## 4 Discussion
Irradiation with CO2 laser can induce physiochemical changes in enamel such as hydrophobicity and zeta potential, which have been found to affect the adhesion of some bacterial species on enamel surfaces [47; 48]. Moreover, it seems that CO2 laser irradiation can directly impact the activity and viability of _Streptococcus mutans_ biofilm on the surfaces of teeth [49]. The topical use of fluoride has been widely acknowledged for its antibacterial properties on cariogenic bacteria in the plaque [50]. The combined use of 5% sodium fluoride varnish and a 10,600 nm CO2 laser was found to reduce bacterial numbers on dentine surfaces [51]. All the studies here used a chemical model rather than a microbiological caries model. Compared to the microbiological model, the chemical model used for the cariogenic challenge allows more control over the experiment conditions, which may generate fewer random errors in the results [52]. Three studies used simple mineralization models (with constant pH around 5) and the other two used pH-cycling models (pH 4.4/7.0). The latter models provide a periodic alternation of pH when sugars are metabolized to form a caries lesion [53]. A main limitation of the chemical models is that they completely ignore the microbiological aspect of the formation of caries [54]. To date, the effect of adjunctively using a 9300 nm CO2 laser and fluoride on the action of cariogenic biofilms on hard dental tissues has not been demonstrated, and further research and exploration using a microbiological model are needed. All of the included studies used a microsecond short-pulsed CO2 laser. The duration of the pulse matches well with the thermal relaxation time of the enamel, which results in less thermal deposit and higher peak power. Only one study included used a CO2 laser accompanied by water cooling [37]. Water cooling is important for controlling temperature. Enamel without water cooling manifested non-uniform surfaces with undesirable white asperities, which are unfavorable to acid resistance [55]. All of the studies included in this systematic review applied fluoride after laser treatment. Researchers found that fluoride application after laser treatment resulted in a significant increase in acid resistance of the enamel with fluoride application before laser irradiation. Fluoride application after laser irradiation produced a greater fluoride uptake in the enamel than fluoride application before laser irradiation [56]. However, there are some opposite findings. A few studies reported that applying fluoride prior to CO2 laser treatment is more effective in preventing enamel demineralization against acid challenge [27; 57; 58]. A high heterogeneity was found in the meta-analysis comparing the effects on mineral loss of the treatment combining fluoride and laser (F + L) versus treatment only with laser (L). We performed a subgroup analysis to look for the source of the heterogeneity. To our knowledge, this grouping method is the best not only because it was determined according to the types of fluoride or methods of assessing mineral loss, but as it can also be regarded as classified according to research group. High heterogeneity was found in the subgroup assessed by cross-sectional microhardness. The high heterogeneity could be due to the sample size and variation in the laser parameters used in the study design. In addition, all the studies in the subgroup in which mineral loss was assessed by cross-sectional microhardness received a treatment of 0.0825% sodium fluoride, and all the studies in the subgroup for which mineral loss was assessed by transverse microradiography received a treatment of 1.23% acidulated phosphate fluoride. Therefore, the subgroup in which the mineral loss was assessed by cross-sectional microhardness can also be regarded as the subgroup using 0.0825% sodium fluoride. For this reason, the results of the subgroup analysis suggested that the additional use of fluoride even in low concentrations still leads to significantly different results from the single use of laser.