MMP-3 Functions As A Key Factor Of Phenotype Loss During The Late Stage Of ASCs’ Chondrogenic Differentiation

Background:Articular cartilage defects is hard to cure and have long held back almost a quarter of the population in our country.In 2017,our government unveiled a notice called "Long-term Plan for the Prevention and Treatment of Chronic Diseases(2017-2025)",in which " bone healthy " was first proposed as a heath promoting action.This notice showed the commitment and determination of the government about solving the degenerative diseases of bone and cartilage.Stem cell based cartilage tissue engineering is an promising treatment of cartilage defection.But all stem cells will suffer from phenotype loss in the late stage(after 3 weeks)of induced chondrogenesis,in vitro.With the development of single cell sequencing technology,researchers can observe and compare the difference of stem cells during differentiation in genetic level.Through single cell RNA sequencing,our team found that the expression level of tumor necrosis factor-α(TNF-α)and matrix metalloproteinase-3(MMP-3)is upregulated along with the progression of phenotype loss,and MMP-3 has been universally confirmed to be a key factor of cartilage degradation.We believe these two phenomena have potential relation.Understanding and interfering the phenotype loss of stem cell during chondrogenesis will promote the development of cartilage tissue engineering and the treatment of articular cartilage defects.Purpose:To testify that hASCs will suffer from phenotype loss in the process of chondrogenesis and the phenotype loss is related to the high expression level of TNFa and MMP-3 through single cell sequencing technology.Furthermore,to confirm that suppressing MMP-3 expression can inhibit the phenotype loss of hASCs.Methods:We treated the hASCs with chondrogenic medium for 4 weeks and then used Western blot to assess the chondrocyte maker protein(Aggrecan and Collagen-II)or confirm the phenotype loss process of hASCs.In order to testify the role of MMP-3 in stem cells’ phenotype loss,we treated hASCs with Etanercept and TIMP1 to reduce the MMP-3 level.Then we testified the chondrogenic ability of hASCs after treating with Etanercept and TIMP1 through WB,IF and Elisa.Results:At the early stage of chondrogenesis,the expression of TNF-α was increased and then up-regulate the expression level of MMP-3.Finally,the highly expressed MMP-3 degraded the extracellular cartilage matrix,leading to hASCs’phenotype loss.IL-1β can slightly upregulate the expression of TNF-α and MMP-3 dependent on the existence of chondrogenesis medium.Single cell RNA sequencing also show the increase of TNF receptor TNFRI/TNFRSF12A.The TNF-α inhibitor Etanercept can inhibit the increase of MMP-3 expression level and reduce the degradation of cartilage matrix.Treating with the MMPs inhibitor TIMP1 can also promote the accumulation of cartilage matrix and reduced the phenotype loss of hASCs eventually.