Effects Of Cyclosporine A And Porphyromonas Gingivalis-Lipopolysaccharide On Periodontal Tissue Regeneration

Objective Earlier research in our group discovered that cyclosporine A（CsA）andPorphyromonas gingivalis-lipopolysaccharide(Pg-LPS) had synergistic action inproliferation and tissue growth factor-β1（TGF-β1） secretion of gingival fibroblasts（GFs）, CsA induced gingival overgrowth was more apparent in the presence ofinflammation. Whether such synergistic effect was unique to gingival, or such effectcould enhance periodontal ligament fibroblasts(PDLFs) in vitro and alveolar boneregeneration in vivo has nerver been reported. The purpose of the study was toexplore the effect of CsA and Pg-LPS combination on PDLFs proliferation andbiological activities, and investigate their effect on periodontal repair in mandibulardefect in rat.Methods1. PDLFs were incubated with CsA (10ng/mL,100ng/mL,200ng/mL,400ng/mL) and Pg-LPS (10ng/mL,100ng/mL,1000ng/mL) respectively, and then toobserve the proliferation of PDLFs.2. The effects of CsA (100ng/mL) plus Pg-LPS(100,1000ng/mL) on the proliferation of PDLFs was observed.3. PDLFs wereincubated with CsA (100ng/mL), Pg-LPS (100ng/mL), or both, then cell cycle wasevaluated with flow cytometry, and the total protein concentration of cells wasassessed with coomassie brilliant blue. Bone morphogenetic protein-2(BMP-2) andalkaline phosphatase(ALP) were determined by enzyme-linked immunosorbentassay(ELISA). The formation of mineralized nodules was assessed with Von kossastaining. Transcription of collagenⅠ and Ⅲ were quantified with real-time PCR.4.The buccal side of the mandibular first molar in Sprague Dawley rats were injectedwith2mg/kg CsA,10,1,0.1μg LPS，or both. The alteration on gingival and alveolarbone was evaluated by haematoxylin and eosin（HE）staining.5. Saline, CsA (2mg/kg)CsA (2mg/kg)+LPS (1μg) were injected into the periodontal defects in Sprague Dawley rats.30day after injection, animals were sacrificed and the formation ofnewly formed alveolar bone, cementum and the width of periodontal ligament wereanalyzed histomorphometrically.Results1. CsA (100ng/mL) enhanced PDLFs proliferation and Pg-LPS (1000ng/mL)inhibited PDLFs proliferation.100or10ng/mL LPS had no effects on PDLFsproliferation.2. PDLFs proliferation was not significantly affected when incubatedwith CsA (100ng/mL) plus Pg-LPS (100ng/mL), whereas significantly inhibitedwhen combining CSA (100ng/mL) with Pg-LPS (1000ng/mL).3. CsA alone orcombined with LPS could promote PDLFs proliferation and the formation ofmineralized islets, and significantly increased ALP, BMP-2secretion and the mRNAexpression levels of COLL-Ⅰof PDLFs, but reduced the mRNA of COLL-Ⅲ ofPDLFs. CsA alone could increase the total protein concentration, but there was nofacilitation when combined with LPS.4. CsA (2mg/kg) did not induce the gingivalovergrowth, and not lead to alveolar bone resorption. Pg-LPS (10μg) could lead toalveolar bone resorption; while combined with CsA, no significant difference wasobserved in the alveolar bone resorption effect. There was still no significantdifference when treated with1,0.1μg LPS alone, or combined with CsA. No obviouschange in gingiva was found following all of the treatments.5.2mg/kg of CsA hadno significant effects on the regeneration of the periodontal tissue in Sprague Dawleyrats, as well as combined with1μg LPS.Conclusions At definite concentration, CsA could enhance PDLFs proliferation,protein synthesis and differentiation. CsA and LPS had synergistic on enhancing thisbiological activity of PDLFs. Low-dose of CsA alone, as well as combined withlow-dose LPS, had no significant effects on the periodontal tissue and theregeneration of the periodontal defects in rats.