Traumatic injuries to newly erupted permanent anterior teeth are common during childhood (1, 2). Avulsion is a traumatic scenario where immediate replantation leads to excellent healing and good prognosis. The type of storage media used is a significant factor that can affect the long-term prognosis for replanted teeth. The storage media maintains the viability of the periodontal ligament cells and thus permits longer extra-alveolar periods prior to replantation (3). Maintenance of the avulsed tooth in an adequate wet medium that can preserve the vitality of the periodontal ligament cells that remain on root surface is the key to success of replantation (4). Current research favors milk, soy milk, saliva, physiologic saline, cell culture media, probiotic media, propolis, coconut water as storage media (3, 5-10)
Host bacteria are a natural surrounding for avulsed teeth. Probiotic bacteria are live microbial food supplements that may benefit the host by influencing the balance between the many species of the commensal flora both in oral cavity and the intestinal tract (11, 12). Recently a host probiotic, L. reuteri DSM 17938 and L. reuteri ATCC PTA 5289, have been shown to preserve vitality of periodontal ligament (PDL) cells in case of dental trauma (3). The key event is that harmless microorganisms, such as strains of lactobacilli or bifidobacteria, can occupy a space in a biofilm that otherwise would be colonized by a pathogen (13). Bifidobacteria are the predominant anaerobic bacteria within the intestinal lumen and play a critical role in maintaining the equilibrium of the normal intestinal flora (14).
Since the late 1980s, a range of dairy products containing bifidobacteria has been marketed in a number of countries worldwide and studies have been performed to validate the survival and positive effects of Bifidobacterium DN-173 010 within the gastrointestinal tract (15). Caglar et al. (16) stated that Bifidobacterium may also have a role in maintaining health by promoting a microbiological balance in the oral cavity and oral defense factors, such as the peroxidase system may inhibit the acidogenicity of bacteria. The aim of the present in vitro study was to investigate the potential of a storage medium, probiotic yogurt (Bifidobacterium animalis DN 173010) in comparison with Hank's balanced salt solution (HBSS), saline and milk in maintaining viable periodontal ligament (PDL) cells on simulated avulsed teeth.
Materials & Methods
Patients received both oral and written information about the study and signed a consent form prior to handling their extracted teeth. Thirty-six freshly extracted single-rooted human permanent second premolar teeth with closed apices were divided into one of the four experimental groups and two control groups (positive and negative) consisting of seven samples each. Teeth extracted from patients with moderate to severe periodontal disease or with extensive caries were excluded. The teeth were extracted as atraumatically as possible and washed in sterile saline solution to eliminate residual blood. Following extractions, the coronal 3 mm of PDL tissues was scraped with a #15 scalpel to remove cells that may have been damaged The positive and negative controls corresponded to 0 minutes and an 8-hour dry time, respectively. After extraction, the positive control teeth were immediately treated with dispase and collagenase. The negative control teeth were bench-dried for 8 h, with no follow-up storage solution time, and then placed in the dispase and collagenase.
The experimental teeth were dried for 30 minutes (during the present time interval, the coronal PDL cells were curetted) followed by a 45 min immersion in one of the four media: Bifidibacterium animalis DN 173010 containing probiotic yogurt (Activia, Danone, Luleburgaz, Turkey), HBSS, saline and milk. Each experimental tooth, after drying and soaking, was incubated for 30 min in 15 ml falcon tubes with a 2.5 ml solution of 0.2 mg ml−1 of collagenase CLS II (Biochrom, Berlin, Germany) and a 2.4 mg ml−1 solution of dispase grade II (Sigma-Aldrich, St.Louis, MO, USA) in phosphate buffer saline (PBS). After incubation, 50 µl of fetal bovine serum (Biowest, Nuaille, France) was added to each tube. All tubes were then centrifuged for 4 min at 1000 r.p.m. The supernatant was then removed with sterile micropipettes, and the cells were labeled with 0.4% Trypan Blue (Sigma-Aldrich, St.Louis, MO, USA) for determination of viability, according to Polverini & Leibovich (17). The number of viable protective least significant difference PDL cells were counted under a light microscope with a hemocytometer at 20× magnification and analyzed. The number of viable cells was obtained by the following mathematical equation: unstained cell count (viable cell) X the dilution of the cell suspension X 104 /number of squares of the hemocytometer counted (3, 10, 18). Statistical analysis of the data was accomplished using Nonparametric ANOVA complemented by Kruskal-Wallis Test and Dunn's Multiple Comparisons Test.
The positive control was found to have a median of 16580000 viable cells and was significantly better than the others, (Table 1) There was only statistically significant differences between positive control and negative control (p<0.001). Besides positive control, the teeth stored in Bifidibacterium animalis DN 173010 containing yogurt demonstrated the highest number of viable PDL cells (N:1 830 000) followed in rank order by HBSS, saline and milk. However there was no significant difference in the number of viable PDL cells between HBSS, milk, Bifidibacterium animalis DN 173010 containing yogurt and saline (p>0.05). All experimental groups were significantly lower than positive controls (p<0.001).
The prevention or limitation of replacement root resorption is the key point in early reestablishment of PDL cellular physiology in case of dental trauma. The critical extraoral dry time for PDL cell viability and the nature of storage media play a very special role at this cellular balance. Blomlof, a pioneer in storage media research, was one of the first to investigate the effect of milk with that of saliva on human PDL cells in simulated conditions (19-22). Milk may keep the cells viable for up to 6 h (hours), saliva may keep them up to 2 h while saline for 1 h (23). Hank’s Balanced Salt Solutions (HBSS) (Save a tooth®, EMT Tooth Saver®) or Viaspan® are cell culture media introduced to North America. They have been successful in preserving the viability of the PDL fibers for extended periods (24). Whereas storage media was a topic of research interest (25-31), the critical extraoral dry time of PDL had been investigated in several studies (3, 10, 30, 32-36) where time frames of 30 min or 45 min are appreciable. Forty-five minute time period was chosen as it allows for comparison with previous investigations. It should be noted that the storage media are primarily used to maintain the viability of the cells and that infection is a secondary consideration (3). The PDL fluid supplies the tooth with the nutrition necessary for the PDL cells to survive. The PDL remaining on the root after injury is dependent on a supply of vital metabolites. Cell destruction begins when these metabolites are withheld. To preserve optimal cell metabolism, the supply should be renewed within 60 minutes from the time of injury. If these cells survive, they will catalyze the reproduction of new cells, which can differentiate and reinstate the supporting tissues. Main philosophy of this survival may involve prevention of protein synthesis in the bacterial cell, encouraging the action of fibroblasts and healing of connective tissue, which contributes to the recovery of the PDL after injury (37).
Probiotics facilitate intestinal epithelial homeostasis through a number of biological responses, including promoting proliferation, migration, survival, barrier integrity, antimicrobial substance secretion, and competition for pathogen interaction with epithelial cells (38, 39).
In the present study, the teeth stored in positive control demonstrated the highest number of viable PDL cells followed in rank order by Bifidibacterium animalis DN 173010 containing yogurt, HBSS, saline and milk. However, there was no significant difference among them. This result is in accordance with Caglar et al. (3) where L reuteri was found to be as efficient as the latter storage media. It was previously confirmed that in periodontal pockets treated with beneficial bacteria, subgingival re-colonization of periodontopathogens was delayed and reduced, as was the degree of inflammation (40). This might have a protective storage media effect of current probiotic species.
The number of viable PDL cells have been detected through root surface treated by collagenase and dispase grade II to minimize the exposure of cells to active trypsin and to preserve maximum cell viability. This procedure allowed rapid cell retrieval and maintained maximum cellular integrity, as was demonstrated by the positive control samples (3, 10, 41).
In conclusion, Bifidibacterium animalis DN 173010 seems to be an alternative for the temporary storage of avulsed teeth due to high number of viable PDL cells. Probiotics may be a suitable transport media for avulsed teeth, but further research is warranted using the commercially available products.