Effects of SLPI on the attachment and inflammatory response of periodontal ligament fibroblasts cultured on titanium surface for development of periodontio-integrated implant

Abstract

The periodontal ligament (PDL) is involved not only in tooth support but also in the repair and regeneration of the alveolar bone and cementum. Titanium (Ti) is a widely used material as an implant fixture and an orthodontic mini-screw because of its high success rate of osseointegration demonstrating excellent mechanical strength and biocompatibility. Because of the removed PDL, this osseointegrated implant has a lower buffering effect on the occlusal force and lower immunity to external inflammation compared to the natural teeth. Recently, various studies have been conducted examining periodontio-integrated implants for the regeneration of periodontal tissue around implants. Secretory leukocyte protease inhibitor (SLPI) promotes wound healing and proliferation. In addition, it reduces alveolar bone resorption in periodontitis and suppresses the expression of proinflammatory cytokines in osteoblasts. SLPI also increases the adhesion, differentiation, and mineralization of osteoblasts on the Ti surface. To date, even though many studies have been reported on the relationship between Ti and periodontal ligament fibroblasts (PDLF), only a few studies have investigated the molecules that promote adhesion and activity in PDLF. Furthermore, no study has been published examining the function of SLPI in PDLF cultured on Ti. Therefore, this study aimed to examine the function of SLPI in the adhesion of PDLF and inhibition of the inflammatory response in PDLF on the Ti surface, for the modeling of periodontio-integrated implants. After treatment of cultured PDLF on the Ti surface with SLPI, MTT assay, immunofluorescence staining, and western blotting were performed to confirm the function of SLPI in the adhesion and proliferation of PDLF. After SLPI and LPS treatment of the PDLF on the Ti surface, the inhibitory effect of SLPI on inflammation was analyzed by PCR, western blotting, and by analyzing the expression of nitric oxide (NO) and prostaglandin E2 (PGE2). The adhesion, viability, and proliferation of SLPI-treated PDLF were higher than those observed in the control cells. The formation of actin stress fibers, paxillin expression, and FAK phosphorylation was increased in the SLPI-treated PDLF compared to that in the control. The expression of Grb2 and Ras proteins and phosphorylation of ERK1/2 was increased in the SLPI-treated PDLF compared to that in the control. The mRNA and protein expression corresponding to TNF-α and IL-1β were lower in the SLPI-treated PDLF after LPS exposure than those observed in the LPS-treated PDLF. In addition, SLPI decreased the protein expression of iNOS and COX-2 and inhibited secretion of NO and PGE2 in LPS-treated PDLF on the Ti surface. Therefore, the results of this study demonstrated that SLPI promoted the adhesion and proliferation of PDLF on the surface of Ti, and reduced the secretion of inflammatory cytokines and mediators, suggesting an effective molecule that can increase the clinical success rate after implantation by providing biological and mechanical stability during the osseointeration process. Further studies should be performed to determine the effect of SLPI on collagen expression and mineralization between PDLF and osteoblasts on Ti. | 치주인대는 치아를 지지할 뿐만 아니라 인접 이틀뼈 및 시멘트질의 수복과 재생에도 관여하는 것으로 알려져 있다. 티타늄은 뛰어난 물리적 강도 및 생체적합성으로 인해 뼈유착률이 높아 임플란트 매식체와 교정용 미니스크류로 널리 사용되고 있다. 임플란트는 치주인대의 제거로 인하여 교합력에 대한 완충효과 및 외부 염증에 대한 면역력이 자연치에 비해 낮다. 분비백혈구단백분해효소억제제(Secretory leukocyte protease inhibitor, SLPI)는 상처치유와 세포증식을 촉진하고, 치주염에서 이틀뼈 흡수를 감소시키며 뼈모세포의 전염증성사이토카인 발현을 억제한다. 또한, 티타늄 표면에서 뼈모세포의 부착 및 분화와 광화를 촉진한다. 따라서, 본 연구에서는 치주인대를 구성하는 세포 중 치주인대 섬유모세포를 이용하여 티타늄 표면 부착과 염증반응에 대한 SLPI의 효과 및 작용기전을 규명하고자 하였다. 티타늄 표면에 배양된 치주인대 섬유모세포에 SLPI를 처리한 후 MTT분석, 면역형광염색, western blotting을 시행하여 치주인대 섬유모세포의 부착과 증식에 대한 SLPI의 기능을 확인하였다. 또한, LPS에 노출된 티타늄 표면의 치주인대 섬유모세포에 SLPI를 처리한 후 RT-PCR, western blotting, NO, PGE2 분석을 통하여 SLPI의 염증억제 효과를 분석하였다. SLPI는 티타늄 표면에서 치주인대 섬유모세포의 부착과 생존 및 증식을 촉진시켰다. SLPI를 처리한 치주인대 섬유모세포에서 F-actin형성과 paxillin의 발현 그리고 FAK의 인산화가 대조군에 비하여 증가하였다. 그리고, SLPI를 처리한 치주인대 섬유모세포에서 Grb2, Ras의 발현 및 ERK1/2의 인산화가 대조군에 비하여 증가하였다. 또한, SLPI는 LPS를 처리한 티타늄 표면의 치주인대 섬유모세포에서 TNF-α, IL-1β의 RNA와 단백질 발현을 감소시켰다. 또한, iNOS와 COX-2의 발현을 억제하였으며 NO와 PGE2의 분비를 억제하였다. 따라서, 본 연구 결과를 토대로 SLPI는 티타늄 표면에서 치주인대 섬유모세포의 부착과 증식을 촉진하고, 염증성 사이토카인 및 매개분자의 분비를 감소시킴으로써 티타늄과 치주인대 섬유모세포의 부착을 강화시켜 임플란트 식립 후 뼈유착과정에서 생물학 및 기계적 안정성을 제공하여 임상적 성공률을 높일 수 있는 효과적인 분자일 것으로 생각된다.