Effect Of Low Intensity Pulsed Ultrasound On The BMP Regulation Mechanism Of Human Periodontal Ligament Cells

There are many complex stomatognathic diseases in adult orthodontic patients, and the most common is periodontal disease, it is difficult to move the teeth directionally in the case of poor periodontium, and resulting in loose teeth or even fall off. Therefore treating and controlling the periodontal disease effectively and repair the periodontal tissue were the prior problem to solve before the orthodontic treatment. Human periodontal ligament cells(HPDLCs) which was the precurosor cell for the regeneration of the periodontal tissue.It was a complex cell group and composed of fibroblast, osteoblast, osteoclast and mesenchymal cell, Some studies showed that the HPDLCs has the potential to differentiate into the osteoblast, which was very important to the regeneration of the periodontal tissue. The Low Intensity Pulsed Ultrasound, as a noninvasive device was able to stimulate the collagen synthesis secretion in cells, accelerate the deposition of calcium salts, and also had a powerful osteogenic ability in repairing long bone fracture or peridontal bone defect. The early studies of this research subject proved that LIPUS was able to improve the activity of ALP in the HPDLC effectively, and enhance the formation of the Calcified nodule. However further study was needed on what the regulation mechanism of these osteogenic indexes showed. The control factor of the osteogenic differentiation: bone morphogenetic protein, which was a multifunctional growth factor and belonged to the TGF-βgroup. This kind of surfactant protein enables undifferentiated cells differentiating into osteoblast by regulation, and then induced the rising of the osteogenic indexes, calcification of extracellular matrix, and taking part in the repair of bone regeneration. The BMPs can be perceived by cells, which can regulate osteogenesis through a series of signal transduction pathways. BMP synthesize of cells with differentiation potentiality increased after they were stimulated by the external factor, then the BMP receptors on the cell membrane would combine with BMP rapidly, in order to stimulate the signal transduction. The activated BMPR acted on the Smad1/5/8 which was in the cytoplasm, this would lead the end of the serine to be phosphorylation. Then Smad1/5/8 would enter in the cell nucleus in the form of R-Smad-Co-Smad in order to act on the osteogenic transcription factor in the cell nucleus, which would control the expression of all forms of osteogenic factors.To Base on the potential osteogenesis effect of LIPUS on HPDLCs, and the regulation mechanism of BMP classic osteogenic pathway. The HPDLCs were cultured in vitro and cell line was established under this studies. the fifth generation of HPDLCs were taken as the object of this study, the LIPUS was used as the stimulus factor, the most appropriate osteogenesis parameter from the early studies was selected: frequency pulses 1.5 MHz, with a pulse width of 200μs and a duty ratio of 1:4, repeated at 1 KHz, at an intensity of 90mW/cm2, 20 minutes/day. By treating the HPDLCs for 2 weeks, the total RNA of HPDLCs was extracted after the first, third, fifth, seventh, ninth, eleventh, and thirteenth day, and to get the cDNA by Reverse Transcription, the dynamic change trend of expression of BMP-2, BMP-4, BMP-6, BMP-9 in the HPDLCs were detected through the Real time-PCR technique. The result showed that the BMP-2 gene expression increased daily after the stimulus of LIPUS, it reached the peak on the third day, and then decreased gradually after peak. But it appeared a obvious increase again on the eleventh day; BMP-4 Gene expression had no obvious change; BMP-6 Gene expression increased considerably on the first day, reached the peak on the third day, and returned to the initial level gradually; BMP-9 had no expression. This indicated that LIPUS has the ability of accelerating the Gene expression of BMP-2 and BMP-6 in the HPDLCs to improve. Moreover the Gene of BMP-2 and BMP-6 showed a band of changes after the stimulus. Besides, the LIPUS might have little effect on the BMP-4, and BMP-9 could not expressed in HPDLCs.A further exploration on the osteogenesis regulation mechanism of LIPUS on HPDLCs was continued: through the Smads of BMP classic osteogenesis pathway as a starting point to verify the response of signal molecule Smads by the stimulation of LIPUS and the process of signal transduction. The fifth generation of HPDLCs was taken as the object of studies, 1.5 MHz frequency pulses, with a pulse width of 200μs and a duty ratio of 1:4, repeated at 1 KHz, at an intensity of 90mW/cm2, duration time of 20 minutes. The total protein after 5, 30, 60,120 and 4 hours was extracted, contemporary cultured cells without treatment as the control group. Phosphorylation of Smad1/5/8 was detected through Western-Blot technique. The result showed that Phospho-Smad/1/5/8 increased obviously at 30 minutes after LIPUS, and continued increased in 60 minutes till the peak at 120minutes, then it decreased considerably after 4 hours. However the total protein of Smad1/5/8 increased more at 60 min, there was no obvious change at the other time point. This part of studies proved that Smads of BMP classic osteogenesis pathway in HPDLCs was activated after the stimulation of LIPUS, and a transient process of Smad 1/5/8 phosphorylation and dephosphorylation was appeared.Based on the previous studies, the signal transmission of Smads was further investigated. The fifth generation of HPDLCs were taken and treated by LIPUS, which of 1.5 MHz frequency pulses, with a pulse width of 200μs and a duty ratio of 1:4, repeated at 1 KHz, at an intensity of 90mW/cm2, duration time of 20 minutes. The signal molecule Smads of HPDLCs was labeled by immunoflourescence at the time of no treatment, 1hour, 2 hours, 4 hours and 8 hours after the treatment. The result through laser scanning confocal microscope showed that the Smads protein were in the cytoplasm in control group. 1 hour After LIPUS the Smads protein started to appear in nuclei. All of the Smads protein was in the nuclei at 2 hours after LIPUS. At 4 hours After LIPUS treatment, most of Smads protein still stayed in the nuclei, but small amount of Smads can be found in the cytoplasm at this time. At the eighth hour after LIPUS, the Smads protein nearly all been returned to the cytoplasm. This study proved that osteogenesis signal transmission pathway of BMP in HPDLCs could be activated under the LIPUS. And Smads as a signaling molecule was transducted between cytoplasm and nuclei.In conclusion, this study used the LIPUS whth a 1.5 MHz frequency pulses, a pulse width of 200μs and a duty ratio of 1:4, repeated at 1 KHz, at an intensity of 90mW/cm2, 20 minutes/day to affect the HPDLCs, and discovered that expression of BMP-2,BMP-6 increased sharply, and represented a dynamic changing process over time. The further study indicated that osteogenesis signal transmission pathway of BMP was activated, and the Smad 1/5/8 protein temporarily displayed a process of phosphorylation and dephosphorylation. The signal molecule Smads protein was transferred from the cytoplasm to the nuclei in HPDLCs; this led to the response from the transcription factor in the nuclei, so that the expression of the osteogenic related gene can be regulated. These above results determined the osteogenesis effect of LIPUS to HPDLCs, and also found that the osteogenesis mechanism of LIPUS to HPDLCs could possibly realized through the Smads signal transduction pathway of BMP classic osteogenesis pathway. This finding provided a reference on the mechanism of LIPUS improving the periodontal tissue reparation.