Many studies have shown that surface topography and the chemical composition of an implant surface can influence the behavior of cells in vitro, including cell attachment, differentiation, and proliferation rates. Today, there are new strategies in the field of implantology to deliberately alter these rates.
In past decades, lasers have been used for several therapeutic purposes. Many beneficial effects have been demonstrated with in vitro
and in vivo
test systems, including antibacterial, antiviral, antitumor, cell differentiation, immunopotentiating, and tissue repair activities 13
. The rapid development of high-brightness LEDs has made feasible the use of LEDs among other light sources, such as lasers, intense pulse light, and other incoherent light systems, for medical treatment and light therapy. In particular, LLL therapy has been increasingly used to treat hard tissue injuries, promoting wound healing and reducing pain. This type of laser has been demonstrated as a noninvasive method for the stimulation of osteogenesis and the reduction of fracture consolidation time through bioenergetic, bioelectrical, biochemical, and biostimulatory effects on cells 14,15
Li et al. 15
found that LED application in three different doses (5, 10, 15 mW/cm2
) did not cause a significant difference over a 3-d period. Kim et al. 16
used an LED device with an intensity of 9.29 mW/cm2
. In the present study, our LED applications had an intensity of 20 mW/cm2
Khadra et al. 17
stated that during the early stages of wound healing the energy requirements of the cells are increased and photostimulation might play an important role in this phase. They found that irradiation on three consecutive days enhanced production of osteocalcin and TGF-β1. We also decided to have application times of LED for 0h, 24h and 48h evaluating these findings of Khadra et al.
While Stein et al. 18
applied laser from a distance of 11.5 cm, Khadra et al. 17
applied it from a distance of 9 cm. We applied LED from a distance of 10 cm due to the manufacturer's instructions which is stated to be the most effective distance.
Aybar et al. 19
investigated DNA synthesis in osteoblasts using BrdU analysis. Mustafa et al. 11
and Aybar et al. used BrdU analyses to evaluate cellular DNA activity in their studies examining the surface properties of titanium discs. We also used the BrdU method for the detection of DNA activity.
In our study, BrdU activity in the SLA group treated once with the LED was higher after 72 h than in the SLActive group, although the opposite result was observed with three LED applications. When cell count and BrdU results are taken into consideration, LED application at 0, 24, and 48 h appeared to slow down the osteoblast-like proliferation process and DNA activity. However, when LED was applied only once, cell proliferation and DNA activity in the early stages were significantly higher and a higher cell count was reached in all groups after 72 h, but DNA activity and live cell counts were found to be lower than in the SLA group. In our study, the high cell count after 72 h despite a decrease in DNA activity suggested that rapid cell proliferation occurred in the early phase. Aybar et al. 19
reached the same conclusion.
In light of these findings, it appears that the application of a single LED treatment raised early-phase DNA activity and promoted cell proliferation of osteoblast-like cells in vitro.
Kim et al. 16
reported that after LED treatment, differentiation was much faster in early-phase osteoblast cultures. They also found that early differentiation of progenitor cells was demonstrated by increased ALP activity and that this increased activity stimulated cell proliferation, resulting in a high cell count in the early phase. In our study, the result of one LED application was consistent with the findings of Kim et al. 16
. We saw enhanced cell proliferation and DNA activity in the early phase of LED application.
Our SEM images showed osteoblast-like cells that tended to be elongated and squamous. This cell morphology on the surface of SLA was consistent with that observed by Aybar et al. 19
. In the SEM images of the SLActive group at a high cell count, few squamous cells were seen. However, in the images of SLA group at a lower cell count, many squamous and elongated cells covered the surface, with intermittent mitosis.
Evaluations of DNA activity, cell counts, and SEM findings indicated that a significant increase in cell count occurred after 72 h due to the proliferation rate and the inhibition of cell death on the SLActive surfaces exposed once to the LED. We believe that the reduced numbers of cells on the titanium surfaces was due to the inability of cells that had undergone apoptosis to adhere to the surfaces.
In conclusion, one-time LED application in the SLActive group resulted in significantly increased cell numbers. However, these findings were not exactly compatible with the SEM findings, which demonstrated fewer cells and weak cell attachment between cells and to the surface. Thus, we suggest that further studies using different LED application intervals are needed to clarify the reason for the increased cell numbers that are apparently incapable of attaching to the titanium surfaces after 72 h.