On day 7, non-lased sockets showed multiple areas of inflammation and granulation tissue with limited signs of primary bone formation. In contrast, extracted sockets irradiated either at 660 or 808nm showed less inflammatory cells infiltration, with more amount of new bone formation. Dual exposure led to minimum signs of inflammation and fibrosis with noticeable woven bone ossification centres. Active osteoblasts, lining the periphery of developing bone tissue, were clearly observed in all the lased sockets. In addition, formation of vaso-dilated blood vessels was obvious in laser-treated sockets, with more dominancy in dual-lased sockets.
On day 14, inflammation reduction along with revealing spots of bone formation and fibrosis areas were evident in non-lased sockets. However, sockets irradiated either at 808 or 660nm revealed considerably reduced signs of inflammation along with enhanced establishment of new blood vessels. However, only single irradiation at 808nm, as well as dual irradiation, led to formation of multiple areas of extending bone tissue, surrounded by epithelial-like rows of osteoblasts (Fig. 1).
3.1. Photobiomodulation at 808nm, either in form of single or combined irradiation, contributed to improved bone regeneration.
Histomorphometrical analysis of osseous tissue showed that the mean (± SD) percentage of bone tissue surface area in groups DM-control, DM-660, DM-808 and DM-dual was (1.32%±1.75), (14.21%±4.47), (41.31%±2.81), (44.42%±2.35) on day 7, and (4.24%±2.04), (17.53%±2.92), (64.41%±2.67), (71.22%±3.41) on day 14, respectively. A statistically significant increase in the osseous mean surface area on day 14 was observed as compared to day 7 in groups DM-808 and DM-dual (P < 0.05); yet, these two groups showed no significant difference with each other over the whole experiment (P > 0.05). On either day 7 or 14, the osseous mean surface area in groups DM-808 and DM-dual was significantly higher as compared to groups DM-660 (P < 0.05) and DM-control (P < 0.01) on the same day. No significant difference in the mean of osseous surface area was watched in group DM-660 in comparison to DM-control (P > 0.05) (Fig. 2A).
The relative levels of OCN mRNA expression in groups DM-control, DM-660, DM-808 and DM-dual were (21.34 ± 6.54), (39.34 ± 4.55), (103.24 ± 4.16), (112.12 ± 4.57) on day 7 and (26.62 ± 4.59), (49.51 ± 5.35), (271.11 ± 6.41), (302.68 ± 5.34) on day 14, respectively. OCN mRNA expression level experienced a significant increase (nearly 3-fold) on day 14 in comparison to day 7 in groups DM-808 and DM-dual (P < 0.01). Relative density of OCN expression level was significantly increased in groups DM-808 and DM-dual as compared to DM-660 and DM-control on either day 7 (P < 0.05) or 14 (P < 0.01) (Fig. 2A).
Quantity (± SD) of osteoblasts per unit area (mm2) in groups DM-control, DM660, DM808 and DM-dual was (0.09 ± 0.03), (0.21 ± 0.77), (3.31 ± 1.66), (3.42 ± 0.68) on day 7, and (0.22 ± 0.15), (2.12 ± 0.52), (6.71 ± 0.87), (7.11 ± 0.84) on day 14, respectively. A significant increase in the osteoblast number was revealed on day 14 as compared to day 7 in all groups, except DM-control (P < 0.01). Mean of osteoblasts number in groups DM-dual and DM-808 was significantly higher when compared to DM-control and DM-660 on either day 7 (P < 0.01) or 14 (P < 0.001). It showed no significant difference in the osteoblast number in groups DM-808 or DM-dual with the other one (P > 0.05). A significant increase in osteoblasts number was observed in group DM-660 in comparison with DM-control on day 14 (P < 0.05) (Fig. 2B).
Analysis of the relative density of RunX2 gene expression in groups DM-control, DM-660, DM-808 and DM-dual was (29.15 ± 8.01), (55.09 ± 5.04), (153.24 ± 8.18), (172.48 ± 7.35) on day 7 and (25.36 ± 6.91), (105.29 ± 6.58), (226.11 ± 5.16), (263.57 ± 6.43) on day 14, respectively. In all groups, the relative levels of Runx2 mRNA expression
showed no significant difference over the whole experiment (P > 0.05), except for group DM-dual presenting a significant increase on day 14 in comparison with day 7 (P < 0.05). A significant change was observed in groups DM-808 and DM-dual as compared to groups DM-660 and DM-control (a factor of 4-5-fold) (P < 0.01) (Fig. 2B).
3.2. Photobiomodulation at 660 or 808nm, either in form of single or combined irradiation, led to diminish fibrous tissue.
The mean (± SD) percentage of fibrosis surface area in groups DM-control, DM-660, DM-808 and DM-dual was (94.42%±3.92), (59.35%±3.62), (58.31%±2.53), (25.62%±1.23) on day 7, and (88.19%±1.24), (54.53%±2.79), (50.41%±1.76), (8.29%±0.64) on day 14, respectively. This amount, only in group DM-dual, exhibited a significant reduction on day 14 in comparison with day 7 (P < 0.05). Group DM-dual exhibited the least mean percentage of fibrosis surface area as compared to the other groups including DM-808 and DM-660 (P < 0.05) and DM-control (P < 0.01). The significantly highest percentage of fibrosis surface area was observed in group DM-control over the whole experiment (P < 0.05) (Fig. 2C).
The relative density of COLI gene expression in groups DM-control, DM-660, DM-808 and DM-dual were (94.17 ± 5.35), (56.13 ± 4.23), (57.73 ± 6.16), (42.01 ± 3.54) on day 7 and (83.03 ± 4.37), (39.65 ± 7.34), (41.52 ± 4.34), (13.36 ± 2.23) on day 14, respectively. Statistical analysis showed that COLI mRNA expression experienced a significant reduction in group DM-dual as compared to groups DM-808 and DM-660 on day 14 (P < 0.05), and DM-control on either day 7 (P < 0.01) or 14 (P < 0.001). Groups DM-808 and DM-660 succeeded to decrease COLI mRNA expression in comparison with DM- (P < 0.01) (Fig. 2D).
3.3. Photobiomodulation, either at single (660 or 808nm) or combined form (660 plus 808nm), boosted blood vessel establishment.
The mean (± SD) percentage of blood vessels surface area in groups DM-control, DM-660, DM-808 and DM-dual was (0.11%±0.01), (0.92%±0.04), (1.24%±0.18), (4.84%±0.53) on day 7, and (0.19%±0.06), (2.41%±0.06), (4.91%±0.14), (8.15%±0.65) on day 14, respectively. Statistical analysis showed a significant increase in the mean percentage of blood vessels surface area on day 14 in comparison with day 7 in all the experimental groups (P < 0.05), excluding group DM-control (P > 0.05). The highest percentage of blood vessels surface area was presented by group DM-dual as compared to groups DM-808 and DM-660 (P < 0.01) and DM-control (P < 0.001). Mean percentage of blood vessels surface area was significantly higher in group DM-808 when compared to DM-660 on day 14 (P < 0.05) (Fig. 2D).
The relative density of VEGF mRNA expression in groups DM-control, DM-660, DM-808 and DM-dual were (15.72 ± 2.13), (61.23 ± 4.23), (72.53 ± 5.51), (173.06 ± 7.45) on day 7 and (33.03 ± 2.53), (114.66 ± 8.66), (169.35 ± 7.94), (281.53 ± 7.57) on day 14, respectively. All groups presented a statistically significant increase (nearly 2-fold) in VEGF mRNA expression on day 14 in comparison with day 7 (P < 0.05), excluding DM-control that showed no significant difference (P > 0.05). Over the whole experiment, group DM-dual exhibited the highest level of VEGF mRNA expression as compared to groups DM-660 and DM-808 (nearly 2-fold) (P < 0.05) and DM-control (nearly 5-fold) (P < 0.01). In addition, VEGF mRNA expression was noticeably upsurged in group DM-808 when compared to DM-660 on day 14 (P < 0.05). As compared to that of group DM-control, VEGF mRNA expression was significantly increased in groups DM-660 and DM-808 (P < 0.05) over the experiment (Fig. 2D).