Effect Of Serum Concentration On The Biological Activity Of Human Nucleus Pulposus Mesenchymal Stem Cells

Background:Neck and low back pain is a series of clinical symptoms caused by intervertebral disc degeneration.It is one of the common diseases in clinical practice.It can cause long-term disability and affect the daily life of patients,increasing personal and social medical costs.The structure of the intervertebral disc is special,the surrounding environment is in a state of hypertonicity and low acidity,and because there is no direct supply of blood vessels,its nutrient and oxygen supply is much lower than other tissues.As the degree of intervertebral disc degeneration increases,the cellular microenvironment in the intervertebral disc will change,cartilage endplates appear to be calcified to damage the metabolic pathways,and nutrient supply is further reduced.These changes will further aggravate the degeneration of the intervertebral disc and form a vicious circle.The exact mechanism leading to the degeneration of the intervertebral disc has not been fully elucidated.Studies have shown that the key factor in intervertebral disc degeneration is the decrease in the number and quality of nucleus pulposus cells.Therefore,the current treatment for intervertebral disc degeneration is mainly focused on increasing and restoring the number and quality of nucleus pulposus cells.Studies have shown that there are nucleus pulposus mesenchymal stem cells(NP-MSCs)in human degenerated nucleus pulposus tissues,which have multidirectional differentiation and self-renewal,making them useful for cell therapy in intervertebral disc degeneration.Stimulating the regeneration and repair of endogenous NP-MSCs can bring a new approach to the treatment of intervertebral disc degeneration.However,due to the low-nutrient microenvironment of the intervertebral disc,it is difficult to regenerate and repair the nucleus pulposus mesenchymal stem cells.Therefore,it is particularly important to explore the effect of changes in the nutrient supply in the intervertebral disc on the biological activity of nucleus pulposus mesenchymal stem cells.Objective: This study aims to observe the effects of different serum concentrations on the biological activity of human normal nucleus pulporeal mesenchymal stem cells in vitro,and to explore the mechanism of intervertebral disc degeneration involved in the changes of intervertebral disc nutritional supply.Methods: The nucleus pulposus tissue of patients with congenital scoliosis correction(Pfirrmann grade I or II)was selected and NPMSCs were isolated and cultured in vitro.First,Flow cytometry was used to detect cells phenotypes(CD105,CD90,CD73,CD45,CD34,HLA-DR).Then observed its ability to induce differentiation into osteogenic,adipogenic and chondrogenic cells.After that,third passage NPMSCs were cultured in DMEM medium containing different concentrations of fetal bovine serum(0%,5%,and 10%)to determine their cell proliferation capacity,cell cycle,apoptosis rate,and stemness genes(OCT4,Nanog,Jag1,Notch1)and functional genes(collagen I,collagen II,aggrecan,SOX9))expression were detected,respectively.Results: The cells isolated and cultured from normal nucleus pulposus tissues could grow in a spindle shape,adhere to the wall,and could be passaged in vitro.The immunophenotype results showed the cells highly expressed CD73(98.9%),CD90(97.9%),CD105(98.9%),lowly expressed HLA-DR(3.11%),CD34(1.83%),CD45(1.19%).The isolated cells could be differentiated into osteoblasts,adipocytes and chondrocytes.According to the criteria of the International Society for Cellular Therapy(ISCT),the isolated and cultured cells were NPMSCs.With the decrease of serum concentration,the cell proliferation rate decreased,cell apoptosis rate increased significantly,and could prolong the G1 phase length(P < 0.05).The QRT-PCR results showed that with the decrease in serum concentration,the relative expression level of stemness genes(OCT4,Nanog,Jag1,Notch1)decreased significantly,the relative expression level of type I collagen increased,and the relative expression level of type II collagen,aggrecan and SOX9 decreased.There are differences statistically significant(P<0.05).Conclusion: The serum concentration has an important influence on the biological activity of human NPMSCs.Low serum concentration affects the culture of human NPMSCs,suppresses the proliferation of NPMSCs,and inhibits the expression of stemness genes and related functional genes.This study not only provides a theoretical basis for NPMSCs to keep silent during the process of intervertebral disc degeneration,but also supports new ideas for their biological therapy.