1. Introduction
Adequate tissue specimen fixation is an essential step for successful and precise microscopic interpretation. Fixation is defined as a series of chemical processes that preserve tissue from deterioration and prevent it from autolysis (tissue destruction by enzymatic activity) and putrefaction (tissue breakdown by bacterial action) [
1,
2]. Fixing the tissue before processing and staining is crucial to maintain the cellular morphology and composition as “lifelike” as possible by crosslinking the protein molecules to convert the tissue from a semi-liquid state to a semi-solid state [
2,
3]. Hardening the tissue specimen prevents its deterioration during preparation, facilitates its manipulation, and allows thin cutting (4–5 μm) before staining [
2,
3,
4].
Tissue fixation can be achieved using physical and chemical methods. The major agents available for tissue fixation include aldehydes, oxidizing agents, alcohol-based agents, and metallic group fixative agents [
3]. An example of an aldehyde-based solution is 10% formalin, which consists of 3.7% formaldehyde in water with 1% methanol [
5]. It is a fixative agent used worldwide owing to its commercial availability, ease of handling, low cost, and effective tissue preservation ability with minimal cellular disruption [
5,
6]. However, 10% formalin has some concerning toxic side effects, such as allergic reactions, skin irritation, and eye burning sensation [
6,
7,
8]. Moreover, formaldehyde has potential human carcinogenicity that can cause nasopharyngeal cancer, myeloid leukemia, and brain tumors [
9,
10]. Therefore, non-toxic natural fixative substitutes should be investigated.
Propolis, also known as “bee glue”, is a natural resinous material produced by bees from substances in plants and flower buds [
11,
12]. The hue of propolis varies according to the region and the plant it originates from [
13]. Its diverse types have a wide melting range of 60–100 °C. It is rigid at low temperatures and pliable at high temperatures [
14]. In contrast to solvents such as water and oil, ethanol rapidly dissolves the primary biologically active components of propolis [
15]. Propolis primarily consists of resin (50–70%), oil and wax (30–50%), and pollen (5–10%), as well as chemical elements such as phenol; amino acids; minerals; carbohydrates; vitamins B, C, and E; flavonoids; and carbohydrates [
16,
17]. Propolis has numerous biological and pharmacological properties, such as antimicrobial, anti-inflammatory, immunomodulatory, antitumor, and even anti-ulcer effects [
18]. These beneficial properties of propolis are attributed to the scavenging activity of free radicals and superoxide anions by butylated hydroxytoluene [
19].
As evident from previous studies, propolis decreases the apoptosis of periodontal ligament cells and can be used as a storage medium for avulsed teeth [
20]. However, there is a lack of studies in the available literature regarding the tissue fixation ability of propolis. Therefore, we aimed to assess the fixative properties of propolis, as it is relatively non-toxic and has attractive biological and pharmacological properties, leading us to believe that propolis might be a safer and more effective natural substitute for formalin.
4. Discussion
Formalin (10%) is the leading tissue fixative agent worldwide for histology and research laboratories. However, owing to the serious and harmful side effects of formaldehyde-based solutions such as 10% formalin, other superior and safer alternative natural fixative agents should be explored and researched.
In this study, propolis displayed promising results in terms of tissue fixation at all time points. Propolis composition may play a role in its effectiveness as tissue fixative and its superiority over 10% formalin. Propolis is composed mainly of a mixture of resin (40–50%), wax (25–30%), essential oils (10%), pollen (5%), and organic compounds such as steroids, amino acids, and polyphenols [
24]. The stickiness of propolis may protect tissue samples from the invasion of various pathogens (putrefaction) and reinforce cellular structure integrity [
24]. Furthermore, the presence of a mixture of resin and wax in the propolis composition could contribute to the crosslinking of tissue molecular proteins, which accelerates and enhances its fixation ability. Organic compounds, by their scavenging action, prevent tissue putrefaction and autolysis due to their antimicrobial and antioxidant effects, and this may also improve propolis fixation properties [
19]. This could explain why the tissue samples that were placed in propolis were firmer during grossing and fixed faster at 12 h and improved over time when compared to 10% formalin, where its fixation capacity improved after 24 h.
To the best of our knowledge, no study in the current literature has been conducted to investigate the fixative properties of propolis. However, several other investigations have been conducted to find alternative natural solutions to be used as substitutes for formalin. For example, honey has demonstrated promising fixative properties [
25]. Sugar syrup and jaggery have also shown encouraging results as alternative natural fixative agents [
8].
Patil et al. conducted an experimental in vitro study on five commercially available fresh goat meats (buccal mucosa) that were placed in 20% buffered formalin, distilled water, 20% honey, 20% sugar syrup, and 30% jaggery syrup. After storing the tissues for 24 h, they reported that honey, sugar, and jaggery syrup fixation ability were similar to each other and closer to formalin, with jaggery syrup showing the best results [
26]. These results are consistent with the findings from our study where honey, sugar, and jaggary syrup can be used as alternative natural fixative agents. However, our study showed that propolis had superior fixation ability compared to 10% formalin after 24 h. The same research group conducted another study to evaluate the fixation properties of 20% honey and 30% jaggery syrup over 6 months and concluded that jaggery and honey demonstrated satisfactory results after 6 months, and jaggery was comparable to formalin in tissue fixation [
27]. Al-Maaini et al. investigated the effectiveness of honey as a natural substitute for formalin by fixing kidney tissue and rat liver in honey and formalin for 24 h; they found that tissues fixed in low honey concentrations (10% and 20%) had comparable results to formalin-fixed control tissues, and increased concentrations of honey produced slower penetration rates and increased tissue hardening, making them difficult to section [
28]. A similar study was conducted by Lalwani et al., who tested the fixation and preservation efficacy of 10% processed and unprocessed honey for 24 h compared to 10% formalin. All groups had an adequate fixation for diagnosis, as there were no statistically significant differences in the scores between the groups. However, they found that the presence of staining artifacts was higher in the honey groups. In concordance with our study’s finding of propolis fixation ability, they concluded that both processed and unprocessed honey could be safe alternatives to formalin [
29].
Kuriachan et al. evaluated the fixative properties of 20% honey, 20% jaggery, and 20% sugar compared to 10% formalin. Human gingival tissues were placed in these solutions for 24 h. Honey and jaggery gave better results than formalin, which aligns with our results as propolis was superior to 10% formalin. However, contrary to our results, sugar showed inferior fixation ability compared to 10% formalin [
30].
Interestingly, propolis showed excellent preservation and fixation properties at 12 and 24 h compared with formalin. Furthermore, subsequent transfer of the tissue samples placed in propolis to formalin showed better fixation ability than formalin alone at all time points. These observations suggest that propolis could be an emergency fixative solution, especially in the absence of formalin. In addition, Kasetty et al. found that local anesthesia showed comparable morphological features as tissues fixed using formalin and can be utilized as an emergency fixative solution [
22].
Our research is the first study using propolis for tissue fixation, with no existing studies in the literature on the use of propolis as a natural formalin substitute. Propolis is non-toxic, non-carcinogenic, readily available, and can be a natural substitute for formalin. Moreover, it can also be naturally extracted using various extraction techniques. Our results showed that propolis is effective in tissue fixation and can be used as a safe substitute for formalin.
4.1. Study Limitations
The evaluators’ subjectivity in scoring different microscopic parameters in this study limited their ability to obtain accurate and highly standardized information. The scale used to score the slides should be more objective. Wide varieties of propolis with varied content could result in an unpredictable outcome while being used in this type of study. Thus, this study requires complete chemical analysis of the propolis extract used. Raw propolis is a non-homogenous resinous mass that cannot be directly used [
31]. It needs to undergo solvent extraction for purification, which can be technique-sensitive [
17]. Moreover, developing a standardized concentration of propolis is challenging because no research has previously examined its usage as a fixative agent. This study warrants further investigation to assess the application of propolis in histopathology.
4.2. Conclusions
Based on the results of this investigation, we conclude that propolis may be a promising natural fixative that could be useful in histopathology. Moreover, propolis can be used as an interim medium to preserve tissue biopsies before formalin fixation, as our study showed that propolis enhances the quality of formalin tissue preservation and fixation.