Differentiation of myofibroblasts in wounds after topical use of metronidazole : an experimental study

1 Federal University of Paraná, Post-Graduation in Surgical Clinics, Curitiba, PR, Brazil. 2 Federal University of Juiz de Fora, Biological Sciences Institute, Departament of Pathology, Juiz de Fora, MG, Brazil. Trindade Differentiation of myofibroblasts in wounds after topical use of metronidazole: an experimental study. 2 Rev Col Bras Cir 46(1):e2015 Recent papers8,9 stated that the knowledge of the myofibroblasts’ activating and blocking factors is a possible key for the treatment and control of neoplasms of stromal origin, such as sarcomas. Topical metronidazole has been used to odor control in malignant fungating wounds. These are elevated over the skin in a mushroomlike fashion, with large folds, similar to villi and crypts, which provide marked proliferation of anaerobic bacteria, resulting in foul odor lesions10. This study aimed to evaluate, in an experimental model of wound healing by secondary intention, the influence of topical metronidazole solution at different concentrations on the amount of protomyofibroblasts and myofibroblasts and its potential role in the contraction phase of wound healing.


INTRODUCTION
T he wound healing process is the subject of research for centuries, but in the last four decades, there has been intensified work on fibroplasia with interest in functional or cosmetic rehabilitation [1][2][3] .
After skin trauma, accidental or surgical, factors related to tissue repair are activated, evolving in stages: coagulation, inflammation, proliferation, wound contraction and remodeling.But these phases may be modified in various situations, such as when there is infection, which perpetuates the inflammatory process and causes the chronification of a skin lesion, such leg ulcers or, conversely, when using topical creams and special dressings that accelerate the process of fibroplasia, in aesthetic procedures 4 .
The phenomenon of wound contraction, that is, the relationship between wound size and reduction rate during healing, completed 100 years of its description 5 .However, the main cell responsible for this phenomenon, the myofibroblast, was only described after decades.In 1971, Gabbiani 6 observed fibroblasts present in granulation tissue that had their cytoplasm similar to those found in smooth muscle cells, thus describing the myofibroblast.In 1990, an experimental study with open wounds in rat skin demonstrated that the myofibroblasts derived from the local fibroblasts, after developing muscle fibrils bands, called alpha smooth muscle actin (α-SMA), a characteristic that identifies the myofibroblast by immunohistochemistry 7 .

Original Article
Differentiation of myofibroblasts in wounds after topical use of metronidazole: an experimental study.

Diferenciação de miofibroblastos em feridas após uso tópico do metronidazol: estudo experimental.
Trindade Differentiation of myofibroblasts in wounds after topical use of metronidazole: an experimental study.

2
Rev Col Bras Cir 46 (1):e2015 Recent papers 8,9 stated that the knowledge of the myofibroblasts' activating and blocking factors is a possible key for the treatment and control of neoplasms of stromal origin, such as sarcomas.
Topical metronidazole has been used to odor control in malignant fungating wounds.
These are elevated over the skin in a mushroomlike fashion, with large folds, similar to villi and crypts, which provide marked proliferation of anaerobic bacteria, resulting in foul odor lesions 10 .
This study aimed to evaluate, in an experimental model of wound healing by secondary intention, the influence of topical metronidazole solution at different concentrations on the amount of protomyofibroblasts and myofibroblasts and its potential role in the contraction phase of wound healing.We randomly divided the animals into six groups.With the exception of the animals in the control group (CG), all the other experimental groups (EG), I to V, underwent dressings with the use of metronidazole in topical solution to 4%, 6%, 8%, 10% and 12%, once a day.Each group was subdivided into three subgroups of six animals for evaluation at three, seven and fourteen days after the skin lesion (Table 1).After the photographic records, we resected the wounds with a margin of 1cm of whole skin around the lesion, with depth to the dorsal musculature of the rat.The segment destined for histology was extended onto a paper filter identified and fixed in 10% formaldehyde for 24 hours.After this period, it was submitted to the conventional histological preparation, included in paraffin block and cuts of 5µm.

METHODS
We used the tissue array method for immunohistochemistry. From the paraffin block, we removed a fragment with a punch number 3 from the central surface area of the wound.The samples taken were deposited in a cassette, according to determination of the map previously elaborated.The material was then sent for immunohistochemical processing using α-SMA and CD34 antibody.The tissue array method allowed the complete analysis of the central area of the wound, with an average of 14 fields evaluated at 400x magnification, considering only the nucleated cells, the research target, ie, the protomyofibroblasts and myofibroblasts.
We used the non-parametric Kruskal-Wallis test to compare the groups at each evaluation moment and to compare the moments of evaluation within each group.Values of p<0.05 indicated statistical significance.We analyzed the data using the IBM SPSS v.20.0 software.
There was a progressive reduction of the wound on the third, seventh and 14th day in all groups analyzed.In the comparison between the experimental groups and the control one, there was no significant difference in the periods evaluated.
The CD34 immunohistochemistry, used to identify neovascularization, marked stromal cells in the matrix, unrelated to neovessels, α-SMA negative, suggestive of being protomyofibroblasts.The use of the tissue array method allowed the comparison of the slides with α-SMA and CD34 antibodies, ensuring that they were not the same cells.In the evaluation of the third day, none of the groups examined presented protomyofibroblasts in the wounds.On day 14, there was no difference between groups.On the seventh day, there was difference between the groups (p=0.022),according to table 2.
By comparing groups two to two as to the presence of protomyofibroblasts on the seventh day, there was a significant difference, as shown in table 3.With α-SMA immunohistochemistry, at the third day evaluation none of the groups presented myofibroblasts in the wounds.On the seventh day, the difference was not significant.On the 14th day, there was difference between groups (p<0.010),according to table 4.

Rev Col Bras Cir 46(1):e2015
When comparing groups two to two as to the presence of myofibroblasts on the 14th day, there was a significant difference (Table 5).
Conversely, Borden et al. 14 used intraperitoneal metronidazole at the dose of 20mg/kg/day, and Trindade et al. 4 , a topical dose of 50mg/kg/day, and they did not find significant difference in wound contraction when comparing the groups.
Wrobel et al. 15  Berry et al. 17  In our study, we observed that the wounds of the experimental and control groups had their area significantly decreased as time went by.In the extracellular matrix, there are fibroblasts that present in their cytoplasm bands of microfilaments known as contractile fibers that express actin but are α-SMA-negative, and may be labeled by CD34 1,18,19 .In the early stages of wound healing granulation tissue formation, the fibroblasts around the wound migrate to the center of the lesion and acquire, in their cytoplasms, microfilament bands similar to the contractile fibers beta and gamma actin, becoming protomyofibroblasts, which initiate the synthesis of extracellular matrix components, such as fibronectin and collagen types I and III 18,20 .These immature myofibroblasts appear in the granulation tissue between fifth and sixth days after wound infliction 21 .
Protomyofibroblasts secrete a form of fibronectin called ED-A fibronectin, which has been deemed important for the expression of the myofibroblast phenotype in the wound 5,20 .
The transformation of the fibroblast into protomyofibroblast seems to depend on the change in the mechanical stress of the wound as compared to the increase in stiffness of the normal skin.However, complete differentiation in myofibroblasts will only occur with the stimulation of the transforming growth factor beta (TGF-β) in the presence of ED-A fibronectin 5,20 .

Platelet-derived growth factor (PDGF) and Tumor
Necrosis Factor-alpha ply roles on protomyofibroblast formation, but these cytokines isolated fail to induce α-SMA expression and differentiation into myofibroblast in vitro or in vivo 1,21 .
Hinz et al. 22 , in an experiment with silicon substrate and TGF-β-treated collagen matrix and culture of rat subcutaneous fibroblasts, achieved an increase in α-SMA expression, demonstrating the action of this factor on the contractile activity of fibroblasts.TGF-β acts on the proliferation and differentiation of fibroblasts into myofibroblasts, activation of keratinocytes, matrix deposition and angiogenesis 23 .
There are factors that inhibit the expression of α-SMA, such as the basic fibroblast growth factor (bFGF), which antagonizes TGF-β1 24 .
The relationship between the extracellular matrix and TGF-β determines normal scarring or a fibrosis process 21,23 .
In our study, the experimental groups with the application of 4%, 6% and 8% metronidazole had a higher number of protomyofibroblasts in the wounds.The experimental groups with 10% and 12% presented lower density of these cells than the control group.This suggests that the action of topical metronidazole in the wound in inducing differentiation of the fibroblast into protomyofibroblast is dose dependent.

Rev Col Bras Cir 46(1):e2015
Knowing the factors of recruitment of fibroblasts and mesenchymal cells, differentiation, proliferation and apoptosis of myofibroblasts are fundamental to understand normal and pathological tissue repair 5 .With regard to myofibroblasts, studies with antibody-labeled immunofluorescence to detect all actin isoforms have shown that these cells originate from the fibroblasts that migrate to the wound 7 .In these studies, myofibroblasts appeared on the sixth day of wound evaluation, and were present from day 12 to day 15.From the 16th to the 20th day, there was an intense decline in the presence of myofibroblasts, and by the 30th day there were no such cells in the wounds.They also observed that fibroblasts apoptotic figures appeared between the 20th and 25th day of the lesion, suggesting that these cells have been programmed to die in cases of wound healing.The authors emphasized that the first phase of wound contraction is independent of the myofibroblast phenotype 7 .
Myofibroblasts are cells that have in According to Hinz et al. 21, myofibroblasts are characterized by the development of α-SMA contractile fibers and increased production of extracellular matrix proteins.These cells connect through focal adhesions to the matrix and with each other by adherent junctions (Figure 1).The main cell to originate the myofibroblast after injury to different tissues seems to be the fibroblast residing in the dermis around the lesion, initially differentiating into protomyofibroblast, characterized by being α-SMA negative.
Recent studies have shown that the extracellular matrix develops and maintains tissue homeostasis, and its dysfunction favors the appearance of fibrotic diseases and stromal neoplasms 5,23,30 .Tissue degeneration in malignancy may arise from cases of fibrotic diseases, such as cirrhosis of the liver, pulmonary and renal fibrosis, characterized by hyperproliferation of fibroblasts, their differentiations into myofibroblasts, and abnormal accumulation of collagen 9,21,30 .Knowledge of the complex process of production, modification and remodeling of the extracellular matrix is the key to acting on cellular responses and on antifibrotic and antineoplastic therapies 2,5,9,19,30,31 .Hinz and Gabbiani 32 stated that the myofibroblast is the main cell involved in fibrotic diseases, and the therapeutic strategy would be to interfere in the differentiation of this cell by controlling TGF-β1 and ED-A fibronectin.
There are reports that the mechanical tension in the wound extracellular matrix would be responsible for maintaining the presence of myofibroblasts 33,34 .Considering the possible myofibroblasts precursors, Hinz 35 raises the hypothesis that the myofibroblasts do not display the same response to injuries' mechanical factors, and that probably there are other factors for the maintenance of the myofibroblast phenotype other than change in wound tension.Protomyofibroblasts and myofibroblasts are present in normal tissues, such as alveolar septum and intestinal crypts, where tissue tension is stable 20 .
In the present study, the experimental groups with a topical application of metronidazole solution at 4%, 6% and 8% presented a higher density of myofibroblasts in the wounds, suggesting that the differentiation of protomyofibroblast into myofibroblast, i.e., the expression of α-SMA, The project was approved by the Committee on Ethics in the Use of Animals of the Pontifical Catholic University of Paraná -CEUA PUCPR, under protocol # 655.The experimental study used 108 rats (Rattus norvegicus, Rodentia mammalia) of the Wistar strain, aged 110 days and average weight between 300g and 315g.They were kept in appropriate individual cages for the species under controlled environmental conditions (12 hours light/dark cycle), with free access to water and standard chow for the species.We estimated the sample size through studies conducted in the researched literature4,11 , and the project followed the guidelines of Law 11794/2008, regulated by decree number 6899, and the recommendations of the Brazilian College of Animal Experimentation.

June 20, 2002 .
photograph, which allowed the conversion of the electronic image to a scale in centimeters.
studied cultures of human fibroblasts and myofibroblasts on a contractile substrate and observed similar contractile strengths, suggesting that in the absence of α-SMA expression, the fibroblast could produce sufficient contractile strength for the closure of an open wound.Ibrahim et al.16 evaluated contraction of open wounds in healing by secondary intention in humans and in male and female rats, without drug interference.The authors demonstrated that α-SMA expression of fibroblasts, ie myofibroblasts in granulation tissue, contributed but were not required for wound contraction, and concluded that fibroblasts generate contractile strengths in vivo.
reported that there was effective contraction of large wounds in the absence of high density myofibroblasts.They have suggested that the contractile unit may be the organization that fibroblasts promote of the thin collagen fibers with the thick ones and the compaction of the connective tissue within the granulation tissue, retracting the dermis and adipose tissue around the wound.
their cytoplasm bands of organized α-SMA microfilaments.But other markers of muscle fibers, heavy chain myosin, desmin, h-caldesmon and smoothelin are negative3,8,28 .Tomasek et al. 1 stated that the myofibroblast plays roles in the synthesis of the extracellular matrix and in the generation of strength responsible for the reorganization of the matrix and contraction of the wound.The increase in cell line studies and genetic tools has made possible to identify other precursors of myofibroblasts besides fibroblasts from the adjacent intact dermis.Precursors such as vascular smooth muscle cells, pericytes, mesenchymal cells, fibrocytes, stellate hepatic cells, bone marrow cells have been proposed, among others 8,27-29 .

Figure 1 .
Figure 1.One cell, multiple origins.Source: Image obtained under license # 3927950953808 from Elsevier's publication in The American Journal of Pathology, The Myofibroblast, volume 170, issue 6, June 2007, in agreement with author Dr. Boris Hinz.

Table 1 .
Division of animals by groups and evaluation days.

Table 2 .
Number of protomyofibroblasts in the wounds of the control and experimental groups on the seventh day of evaluation.

Table 3 .
Comparison between groups two to two in relation to the presence of protomiofibroblast on the seventh day.

Table 4 .
Number of myofibroblasts in the wounds of the control and experiment groups on the 14th day of evaluation.

Table 5 .
Comparison between groups two to two in relation to the presence of myofibroblast on the 14th day.