Abstract
This study evaluated the early recovery process of the palatal wounds of dogs using bismuth subgallate. Five healthy adult male dogs underwent eight 5-mm partial-thickness punch biopsies in two paired columns on the palatal mastigatory mucosa. For the haemostasis, one side received moistened gauze pressure (test group 1), and the other received bismuth subgallate (test group 2). A description of the epithelium and connective tissue repair was made at 3, 7, 14 and 21 days. During the first days, a mass of disorganized tissue covered the connective tissue, in which there was intense chronic inflammation, and migration of epithelium cells from the edges towards the central region to close to the wound was seen. The final evaluation demonstrated well organized epithelial and connective tissues in all the samples. Epithelium thickness was measured at 0, 14 and 21 days, from images of the digitalized histological sections. In comparisons between the test groups, the bismuth subgallate group was slightly better than the saline group, but no statistically significant difference was found at 21 days. It was possible to conclude that bismuth subgallate did not interfere in the tissue repair of the palatal mastigatory mucosa in dogs.
Haemostasis is a fundamental principle in surgical procedures, and can be obtained through the careful handling of soft tissue and by closing the surgical wound. Some situations can present prolonged bleeding, especially in excisional wounds. The concern regarding excessive bleeding from the palate after harvesting tissue has promoted a number of studies with various substances and materials for topical haemostatic action. Preliminary studies with the use of haemostatic agents in periodontal surgery show a faster and safer haemostasis, compared to the usual compression method.
Bismuth compounds have been used topically in medicine for almost three centuries, as an emollient, astringent and antiseptic agent. Bismuth subgallate, or dermatol, is an insoluble compound in water, alcohol, chloroform and ether and has a bright yellow, odourless, tasteless powder form. The use of bismuth subgallate paste in adenotonsillectomy procedures has improved the prevention of postoperative bleeding. It has been reported that bismuth subgallate is able to reduce the clotting time and its localized action has an effect on the early steps of the intrinsic pathway. In periodontal surgery, bismuth subgallate paste was used as a simplified and efficient method for hemostasis. Kim et al. compared two different hemostasis methods at the palatal donor sites in 20 patients. The application of bismuth subgallate paste to wounds demonstrated significant benefit in achieving hemostasis, when compared to the pressure method only. The average time to hemostasis was significantly shorter for the bismuth subgallate paste group, when compared to the moistened gauze group, and the difference between the groups was statistically significant.
One experimental study using bismuth subgallate on skin wounds in Wistar rats revealed that the qualitative evolution of the granulation tissues showed no significant difference when compared to the control group treated with saline, thereby concluding that the substance did not interfere with the normal development of skin wound healing. Recently, bismuth subgallate paste was evaluated on bone repair in rats, and the study demonstrated that the material presence did not interfere with post-tooth extraction bone repair.
Although there have been favourable reports of its use as a local haemostatic in periodontal surgery, there are no reports on the healing process of oral mucosa wounds using this material. This study evaluates the early recovery process of palatal wounds in dogs using bismuth subgallate.
Materials and methods
Five healthy male mongrel dogs with permanent dentition and no periodontal disease were used in this study. The animals were anaesthetized intravenously with 0.1 mg/kg acepromazine maleate (Acepran, Univet, São Paulo, Brazil) and 12.5 mg/kg thiopental sodium (Thiopentax, Cristália, Itapira, Brazil), and then were connected to the circuit anaesthetic inhalant and maintained anaesthetized with 1.5% halothane diluted in oxygen at 100% (Halotano, Cristália, Itapira, Brazil).
For each animal, eight 5-mm partial-thickness punch biopsies (Otemac Laboratory, Curitiba, Brazil) were made in two uniform columns of the palatal mastigatory mucosa, between the canine and first molar areas. Initially, as test group 1, 4 biopsies were performed on one side, and haemostasis was achieved by compression with moistened gauze pressure. After the complete haemostasis, the biopsies on the opposite side were carried out, as test group 2. For this group, bismuth subgallate paste (Farmanilquima, Curitiba, Brazil) was applied to the wounds with a dry gauze compression ( Fig. 1 ). Sterile bismuth subgallate powder was added to the saline to obtain a consistency similar to that of toothpaste. During the experimental period, all the animals were kept on a controlled soft-diet, and 0.5 mg/kg butorphanol tartrate (Torbugesic, Wyeth-Whitehall, Itapevi, Brazil) was administered intramuscularly once a day for 3 days to control pain.
At 3, 7, 14 and 21 postoperative days, the animals were anaesthetized again in order to obtain new biopsies. The soft tissue blocks were removed with a sharp surgical number 15 blade (Becton Dickinson, Curitiba, Brazil) from the anterior to posterior surgical palatal wounds, in order to include all created wounds. The samples were always collected bilaterally for each period of evaluation.
Six 6 μm histological central sections from each sample were stained with haematoxylin and eosin (H–E) for evaluation. A histologic description of tissue repair was made in all periods, and the epithelium thickness was measured at 0 h as a control, and at 14 and 21 days for test groups 1 and 2. The images of the digitalized histological sections with 4× magnification were obtained using an optical microscope (Olympus BX 50, Japan) and analysed by the Image J programme (National Institutes of Health, Bethesda, USA), previously adjusted in millimetres. The results are given as means and standard deviation to describe the dispersion of the data, and the groups were compared using the Kruskall–Wallis rank test. The comparisons between the pairs of means were made using the Dunn test.
Results
For the 3-day period samples, areas of discontinued epithelium at the edges of the wound and a mass of disorganized tissue including clotted blood and a fibrin layer covering the connective tissue were observed. An infiltrate of chronic inflammatory cells, predominantly macrophages and lymphocytes was present in the subepithelial connective tissue of the wound margins, although permeated by a neutrophil infiltrate. These observations were similar in both groups but differentiated by an intense inflammatory response and the reduced quantity of clotted blood in test group 2 ( Fig. 2 a and b ).
At 7 days, there was an increase in the volume of epithelial tissue with intense proliferation of the basal and spinosum stratum. Weak intercellular junctions and widened intercellular spaces were seen. The migration of epithelial cells from the edges towards the central region was not sufficient to close the wound. A moderate infiltrate of chronic inflammatory cells was seen in both groups, but was more evident in test group 2. Macrophages and lymphocytes were present, but no neutrophil infiltrate was observed. Granulation tissue and numerous fibroblasts were seen in all of the samples ( Fig. 2 c and d).
At 14 days, a well-organized epithelial tissue was observed. Even though the cellular strata were defined, the corneum stratum was fine and parakeratinized, and there were no signs of epithelial ridges. Few chronic inflammatory cells were present in test group 2; nevertheless, clear repair signs with intense deposition of collagen fibres were observed ( Fig. 2 e and f).
21 days later, the repair process was almost completed. A well-defined and parakeratinized epithelium was present. Epithelial ridges were noted, although they were less noticeable and the epithelium thickness was thin when compared to the control group. The connective tissue had an increased density of collagen fibres and the inflammatory response in the subepithelial connective tissue had almost completely disappeared in all of the samples (Figs. 2 g, h, 3 , 4 and 5 ).
