الفهرس 
Laser Fundamentals and SafetyOnly 14 pages are availabe for public view
 |  | from | 153 |  |  |
| | | from | 153 | | |
Abstract
This study was designed to investigate the effect of single dose of low level laser (Diode) on odontoblast-like cells derived from human bone marrow mesenchymal stem cells. Ten Human bone marrow mesenchymal stem cells (h-BMMSCs) samples were collected from the clinical pathology laboratories leftover of Sheikh-Zayed Specialized hospital- Bone Marrow Transplant Department after obtaining patient consent and acceptance from Research Ethics Committee-Faculty of Dentistry-Ain Shams University.
Each sample was isolated and subcultured to produce 4 flasks of BMMSCS which were then induced after 21 days into 4 flasks of odontoblast-like cells, then verification of odontoblastic differentiation was performed by positive results of gene expression of DSPP,DMP1and ALP by RT-PCR. The samples were then grouped to be examined after odontoblastic differentiation by 3 and 14 days.
The 40 flasks of odontoblast-like cells obtained from the 10 collected samples were divided into 2 groups; control group A and LLL irradiated group B. Each group was further subdivided into 2 subgroups (A1,A2 and B1,B2), where A1, B1 were examined after 3 days of odontoblastic differentiation and A2, B2 were examined after 14 days of odontoblastic differentiation. group B was irradiated by single dose of Diode laser (GaAlAs), 940nm Wave length with the following parameters; 0.9 W (Power), continuous wave, non-contact mode for 15 seconds and 13.5 j (Energy delivered).
Each subgroup was examined by Inverted Phase Contast Light Microscope to detect cell count, viability after being stained with trypan blue within a hemacytometer. Negative stain indicates viable cells and hemacytometer gives cell count per flask. RT-PCR was performed for each subgroup to detect the amount of gene expression of DMP1 (which plays a vital role in biomineralization and has a potent binding capacity to collagen fibrils and calcium) and ALP (which is found in organic matrix, associated with matrix vesicles and occurring free within matrix). The data of cell count and RT-PCR were statistically analyzed. Then matrix mineralization was examined by inverted phase contast light microscope after being stained by alizarin red stain for further confirmation of matrix mineralization, where calcific deposits were stained red.
Confirmation of odontoblastic differentiation from h-BMMSCs was revealed by obtaining RT-PCR positive results of DMP1, DSPP and ALP genes which were expressed with high amounts at day 21 from the beginning of cell culturing. Meanwhile DSPP gene expression indicates complete odontoblastic differentiation using RT-PCR test.
Cell count and viability statistical results of the present study showed that the control subgroup A1 (3 days) was significantly higher than control subgroup A2 (14 days). However, irradiated subgroup B1 (3 days) showed statistical non-significant higher cell count than irradiated subgroup B2 (14 days). Both irradiated subgroups (B1 and B2) showed non-significant increase in cell count measurements compared to their control subgroups (A1 and A2) respectively, with the highest mean in 3 days after LLLT subgroup (B1).
In the present study, DMP1 gene expression statistical results in control subgroups showed a statistically significant increased expression at 14 days (A2) compared to 3 days (A1) subgroups after odontoblastic differentiation. In LLLT group, DMP1 was expressed with statistically significant increase at 14 days (B2) than 3 days (B1) subgroups after odontoblastic differentiation. The LLLT irradiated group showed significant increase in DMP1 expression in comparison to their control groups with the highest mean in 14 days after LLLT subgroup (B2).
In the present study, ALP gene expression statistical results in control subgroups showed a statistically significant increased expression at 3 days (A1) compared to 14 days (A2) subgroups after odontoblastic differentiation. In LLLT group, ALP was expressed with statistically significant increase at 14 days (B2) than 3 days (B1) subgroups after odontoblastic differentiation. The LLLT irradiated group showed significant increase in ALP expression in comparison to their control groups with the highest mean in 14 days after LLLT subgroup (B2).
At the periphery of the flask; mineralized matrix formation by ARS results showed few meshwork-like calcific deposits at 3 days after odontoblastic differentiation in subgroup A1. Large globules of calcific deposits were detected 14 days after odontoblastic differentiation in subgroup A2. LLLT group showed meshwork-like calcific deposits, apparently heavier in amount and stain as well as some globules at 3 days after odontoblastic differentiation in subgroup B1. Large, deeply stained calcific area of fused globules was detected 14 days after odontoblastic differentiation in subgroup B2.
At the center of the flask; matrix formation by ARS results showed negative reults at 3 days after odontoblastic differentiation in subgroup A1. Positive results were detected 14 days after odontoblastic differentiation in subgroup A2. LLLT group showed negative results at 3 days after odontoblastic differentiation in subgroup B1. Positive results were detected 14 days after odontoblastic differentiation in subgroup B2.