fibres in the cutaneous wounds after the proposed dermatological treatments. A total of 90 images were obtained and analysed. No statistically significant differences (P > 0.01) were observed in the volume or area of type 1 collagen fibres between the treatment groups. Significant differences (P < 0.01) were only identified for the volumes and areas of type 3 collagen, with treated animals also presenting lower mean values for the volume and area of elastic fibres compared to the control group.Discussion: The preponderance of type 3 immature collagen in the cutaneous wounds of animals treated with stem cells indicates active collagenase and greater fibroblastic activity, which is probably induced by stem cells. Diametrically, the identification of lower levels of elastic fibres in the cutaneous fragments treated with stem cells suggests that cell therapy does not contribute satisfactorily to elastogenesis. Previous reports suggested that mesenchymal stem cells may decrease elastin synthesis, and such a situation may have occurred in this study. The autologous mesenchymal stem cells increased the formation of collagen fibres in diabetic mice at the detriment of the formation of elastic fibres, thus suggesting active early collagen in the first 2 weeks of the cicatricial process

Xenogeneic Mesenchymal Stem Cells in the Formation of Hyaline Cartilage in Osteochondral Goat Failure

Background: Osteochondral knee failures are among the most common causes of disability among the elderly human population and animal athletes. The xenogeneic transplantation of mesenchymal stem cells is a questionable therapeutic alternative that, despite the low expression of Major Histocompatibility Complex type II by these cells, still has relevantuncertainties about the safety and clinical efficacy. The main objective of the present study was to investigate whether the xenogeneic transplantation of mesenchymal stem cells induces hyaline cartilage formation, without histopathological evidence of rejection, in osteochondralfailures of goats.Materials, Methods & Results: Five female goats were used, submitted to three surgical osteocondral failures in the right knee, treated with xenogenic mesenchymal stem cells of dental pulp, xenogenic platelet-rich plasma and hemostatic sponge of hydrolyzed collagen, respectively. The lesions were evaluated after 60 days of treatment, aiming to identify thepresence of hyaline cartilage or fibrocartilage and the subchondral bone pattern (regenerated or disorganized). Transplantation of xenogenic mesenchymal stem cells induced predominant formation of hyaline cartilage (P 0.05). Macroscopically, the lesions of the stem cell treated group showed formation of firm repair tissue, opaque staining, integrated with adjacent cartilage and with the failure filling almost completely. The groups treated with PRP and hemostatic sponge of hydrolyzed collagen presented, on average, partial filling of the lesion, with irregular shape and darkened coloration.Discussion. The absence of macroscopic and histopathological evidences of an inflammatory process on the surface and in the internal portion of the osteochondral lesions treated with xenogeneic stem cells, probably due to the low expression of Major Histocompatibility Complex type II by these cells, which would theoretically induce low rejection response. Such observations are of great importance, since graft-versus- host disease syndrome is a serious condition, responsible for the low therapeutic efficacy with transplantation of cells or grafts in humans. The formation of fibrocartilage, although without macro and microscopic evidence of degeneration or necrosis, in the osteochondral failures treated with PRP and hemostatic collagen sponge suggest that paracrine factors of the local microenvironment of the osteochondral failure are possibly responsible for the formation of fibrocartilaginous tissue or by inhibition of normal cartilage formation. The fibrocartilage formed in the Plasmaand Control groups, contributed to the commitment in the filling of the lesion, contrasting with the almost complete fill of the lesions treated with stem cells. The xenotransplantation of mesenchymal stem cells induced formation of hyaline cartilage and did not promote histopathological evidence of rejection in osteochondral lesions of goat knees. The treatments with PRP and hemostatic sponge of hydrolyzed collagen induced greater formation of fibrocartilaginous cartilaginous surface in the osteochondral failures

Isolation, Expansion, Differentiation and Growth Kinetics Essay in Mesenchymal Stem Cells Culture from the Bone Marrow of Collared Peccaries (Tayassu tajacu)

Background: There are few studies on stem cell isolation in wild animals that provide isolation and culture protocols of these cells in vitro. Among the wild species studied, we present the collared peccary (Tayassu tajacu) as a model with potential to obtain and use MSC in preclinical studies. These animals are phylogenetically close to the domestic pig, popularly known as peccaries and found naturally in South America, Central America and the South of the United States. The aim of the present study was to establish a protocol for the isolation, in vitro cell expansion, differentiation and assessment of the stromal MSC growth curve before and after thawing.Materials, Methods &amp; Results: Mesenchymal stem cells (MSC) from collared peccary bone marrow (Tayassu tajacu) were isolated and expanded by centrifuge in Ficoll® solution and cultured in DMEM® High Glucose medium. The culture was assessed by assays of colony forming units CFU-F and growth curve by saturation (GCS). Cultures in the third passage, with 70% confluence, were replicated at 105 cells/mL concentration in the culture media to induce osteogenic cell differentiation and adipogenic cell differentiation, respectively. The MSC were frozen in nitrogen for 40 days, thawed and re-assessed for cell viability and GCS.Discussion: The bone marrow collected presented high mononuclear cellularity, with a mean variability of 94.5% and 60.83 ± 4.27 UFC were identified in the samples and cells with fibroblast-like-cell morphology were observed. When they were expanded, the mean cell viability was 95%, the mean cell concentration obtained was 233.31 ± 20.04 cells per 25cm2 bottle and the culture reached the growth plateau in GCS between the 13th and 16th day. The osteoblastic cell differentiation assay showed after 18 days, morphology similar to osteoblasts, with irregular cytoplasm limits, cell prolongation formation and flattened appearance. After staining with Alizarin Red, the nucleus presented a wine red coloring and the cytoplasm, more basophilic and well-defined, with calcium deposits inside the cells. The cultures submitted to adipogenic differentiation were large, hexagonal, irregular and presented birrefringent cytoplasm granules after the third week of culture. When stained with Oil Red it was observed that the cytoplasm granules were scattered small fat vacuoles and stained maroon. The viability after thawing was 78% and the mean cell concentration obtained in GCS was 199.71 ± 14.72 cells per 25 cm2 bottle. The curves reached the saturation plateau early, on the eighth day of observation. From then onwards the cultures entered became exhausted and the cell concentration of the samples decreased progressively until minimum values. These results showed the presence of a well-defined MSC population in the collared peccary bone marrow with a high rate of replication in vitro and potential for differentiation confirmed by the adipogenic and osteogenic lines. The cryopreservation technique adopted presented satisfactory results, but indicated a significant cell stress after thawing that justifies investigation of the apoptosis rates induced post thawing in the species. Furthermore, the bone marrow collection did not harm the animals and the facility of stromal MSC isolation and culture qualifies the collared peccary as a viable alternative model to obtain MSC and for studies in the area of cell therapy

Characterization and plasticity of wharton's jelly mesenchymal stem cells of goat

Mesenchymal stem cells (MSCs), obtained from several anatomical sites, have already been described, characterized and used in therapeutic models for tissue repair. The umbilical cord mesenchymal stem cells, represented by cells from arteries and veins walls, as well as Wharton's jelly are easy to be obtained, highly available, require no invasive procedure, do not present risk to donors and do not present ethical limitation. The aim of this research was to analyze the plasticity of Wharton's jelly mesenchymal stem cells (WJ-MSCs) of goat, evaluating their behavior in vitro and characterizing them immunophenotypically. Thus, tests were performed on colony forming units, viability and cell growth curve, flow cytometry analysis and plasticity potential. Goat umbilical cord matrix cells exhibited fibroblastoid morphology with colony formation and self-renewal ability, always maintaining their undifferentiated state up to the eighth passage (P8). The growth curve kinetics exhibited the LAG, LOG, and DECAY phases, without displaying a PLATEAU phase. The plasticity assay demonstrated positive differentiation for osteogenic, adipogenic and chondrogenic lines, characterized by the synthesis of intracytoplasmic granules or extracellular matrix with the presence of calcium, lipids and proteoglycans. Flow cytometry demonstrated the expression of CD90 and CD105; absence of CD14 expression.  It is concluded that the cell population isolated from the Wharton's  jelly of goat constitutes a representative sample of mesenchymal stem cells, with great possibilities in the field of regenerative and reproductive medicine

Treatment of experimental cutaneous wounds in diabeties mice with mesenchymal stem-cells associated or not to the platelet-rich plasma

Avaliou-se clinicamente a eficácia do tratamento de feridas cutâneas experimentais de camundongos (Mus musculus CS7BL/6) diabéticos com células-tronco mesenquimais (CTM), plasma autólogo rico em plaquetas (PRP) e CTM associadas ao PRP. O quadro clínico de diabetes foi induzido por meio da administração de estreptozootocina diluída em tampão citrato de sódio. As CTM foram coletadas a partir da medula óssea de camundongos isogênicos doadores, saudáveis, expandidas em laboratório e aplicadas sobre as lesões dos animais avaliados. O PRP foi obtido após processamento do sangue total de camundongos isogênicos doadores, saudáveis, e aplicado juntamente com as CTM no leito lesional. Observaram-se diferenças (p0,01) entre estes tratamentos para o aumento do percentual de colágenos tipos I e III no leito lesional. A avaliação da influência do tempo para a cicatrização completa das feridas cutâneas entre os grupos estudados revelou uma tendência mais acentuada (p0,01) among these treatments for the increase of the percentage of collagens types I and III in the wound. The evaluation of the influence of the time for the complete cicatrization of the cutaneous wounds among the studied groups revealed a tendency more accentuated (p <0,01) for null values, which represent complete cicatrization of the lesion, among the animals of the groups treated with isolated MSC or associated to PRP, following by the groups just treated with PRP, semi-permeable adhcrent curative and group witness, respectively. These results allow to infer that the transplant of autogenous MSC is a more effective treatment for the cicatrization of cutaneous wounds of diabetic mice that the cutaneous application of PRP, daily asepsis of the cutaneous wounds with physiologic solution and covering of the lesions with semi-penneable adherent curative. However, the association between MSC and PRP does not accelerate the cicatrization of the cutaneous wounds of diabetic mice, when compared to the treatment with isolated MSC.Coordenação de Aperfeiçoamento de Pessoal de Nível Superio

Topical treatment with honey, propolis in gel and allantoin cream in rabbit wounds experimentally infected

O efeito cicatrizante e antimicrobiano do mel, própolis em gel e creme a base de alantoína foram avaliados durante o tratamento de feridas de coelhos, infectadas experimentalmente com Staphilococcus aureus coagulase positivos, Pseudomonas aeruginosa e Pasteurella multocida na concentração de 108 unidades formadoras de colônias (UFC)/mL. Não foram observadas diferenças significativas (p<0,05) entre os tratamentos para as variáveis intensidade do edema e tamanho do halo eritematoso. Os tratamentos com solução salina, mel e creme a base de alantoína também não apresentaram diferença (p<0,05) para a variável tempo de cicatrização. Estes resultados não contestam a atividade antiflogística do mel, da própolis e do creme a base de alantoína, mas demonstra contribuição equivalente com a limpeza das lesões para a redução da inflamação local. De forma contrária, o gel de própolis resultou em maior agravamento da infecção e retardo da cicatrização das feridas, quando comparado aos demais tratamentos. A sensibilidade, in vitro, das amostras de S. aureus coagulase positivos, P. aeruginosa e P. multocida, ao mel e à própolis, na mesma concentração inoculada nas lesões dos coelhos, também foi avaliada pela técnica de formação de halo de inibição em meio de cultura. Os resultados mostraram que todas as amostras bacterianas foram sensíveis ao mel e à própolis. Entretanto, estes resultados divergem da maior parte dos trabalhos que utilizam o mel e a própolis, descrevendo-os como possuindo baixo efeito antimicrobiano contra P. aeruginosa e P. multocida. Todos estes resultados estimulam o prosseguimento de novas pesquisas sobre a utilização do mel e da própolis na cicatrização de feridas infectadas, que elucidem o real espectro de ação antibacteriano destes compostos e sua relação com a diferenciação celular durante o processo de reepitelização.The healing and antimicrobial effects of the honey, propolis in gel and allantoin cream were evaluated during the treatment of the rabbit s wounds. The lesions were infected experimentally with Staphilococcus aureus coagulase positive, Pseudomonas aeruginosa and Pasteurella multocida in the concentration of 108 colony forming units (CFU)/mL. Significant differences were not observed (p < 0.05) among the treatments for the variables intensity of the edema and size of the erythematosus halo. The treatments with saline solution, honey and allantoin cream did not also present difference (p < 0.05) for the variable time of cicatrization. These results do not contest the antiphlogistic activity of the honey, propolis and allantoin cream, but demonstrates equivalent contribution with the cleaning of the lesions for the reduction of the local inflammation. In a contrary way, the propolis gel resulted in larger worsening of the infection and retard of the cicatrization of the wounds, when compared to the other treatments. The sensibility in vitro of the samples of S. aureus coagulase positive, P. aeruginosa and P. multocida to honey and propolis, in the same concentration inoculated in the lesions of the rabbits, was also evaluated by the technique of formation of the inhibition halo in middle of culture. The results showed that all the bacterial samples were sensitive to honey and propolis. However, these results diverge of most of the works that use honey and propolis, describing them as possessing low effect antimicrobial against P. aeruginosa and P. multocida. All these results stimulate the pursuit of new researches about the use of honey and propolis in the cicatrization of infected wounds, elucidating the real spectrum of antibacterial action of these composed and their affinity with the cellular differentiation during the reepithelization process.Coordenação de Aperfeiçoamento de Pessoal de Nível Superio

Uso tópico do mel de abelha, oxitetraciclina e hidrocortisona, na reparação de feridas cutâneas, por segunda intenção, em coelhos

O interesse médico nas propriedades antiinflamatórias e cicatrizantes do mel de abelhas, para o tratamento de feridas cutâneas em humanos e animais tem sido crescente. Nesse estudo, avaliou-se o efeito terapêutico do mel de abelhas, oxitetraciclina 3%, hidrocortisona 1% e da associação entre oxitetraciclina e hidrocortisona, na reparação, por segunda intenção, de feridas cutâneas de coelhos. Foram criadas cirurgicamente seis lesões sagitais dorsais, três em cada antímero, que foram tratadas com solução salina (NaCl 0,9%), pomada de hidrocortisona 1%, pomada de oxitetraciclina 3%, vaselina e pomada com associação de hidrocortisona 1% e oxitetraciclina 3%, respectivamente. Não foi observada contribuição terapêutica quanto à aceleração da redução do tamanho das feridas entre os tratamentos, quando comparados ao grupo controle (solução salina). Contudo, os animais tratados com associação entre oxitetraciclina e hidrocortisona e apenas com hidrocortisona, apresentaram retardo na cicatrização quando comparados aos demais grupos.The medical interest in anti-inflammatory and healing properties of bees honey used for the treatment of cutaneous wound’s in humans and animals has been increasing. This study investigated the therapeutics effects of bee (Apis mellifera) honey, oxitetracicline 3%, hydrocortisone 1% and oxitetracicline 3%, plus hydrocortisone 1% on cutaneous wound’s repair process by second intention, in rabbits. Six saggital dorsal lesions were created surgically, three in each antimere, which were treated using saline solution NaCl 0,9% (control), hydrocortisone 1%, oxitetracicline 3%, petrolatum (vehicle) and hydrocortisone 1% plus oxitetracicline 3%, respectively. Considering acceleration on the wound’s repair (diminishing in the size of the wounds) there weren’t differences among the treated groups comparing to the control (saline solution). However, animals treated with only hydrocortisone and hydrocortisone associated with oxitetracicline showed delayed cicatrisation process when compared to the other groups