Abstract

Introduction

Healing is the natural restorative response of a tissue lesion. The healing process has three distinct phases: inflammation, proliferation and maturation¹1 . Witte MB, Barbul A. General principles of wound healing. Surg Clin North Am. 1997 Jun;77(8):509-28. PMID: 9194878.. If one of these phases fails or is prolonged, there may be a delay in or failure of wound occlusion. For surgeons, the healing of a surgical wound must follow the process of restitutio ad integrum, so as to make the scar as inconspicuous as possible and avoid atrophic or hypertrophic reactions such as keloid.

In otorhinolaryngology, surgical treatment of congenital or acquired stenosis of the external acoustic meatus, choanae and larynx is a challenge because of the risk of postoperative relapse, and the use of a scar-inhibiting agent would be a useful and efficient alternative. Two topical medications that act on the healing of wounds are Mitomycin C (MMC) and Clobetasol propionate (CP). MMC, produced by the fungus Streptomyces caespitosus, not only is a potent antibiotic and chemotherapeutic agent, but also is an alkylating agent that blocks RNA transcription for formation of extracellular matrix (ECM) components and induces apoptosis of fibroblasts, with a marked reduction in collagen and ECM protein levels²2 . Ribeiro FAQ, Guaraldo L, Borges JP, Zacchi FFS, Eckley CA. Clinical and histological healing of surgical wounds treated with mitomycin c. Laryngoscope. 2004 Jan;114(1):148-52. doi: 10.1097/00005537-200401000-00027.
https://doi.org/10.1097/00005537-2004010... ^{, 3}3 . Porter GT, Gadre SA, Calhoun KH. The effects of intradermal and topical mitomycin C on wound healing. Otolaryngol Head Neck Surg. 2006 Jul;135(1):56-60. PMID: 1681583.. CP is a glucocorticoid with high anti-inflammatory power and acts on keratinocytes, thereby causing reductions in ECM volume and cell proliferation. This results in epidermal thinning and, through inhibition of mitoses in fibroblasts, reduction of the amount of collagen and ECM volume⁴4 . Schoepe S, Schäcke H, May E, Asadullah K. Glucocorticoid therapy-induced skin atrophy. Exp Dermatol. 2006 Jun;15(6):40620. PMID: 16689857. in the dermis.

Experimental studies on animals have enabled observations on the topical effect of these medications towards inhibition of the scar response, in relation to MMC²2 . Ribeiro FAQ, Guaraldo L, Borges JP, Zacchi FFS, Eckley CA. Clinical and histological healing of surgical wounds treated with mitomycin c. Laryngoscope. 2004 Jan;114(1):148-52. doi: 10.1097/00005537-200401000-00027.
https://doi.org/10.1097/00005537-2004010... ^{, 5}5 . Ribeiro FAQ, Borges JP, Zacchi FFS, Guaraldo L. The clinical and histological behavior of rat skin submitted to topical and injected Mitomycin C. Braz J Otorhinolaringol. 2003 Mar-Apr; 69(2):151-8. doi: 10.1590/50034-72992003000200002.
https://doi.org/10.1590/50034-7299200300...

6 . Gray SD, Tritle N, Li W. The effect of mitomycin on extracellular matrix proteins in a rat wound model. Laryngoscope. 2003 Feb;113(2):237-42. PMID: 12567075.

7 . Ribeiro FAQ, Guaraldo L, Borges JP, Vianna MR, Eckley CA. Study of wound healing in rats treated with topical and injected Mitomycin C. Ann Otol Rhinol Laryngol. 2008 Oct;117(10):786-90. doi: 10.1177/000348940811701015.
https://doi.org/10.1177/0003489408117010...

8 . Baptistella E, Malafaia O, Czeczko NG, Ribas-filho JM, Nassif PAN, Nascimento MM, Pachnicki JP. Comparative study in swines' vocal cords healing after excision of fragment with CO2 laser with mitomycin and 5-fluorouracil postoperative topical application. Acta Cir Bras. 2009 Jan-Feb;24(1):13-8. PMID: 19169536.

9 . Yoon YH, Park JY, Park YH. The preventive effect of mitomycin-C on the external auditory canal fibrosis in an experimentally induced animal model. Am J Otolaryngol. 2010 Jan-Feb;31(1):9-13. PMID: 19944892.

10 . Warner D, Brietzke SE. Mitomycin c and airway surgery: how well does it work? Otolaryngol Head Neck Surg. 2008 Jun;138(6):700-9. PMID: 18503839.

11 . Martins MD, Marques MM, Bussadori SK, Martins MA, Pavesi VC, Mesquita-Ferrari RA, Fernandes KPS. Comparative Analysis between Chamomilla recutita and corticosteroids on wound healing. An in vitro and in vivo study. Phytother Res. 2009 Feb;23(2):274-8. PMID: 18803230. ^{- 12}12 . Roh JL, Koo BS, Yoon YH, Rha KS, Park CI. Effect of topical mitomycin C on the healing of surgical and laser wounds: a hint on clinical application. Otolaryngol Head Neck Surg. 2005 Dec;133(6):851-6. PMID: 16360502. and PC¹³13 . Schäcke H, Döcke WD, Asadullah K. Mechanisms involved in the side effects of glucocorticoids. Pharmacol Ther. 2002 Oct;96(1):2343. PMID: 12441176.. A variety of methodological criteria has been used, especially regarding the times for evaluating and reevaluating the wounds, which have often generated conflicting or unenlightening results.

The present study had the objective of using histological techniques to study the effects of the topical action of MMC and CP on the healing process of surgical wounds on the backs of rats, on the 30^th and 60^th days after the injury.

Methods

The present study was fully approved by the ethics committee for animal experimentation of the Institute of Advanced Otorhinolaryngological Sciences (ICAO number 2/11) and was carried out under veterinarian supervision and follow-up.

A controlled experimental design was selected for this study, which was conducted on 57 Wistar rats: all male adults weighing over 200g. The animals were kept in individual cages, with food and water ad libitum, in an environment with a constant temperature of 21°C.

The rats were anesthetized by means of an intraperitoneal injection of ketamine (40 mg/kg) and xylazine hydrochloride (10 mg/kg). Afterwards, the rats were depilated on their backs so that a circular surgical wound, of 1 cm in diameter, could be made in that region, with removal of the skin and subcutaneous tissue. Once the surgical procedure had been concluded, all the wounds were washed with 10 ml of 0.9% physiological solution.

In the control group (CG), composed of 13 rats, no medication was applied to the surgical wound. Six rats were euthanized after 30 days and the remaining seven, after 60 days.

The group treated with MMC (MMCG), with 22 rats, received a single topical application of 2 ml of MMC at a concentration of 0.5 mg/ml (Mitocin, Bristol-Myers Squibb, São Paulo, Brazil), which was dripped onto a gauze over the wound, for a period of five minutes. After this period, the wound was washed again with 10 ml of 0.9% physiological serum. Ten animals were euthanized after 30 days and 12, after 60 days.

The group treated with CP (CPG), composed by 22 rats, received applications of clobetasol propionate in the form of a 0.05% cream (Psorex, Glaxo Smith Kline, São Paulo, Brazil), at a dosage of 0.25 g/kg, once a day, for 15 days. This dosage was used in accordance with the literature¹⁴14 . Shimo T, Takahara Y, Noguchi Y, Mukawa A, Kato H, Ito Y. Comparative toxicity test of dexamethasone valerate (DV-17) and other steroid ointments in rats. J Toxicol Sci. 1982 Jul;7(suppl 1):1533. PMID: 7154134. ^{, 15}15 . Kimura M, Tarumoto Y, Nakane S, Otomo S. Comparative toxicity study of hydrocortisone 17-butyrate 21-propionate (HBP) ointment and other topical corticosteroids in rats. Drugs Exp Clin Res. 1986;12(8):643-52. PMID: 3757761., since CP has a high degree of toxicity for rats. Eleven rats were euthanized after 30 days and 11 after 60 days.

The reason for creating a control group of animals that were not treated instead of using a region where there was no application of the medication on the backs of the treated animals themselves was to avoid possible systemic interferences from topical administration, in relation both to MMC¹⁶16 . Bradner WT. Mitomycin C: a clinical update. Cancer Treat Rev. 2001 Feb;27(1):35-50. PMID: 11237776. and to CP¹⁴14 . Shimo T, Takahara Y, Noguchi Y, Mukawa A, Kato H, Ito Y. Comparative toxicity test of dexamethasone valerate (DV-17) and other steroid ointments in rats. J Toxicol Sci. 1982 Jul;7(suppl 1):1533. PMID: 7154134. ^{, 15}15 . Kimura M, Tarumoto Y, Nakane S, Otomo S. Comparative toxicity study of hydrocortisone 17-butyrate 21-propionate (HBP) ointment and other topical corticosteroids in rats. Drugs Exp Clin Res. 1986;12(8):643-52. PMID: 3757761..

After anesthetization using ketamine and xylazine hydrochloride intraperitoneally, the animals were euthanized by means of an intracardiac injection of sodium thiopental (24 mg/kg) and potassium chloride (0.5 ml). The skin of the dorsal region of the animals, adjacent to the scar, was depilated. The whole area, including the scar, was photographed and then removed surgically. The fragments were fixed in 10% formol and processed using standard histological techniques, with embedding in paraffin and preparation of slides with sections of thickness 3 µm, for histological examination by means of HE and picrosirius red stainings.

The following parameters were taken into consideration for the histological evaluation of the slides stained with HE: presence of fibroblasts and fibrocytes and vascular proliferation in terms of the quantity of blood vessels in the dermis. The evaluations were carried out qualitatively (presence or absence) and semi-quantitatively (weak, moderate or high intensity) by two experienced histologists, and the variables were classified according to their intensity, as degree 1 (absent or weak intensity) and degree 2 (moderate or high intensity). We observed all field In the microscopic evaluation with x10, x20, x40 magnification lenses.

The slides stained with picrosirius red were used to evaluate the concentration of collagen fibers present in the connective tissue underlying the epithelium. On these slides, with the aid of an optical microscope coupled to an Axiocam MRc 5 camera (Zeiss), the area of the scar was located and photographed between two and eight times, depending on its extent when magnified x200. The Image Pro Plus 4.5^(r) software was used to obtain a quantitative reading of the collagen deposits in selected areas of the skin, by means of digital recognition of the regions stained in red (areas of collagen deposition) and the intensity of the staining (the stronger the staining was, the greater the deposition of collagen fibers was), thus calculating the collagen concentration.

After gathering the data, a computerized database was set up, which was subjected to statistical analysis with the aid of the Statistical Package for the Social Sciences (SPSS) version 13.0 software.

The study population was characterized by means of

descriptive statistics. Fisher's exact test was used to compare proportions. Mean values were compared using the KruskalWallis test. Post-hoc analysis was performed using Mann-Whitney test (two by two comparisons), with Bonferroni correction for the significance level. In that case, statistical tests were considered to be significant when the P value was less than 0.017. In the other tests, the significance level was set at 5%.

Results

The wounds on the backs of the rats healed by second

intention. In all the rats, a crust was seen to form, and this fell off spontaneously. In the control group, the scar crusts fell off, on average, on the 15^th day; in MMCG they fell off around the 25^th day; and in CPG, on the 26^th day. In the latter group, some rats still presented crusts on the wound until the 30^thday.

The histological results from analyses carried out on the

fragments from the rats euthanized after 30 days are presented in Table 1, and the results of those after 60 days are in Table 2.

Discussion

The macroscopic evaluation on the wounds of the animals in the three groups showed that there were differences in the time that the crusts took to fall off. This demonstrated that the healing was slower among the animals that received topical medication, especially those of the CP group. This difference may have been due to the ways in which the two medications were applied: MMC in a single dose and CP, daily for 15 days.

Regarding the quantities of fibroblasts and fibrocytes, there was no statistically significant difference among the three groups in the 30 and 60-day evaluations, thus demonstrating that MMC and CP did not interfere in fibroplasia. This result differed from what was found by Ribeiro et al. ⁷7 . Ribeiro FAQ, Guaraldo L, Borges JP, Vianna MR, Eckley CA. Study of wound healing in rats treated with topical and injected Mitomycin C. Ann Otol Rhinol Laryngol. 2008 Oct;117(10):786-90. doi: 10.1177/000348940811701015.
https://doi.org/10.1177/0003489408117010... : over an observation period of 90 days after using topical MMC at a concentration of 0.5 mg/ ml on wounds on the backs of six rats for five minutes, these authors reported that there was a significantly greater quantity of fibroblasts and lower quantity of fibrocytes in the fragments removed from these animals, in relation to the control group, which was also composed of six rats.

Vascular proliferation (angiogenesis) is part of the proliferative phase of healing and, in the present study, presented moderate degree in the three groups, in the evaluation carried out after 30 days. No statistical analysis was possible at this time. In the analysis after 60 days, on the other hand, there was no statistically significant difference among the three groups. The findings from the present study therefore indicate that administration of MMC and CP at the specified dosages and posology did not alter the angiogenesis at the times of 30 and 60 days. On the other hand, Ribeiro et al. ⁷7 . Ribeiro FAQ, Guaraldo L, Borges JP, Vianna MR, Eckley CA. Study of wound healing in rats treated with topical and injected Mitomycin C. Ann Otol Rhinol Laryngol. 2008 Oct;117(10):786-90. doi: 10.1177/000348940811701015.
https://doi.org/10.1177/0003489408117010... demonstrated that there was a significant increase in vascular proliferation in scar fragments on the backs of rats, in a group treated with topical MMC at a concentration of 0.5 mg/ml for 5 minutes, after a period of 90 days.

A decrease in the collagen concentration in scar fragments from the vocal folds of pigs stained with picrosirius red was observed by Camargo et al. ¹⁷17 . Camargo PA, Campos AC, Matias JE, Rispoli DZ, Przysiezny PE, Fonseca VR. Topical mitomycin C effect on swine vocal folds healing. Braz J Otorhinolaringol. 2006 Sep-Oct;72(5):601-4. PMID: 17221051. and Baptistella et al.⁸8 . Baptistella E, Malafaia O, Czeczko NG, Ribas-filho JM, Nassif PAN, Nascimento MM, Pachnicki JP. Comparative study in swines' vocal cords healing after excision of fragment with CO2 laser with mitomycin and 5-fluorouracil postoperative topical application. Acta Cir Bras. 2009 Jan-Feb;24(1):13-8. PMID: 19169536.. Both of these authors applied MMC soaked in cotton to the wound for three minutes and analyzed the effect of the medication on healing after 30 days. However, Camargo et al. ¹⁷17 . Camargo PA, Campos AC, Matias JE, Rispoli DZ, Przysiezny PE, Fonseca VR. Topical mitomycin C effect on swine vocal folds healing. Braz J Otorhinolaringol. 2006 Sep-Oct;72(5):601-4. PMID: 17221051. used MMC at a concentration of 0.4 mg/ml and Baptistella et al.⁸8 . Baptistella E, Malafaia O, Czeczko NG, Ribas-filho JM, Nassif PAN, Nascimento MM, Pachnicki JP. Comparative study in swines' vocal cords healing after excision of fragment with CO2 laser with mitomycin and 5-fluorouracil postoperative topical application. Acta Cir Bras. 2009 Jan-Feb;24(1):13-8. PMID: 19169536., 0.04%.

No other studies demonstrating inversion of the concentration of collagen fibers in scars between 30 and 60 days, as reported in the present study, were found in the literature. This was probably because observations tend to be made preferentially up to 30 days after the injury. A decrease in collagen concentration over a 30-day period has also been observed in the injuries treated

with both medications. The increase in the collagen concentration of collagen in the scars of the treated groups in relation to the control group seen after 60 days demonstrates that these drugs retard healing during the initial period. Nevertheless, an increase in collagen deposition was observed on the 60^th day after the date of the procedure. The remodeling phase of the healing process begins 21 days after the injury and can continue indefinitely.

Conclusions

In the rats treated with Mitomycin C and Clobetasol there was a late fall in the scar crusts (delay in the initial healing). The medications did not interfere in fibroplasias and in angiogenesis. In the rats treated with Mitomycin C and Clobetasol there was a decrease in collagen over a 30-day period and an increase over a 60-day period, demonstrating a delay in the wound healing.

References

Publication Dates

History