Objective: To develop an in vitro model for measuring oral substantivity that is predictive of in vivo behaviour. Methods: Three in vitro models were developed using hard (bovine enamel [EN], hydroxyapatite powder [HA]) or soft (human buccal epithelial cells [BEC]) tissue and characterized using eugenol as a marker of uptake. Substrates were incubated with artificial saliva for 2h, exposed to experimental mouthrinse for 20min then washed twice with deionised water. Bound eugenol was desorbed with methanol and quantitated by HPLC. The behaviour of three experimental mouthrinses (MW1-3), formulated to differing degrees of predicted substantivity, was determined in each model. The same experimental rinses were used in a randomised, double-blind, three-way cross over clinical study (n=16). Volunteers rinsed with MW1-3 for 20s, rinsed with deionized water for 10s and unstimulated saliva samples were collected over 2h and analysed by HPLC. Results: Relative uptake of eugenol for each in vitro model, and AUC for eugenol in saliva post-rinse are shown below:
|
In vitro model |
Relative Uptake (±SD, n=X) |
||
|
MW1 |
MW2 |
MW3 |
|
|
BEC |
42 (28)b |
51 (21)b |
100 (30)a |
|
EN |
83 (75)a |
58 (32)a |
100 (51)a |
|
HA |
25 (2)c |
53 (5)b |
100 (7)a |
|
In situ model |
AUC (μg*min/ml) (±SD, n=16) |
||
|
154 (17)b |
198 (21)a |
207 (22)a |
|
Significant differences are indicated by different letters (p < 0.05).
The in vitro models behaved differently, although there was directional agreement between BEC and HA. HA was the only model to show significant differences between MW1-3. In situ, there was no statistical difference in total salivary eugenol levels between MW2 and MW3, however both were significantly greater than MW1. Conclusions: None of the in vitro models exactly resembled the in situ situation, however directional agreement was observed for the HA model. Further work is required on these models to better replicate the oral situation.