The Effect Of Acidic Microenvironment Of The Degenerated Intervertebral Disc On The Biological Behavior Of Mesenchymal Stem Cells

Background. Low back pain is a public health problem that has significant socioeconomic impact. According to the epidemiological survey, about70%of the adult population of the world had experienced low back pain at least one time during their lifetime. The economic loss due to lower back pain in the United States is about90billion dollars each year. China Health Statistics Yearbook2008revealed that chronic lower back pain located at six in the prevalence ranking of all chronic diseases. So it seems that social productivity losses caused by low back pain are also huge in our country. Although the exact etiology of low back pain remains elusive, intervertebral disc (IVD) degeneration has been proposed as one of the major causes. The disc cells synthesize the matrix components which play an important role in the regulation of the biochemical and biomechanical functions of IVD. IVD degeneration involves a decrease in the number of disc cells that leads to the loss of extracellular matrix. Cell-based therapy has emerged as a promising approach for the treatment of IVD degeneration. With the ability to self-renew and differentiate into specialized cells, mesenchymal stem cells (MSCs) have gained more attention as the sources of cells for IVD regeneration. However, the harsh chemical microenvironment of IVD, which is characterized by acidity, hypoxia, high osmolarity and limited nutrition, may significantly influence the biological and metabolic vitality of MSCs and impair their repair potential. Among the microenvironment factors that do harm to the regenerative process, the acidic microenvironment of the degenerated intervertebral disc has the most profound negative effect on MSCs. Recent studies revealed that the matrix acidity of the human intervertebral disc gets worse with the severity of disc degeneration. Human adipose tissue derived mesenchymal stem cells (ADMSCs) may be an ideal source of cells for IVD regeneration because of their ease of access, little donor site morbidity, comparative abundance and high proliferation rate. The age of the donor may have an important effect on the viability of ADMSCs and there may be a certain pH threshold under which the viability, proliferation and matrix metabolism of ADMSCs would be significantly affected. This study aimed to investigate the viability, proliferation and the expression of main matrix proteins of human ADMSCs in the acidic microenvironment of IVD under normal and degenerated conditions. Clarification on this point will provide information for the development of human ADMSCs-based regeneration of intervertebral disc. Our previous study had found that, through cell transplantation and in combination with growth factors and anti-inflammatory drug injection, ADMSCs have some regenerative effect on degenerated intervertebral disc. However, the harsh microenvironment in degenerated intervertebral disc makes it difficult for ADMSCs to maintain long-term viability after implantation. In recent years, some scholars have found evidence for the existence of mesenchymal stem cells in intervertebral disc. Further studies confirmed the presence of multipotent progenitor cells in degenerated human annulus fibrosus and mesenchymal stem cells in human degenerated nucleus pulposus and cartilage endplate. Meanwhile, some other studies also confirmed the presence of mesenchymal stem, cells in the nucleus pulposus of some animals and found that these endogenous mesenchymal stem cells have the potential of migration and regeneration in IVD, suggesting that these novel tissue-specific mesenchymal stem cells may have potential value for research and application in IVD regeneration. Currently, the existence of nucleus pulposus mesenchymal stem cells (NPMSCs) has been verified by a series of studies. Our research team have successfully isolated rat NPMSCs in previous work, which have the general features of MSCs and could be implemented as cartilage, adipogenic and osteogenic differentiation, in full compliance with the standard for MSCs proposed by International Association of Cell Therapy. However, as novel tissue-specific MSCs, whether NPMSCs is suitable for intervertebral disc tissue engineering is still unknown. The comparison of the influence of acidic microenvironment of the degenerative disc on the biological behavior of NPMSCs and ADMSCs (survival, proliferation and extracellular matrix metabolism) will provide us with a better understanding of the biological characteristics of these two kinds of MSCs and help the correct evaluation of their potential value for application in the field of biological repair of the degenerated IVD. This would eventually make contributions to the development of novel applications of MSCs to IVD regeneration. Based on the progress aforementioned and our previous work in this field, this paper mainly carried out research work in the following two aspects:(1) to investigate the influence of different acidity of IVD microenvironment on the viability, proliferation and the expression of main matrix proteins of human ADMSCs originated from two different age groups;(2) to examine the viability, proliferation and extracellular matrix metabolism of rat NPMSCs compared with ADMSCs under different acidity of IVD microenvironment. Part I:The Influence of Acidic pH Conditions Simulating the Microenvironment of Degenerated Intervertebral Discs on the Biological Behavior of Human Adipose Tissue Derived Mesenchymal Stem CellsObjective. This study was designed to examine the survival and biological behavior of human adipose tissue derived mesenchymal stem cells (ADMSCs) under acidic conditions simulating the microenvironment of degenerated intervertebral disc.Methods. Human ADMSCs were isolated from two age groups, including six young male donors aged8-12years and six mature male donors aged33-42years, and verified for pluripotent. These ADMSCs were cultured under four different pH levels, including pH7.4,7.1,6.8, and6.5, which simulated the standard condition and the normal, mildly degenerated and severely degenerated IVD. Cell viability was measured by FITC-AV/PI (Fluorescein isothiocyanate-Annexin-V/propidium iodide) staining, and cell proliferation was measured by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay. The expression of aggrecan, collagen-I, collagen-II, matrix metalloproteinase-2(MMP-2), tissue inhibitor of metalloproteinase-3(TTMP-3), p53and caspase-3at the mRNA level was examined by RT-PCR (real-time quantitative polymerase chain reaction), and the expression of aggrecan, collagen-I, collagen-II, MMP-2and TIMP-3at the protein level was measured by ELISA (enzyme-linked immunosorbent assay).Results. Acidic pH inhibited the viability and proliferation of human ADMSCs from both age groups. The expression of aggrecan, collagen-I and collagen-II of ADMSCs from both age groups was suppressed by acidic pH. ADMSCs harvested from young and mature donors exhibited similar responses to the acidic pH. However, ADMSCs from young donors appeared less sensitive to the low pH levels, as revealed by less suppression in cell viability, proliferation and anabolic metabolism of extracellular matrix. Generally, human ADMSCs appeared less sensitive to the matrix acidity of the degenerated IVD with a pH value higher than6.8.Conclusion. These results indicated that acidic pH in the microenvironment of degenerated IVD may represent an important detrimental factor for IVD regeneration based on human ADMSCs. Human ADMSCs harvested from young donors may be more appropriate to be transplanted into degenerated IVD, and the implantations might be more effective at an early stage of degeneration with a mildly degenerated IVD when the pH of matrix acidity is higher than6.8. Part Ⅱ:The Influence of Acidic pH Conditions Simulating theMicroenvironment of Degenerated Intervertebral Discs on the Biological Behavior of Rat Nucleus Pulposus Mesenchymal Stem Cells and Adipose Tissue Derived Mesenchymal Stem CellsObjective. This study was designed to examine the viability, proliferation and matrix metabolism of rat nucleus pulposus mesenchymal stem cells (NPMSCs) compared with adipose tissue derived mesenchymal stem cells (ADMSCs) under acidic conditions simulating the microenvironment of degenerated intervertebral disc (IVD).Methods. NPMSCs and ADMSCs isolated from Sprague Dawley rats were verified for pluripotent and cultured under four different pH levels representing the standard condition (pH7.4) and the normal, mildly degenerated and severely degenerated intervertebral disc (pH7.1,6.8and6.5, respectively). Cell viability was examined by FITC-AV/PI (Fluorescein isothiocyanate-Annexin-V/propidium iodide) staining. Cell proliferation was measured using a CCK-8(Cell counting kit-8) assay. The expression of aggrecan, collagen-Ⅰ, collagen-Ⅱ, matrix metalloproteinase-2(MMP-2), a disintegrin and metalloproteinase with thrombospondin motifs-4(ADAMTS4) and tissue inhibitor of metalloproteinase-3(TIMP-3) at both mRNA and protein levels were measured by RT-PCR (real-time quantitative polymerase chain reaction) and western blotting.Results. In both NPMSCs and ADMSCs, acidic pH conditions simulating the microenvironment of degenerated IVD inhibited cell viability and proliferation. Acidic pH conditions also downregulated the expression of aggrecan, collagen-Ⅰ, collagen-Ⅱ and TIMP-3, and upregulated the expression of MMP-2and ADAMTS4in both cell types. However, compared with ADMSCs, NPMSCs were significantly less suppressed in cell viability and proliferation by acidic pH, as demonstrated by the expression of significantly higher levels of aggrecan and collagen-Ⅱ, and lower levels of MMP-2and ADAMTS4.Conclusion. The acidic pH conditions in the microenvironment of degenerated IVD represent a major obstacle for IVD regeneration by ADMSCs and NPMSCs. It seems that NPMSCs are less sensitive to the inhibition effect induced by acidic pH conditions. NPMSCs could be promising candidates for stem cells-based IVD regeneration. These findings may improve the translational study of mesenchymal stem cells in IVD regeneration.