Soy Milk as a Storage Medium to Preserve Human Fibroblast Cell Viability : An In Vitro Study

Mature tooth avulsion results in attachment damage and pulp necrosis (1,2). Ideally, the tooth should be replanted immediately after the injury (1,3). Unfortunately, immediate repositioning of the tooth is not always possible (3,4). In such situations, the prognosis is strongly influenced by extraoral drying time and by the transportation medium (5). Transportation in wet physiological media prevents the desiccation of periodontal cells, which reduces the cell damage (1) and prevents ankylosis and replacement resorption (6). Soy Milk as a Storage Medium to Preserve Human Fibroblast Cell Viability: An In Vitro Study


INTRODUCTION
Mature tooth avulsion results in attachment damage and pulp necrosis (1,2).Ideally, the tooth should be replanted immediately after the injury (1,3).Unfortunately, immediate repositioning of the tooth is not always possible (3,4).In such situations, the prognosis is strongly influenced by extraoral drying time and by the transportation medium (5).Transportation in wet physiological media prevents the desiccation of periodontal cells, which reduces the cell damage (1) and prevents ankylosis and replacement resorption (6).media that are easily available at the site of an accident (8,9,18).This solution provides nutritive substances that are able to nourish the cells (18,19), such as amino acids, carbohydrates and growth factors (20), a neutral pH and physiological osmolality (18,19).Although several studies have been conducted evaluating the cow milk as a storage medium (10)(11)(12)(13)(14)18), the potential use of soy milk for tooth storage has been poorly evaluated (21).Whereas a growing number of people are using this alternative to cow milk, which reflects an increase in its accessibility, this study evaluated a milk-like soy beverage compared with whole cow milk, skim cow milk and powdered cow milk regarding their ability to preserve fibroblasts.The null hypothesis was that soy milk is as effective as cow milk for preservation of fibroblast cell viability.

MATERIAL AND METHODS
Immortalized human mouth fibroblasts (Cell Bank of Rio de Janeiro, Rio de Janeiro, RJ, Brazil) were cultured in T-25 cell culture flasks containing Dulbecco's Modified Eagle Medium (DMEM) (Sigma Chemical Co., St. Louis, MO, USA) supplemented with 10% fetal calf serum (Invitrogen, Branchburg, NJ, USA), 100 units mL -1 of penicillin/streptomycin (Sigma Chemical Co.) in a humidifier incubator with 5% CO 2 and 95% air at 37 o C. Growth was permitted until the cells achieved confluence.These cells were detached, counted using a hemocytometer and plated in 96-well plates (Coastar Corp., Cambridge, MA, USA) at an initial density of 1x10 4 cells.well - in 100 µL of culture medium.The plates were returned to the incubator for 24 h.Subsequently, the culture medium was drained from each well and the cells were exposed to 100 µL of the different experimental solutions at room temperature for 5, 15, 30 and 45 min.The storage solutions used in the experiments were one soy milk fortified with vitamins -SM (Sollys Original; Nestlé, Araras, SP, Brazil) and three presentations of cow milk: long shelf-life ultra-high temperature whole milk -WM (Italac Integral; Italac, Corumbaíba, GO, Brazil); long shelf-life ultra high temperature skim milk -SKM (Italac Desnatado; Italac) and powdered milk -PM (Molico; Nestlé, Araçatuba, SP, Brazil).The positive control corresponded to cells maintained in 10% DMEM, without additional treatment.After the experimental times ran out, the storage solutions were replaced by 10% DMEM and incubated with MTT solution (5 mg.mL -1 ) prior to cell viability evaluation.The same protocol was used for the positive control group (DMEM).After 4 h, the MTT solution was removed and 100 µL of dimethyl sulfoxide (Sigma Chemical Co.) were added to each well.Cell viability was determined by measuring the optical density at 540 nm on a microplate reader.The experiment was done in quadruplicate on two different days, with 10 samples per group.The absolute values of absorbance obtained from each well for each group at all experimental times were subjected to analysis of normality.Data were analyzed statistically by Kruskal-Wallis and Dunn's test at a 5% significance level.

RESULTS
The absorbance values, which represent fibroblast cell viability for each tested medium and for each tested period are shown in Figure 1.There were time-dependent results according to the analyzed experimental solution.SKM had the lowest capacity to maintain cell viability in all analyzed times (p<0.05).At 5 min, SKM had lower indices of absorbance than the PM and the control group (DMEM) (p=0.001,Fig. 1A).At 15 min, SKM remained as the worst analyzed storage solution, with absorbance rates lower than the WM, SM and control group (DMEM) (p=0.020,Fig. 1B).Even though at 30 and 45 min the cells preserved in SKM showed viability comparable to that of PM and WM (p>0.05), its absorbance indices were inferior to those of the SM and control group (DMEM) (30 min, p=0.006; 45 min, p=0.001,Fig. 1C and Fig. 1D, respectively).Although there is a tendency to reduce the levels of absorbance presented in each group over time, there were no significant differences in viability rates between 5 and 45 min for each storage solution (WM, p=0.154;SKM, p=0.270;PM, p=0.070;SM, p=0.241).

DISCUSSION
In the current study, the null hypothesis was rejected.SM was able to preserve fibroblast cell viability, showing similar results to those of the control group (DMEM culture medium), cow milk and PM.The potential benefits of SM as a storage medium for avulsed teeth has recently been demonstrated.In agreement with the present study, Moazami et al. (21) showed that HBSS, powdered baby formula and SM maintained equally the cell viability.Despite the similar results, it is important to address the differences of the cell culture model, the method used for viability analysis, type of soy powered milk used and periods of evaluation.The improved viability of cells stored in SM may be due to the cytoprotective effects of nutrient constituents such as carbohydrates, sugars, proteins, fats, calcium, vitamins A, C, D and E, as well as the physiological pH.Such components may help nourishing the cells and maintaining their viability, which makes SM an alternative to other milk formulations as a storage medium.There is strong evidence that milk effectively maintains the viability of periodontal ligament cells (6,(11)(12)(13)(14)18).However, concern has been raised over its temperature (4,6,11) and fat content (4,12).
Avulsed teeth are normally stored in a medium at room temperature (10), although some studies suggest better results when they are stored in chilled milk (6,11,22).The current study was performed at room temperature to reproduce the clinical conditions.Regarding the fat content, a previous study indicated that milk with a lower fat content might be more appropriate for maintaining cell viability than whole milk, which has a higher fat content (12).However, in the present study, there was no significant difference between WM and SKM.The lack of difference between the groups maintained in WM and SM might be related to the experimental design, the type of milk and the temperature of the milk.Interestingly, the milk with a lower fat content presented worse results compared with SM and the positive control (DMEM).Given these results, it may be speculated that the reduction in fat and carbohydrates during the production of SKM reduced its capacity to nourish the cells.
This study also investigated powdered milk, which presented good results when compared with the long shelf-life milks.Similar results have been reported elsewhere (14,21,23), but differences in cell type and experiment design do not allow direct comparisons.Despite PM potential as a storage media, the favorable results obtained with SM, were a point of great interest in the present study.This soy-based drink enriched with vitamins represents a promising alternative for Despite the interesting results found in the present study, considerations must be addressed regarding the methodologies used in in vitro studies to evaluate storage media for avulsed teeth (2,10,24,25).Immortalized cell cultures, such as those used in this study, differ from primary cell cultures by their ability to proliferate indefinitely; thus, they have a different life cycle, which is a limitation.Experimental designs using newly extracted teeth, which simulate avulsion, should be carried out because they reproduce more accurately the clinical situation.However, we consider the model using extracted teeth as appropriated after the preliminary studies, which tend to be better designed with immortalized cells, as a way to validate the results.Thus, in vitro studies using immortalized cell cultures represent a complementary model to tooth models, with easy reproducibility, which justifies the choice of methods in this study.
Regarding the method used to evaluate cell viability, studies using trypan blue staining have shown less sensitivity because it does not characterize the metabolic condition of the cell or its actual physiological health (2).In this way, an MTT assay should be a more sensitive tool for cell viability analysis than trypan blue staining (4).Considering that the relative opacity of milk may interfere in optical density reading (4), after exposure to milk, the wells were washed twice with PBS and filled with fresh medium to avoid interference in the optical readings.
Studies have demonstrated that time can influence the effectiveness of different storage media (1,4,11).The time over which cell viability is preserved in milk is controversial (4).The present study used time periods (from 5 to 45 min) because this allowed monitoring small changes over time.At the evaluated times, WM, PM and SM stored at room temperature showed a similar capacity to preserve human gingival fibroblasts.However, what drew most attention in this study were the results for the SM and SKM.While the former was similar to the positive control at all experimental times, the latter was significantly less effective than the control group (DMEM).Further in vitro and in vivo studies should be conducted with variable dry periods and longer storage times to determine the capacity of SM for storage of the avulsed teeth.
The favorable results obtained in this preliminary study suggest that SM might be a promising alternative medium for transportation of avulsed teeth.In contrast, SKM tends to be not adequate as a storage medium for avulsed teeth.