Nucleus Pulposus Chondrocytes Research Articles

GREM1, LRPPRC and SLC39A4 as potential biomarkers of intervertebral disc degeneration: a bioinformatics analysis based on multiple microarray and single-cell sequencing data

BackgroundLow back pain (LBP) has drawn much widespread attention and is a major global health concern. In this field, intervertebral disc degeneration (IVDD) is frequently the focus of classic studies. However, the mechanistic foundation of IVDD is unclear and has led to conflicting outcomes.MethodsGene expression profiles (GSE34095, GSE147383) of IVDD patients alongside control groups were analyzed to identify differentially expressed genes (DEGs) in the GEO database. GSE23130 and GSE70362 were applied to validate the yielded key genes from DEGs by means of a best subset selection regression. Four machine-learning models were established to assess their predictive ability. Single-sample gene set enrichment analysis (ssGSEA) was used to profile the correlation between overall immune infiltration levels with Thompson grades and key genes. The upstream targeting miRNAs of key genes (GSE63492) were also analyzed. A single-cell transcriptome sequencing data (GSE160756) was used to define several cell clusters of nucleus pulposus (NP), annulus fibrosus (AF), and cartilaginous endplate (CEP) of human intervertebral discs and the distribution of key genes in different cell clusters was yielded.ResultsBy developing appropriate p-values and logFC values, a total of 6 DEGs was obtained. 3 key genes (LRPPRC, GREM1, and SLC39A4) were validated by an externally validated predictive modeling method. The ssGSEA results indicated that key genes were correlated with the infiltration abundance of multiple immune cells, such as dendritic cells and macrophages. Accordingly, these 4 key miRNAs (miR-103a-3p, miR-484, miR-665, miR-107) were identified as upstream regulators targeting key genes using the miRNet database and external GEO datasets. Finally, the spatial distribution of key genes in AF, CEP, and NP was plotted. Pseudo-time series and GSEA analysis indicated that the expression level of GREM1 and the differentiation trajectory of NP chondrocytes are generally consistent. GREM1 may mainly exacerbate the degeneration of NP cells in IVDD.ConclusionsOur study gives a novel perspective for identifying reliable and effective gene therapy targets in IVDD.

TREATMENT WITH MESENCHYMAL STEM CELL AND CHONDROITIN SULFATE IN RABBITS WITH INVERTEBRAL DISC DEGENERATION

<h3>Background</h3> Bone marrow-derived mesenchymal stromal cells (BMMSC) can be expanded ex vivo, which have the ability to reduce changes resulting from intervertebral disc degeneration. In addition to cell therapy, nutraceuticals such as chondroitin sulfate are being studied, due to possible influence on the regeneration of osteoarticular tissues. This study evaluated histologically and morphometrically the effects of BMMSC and chondroitin sulfate (CS), as well as the association of both, in rabbits with intervertebral disc degeneration (IDD). <h3>Methods</h3> Forty four New Zealand rabbits, weighing around 3.5 and 4.5 kg, approximately 1 year old, were selected and randomly divided into four distinct groups (n = 11): Group A (control), Group B (BMMSC), Group C (CS), Group D (BMMSC and CS). At seven and 14 days after injury, culture medium was infused in group A, and 1.2 × 10⁶ BMMSC were transplanted in groups B and D. The animals from group C and D received eight subcutaneous applications of CS at a dose of 5 mg/kg, one application every seven days. <h3>Results</h3> Histopathological alterations were observed: size; morphology of annulus fibrosus (AF); chondrocytes and AF metaplasia; holes and cracks in the nucleus pulposus (NP); proliferation of NP chondrocytes; presence of notochordal cells in the NP; endplate morphology; new bone formation, and final score according to Bergknut et al. (2013). A benefit was noticed in the use of BMMSC, CS or both, the final score in Groups B, C and D was higher than in Group A. <h3>Conclusion</h3> The three treatments evaluated indicated a lower degeneration score when compared to the control group. However, Group D was not superior to Group B or C, which suggests that the combination of the two therapies does not provide additional benefits.

A Membranome-Centered Approach Defines Novel Biomarkers for Cellular Subtypes in the Intervertebral Disc.

Lack of specific marker-sets prohibits definition and functional distinction of cellular subtypes in the intervertebral disc (IVD), such as those from the annulus fibrosus (AF) and the nucleus pulposus (NP). We recently generated immortalized cell lines from human NP and AF tissues; these comprise a set of functionally distinct clonal subtypes. Whole transcriptome analyses were performed of 12 phenotypically distinct clonal cell lines (4× NP-Responder, 4× NP-nonResponder, 2× AF-Sheet forming, and 2× AF-nonSheet forming). Data sets were filtered for membrane-associated marker genes and compared to literature. Comparison of our immortal cell lines to published primary NP, AF, and articular chondrocytes (AC) transcriptome datasets revealed preservation of AF and NP phenotypes. NP-specific membrane-associated genes were defined by comparison to AF cells in both the primary dataset (46 genes) and immortal cell-lines (161 genes). Definition of AF-specific membrane-associated genes yielded 125 primary AF cell and 92 immortal cell-line markers. Overlap between primary and immortal NP cells yielded high-confidence NP-specific marker genes for NP-R (CLDN11, TMEFF2, CA12, ANXA2, CD44) and NP-nR (EFNA1, NETO2, SLC2A1). Overlap between AF and immortal AF subtypes yielded specific markers for AF-S (COLEC12, LPAR1) and AF-nS (CHIC1). The current study provides a reference platform for preclinical evaluation of novel membrane-associated cell type-specific markers in the IVD. Future research will focus on their biological relevance for IVD function in development, homeostasis, and degenerate conditions.

Non-invasive Raman Spectroscopy and Quantitative Real-Time PCR Distinguish Among Undifferentiated Human Mesenchymal Stem Cells and Redifferentiated Nucleus Pulposus Cells and Chondrocytes In Vitro.

Background:The most common cause of lower back pain is the pathological degeneration of the nucleus pulposus (NP). Promising NP regeneration strategies involving human mesenchymal stem cells (hMSCs) would require specific markers to confirm successful differentiation into the NP lineage and to distinguish the articular cartilage (AC).Objective:We sought specific NP mRNA markers that are upregulated in native NP cells but not in dedifferentiated NP cells, undifferentiated hMSCs or chondrocytes. We also considered the suitability of non-invasive Raman spectroscopy to distinguish among these classes of cells.Method:We used quantitative real-time PCR and Raman spectroscopy to analyse undifferentiated hMSCs in monolayers and embedded in hydrogels, and compared the results with dedifferentiated and redifferentiated human NP and AC cells.Results:The redifferentiation of NP cells induced the expression of annexin A3 (ANXA3), collagen type II (COL2) and proteoglycan mRNAs, whereas the redifferentiation of AC cells only induced proteoglycan expression. Redifferentiated NP cells expressed higher levels of ANXA3, COL2, paired box 1 (PAX1) and OCT4 mRNA than redifferentiated AC cells. Redifferentiated NP cells and undifferentiated hMSC-TERT cells expressed similar amount of OCT4 mRNA, indicating that only ANXA3, COL2 and PAX1 are promising markers for redifferentiated NP cells. Raman spectra clearly differed among the three cell types and highlighted their differentiation status.Conclusion:We recommend ANXA3, COL2 and PAX1 as markers to determine the success of hMSC-based differentiation to regenerate NP cells. Raman spectroscopy can be used to determine cell type and differentiation status especially in the context of clinical trials.

Unique glycosignature for intervertebral disc and articular cartilage cells and tissues in immaturity and maturity.

In this study, on/off markers for intervertebral disc (IVD) and articular cartilage (AC) cells (chondrocytes) and distinct glycoprofiles of cell and tissue-types were identified from immaturity to maturity. Three and eleven month-old ovine IVD and AC tissues were histochemically profiled with a panel of lectins and antibodies. Relationships between tissue and cell types were analysed by hierarchical clustering. Chondroitin sulfate (CS) composition of annulus fibrosus (AF), nucleus pulposus (NP) and AC tissues was determined by HPLC analysis. Clear on/off cell type markers were identified, which enabled the discrimination of chondrocytes, AF and NP cells. AF and NP cells were distinguishable using MAA, SNA-I, SBA and WFA lectins, which bound to both NP cells and chondrocytes but not AF cells. Chondrocytes were distinguished from NP and AF cells with a specific binding of LTA and PNA lectins to chondrocytes. Each tissue showed a unique CS composition with a distinct switch in sulfation pattern in AF and NP tissues upon disc maturity while cartilage maintained the same sulfation pattern over time. In conclusion, distinct glycoprofiles for cell and tissue-types across age groups were identified in addition to altered CS composition and sulfation patterns for tissue types upon maturity.

Relationship between Initial Telomere Length, Initial Telomerase Activity, Age, and Replicative Capacity of Nucleus Pulposus Chondrocytes in Human Intervertebral Discs: What Is a Predictor of Replicative Potential?

There is evidence that telomere length (TL), telomerase activity (TA), and age are related to the replicative potential of human nucleus pulposus chondrocytes (NPCs). However, it has not yet been established if any of these factors can serve as predictors of the replicative potential of NPCs. To establish predictors of the replicative potential of NPCs, we evaluated potential relationships between replicative capacity of NPCs, initial TL (telomere length at the first passage), initial TA (telomerase activity at the first passage), and age. Nucleus pulposus specimens were obtained from 14 patients of various ages undergoing discectomy. NPCs were serially cultivated until the end of their replicative lifespans. Relationships among cumulative population doubling level (PDL), initial TL, initial TA, and age were analyzed. Initial TA was negatively correlated with age (r = -0.674, P = 0.008). However, no correlation between initial TL and age was observed. Cumulative PDL was also negatively correlated with age (r = -0.585, P = 0.028). Although the cumulative PDL appeared to increase with initial TL or initial TA, this trend was not statistically significant. In conclusion, age is the sole predictor of the replicative potential of human NPCs, and replicative potential decreases with age. Initial TL and initial TA are not predictors of replicative potential, and can serve only as reference values.

In vitro lifespan and senescence mechanisms of human nucleus pulposus chondrocytes

Background contextOur previous in vivo study demonstrated that human nucleus pulposus chondrocytes (NPCs) in aging discs exhibited characteristic senescent features such as an increased senescence-associated β-galactosidase (SA-β-gal) expression, shortened telomere, and decreased telomerase activity. The replicative p53-p21-pRB pathway, rather than the stress-induced p16-pRB pathway, played a more important role in the senescence of NPCs in an in vivo condition, although there is a situation in which both the pathways can be activated simultaneously. However, the in vitro lifespan and senescence mechanisms of human NPCs remain unclear. PurposeTo evaluate the underlying mechanisms of in vitro lifespan and in vitro senescence of the human NPCs and to verify whether the in vitro senescence mechanisms of the human NPC can represent the in vivo aging mechanisms of the cell. Study design/settingAn in vitro study. MethodsWe serially cultivated human NPCs from patients of different ages (35, 42, 55, 66, and 76 years) until the cells reached the end of their in vitro lifespan. During each subcultivation, we calculated NPCs cumulative population doubling level (PDL) and examined senescence markers (SA-β-gal, telomere length, telomerase activity, and p53, p21, pRB, and p16 expressions). ResultsThe cumulative PDLs of the NPCs from patients aged 35, 42 55, 66, and 76 years were 32, 29, 11, 9, and 11, respectively. The younger patients (35 and 42 years) had a higher mean of cumulative PDLs than the elderly patients did (55, 66, and 76 years; 30.5 vs. 10.3; p=.001). In addition, there was a significant, negative correlation between the cumulative PDLs and patient's age (r=−0.89; p=.04). With advancing culture passages, the NPCs irrespective of patient's age exhibited characteristic senescent features, such as an increase in SA-β-gal expression, shortening of telomeres, decrease in telomerase activity, and activation of both replicative p53-p21-pRB and stress-induced p16-pRB pathways. However, all the senescent features occurred at the earlier passages in elderly compared with younger patients. ConclusionsThe present study demonstrated that the human NPCs had a finite in vitro lifespan, which declined with host aging. The in vitro lifespan was determined by both replicative and stress-induced senescence mechanisms. The similarity in the in vitro senescent features with those apparent in the previous in vivo study suggests a possibility of the in vitro senescence mechanisms of the human NPC as a model of the in vivo aging mechanisms of the cell for future studies.

Ex VivoObservation of Human Nucleus Pulposus Chondrocytes Isolated From Degenerated Intervertebral Discs

Study DesignWe performed an ex vivo study to observe cell morphology and viability of human nucleus pulposus (NP) chondrocytes isolated from degenerated intervertebral discs (IVD).PurposeTo better understand the biological behavior of NP chondrocytes in monolayer cultures.Overview of LiteratureBiological repair of IVDs by cell-based therapy has been shown to be feasible in clinical trials. As one of the most promising transplanting seeds, how the isolated NP chondrocytes behavior ex vivo has not been fully understood.MethodsHuman NP chondrocytes were harvested from 20 degenerated IVDs and cultured in monolayers. Histological and immunochemistry staining was used to detect cell morphology change. Cell viability was studied by analyzing cell cycle distribution and apoptotic rate in the primary and subculuted cells.ResultsThe round or polygonal primary NP chondrocytes had an average adherence time of 7 days and took nearly 31 days to reach 95% confluence. The spindle-shaped P1 NP chondrocytes increased growth kinetics and took about 12 hours to adhere and 6.6 days to get 95% confluent. Immunochemistry staining of collagen II was positive in the cell cytoplasm. Nearly 90% of the confluent NP chondrocytes stayed in G1 phase while 16% underwent apoptosis. No significant difference of the collagen II expression, cell cycle distribution or the apoptosis indices were detected between the primary and subcultured NP chondrocytes.ConclusionsHuman NP chondrocytes undergo significant morphological change in monolayer cultures. Cell cycle distribution pattern and apoptosis index of the cutured NP chondrocytes potentially influence their clinical transplantation or laboratory use.

Differential Gene Expression Profiling of Metalloproteinases and Their Inhibitors

A comparative in vitro metalloproteinases and their inhibitors gene expression profile. To obtain a complete expression profile of matrix metalloproteinases (MMPs), family of proteases with a disintegrin and metalloproteinase domain with thrombospondin motifs (ADAMTS), and tissue inhibitors of metalloproteinases (TIMPs) in bovine adult nucleus pulposus (NP) cells and to compare this profile with the expression profile obtained from bovine adult articular chondrocytes cultured under identical conditions. The cells of the NP resemble articular chondrocytes morphologically but produce a matrix which, though consisting of similar components, has very different biomechanical properties. No specific markers for NP cells have yet been identified; they can be distinguished from chondrocytes only by differences in gene expression. Here we compare profiles of gene expression of metalloproteinases and their inhibitors between NP cells and chondrocytes to improve understanding of the differences between these cell types. NP cells and articular chondrocytes were harvested respectively from bovine caudal discs and the articular cartilage of metacarpal-phalangeal joints of 18- to 24-month-old steers. These cells were cultured under identical conditions for 96 hours in alginate beads. Expression levels of MMPs, ADAMTSs, and TIMPs were detected by real-time RT-PCR. Gene profiling demonstrated distinct differences between levels of MMPs, ADAMTSs, and TIMPs produced by chondrocytes and NP cells. In particular, NP cells expressed considerably more MMP-2 and MMP-14 than chondrocytes, and expression of ADAMTS-1,-2,-17 and TIMP-1 was also higher. However, expression of MMP-1,-3,-7,-8,-10,-11,-13,-16,-19,-20,-21,-23,-24,-28, ADAMTS-4,-5,-6,-14,-18,-19, and TIMP-3 was lower in NP cells than in chondrocytes. Chondrocytes but not NP cells expressed MMP12 and MMP27; this difference is a potential marker for distinguishing between NP cells and chondrocytes. Because culture conditions and animal age were identical, differences in metalloproteinase and inhibitor expression between NP cells and chondrocytes were intrinsic to cell phenotype and not induced by differences in the in situ extracellular environment.

Senescence mechanisms of nucleus pulposus chondrocytes in human intervertebral discs

Background context The population of senescent disc cells has been shown to increase in degenerated or herniated discs. However, the mechanism and signaling pathway involved in the senescence of nucleus pulposus (NP) chondrocytes are unknown. Purpose To demonstrate the mechanisms involved in the senescence of NP chondrocytes. Study design/setting Senescence-related markers were assessed in the surgically obtained human NP specimens. Patient sample NP specimens remaining in the central region of the intervertebral disc were obtained from 25 patients (mean: 49 years, range: 20–75 years) undergoing discectomy. Based on the preoperative magnetic resonance images, there were 3 patients with Grade II degeneration, 17 patients with Grade III degeneration, and 5 patients with Grade IV degeneration. Outcome measures We examined cell senescence markers (senescence-associated β-galactosidase [SA-β-gal], telomere length, telomerase activity, p53, p21, pRB, and p16) and the hydrogen peroxide (H 2O 2) content as a marker for an oxidative stress in the human NP specimens. Methods SA-β-gal expression, telomere length, telomerase activity, and H 2O 2 content as well as their relationships with age and degeneration grades were analyzed. For the mechanism involved in the senescence of NP chondrocytes, expressions of p53, p21, pRB, and p16 in these cells were assessed with immunohistochemistry and Western blotting. Results The percentages of SA-β-gal-positive NP chondrocytes increased with age (r=.82, p<.001), whereas the telomere length and telomerase activity declined (r=−.41, p=.045; r=−.52, p=.008, respectively) However, there was no significant correlation between age and H 2O 2 contents (p=.18). The NP specimens with Grade III or Grade IV degeneration showed significantly higher percentages of SA-β-gal-positive NP chondrocytes than those with Grade II degeneration (p=.01 and p=.025, respectively). Immunohistochemistry showed that the senescent NP chondrocytes in all the specimens expressed p53, p21, and pRB, but a few NP chondrocytes in only two specimens expressed p16. Western blotting showed that the expressions of p53, p21, and pRB displayed a corresponding pattern, that is, a strong p53 expression led to strong p21 and pRB expressions and vice versa. Conclusions Our in vivo study demonstrated that senescent NP chondrocytes increased or accumulated in the NP with increasing age and advancing disc degeneration. The NP chondrocytes in the aging discs exhibited characteristic senescent features such as an increased SA-β-gal expression, shortened telomeres, and decreased telomerase activity. We further demonstrated that the telomere-based p53-p21-pRB pathway, rather than the stress-based p16-pRB pathway, plays a more important role in the senescence of NP chondrocytes in an in vivo condition. Our results suggest that prevention or reversal of the senescence of NP chondrocytes can be a novel therapeutic target for human disc degeneration.

Chondrogenic Differentiation of Human Mesenchymal Stem Cells in Three-Dimensional Alginate Gels

We characterized the temporal changes in chondrogenic genes and developed a staging scheme for in vitro chondrogenic differentiation of human mesenchymal stem cells (hMSCs) in three-dimensional (3D) alginate gels. A time-dependent accumulation of glycosaminoglycans, aggrecan, and type II collagen was observed in chondrogenic but not in basal constructs over 24 days. qRT-PCR demonstrated a largely characteristic temporal pattern of chondrogenic markers and provided a basis for staging the cellular phenotype into four stages. Stage I (days 0-6) was defined by collagen types I and VI, Sox 4, and BMP-2 showing peak expression levels. In stage II (days 6-12), gene expression for cartilage oligomeric matrix protein, HAPLN1, collagen type XI, and Sox 9 reached peak levels, while gene expression of matrilin 3, Ihh, Homeobox 7, chondroadherin, and WNT 11 peaked at stage III (days 12-18). Finally, cells in stage IV (days 18-24) attained peak levels of aggrecan; collagen IX, II, and X; osteocalcin; fibromodulin; PTHrP; and alkaline phosphatase. Gene profiles at stages III and IV were analogous to those in juvenile articular and adult nucleus pulposus chondrocytes. Gene ontology analyses also demonstrated a specific expression pattern of several putative novel marker genes. These data provide comprehensive insights on chondrogenesis of hMSCs in 3D gels. The derivation of this staging scheme may aid in defining maximally responsive time points for mechanobiological modulation of constructs to produce optimally engineered tissues.

Senescence of Nucleus Pulposus Chondrocytes in Human Intervertebral Discs

Study DesignSenescence-related markers were assessed in surgically obtained human nucleus pulposus (NP) specimens.PurposeTo demonstrate the mechanism and signaling pathway involved in the senescence of NP chondrocytes.Overview of LiteratureThe population of senescent disc cells has been shown to be increased in degenerated or herniated discs. However, the mechanism and signaling pathway involved in the senescence of NP chondrocytes are unknown.MethodsWe examined cell senescence markers [senescence-associated β-galactosidase (SA-β-gal), telomere length, telomerase activity, p53, p21, pRB and p16] and the hydrogen peroxide (H2O2) content in human NP specimens.ResultsThe percentage of SA-β-gal-positive NP chondrocytes increased with age, while the telomere length and telomerase activity declined. However, there was no significant correlation between age and H2O2 content. The NP specimens with grade III or IV degeneration showed significantly higher percentages of SA-β-gal-positive NP chondrocytes than those with grade II degeneration. Immunohistochemistry showed that senescent NP chondrocytes in all specimens expressed p53, p21, and pRB, while a few NP chondrocytes in only two specimens expressed p16.ConclusionsThe present study demonstrates that, with increasing age and advancing disc degeneration, senescent NP chondrocytes increase or accumulate in the NP. Furthermore, the telomere-based p53, p21, pRB pathway, rather than the stress-based p16, pRB pathway, plays a more important role in the senescence of NP chondrocytes in in vivo conditions. Our results suggest that prevention or reversal of senescence of NP chondrocytes can be a novel mechanism by which to prevent human disc degeneration.