Fibroblast like cells predominate in

Fibroblast-like OTX-015 predominate in human PDL cell culture. Several metabolic and morphologic similarities exist between PDL fibroblasts and human skin fibroblasts in vitro (Blomlof, 1981), which make human skin fibroblasts an ideal cell line to test PDL fibroblast storage media. To enumerate viable cells, studies have used either collagenase – dispase enzymes (Martin and Pileggi, 2004; Gopikrishna et al., 2008) or trypan blue exclusion staining (Sigalas et al., 2004; Ozan et al., 2008). Since trypan blue exclusion staining easily and quickly differentiates nonviable from viable cells (Ozan et al., 2008), we chose this technique for our study. On the other hand, the use of human foreskin fibroblasts presents some limitation to our study which should be considered when interpreting the results. Previous studies have recommended using extracted teeth, as a simulation of avulsion (Martin and Pileggi, 2004; Haas et al., 2008). However, trauma induced by different clinicians during extractions, prior to obtaining PDL fibroblasts, could translate to variability of PDL cell vitality counts (Rajendran et al., 2011). Therefore, there are both advantages and disadvantages to using either skin fibroblasts or PDL fibroblasts for in vitro studies. A second limitation of our study was using trypan blue staining to assess cell viability because this technique assesses neither the physiologic health nor the metabolic capabilities of the cells. Furthermore, this method cannot distinguish between necrotic and apoptotic cells (Rajendran et al., 2011). Finally, because homogenized milk is the most efficient and easily available storage medium for avulsed teeth, our study should be considered as the initial step for future investigations of the various clinical applications of S. persica.
HBSS is a standard saline solution that supports the growth of many cell types and is used extensively in biomedical research (Krasner and Person, 1992). In our study, incubation of human skin fibroblasts in HBSS for either 60 or 120min resulted in the lowest percentage of viable cells compared to any other experimental solution. This result contradicts other studies, which report that cell viability in HBSS is better than milk even after 12h (Hiltz and Trope, 1991; Huang et al., 1996). These inconsistent outcomes may be a result of the temperature of HBSS or the type of milk used in each study. Milk is widely accepted as an effective storage medium (Sigalas et al., 2004), and its efficacy can last from 3 to 48h (Ashkenazi et al., 1999). The physiologic osmolality (Elvin-Lewis, 1982) and the nutrients and growth factors in milk (Belford et al., 1995) may contribute to its increased ability to preserve cell viability. In our experiments, incubating human skin fibroblasts in milk resulted in a higher percentage of viable cells than observed in any other experimental solution at all experimental incubation periods. This result concurs with previous studies (Ashkenazi et al., 1999; Sigalas et al., 2004).
In order to determine if there is a threshold exposure to S. OTX-015 persica extracts that becomes toxic to the cells, additional in vitro and in vivo studies should be performed with variable storage times and concentrations of S. persica extracts. Additionally, currently there are no available antimicrobial or anti-inflammatory storage media that can also effectively maintain PDL cell viability. Because of the antimicrobial (Al-Otaibi et al., 2003; Ezoddini-Ardakani, 2010) and anti-inflammatory properties (Ahmad et al., 2011; Ibrahim et al., 2011) of S. persica, future research may establish that these properties improve the prognosis of avulsed teeth by preventing resorption that often leads to the loss of replanted teeth.

Conclusions
We conclude that homogenized milk preserves the viability of human foreskin fibroblasts better than HBSS, DMEM, and S. persica hexane and ethanol extracts. Because storage in S. persica hexane and ethanol extracts resulted in a similar percentage of viable cells as storage in HBSS, these extracts should be considered an alternative storage media to HBSS.