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Changes in the molecular phenotype of nucleus pulposus cells with intervertebral disc aging.

Tang X, Jing L, Chen J - PLoS ONE (2012)

Bottom Line: In addition, CD protein (CD24 and CD90) and Brachyury (T) expression in immature disc cells were also confirmed via flow cytometry.Similar to IHC staining, results revealed a higher percentage of immature NP cells expressing CD24 and Brachyury, while higher percentage of immature AF cells was stained positively for CD90.These age-related phenotype changes provide a new insight for a molecular profile that may be used to characterize NP cells for developing cell-based regenerative therapy for IVD regeneration.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, Duke University, Durham, NC, USA.

ABSTRACT
Intervertebral disc (IVD) disorder and age-related degeneration are believed to contribute to low back pain. Cell-based therapies represent a promising strategy to treat disc degeneration; however, the cellular and molecular characteristics of disc cells during IVD maturation and aging still remain poorly defined. This study investigated novel molecular markers and their age-related changes in the rat IVD. Affymetrix cDNA microarray analysis was conducted to identify a new set of genes characterizing immature nucleus pulposus (NP) cells. Among these markers, select neuronal-related proteins (Basp1, Ncdn and Nrp-1), transcriptional factor (Brachyury T), and cell surface receptors (CD24, CD90, CD155 and CD221) were confirmed by real-time PCR and immunohistochemical (IHC) staining for differential expression between IVD tissue regions and among various ages (1, 12 and 21 months). NP cells generally possessed higher levels of mRNA or protein expression for all aforementioned markers, with the exception of CD90 in anulus fibrosus (AF) cells. In addition, CD protein (CD24 and CD90) and Brachyury (T) expression in immature disc cells were also confirmed via flow cytometry. Similar to IHC staining, results revealed a higher percentage of immature NP cells expressing CD24 and Brachyury, while higher percentage of immature AF cells was stained positively for CD90. Altogether, this study identifies that tissue-specific gene expression and age-related differential expression of the above markers do exist in immature and aged disc cells. These age-related phenotype changes provide a new insight for a molecular profile that may be used to characterize NP cells for developing cell-based regenerative therapy for IVD regeneration.

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Histological characterization of NP tissue from IVD of rats at different ages.Frozen tissue sections from (A, D) 1 m, (B, E) 12 m and (C, F) 21 m-old rats were stained with (A, B, C) Hematoxylin and Eosin (H&E) for cell structure and (D, E, F) Safranin O (SO) for proteoglycan content. Representative images are shown and the inserts of small images shown higher magnifications of interested regions. Bar: 50 µm. m: month. Arrows in subfigures indicate vacuolated morphology. (G) The percentage of cells with vacuolated morphology indicates an age-dependent change in NP tissue. **p<0.01 as compared to 1 m NP (one-factor ANOVA, n = 8). (TIF)
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pone-0052020-g001: Histological characterization of NP tissue from IVD of rats at different ages.Frozen tissue sections from (A, D) 1 m, (B, E) 12 m and (C, F) 21 m-old rats were stained with (A, B, C) Hematoxylin and Eosin (H&E) for cell structure and (D, E, F) Safranin O (SO) for proteoglycan content. Representative images are shown and the inserts of small images shown higher magnifications of interested regions. Bar: 50 µm. m: month. Arrows in subfigures indicate vacuolated morphology. (G) The percentage of cells with vacuolated morphology indicates an age-dependent change in NP tissue. **p<0.01 as compared to 1 m NP (one-factor ANOVA, n = 8). (TIF)

Mentions: Both H&E and Safranin O staining clearly illustrated differences in cellular structure and extracellular matrix for NP tissue among different ages (1 m, 12 m and 21 m, Fig. 1). In general, the NP tissues exhibited an age-dependent decrease in total cell number. Moreover, a significant higher percentage (∼67%) of cells with vacuole-like structure was observed in immature NP (1 m, Fig. 1 A, D, G,) as compared to that in mature and aged NP. About 25% of cells with vacuole-like structures were still observed in the mature NP (12 m, Fig. 1 B, E, G), but only 6% was kept in the aged NP (21 m, Fig. 1 C, F, G). This finding of an age-dependent decrease in vacuole-like cells remains consistent with previous reports of sparse or no notochordal cells found in 1 to 2-year old rats [9], [36]. Additionally, Safranin O staining revealed that 1 m NP tissue displayed a denser matrix structure (Fig. 1 D) compared with 12 m and 21 m NP, which were shown to be more scattered and clustered (Fig. 1 E, F).


Changes in the molecular phenotype of nucleus pulposus cells with intervertebral disc aging.

Tang X, Jing L, Chen J - PLoS ONE (2012)

Histological characterization of NP tissue from IVD of rats at different ages.Frozen tissue sections from (A, D) 1 m, (B, E) 12 m and (C, F) 21 m-old rats were stained with (A, B, C) Hematoxylin and Eosin (H&E) for cell structure and (D, E, F) Safranin O (SO) for proteoglycan content. Representative images are shown and the inserts of small images shown higher magnifications of interested regions. Bar: 50 µm. m: month. Arrows in subfigures indicate vacuolated morphology. (G) The percentage of cells with vacuolated morphology indicates an age-dependent change in NP tissue. **p<0.01 as compared to 1 m NP (one-factor ANOVA, n = 8). (TIF)
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3526492&req=5

pone-0052020-g001: Histological characterization of NP tissue from IVD of rats at different ages.Frozen tissue sections from (A, D) 1 m, (B, E) 12 m and (C, F) 21 m-old rats were stained with (A, B, C) Hematoxylin and Eosin (H&E) for cell structure and (D, E, F) Safranin O (SO) for proteoglycan content. Representative images are shown and the inserts of small images shown higher magnifications of interested regions. Bar: 50 µm. m: month. Arrows in subfigures indicate vacuolated morphology. (G) The percentage of cells with vacuolated morphology indicates an age-dependent change in NP tissue. **p<0.01 as compared to 1 m NP (one-factor ANOVA, n = 8). (TIF)
Mentions: Both H&E and Safranin O staining clearly illustrated differences in cellular structure and extracellular matrix for NP tissue among different ages (1 m, 12 m and 21 m, Fig. 1). In general, the NP tissues exhibited an age-dependent decrease in total cell number. Moreover, a significant higher percentage (∼67%) of cells with vacuole-like structure was observed in immature NP (1 m, Fig. 1 A, D, G,) as compared to that in mature and aged NP. About 25% of cells with vacuole-like structures were still observed in the mature NP (12 m, Fig. 1 B, E, G), but only 6% was kept in the aged NP (21 m, Fig. 1 C, F, G). This finding of an age-dependent decrease in vacuole-like cells remains consistent with previous reports of sparse or no notochordal cells found in 1 to 2-year old rats [9], [36]. Additionally, Safranin O staining revealed that 1 m NP tissue displayed a denser matrix structure (Fig. 1 D) compared with 12 m and 21 m NP, which were shown to be more scattered and clustered (Fig. 1 E, F).

Bottom Line: In addition, CD protein (CD24 and CD90) and Brachyury (T) expression in immature disc cells were also confirmed via flow cytometry.Similar to IHC staining, results revealed a higher percentage of immature NP cells expressing CD24 and Brachyury, while higher percentage of immature AF cells was stained positively for CD90.These age-related phenotype changes provide a new insight for a molecular profile that may be used to characterize NP cells for developing cell-based regenerative therapy for IVD regeneration.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, Duke University, Durham, NC, USA.

ABSTRACT
Intervertebral disc (IVD) disorder and age-related degeneration are believed to contribute to low back pain. Cell-based therapies represent a promising strategy to treat disc degeneration; however, the cellular and molecular characteristics of disc cells during IVD maturation and aging still remain poorly defined. This study investigated novel molecular markers and their age-related changes in the rat IVD. Affymetrix cDNA microarray analysis was conducted to identify a new set of genes characterizing immature nucleus pulposus (NP) cells. Among these markers, select neuronal-related proteins (Basp1, Ncdn and Nrp-1), transcriptional factor (Brachyury T), and cell surface receptors (CD24, CD90, CD155 and CD221) were confirmed by real-time PCR and immunohistochemical (IHC) staining for differential expression between IVD tissue regions and among various ages (1, 12 and 21 months). NP cells generally possessed higher levels of mRNA or protein expression for all aforementioned markers, with the exception of CD90 in anulus fibrosus (AF) cells. In addition, CD protein (CD24 and CD90) and Brachyury (T) expression in immature disc cells were also confirmed via flow cytometry. Similar to IHC staining, results revealed a higher percentage of immature NP cells expressing CD24 and Brachyury, while higher percentage of immature AF cells was stained positively for CD90. Altogether, this study identifies that tissue-specific gene expression and age-related differential expression of the above markers do exist in immature and aged disc cells. These age-related phenotype changes provide a new insight for a molecular profile that may be used to characterize NP cells for developing cell-based regenerative therapy for IVD regeneration.

Show MeSH
Related in: MedlinePlus