Introduction ============ The most important step for all the drugs and drug formulations is their effective administration to the body. The oral cavity is the first step of the digestive system, where most of the drugs or formulations may be easily absorbed from the gastrointestinal tract. As the most important and main port of drug absorption in the oral cavity, the oral mucosa seems to be the best site for formulations like ointments, gels, solutions, pastes, etc. Oral mucosa comprises oral cavity tissues like keratinized and nonkeratinized stratified epithelium, as well as the underlying lamina propria composed of fibrous connective tissue. As reported in the literature, oral mucosa can be divided into two layers; a thicker keratinized squamous cell keratinized stratified epithelium, and a thinner nonkeratinized stratified epithelial which include a large amount of connective tissue ([@B1]-[@B3]). The former is composed of hard, thick, flat, and horny tissues that are covered by thick keratinized epithelium. Oral mucosa is also divided into two parts, the inner soft mucosa and the outer hard mucosa ([@B4]). In normal conditions, the outer mucosa, which is called buccal mucosa, is covered by thick keratinized stratified epithelium. The buccal mucosa forms a part of the lips and borders the teeth. The oral cavity mucosa may serve as a target for both the systemic and local drug delivery because of its permeability. Buccal mucosa is permeable to a variety of drugs ([@B4], [@B5]). Various studies have demonstrated that topical drug absorption is affected by many variables, such as drug formulation, type and surface area, as well as site of administration. Because the buccal mucosa represents the most permeable site in the oral cavity, the drug delivery by buccal route may be regarded as a valuable alternative to the parenteral routes in many cases. The buccal mucosa has been used to deliver different agents with a variety of objectives in oral drug delivery ([@B6]-[@B12]). The transmucosal absorption of drug from buccal epithelium is limited to drugs with relatively low molecular weights. The permeability of the buccal mucosa depends on many factors, such as the formulation and the type of the molecule. Some classes of drugs cannot penetrate the buccal mucosa because of their inability to dissolve within the lipoidal intercellular fluid. However, other drugs are rapidly absorbed in the buccal tissue by passive transport after their topical application ([@B13], [@B14]). In comparison to other mucosae such as palatal and nasal mucosa, the absorption of drugs through the buccal mucosa is relatively higher and is limited to formulations that are soluble in lipids. When drugs do not dissolve in the lipoidal intercellular fluids, they have a poor permeability rate ([@B15]-[@B19]). The objective of this study was to evaluate and compare the effects of different formulations containing metronidazole gel and metronidazole cream on the buccal mucosal epithelial cells after topical administration. Materials and Methods ===================== This experimental study was performed on 8 healthy male volunteers, aged 20-27 years old. All the subjects were informed about the study and consented to the study. The study was approved by the ethic committee of Tehran University of Medical Sciences, Tehran, Iran (ethical code: 90-10-1). A consent form was signed by each patient. Inclusion criteria: 1-normal buccal mucosa (free of any lesion or ulcer), 2-age between 20 to 30 years, 3-no history of sensitivity to metronidazole. Exclusion criteria: 1-allergy to metronidazole, 2-a history of antibiotic consumption, 3-allergy to propylene glycol, 4-allergy to paraben, 5-allergy to propylene glycol, 6-allergy to paraben, 7-current oral disease such as dental caries or periodontal disease, 8-history of systemic diseases. The drug was applied directly to the oral mucosa by a topical manner as a solution, gel or cream. The formulation is listed in the [Table 1](#T1){ref-type="table"}. Metronidazole gel is a suspension that is a mixture of metronidazole, hydroxypropyl methyl cellulose (2%) and isopropyl alcohol (99%) with a concentration of 1%. All volunteers (8 patients) were selected randomly to receive one of the three formulations. The oral mucosal cells were washed with normal saline and divided into 3 groups (solution, gel, and cream). After washing the mouth, cream formulation, as a test solution, was applied to the buccal mucosa in a quantity of 1 g. Then, a 3-cm diameter piece of gel with the same quantity was applied on the mucosa. After 20 min, the buccal mucosal cells were washed with normal saline and transferred to the study room to be immediately fixed by methanol for 24 h. Samples were collected and sent for histological examination to Isfahan University of Medical Sciences, Isfahan, Iran. **Histology** Samples were fixed in 10% formaldehyde. Then, the mucosal cells were prepared in paraffin blocks. Then, 5-μm sections were taken by the slide-making method. Sections were stained with Hematoxylin and Eosin (H&E). A general and morphological study of the sections was done under a light microscope (Leica 4000 DM, Germany) ([@B20], [@B21]). ###### The formulations used in the study -------------------- ----------------- ------------ ------------------ ------------ --------- ------------------ --------------------- Formulation Concentration pH Dosage Dosage\ Active ingredients Manufacturer Unit Metronidazole Gel 1 g gel 100 g Propylene glycol Aladdin, China Metronidazole Gel 1 g gel 1 g gel Propylene glycol Aladdin, China ------------------------------------------------- -------------- ------------- ------------- ----------- ----------- ------------------ --------------------- ###### The criteria used for distinguishing the types of cells in the buccal mucosa ------------------------------------------------------------------------------------------------------------------------------------ ***Type of cells*** ***Characteristics*** ------------------------------------------- ----------------------------------------------------------------------------------------- Mucous Flat, thin, and colorless epithelium Lymphocyte Small and colorless cells with light violet nuclei\ Flattened cells with medium to large nuclei Macrophage Medium-sized cells with light violet nuclei and clear cytoplasm\ These cells may be red, blue, yellow or orange because of their presence in the mucosae Giant multinucleated macrophage Large, multinucleated cells that may also contain different colors\ This may be due to the number of nuclei of these cells Keratinocyte Thin keratinized stratified epithelium\ Cells are polygonal in shape with round nuclei Langerhans cell Plump, large eosinophilic cells\ These cells are scattered throughout the epithelium Melanocytes Small, round or oval cells with dark nuclei\ These cells are usually present in the upper parts of the epidermis and are easily observed under light microscope ------------------------------------------------------------------------------------------------------------------------------------------ {#F1} ###### The mean (± standard deviation) of the cellular variables in the buccal mucosa in each of the treatment groups **Treatment** **Parameters** --------------- ---------------- --------------- ---------------- ---------------- ---------------- Group A Metronidazole 24 (17-30) 3 (2-3) 12 (2-14) 8 (6-12) 4 (2-8) 8 (4-12) Gel 19 (11-25) 4 (2-6) 15 (6-24) 5 (2-6) 3 (1-5) 5 (2-7) Group B Metronidazole 20 (9-29) 3 (1-6) 10 (2-12) 6 (5-10) 4 (1-6) 5 (3-7) Cream 12 (6-19) 2 (1-3) 8 (5-13) 1 (0-3) 1 (0-2) 1 (0-3) t-test (p-value) 0.005\* 0.021\* 0.025\* 0.008\* 0.006\* ---------------------------------------------------------------------------------------------------------------------------------------------- The data were analyzed using descriptive and inferential statistics (the Chi-squared test). Data were expressed as mean ± standard deviation, minimum and maximum (min-max). P-values less than 0.05 were considered statistically significant. All the statistical analyses were done using SPSS 16 software. Results ======= Microscopic examination of the buccal mucosa cells demonstrated that keratinocytes have a regular columnar shape, and they are generally located near the center of the epithelium. There are one or two layers of cells. Also, the basal nuclei are seen in all buccal cells. The nuclei were mostly elongated in shape and smaller in size, compared with other cell types ([Fig. 1](#F1