| IntroductionThe periodontal ligament functions as a cushion to mitigate the mechanical forces of mastication and maintains homeostasis such as remodeling of the adjacent bone. Periodontal ligament retains regenerative capacity to some degrees throughout adulthood, which is attributed to stem cells maintaining their proliferation and differentiation potential in the area. Previous studies have determined that periodontal ligament stem cells (PDLSCs) play a crucial role in regeneration of periodontal defects, contributing to the formation of new cementum, alveolar bone, and periodontal ligament. In addition, several in vitro findings showed that PDLSCs can differentiate into osteoblast-like cells when challenged with dexamethasone,β-glycerophosphate and ascorbic acid. Recently, tissue engineering based on PDLSCs to enhance periodontal regeneration has been the focus of periodontal research. In situ engineered constructs containing in vitro expanded autologous cells have been used to regenerate periodontal defects. While how to rapidly gain plenty of functional seeded cells in vitro is the key technique for bioengineering tissue. A theoretical way to achieve this goal would be to provide a"stimulatory''environment for PDLSCs to expedite tissue engineering of periodontium.With the acceleration of human space exploration, aerospace medicine has been gradually developed recently. Due to payload constraints, flight cost, spaceflight experiments are limited; several ground-based systems have been invented to simulate microgravity. Rotary cell culture system (RCCS), recommended by National Aeronautics and Space Administration (NASA) as an effective tool for analysis of cells characteristics in conditions similar to microgravity in space, is simultaneously a kind of three dimensional (3D) dynamic culture system. Its simulated microgravity (SMG) function is due to the alteration of the cell's perception of a continuously changing gravitational direction as a result of rotational culture conditions. At the same time, the rotational motion of this system prevents sedimentation, creating a suspension culture environment, and seems to be ideal for overcoming some drawbacks associated with static culturing systems. Several researches indicated that RCCS benefit certain type of cellular aggregation, subsequent intercellular adhesion, and gradual formation of 3-dimensional (3D) cell clumps. So we wonder that whether RCCS is an effective tool in vitro for expansion of functional stem cells in 3D form, which can facilitate periodontal tissue engineering.Although, SMG is known to affect the biological behavior of several kinds of cells, the biology of different cells cultured in SMG is different; even a few conflicting findings have recently been reported with respect to the effects of SMG on definite cell types such as osteogenic cells. Up to now, no data exist illustrating the behavior of periodontal ligament stem cells under SMG. Thus, the objective of current study was to clarify the effects of three-dimensional dynamic simulated microgravity created by RCCS on hPDLSCs proliferation, osteoblastic differentiation and morphological changes. For it is the first time to investigate the effects of simulated microgravity on periodontal ligament stem cells, the research may lend insight into variations of cell response in 3D environment, and contribute to achievement of desirable periodontal regeneration utilizing PDLSCs-based tissue engineering approaches.Main results1. Isolation of periodontal ligament stem cells and investigation of multiple differentiation ability.HPDLSCs were isolated, cultured and expanded as previously described. Procedures were performed according to the approval of the institutional review board and the informed consent of the patients. Wisdom and premolar teeth intended for extraction due to orthodontic reasons were used as the cell source. Periodontal ligament tissue was cultured in MesenPRO RS medium HPDLSCs were enriched by collecting multiple colonies. HPDLSCs at passage three were used in experiments. Growth characteristics and multipotent differentiation of the cell were assessed.2. Cell cultures under SMG and biological behavior analysisCells were co-incubated with microcarrier beads of Cytodex3, and were placed in the rotating bioreactor (55-mL rotating wall vessel, Synthecon, U.S.A.) Cells morphology observation was examined such as scanning electron microscopy, transmission electron microscopy, fluorescence staining of microfilaments. Cell proliferation assessment including cell counting, flow cytometry and BrdU incorporation were investigated. And also, osteogenic differentiation potential was assessed. The results showed that simulated microgravity can change cell morphology, enhance cell proliferation and cell osteogenic differentiation ability.3. The mechanism of Smads in hPDLSCs ostegenic differentiation induced by simulated microgravityTGF-β1 can enhance ostegenic differentiation ability of hPDLSCs, as indicated by increased ALP, OCN and COL1 levels. In addition, Smads plays an important role in process of osteogenic differeniation induced by simulated microgravity. The mechanism maybe activated by pathway of TGF-βligament—membrane receptor—Smads proteins—transcriptor—gene expression.ConclussionsIn this article, we have found that this simulated microgravity environment can enhance periodontal ligament stem cells proliferation, viability and osteogenic differentiation. This environment also stimulates morphological changes and tended to aggregate as compared to the static control. In addition, Smads plays an important role in process of osteogenic differeniation induced by simulated microgravity.We conclude that the 3D dynamic simulated microgravity culture system has the potential to be used for the bioengineering reconstruction of the periodontal tissues. And the observation can benefit physiopathology of periodontium in aerospace flight. |
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