Effects of Changes in Osmolarity on the Biological Activity of Human Normal Nucleus Pulposus- Derived Mesenchymal Stem Cells


 Background:The expansion and differentiation of nucleus pulposus-derived mesenchymal stem cells（NPMSCs） are considered as potential therapeutic tools for clinical applications in intervertebral disc tissue engineering and regenerative medicine.However, the harsh microenvironment within the intervertebral disc is the main limitation of its regeneration.The osmolarity of the intervertebral disc is higher than that of other tissues, which has an important influence on the biological characteristics of NPMSCs. In this study,we observed the effect of different osmolarities on the biological characteristics of human normal NPMSCs cultured in vitro and explored the role of osmolarity in intervertebral disc degeneration. Methods: NPMSCs were isolated from the lumbar intervertebral disc (Pfirrmann I or II) of patients with lumbar fractures by collagenase digestion, and nucleus pulposus-derived stem cells were isolated, cultured and passaged in vitro. The isolated cells were identified by immunophenotype and multilineage differentiation. Then, the cells were cultured under conditions with different osmotic pressures. The cell proliferation rate, apoptosis rate ,cell cycle status and cell senescence were detected, and the expression of stemness genes (Nanog, Notch1, Jag1, OCT4), functional genes (Aggrecan, Collagen II) and the P16INK4A and RB genes was detected. siRNA transfection was used to determine the role of the P16INK4A/RB signaling pathway in the effect of changes in osmotic pressure on cells.Results: The experimental results demonstrated that the isolated cells met the standards of the International Society for Cell Therapy. The cells we isolated and cultured from the intervertebral discs are NPMSCs. Our work showed that low and high osmotic pressure can inhibit cell proliferation, promote cell apoptosis, block cell cycle progression, promote cell senescence,downregulate the expression of stemness genes (Nanog, Notch1, Jag1, OCT4) and functional genes (Aggrecan, Collagen II), and upregulate the expression of the P16INK4A and Rb genes.The transfection results showed that after knocking down P16INK4A gene, hypotonic or hypertonicity had no effect on expression of genes (Aggrecan, Collagen II,Rb).Conclusion: The change in osmotic pressure has an important effect on the biological activity of NPMSCs, and this effect may occur through the p16INK4A/Rb pathway.This study provides a theoretical basis for the future treatment of intervertebral disc degeneration.


Introduction
Low back pain is common, and its prevalence is increasing due to the aging of the population, serious affecting the lives and health of patients. This condition is a major burden on society because of its high medical and nancial costs [1,2]. Studies have shown that intervertebral disc degeneration (IVDD) is an important pathological factor in back pain [3,4]. The mechanism underlying this condition is relatively complex, but to date, the pathogenesis of IVDD has not been fully elucidated. Degeneration of the intervertebral disc is characterized by damage to nucleus pulposus (NP) cell function and reduced cell number, which in turn inhibits the synthesis of extracellular matrix [5,6]. The intervertebral disc connects two adjacent vertebral bodies, and no blood vessels directly supply nutrients. This structure is composed of three parts: annulus brosus, NP and cartilage endplate [7]. The NP is located in the center of the intervertebral disc and has a unique biological environment, which is characterized by hyperosmolarity, nutrient de ciency, acidity and hypoxia [8]. Normal intervertebral discs lack blood vessels and a nutrient supply, and metabolites are mainly eliminated through the diffusion of the cartilage endplate pathway [9] because in a hypoxic environment, cells primarily provide energy through glycolytic metabolism [10,11].
As the degree of IVDD increases, the microenvironment of the cells in the intervertebral disc will change, the glucose supply will decrease, the oxygen supply will change, acidity will increase, and osmotic pressure will also change. These changes further aggravate IVDD and form a vicious cycle [12].
Studies have shown that NP cell damage is an important cause of IVDD [13]. Therefore, current research on the treatment of IVDD is mainly focused on preventing the degradation of the extracellular matrix and increasing the number of NP cells [14]. Blanco et al. [15] identi ed NP-derived mesenchymal stem cells (NPMSCs) in human degenerated NP tissues; these cells could replicate and undergo adipogenesis, osteogenesis and chondrogenesis, indicating they are potential candidates for cell therapy in IVDD.
Stimulating the regeneration and repair of endogenous NPMSCs may be a new strategy for the treatment of IVDD. However, due to the complex microenvironment of the intervertebral disc, regeneration and repair of NPMSCs is di cult.
The NP tissue of the intervertebral disc contains a high level of hydrophilic extracellular matrix, and the main component is proteoglycan [16]. Aggrecan increases intervertebral disc tissue permeability and resistance to pressure, and degradation of aggrecan can lead to damage to intervertebral disc function and the occurrence of IVDD. Aggrecan plays an important role in maintaining the hydration state and osmolarity of intervertebral discs [17]. Sulfated glycosaminoglycan is highly negatively charged and adsorbs cations and water molecules, thereby increasing the osmolarity of the tissue, promoting water entry in the intervertebral disc tissue and then increasing the expansion pressure of the tissue to bear the compressive stress load of the spine [18,19]. The various movements and loads of the spine throughout the day lead to dynamic changes in the osmolarity of the NP tissue. When the load stress exceeds the osmolarity of the NP tissue, water is squeezed out of the NP, and the osmolarity increases. When the stress is reduced, water is reabsorbed in the NP tissue to reduce osmolarity; thus, the osmolarity in the NP tissue is dynamically changing [20,21].
Studies have determined that the osmolarity of NP tissue is 430-496 mOsm/kg H 2 O [22,23], which is much higher than that of other tissues. As an important factor for survival in the intervertebral disc tissue, osmolarity has a major effect on the activity of NPMSCs. Current research on the effect of osmotic pressure on NPMSCs mostly focuses on the intervertebral discs of cattle, mice, dogs, etc. In this study, for the rst time, human normal NPMSCs were used to study the effect of changes in osmolarity on their biological activities and to explore the role of osmolarity in IVDD.

Cell isolation and culture
All experiments in this study were approved by the Ethics Committee of the First A liated Hospital of Anhui Medical University. In this study, intervertebral disc samples were collected from patients undergoing lumbar fracture surgery at the First A liated Hospital of Anhui Medical University. According to the P rrmann disc degeneration grading system, the patients with disc degeneration were classi ed as Grade I or Grade II. Under sterile conditions, the tissue cultured in vitro from the annulus brosus was carefully separated using a microscope. Then, scissors were used to cut the NP tissue into pieces, and the tissue was placed in a cell incubator and digested with 0.2 mg/ml collagenase II (Gibco, USA) for 4 to 6 hours. The supernatant was centrifuged at 1500 rpm for 5 min and discarded. The centrifuged tissue was cultivated with the prepared medium, which consisted of Dulbecco's modi ed Eagle's medium-low glucose (HyClone, USA), 10% fetal calf serum (Gibco, USA), and 1% penicillin/streptomycin (Gibco, USA) at 37°C in 5% CO 2 . After 24-48 hours, the cells began to adhere to the wall and underwent the rst uid change, followed by a change of medium every 3 days. When the cell con uence reached approximately 90%, the cells were subcultured at 1:3, and the third generation of cells was selected for the experiments.

Immunophenotype of NPMSCs
After trypsin (Biosharp, USA) digestion of NPMSCs, the cells were washed with phosphate-buffered saline (PBS, Sigma, USA) and centrifuged to generate a 100 µl cell suspension. CD105-PE, CD90-PE, CD73-PE, CD45-PE, CD34-PE, and HLA-DR-PE (eBioscience, USA) monoclonal antibodies were added to each tube. The samples were incubated in the dark for 30 min. After the cells were washed with PBS, they were resuspended in 400 µl of PBS, and then, ow cytometry (BD, USA) was used to detect the percentage of positive cells and the uorescence intensity. An isotype control (eBioscience, USA) was used for each tube.

Multilineage differentiation
The isolated cells were plated in a 6-well plate and then cultured with mesenchymal stem cell differentiation medium (Cyagen Biosciences, Guangzhou, China), including osteogenic differentiation, adipogenic differentiation, and chondrogenic differentiation media. The cells were cultured for 21 days for osteogenic differentiation, 28 days for adipogenic differentiation, and 28 days for chondrogenic differentiation. Then, the culture medium was discarded, and the cells were stained with Alizarin Red, Oil Red O, and Alcian blue. Finally, the staining results were observed under an inverted microscope.

Preparation of four experimental groups
Cells isolated from the intervertebral disc tissue were cultured and passaged.Sterilized NaCl and deionized water were added to the medium to prepare culture medium with different osmotic pressure levels, and the osmotic pressure was monitored using a freezing point osmotic pressure instrument (Fiske, USA). Media with four osmotic pressure levels, namely, 250 mOsm/kg H 2 O (group 1), 300 mOsm/kg H 2 O (group 2), 450 mOsm/kg H 2 O (group 3) and 600 mOsm/kg H 2 O (group 4), were used. Only osmotic pressure was different in each group; the other culture conditions were the same.

Cell proliferation assay
Cell Counting Kit-8 (CCK-8) assays were used to detect the proliferation of the NPMSCs cultured under different osmotic pressure conditions. NPMSCs were seeded in 96-well plates at a density of 5×10 3 cells/ml. These cells were cultured for 1, 3, 5, 7, 9 and 11 days in the four groups (1, 2, 3 and 4). The cells were incubated with 10 µl of CCK-8 reagent (Dojindo, Japan) at 37°C in the dark for 2.5 hours. No cells were added to the isotype group. A SpectraMAX microplate reader was used to measure the absorbance of different groups at 450 nm.

Apoptosis assay
The isolated cells were planted in 6-well plate. These cells were cultured in four groups (1, 2, 3 and 4) for 5 days in different osmotic media. The cells were digested with trypsin without EDTA (Biosharp, USA) and washed twice with PBS. The harvested cells were incubated with 5 µl of Annexin V-FITC and 5 µl of propidium iodide (PI; KeyGEN BioTECH, China) for 5 min at 37°C in the dark. Flow cytometry (BD, USA) was used to detect the percentage of apoptotic cells.
Cell cycle analysis PI (Beyotime, China) staining was used to analyze the cell cycle. Brie y, after NP cells were treated with medium with different osmolarities for 5 days, they were digested with 0.25% trypsin and xed with cold ethanol overnight. The cells were washed with PBS to remove ethanol. The prepared 500 µl PI/RNase A staining working solution was added to the tube and incubated for 30 min in the dark at room temperature. After the cells were washed with PBS, the fractions of each phase of the cell cycle were detected by ow cytometry.

SA-β-Gal staining assay
The cells were seeded in a 6-well plate and then cultured in the above four different osmotic pressure groups for 5 days. SA-β-Gal staining was performed according to the manufacturer's instructions (Beyotime, China). Then, the staining results were observed under an inverted microscope. Senescent cells were stained. The percentage of positive cells to the total number of cells was determined.

Real-time PCR analysis
The cells were cultured for 7 days with the above four types of media with different osmotic pressures. Then, TRIzol reagent was used to extract total RNA. Total RNA was reverse transcribed into cDNA using a reverse transcription reagent (TaKaRa, Japan). GAPDH was used as an internal reference gene. A SYBR Premix Ex Taq PCR kit (TaKaRa, Japan) and LightCycler system (Roche, Switzerland) were used to analyze the obtained cDNA for real-time quantitative PCR (RT-qPCR). The primers used for RT-qPCR are listed in Table 1 and were synthesized by Sangon Biotech (Shanghai, China). The mRNA expression of target genes was calculated with the 2 -△△Ct method.
Western blot analysis After 7 days of culture with media with different osmotic pressures, the four groups of cells were treated with RIPA lysis buffer (Beyotime, China), and total protein was extracted. The extracted protein samples from each group were separated by SDS-PAGE and transferred to PVDF membranes.The PVDF membranes were incubated with primary antibodies (β-actin: Bioworld, BS6007M; Aggrecan: Abcam, ab3778; Collagen II: Abcam, ab188570; P16INK4A: Abcam, ab108349; Rb: Abcam, ab181616) and horseradish peroxidase (HRP)-conjugated secondary antibodies (Beyotime, China). After the protein bands were visualized using an ECL Plus system (Thermo, USA), their gray values were measured by Quantity One software (Bio-Rad, USA). The protein expression of target molecules was normalized to that of β-actin.

Statistical analysis
All data in this study are expressed as the mean ± standard error of three independent replicates. The signi cant difference between groups was analyzed by one-way analysis of variance (ANOVA) using SPSS 19.0 software. A P value < 0.05 was considered statistically signi cant.

Isolation and characterization of NPMSCs
After the primary cells were cultured for 4 to 5 days, the isolated and cultured cells adhered to the wall. Under a microscope, the cells adhered to the wall and had a spindle shape. The cells fused in approximately 20 days, and the speed of fusion increased after passaging. The cell morphology of the P2 and P3 generations remained uniform, and the cells had a spindle shape (Fig. 1). Flow cytometric analysis showed that the cells had high expression of CD73, CD90, and CD105 and low expression of CD34, CD45, and HLA-DR (Fig. 2). After 21 days of osteogenic differentiation, mineralized nodules could be detected by Alizarin Red staining. After 28 days of adipogenic differentiation, Oil Red O staining showed that lipid vesicles were produced in the cells. After 28 days of chondrogenic differentiation, Alcian blue staining showed the presence of sulfated proteoglycan in the cells (Fig. 3).

NPMSCs cultured under different osmotic pressures
We cultured NPMSCs in four groups under different osmotic pressures. After ve days of culture, morphological changes in the cells were observed, and their morphology was recorded by photographs.
As the osmotic pressure changed, the cell morphology also changed. Compared with the iso-osmotic pressure group, the hypertonic group had larger and atter cells, and the greater the increase in osmotic pressure, the more signi cant the changes in the cells were. The morphology of the cells in the hypotonic group also showed the same change (Fig. 4).

Cell proliferation of NPMSCs
After the cells in the four groups (1, 2, 3, and 4) were grown for 5 days, the proliferation rate of group 1 was lower than that of group 2, and the cell proliferation rates of groups 3 and 4 decreased and were lower than that of group 2. The cell proliferation rate of group 4 was lower than that of group 3 (Fig. 5).

Apoptosis assay of NPMSCs
The cells were cultured in four groups, 1, 2, 3, and 4, for 5 days. The apoptosis rate of group 2 was the lowest. The apoptotic rates of groups 1, 3 and 4 were signi cantly higher than that of group 2. The apoptosis rate of group 4 was higher than that of group 3 (Fig. 6).

The cell cycle of NPMSCs
We cultured the above 4 groups of cells for 5 days. The results showed that compared with the isotonic pressure group, the hypotonic group and the hypertonic group showed inhibition of the cell cycle, and the higher the osmotic pressure, the more obvious the inhibitory effect on the cells was (Fig. 7).

SA-β-Gal activity of NPMSCs
The SA-β-Gal activity of the hypotonic group and the hypertonic group was signi cantly higher than that of the isotonic group. Both hypotonic and hypertonic conditions can promote cell senescence. The greater the osmotic pressure, the more signi cant the enhancement of cell senescence was (Fig. 8).

Gene expression of NPMSCs
NPMSCs were cultured under four different osmotic pressures for 7 days. Compared with that in group 2, the expression of stemness genes (Nanog, Notch1, Jag1, OCT4) and functional genes (Collagen II, Aggrecan) was downregulated in group 1, group 3 and group 4, and the expression in group 4 showed greater downregulation than that of group 3. The expression of the P16 INK4A and RB genes was also upregulated, and group 4 showed greater upregulation than group 3 (Fig. 9).

Protein expression of NPMSCs
NPMSCs were cultured in groups 1, 2, 3 and 4 for 7 days, and Western blotting results showed that compared with that in group 2, the expression of Collagen II and Aggrecan in group 1, group 3 and group 4 was downregulated, and group 4 showed greater downregulation than group 3. The expression of P16 INK4A and RB was also upregulated, and group 4 showed greater upregulation than group 3 (Fig. 10).
The effect of P16 INK4A knockdown on the NPMSC activity After knockdown of P16 INK4A by siRNA transfection, the effects were veri ed by PCR and Western blots, which showed that the expression of P16 INK4A was signi cantly lower than that of the control group (Fig. 11).
. After knocking down P16 INK4A , we continued to culture the cells under the above 4 osmotic pressure conditions and then tested them. The mRNA and protein levels of Collagen II, Aggrecan and RB were detected, and the results showed no signi cant differences among the groups (Fig. 12).

Discussion
The intervertebral disc is a tissue with a special structure. Its biological environment is relatively closed, and blood vessels that directly supply nutrients are lacking, which results in this speci c structure. As the degree of IVDD increases, the microenvironment of the intervertebral disc changes, and the osmotic pressure also changes [24]. Moreover, various movements and loads of the spine lead to changes in the osmotic pressure in the NP tissue. A study by Urban et al. [25] showed that the osmotic pressure of NP tissue is between 450 and550 mOsm/kg H 2 O in a day. However, the exact mechanism of the effect of osmotic pressure changes on NPMSCs is still unclear. This study simulated different osmotic pressure environments to culture human normal NPMSCs from intervertebral discs to study the effect of osmotic pressure on the biological activity of these cells.
The NP tissue selected in this study was derived from patients with lumbar vertebral fractures and was from normal intervertebral discs (P rrmann I or II). We isolated and cultured cells in vitro and found that the cells adhered to the wall and grew in a radial spiral shape, with high expression of CD73, CD90, and CD105, low expression of HLA-DR, CD34, and CD45, and differentiation towards osteogenic, adipogenic and chondrogenic lineages. According to the evaluation criteria of the International Association for Stem Cell Therapy (ISCT)[26], the isolated and cultured cells were NPMSCs.
Ishihara et al. [27] cultivated NP tissue in vitro and found that increased osmotic pressure could enhance the synthesis of proteoglycans, and the increase in proteoglycan synthesis was related to the osmotic pressure of the culture medium but unrelated to Na + or Cl − . Neidlinger et al. [28] increased the osmotic pressure of the medium in bovine NP cells from 300 mOsm/kg H 2 O to 500 mOsm/kg H 2 O, and the proteoglycan expression increased. Spillekom et al. [29] found that the synthesis of proteoglycan in dog NP cells was optimal when the osmotic pressure in the culture medium was 400 mOsm/kg H 2 O. Studies have also found that high osmotic pressure can inhibit the proliferation of NP cells and the synthesis of extracellular matrix and promote the apoptosis of NPMSCs [30][31][32]. The above ndings on osmotic pressure on the biological activity of NP cells are different, which may be caused by the method of cell culture, the difference in cell species, and the different exposure conditions. In this study, we simulated different osmotic pressure environments in vitro and cultured NPMSC cells under different osmotic pressure conditions. Experimental results showed that compared with normal osmotic pressure, hypotonic and hypertonic environments inhibited the proliferation of NPMSCs. The higher the osmotic pressure, the more obvious the inhibition of NPMSC proliferation was.
High osmotic pressure disrupts the cell cycle, can arrest cells in the G1 phase and inhibits the cell cycle [33][34][35]. These ndings are similar to our research results. We used ow cytometry to detect the cell cycle of the NPMSCs cultured under different osmotic pressures and found that compared with the normal osmotic pressure group, the high osmotic pressure group showed arrest of cells in the G1 phase. The higher the osmotic pressure, the stronger the inhibitory effect is. Research shows that not only high osmotic pressure but also low osmotic pressure can impair the cell cycle progression of NPMSCs.
Changes in osmotic pressure also affect the expression of extracellular matrix. The results showed that when the osmotic pressure was decreased or increased, the expression of related genes (Nanog, Nocth1, Jag1, OCT4) in NPMSCs decreased, and the expression of Collagen II and Aggrecan was also reduced compared to that in the normal osmotic pressure group. These results suggested that abnormal changes in osmotic pressure will cause dysfunction of NPMSCs, which will lead to IVDD. Messmer[36] found that an increase in tear osmotic pressure is the main cause of ocular in ammation and corneal stem cell apoptosis and aging. Many studies have shown that as cells age, the level of P16 INK4A increases [37][38][39]. The P16 INK4A /Rb signaling pathway plays an important role in cell senescence, and P16 INK4A can cause permanent cell growth arrest and cell senescence [40].
This study also con rmed these ndings.Research shows that compared with the normal osmotic pressure group, the hypotonic and hypertonic groups showed an increased proportion of cell senescence, as demonstrated by galactosidase staining, the expression of the P16 INK4A and Rb genes increased, and the expression of Collagen II and Aggrecan decreased. These results suggested that low and high osmotic pressure induced the upregulation of P16 INK4A gene expression in NPMSCs, causing damage to NPMSCs.
To further clarify the role of P16 INK4A and osmotic pressure in NPMSCs, we used siRNA transfection to knock down the expression of P16 INK4A . We detected the P16 INK4A protein level by Western blots and the P16 INK4A mRNA level by PCR. We found that the expression of P16 INK4A in the siRNA-transfected cells was signi cantly lower than that of the control group. Both methods showed good transfection effects and high e ciency. After knockdown of P16 INK4A , we found that there was no signi cant difference in the expression of the Collagen II, Aggrecan and Rb genes after the osmotic pressure was changed, as shown by PCR and Western blots. Silencing of the P16 INK4A gene blocked the adverse effects of osmotic pressure on the expression of the Collagen II, Aggrecan and RB genes in NPMSCs. P16 INK4A /Rb is an important pathway for cell proliferation. During normal cell proliferation, Cyclin D1 forms a complex with CDK4/6 to phosphorylate the Rb protein, and the phosphorylated Rb protein is dissociated from the transcription factor E2F, which promotes cell transition from G1 to S phase and enhances cell proliferation [41]. The P16 INK4A protein is an inhibitor of CDK4/6, which can compete with Cyclin D1 to bind CDK4/6, inhibit CDK4/6-mediated phosphorylation of RB protein, prevent cells from entering S phase from G1 phase, and inhibit cell proliferation [41,42]. This study con rmed that compared with the normal osmotic pressure group, the low osmotic pressure and high osmotic pressure groups showed activation of the P16 INK4A /Rb signaling pathway. The expression of the P16 INK4A gene was upregulated, and the phosphorylation level of the Rb protein was reduced. Rb combined with the transcription factor E2F and prevented cells from entering the S phase from the G1 phase,and the cell cycle is inhibited. After knockdown of P16 INK4A expression by siRNA, the P16 INK4A /Rb signaling pathway was blocked, the change in osmotic pressure could not activate the P16 INK4A /Rb signaling pathway, the expression of the Rb gene was not affected, and the cell cycle progression of NPMSCs was not blocked. Cell aging was inhibited, and the expression of extracellular matrix components did not decrease. Because of the time limit of siRNA transfection, we did not conduct long-term cell culture on the knockdown cells and did not perform more in-depth research on cell differentiation and proliferation.
We used fetal bovine serum instead of human serum to culture the human NPMSCs. Differences in serum species may affect the test results, but human serum samples are limited. Second, the study was conducted in 2D culture medium, and the response of NPMSCs in a 3D culture system may be different.
Third, the research on the silencing of P16 INK4A gene is mainly carried out in monolayer cultured NPMSCs. Dedifferentiation and short-term effects are inevitable.Although these effects were biologically important, they may not be relevant to in vivo situations. The long-term effect in the body is very important. Although the exact mechanism of P16 INK4A has yet to be determined, knocking down P16 INK4A in NP cells may have some bene cial biological functions.

Conclusion
In summary,the results showed that changes in osmotic pressure have an important effect on the biological activity of NPMSCs, hypotonic or hypertonic can induce cell apoptosis and promote cell senescence.Studies have shown that this effect may occur through the P16 INK4A /Rb pathway and affect the regeneration and restore functions of NPMSCs.By knocking down the P16 INK4A gene, the adverse effects of changes in osmotic pressure on cells can be reversed.This study provides a useful theoretical basis for the treatment of IVDD in the future.

Abbreviations
NPMSCs human nucleus pulposus-derived mesenchymal stem cells IVD