In Vitro Simulation Of The In Situ Osteocytes And It's Microenvironment In Bone

Objective:Establish a model of internal osteocyte in vitro under relative resting conditions,simulate the internal microenvironment and look for marker molecules in different states of osteocyte.Methods:1.Plating osteoblast cell line MC3T3-E1 in type I collagen gel and making osteogenic induction;2.The cell gel layers induced for 14 days,21 days,28 days,and 35 days were embedded in paraffin-embedded sections;3.Observing cell morphology and intercellular connections by HE staining;4.The expression of osteoblast marker RUNX2 and osteocyte marker DMP1 was detected by IH method;5.The model was used to validate markers in different states of bone cells.Results:1.In the process of osteogenic induction,the depth of cell infiltrating into the gel gradually deepened with the culturing days went by,and stellate,osteocyte-like cells were found on the 35 th day in HE staining,and small tubular structures were found.At 14-28 days of induction,gel layer cells expressed RUNX2 gradually attenuated,and then on the 35 th day RUNX2 was not expressed.On the 28 th day of induction,the gel layer cells began to express DMP1,and with the increase of induction days,the expression gradually strengthened;2.The bone cells did not express Notch1 in bone tissue sections,but Notch1 was expressed in bone cells in vitro,while Notch1 expression was consistent in osteocyte model and bone tissue sections.3.In the osteoblast cell line MC3T3-E1,down-regulation of Notch2 increased the expression of downstream target gene NFATc1 of Notch2,downstream target gene Notch1 of ?-cantein and ?-cantein itself,and then affected bone remodeling.4.In the bone cell line MLO-Y4,downregulation of Notch1 decreased Notch2,and then they influenced bone remodeling together.Conclusions:1.Successfully establish an in vitro model of osteocyte in the resting state and simulate the microenvironment of internal osteocyte;2.Notch1 can be used as a marker to determine the different functional states of bone cells;3.Notch1 and Notch2 affected bone remodeling together.