The Effect Of TGF-β1 On Human Periodontal Ligament Fibroblasts Secreting MMP-1

【Introduction】Root Resorption is one of most common orthodontic treatment complications. Root is able to repair its destruction to some degree. However, if we went through the teeth carefully by X-ray exemination, we could found that approximately all of the roots after orthodontic tooth movement present root resorption to a certain extent. Orthodontic tooth movement is achieved by the remodeling of periodontal ligament (PDL) tissue and alveolar bone around the moving teeth in response to orthodontic force application. PDL and its surrounding tissue are composed of fibroblasts, osteoblasts, osteoclasts, cementoblasts and as well as the extracellular matrix (ECM). The major components of which are the type I and III collagens. These collagens connect the alveolar bone and cementum of the surface of the tooth root, while maintaining the PDL space and providing a three-dimensional scaffold for those cells around the PDL. During the process of root resorption, HPDLF cells play pivotal roles in the progress of degradation of ECM. Consequently, as tissue remodeling progresses, ECM components can be degraded by secreted proteolytic enzymes or phagocytosed by cells in the PDL, such as HPDLF. Among host proteases that target the ECM, matrix metalloproteases (MMPs) play a major role in both degradation and remodeling of matrix proteins during orthodontic tooth movemrnt. MMP-1 is active against native triple-helical interstitial collagens and can initiate tissue remodeling. The activation of MMP-1 is regulated by a group of endogenous proteins named tissue inhibitors of metalloproteinases (TIMPs), which are each capable of inhibiting almost every member of the MMP family in a non-specific manner. MMPs and TIMPs are involved in the physiological turnover of periodontal tissues and are thought to play very important roles in orthodontic tooth movement as well as the process of root resorption, and are believed to be mediated by several stimulators, such as TGF-β1, TNF-α, IL-6, OCN, OPG, RANKL and M- CFS .【Objectives】1 To investigate the level of human periodontal ligament fibroblasts (HPDLFs) secreting matrix metalloproteinase-1(MMP-1) in vitro.2 To elucidate the biological effects of transforming growth factor-β1 (TGF-β1) on human periodontal ligament fibroblasts (HPDLFs) secreting matrix metalloproteinase-1 (MMP-1).【Methods】1 Human PDL cells were isolated from healthy periodontal ligaments of the first premolars of individuals undergoing tooth extraction for orthodontic treatment. Healthy periodontal tissue was removed from the middle third of the root surface, and then transferred to 100 mm plastic culture dishes. The explants were cultured in alpha-modified minimum essential medium (a-MEM), supplemented with 10% fetal calf serum, 100 U/ml of penicillin and 100 lg/ml of streptomycin in a humidified atmosphere of 95% air and 5% CO2 at 37C0. When the cells growing from the explants became confluent, they were harvested with 0.125% trypsin in phosphate-buffered saline (PBS) and transferred to plastic culture flask at a 1 : 3 split ratio.2 For first experiment, the cells were cultured in 25ml culture flasks at a density of 2 x 105 cells/ml in a-MEM supplemented with 10% FCS and antibiotics until confluence. The levels of MMP-1 secreted by the HPDLFs were measured by using an enzyme-linked immunosorbent assay (ELISA). Experiments were carried out with cells in fifth passaged cultures.3 For second experiment, after cells confluence, HPDLFs were exposed to TGF-β1 (PeproTech, U.S.A ) at various concentrations (0.03ng/ml,0.05ng/ml,0.07ng/ml,0.09ng/ml) in monolayer cultures. The levels of MMP-1 secreted by the HPDLFs treated with TGF-β1 were measured by ELISA. Differences between experimental and contol groups were tested by using multiple linear regression analysis. Experiments were carried out with cells in fifth passaged cultures as well.【Results】1 HPDLFs secreted MMP-1 in vitro at level: 6.542ng/ml.2 The data of these experiments demonstrate TGF-β1 treatment significantly increased the secretion of MMP-1 in a time-dependent manner (P<0.05). However,differently concentrated TGF-β1 effect on HPDLFs secreting MMP-1 differently at each moment.【Conclusion】TGF-β1 plays a pivotal role in the process of root resorption during orthodontic tooth movement. HPDLFs secreting MMP-1 is regulated by TGF-β1. TGF-β1 treatment could significantly increase the secretion of MMP-1 in a time-dependent manner. Though different concentration of TGF-β1 effect on HPDLFs secreting MMP-1 differently at each moment, long acting of TGF-β1 can increase the secreting level of MMP-1 in general and result in the development of roots resorption. Consequently, decreaseing the local concentration of MMP-1 via inhibiting the bioactiveness of TGF-β1 or reducing its expose time to root surface, could be regarded as one of effective methods of preventing the development of roots resorption during orthodontic tooth movement.