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. 2023 Mar 1;48(10):728–736. doi: 10.1097/BRS.0000000000004530

Regenerative Capability of Human Nucleus Pulposus Cells in Degenerated Disc Under Hydrostatic Pressure Mimicking Physiologically Relevant Intradiscal Pressure In Vitro

Yoshiki Takeoka 1, Yutaro Kanda 1, James D Kang 1, Shuichi Mizuno 1,✉
PMCID: PMC10118243  PMID: 36856558

Study Design.

Isolated human nucleus pulposus (hNP) cells from the degenerated intervertebral disc (IVD) were incubated under hydrostatic pressure (HP) and evaluated for regenerative potential.

Objectives.

To characterize metabolic turnover in hNP cells isolated from degenerated IVDs classified by Pfirrmann grade under physiologically relevant HP at high osmolality in vitro.

Summary of Background Data.

We demonstrated that bovine caudal nucleus pulposus cells isolated from healthy cows produced more extracellular matrix under cyclic HP followed by constant pressure (mimicking physiological intradiscal pressure in humans) than under no pressure in vitro. We assessed the effects of pressure on human degenerated cells isolated under the same regimen of pressure used for bovine cells.

Materials and Methods.

hNP cells isolated from discarded tissue classified as Pfirrmann grade 2 to 3 (n = 13: age, 46.7 ± 14.0) and grade 4 (n = 13: age, 53.0 ± 11.5) were incubated under cyclic HP at 0.2 to 0.7 MPa, 0.5 Hz for 2 days followed by constant pressure at 0.3 MPa for 1 day, repeated twice over 6 days. The gene expression and immunohistology of matrix molecules and catabolic and anticatabolic proteins were evaluated.

Results.

Aggrecan and collagen type II expression were significantly more upregulated under HP in grades 2 to 3 than in grade 4 tissues (both, P < 0.01). Linear regression analysis showed a positive correlation between matrix metalloproteinase 13 and tissue inhibitor for metalloproteinase 2 expression in grades 2 to 3, whereas a negative correlation was found in grade 4 (P < 0.05). Immunohistological staining revealed the activation of a mechanoreceptor, transient receptor potential vanilloid 4, under HP.

Conclusions.

Resident cells in mild-moderate degenerated discs classified as Pfirrmann grade 2 to 3 have the potential to promote extracellular matrix production and maintain adequate cell viability under physiological spinal loading.

Relevance.

This study explored the potential of degenerated remnant nucleus pulposus cells under a physiological environment, possibly leading to establishing strategies for IVD regeneration.

Key words: cervical spine, extracellular matrix, human, hydrostatic pressure, intervertebral disc degeneration, lumbar spine, nucleus pulposus cell, osmolality, Pfirrmann degeneration grade, regenerative therapy

Back pain is a global health problem with a heavy socioeconomic burden, and intervertebral disc degeneration (IVDD) is one of the major independent risk factors.1 None of the current surgical treatments for IVDD addresses the regeneration of the disc. When we develop regenerative therapeutic strategies, it is imperative to understand the capability of the resident nucleus pulposus (NP) cells in the degenerated disc for cell proliferation and anabolic turnover. In fact, for any regenerative therapy, it is important to know whether the resident NP cells synergistically promote regeneration under daily variations in spinal loading.2,3 This physiology includes changes in intradiscal hydrostatic pressure (HP) in daily cycles4,5 and high osmotic pressure (OP) generated by the highly negatively charged amorphous extracellular matrix (ECM).6,7 We have demonstrated healthy bovine NP cells expressed anabolic turnover under cyclic HP followed by constant HP.2,3 Thus, we hypothesized that human nucleus pulposus (hNP) cells in the mild-moderately degenerated intervertebral disc (IVD) are capable of producing greater ECM with higher cell viability than those in the advanced degenerated IVD under physiologically relevant HP and OP. We tested this hypothesis using hNP cells isolated from IVD classified as Pfirrmann grade 2 to 3 and 4,8 and incubated under HP at high OP. We evaluated cell viability and gene expression of metabolic molecules as well as the accumulation of ECM immunohistologically. These biomarkers were analyzed along with the age of the subjects, cell origin in the cervical or lumbar spine, and degenerative grades of IVD. In addition, we confirmed the effects of HP through the transient receptor potential vanilloid 4 (TRPV4) immunohistologically.9–11

MATERIALS AND METHODS

Ethics Statement

All experiments using discarded tissues were performed under the approval and guidance of the Institutional Review Board at the authors' institution. Written informed consent was obtained from each patient in accordance with the principles of the Declaration of Helsinki.

mRNA Probes, Antibodies, and Reagents

TaqMan® probes, antibodies, and reagents are listed in Supplementary Table 1 (Supplemental Digital Content 1, http://links.lww.com/BRS/C29).

Correlation Between Sulfated Glycosaminoglycan of Extracellular Matrix and Pfirrmann Grade of Human Nucleus Pulposus Tissue

We collected discarded hNP tissues from patients who underwent lumbar discectomy, lumbar interbody fusion, cervical discectomy, or cervical fusion surgery [n = 26: 11 males and 15 females; 49.8 ± 12.9 (22–75) years; Pfirrmann grade,8 median 3.5 ± 0.7 (2–4)]. hNP tissues were carefully obtained from the first bite of tissue using a laminectomy rongeur without any violation of vertebral endplates. Before the HP loading experiment, the correlation between Pfirrmann grade and sulfated glycosaminoglycan (sGAG) concentration of the tissue was validated. hNP tissue (40–50 mg) was digested in 125 µg/mL papain solution at 60°C overnight. The concentration of sGAG was measured with a dimethylmethylene blue assay.

Isolation and Preculture of Human Nucleus Pulposus Cells/Clusters

For loading experiments, hNP tissues were digested in 0.15% collagenase type 1 with antibiotics at 37°C overnight. Then hNP cells were rinsed with Dulbecco phosphate-buffered saline, seeded onto 1.5% cell-culture-grade agarose-coated 6-well plates, and preincubated in Dulbecco modified Eagle medium/Ham F-12 (1:1) supplemented with 10% fetal bovine serum, 1% insulin-transferrin-selenium, and antibiotics at 37 °C, 5% CO2 for 2 to 3 days. During this preincubation, undigested debris and contaminated erythrocytes were removed. The cells/clusters were allocated into 2 groups: grades 2 to 3 corresponding to Pfirrmann grade8 [n = 13: 4 males and 9 females; age, 46.7 ± 14.0 (22⁠–71)]; and grade 4 [n = 13: 7 males and 6 females; age, 53.0 ± 11.5 (37⁠–75)] (Supplementary Table 2, Supplemental Digital Content 2, http://links.lww.com/BRS/C30). The phenotype of hNP cells was validated immunohistologically with antibodies against Brachyury in both groups (Supplementary Figure 1, Supplemental Digital Content 3, http://links.lww.com/BRS/C31).

Cell Suspension Culture Within Pouches

After preculture, the hNP cells/clusters were suspended within a hollow fiber tubing described previously.3 Briefly, the tubing was cut into 35 mm lengths, immersed in pure ethyl alcohol (200 proof), and autoclaved in Dulbecco phosphate-buffered saline. The hNP cells/clusters (1.0×105/25 µL) were injected into the tubing with a pipette, and both ends of the tubing were closed with stainless-steel clips to form a pouch (Figure 1A).

Figure 1.

Figure 1

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Our semipermeable membrane pouch culture module and pressure/perfusion culture system (A–C) and validation of hNP cells (D and E). A, Semipermeable membrane pouches and a pressure-proof chamber. B, Repetitive regimen of 2-day cyclic and 1-day constant HP. C, Macroscopic appearance of pressure/perfusion culture module and system. D, Validation of hNP tissues with sGAG normalized to DNA amount in Pfirrmann grade 2 to 3 and 4. E, Cell viability of hNP cells in our culture system. The cultured hNP cells/clusters were stained with Calcein-AM for live cells and EthD1 for dead cells in grades 2 to 3 and 4 under no HP and HP at 6 days. The number of positive cells was counted in four random high-power fields (×400). The immunopositivity for Calcein-AM was calculated as relative to the sum of Calcein-AM- and EthD1-positive cells. The bar indicates 100 μm. Data are presented with box plots (n = 4 for each). Multiway repeated measures ANOVA with the Bonferroni post hoc test was used. ANOVA indicates analysis of variance; EthD1, ethidium homodimer-1; hNP, human nucleus pulposus; HP, hydrostatic pressure; sGAG, sulfated glycosaminoglycan.

Culture Regimen of Hydrostatic Pressure and Medium Osmolality

The pouches were incubated under one of 2 regimens based on our previous studies3: (1) No HP, in which pouches were placed in culture medium under atmospheric pressure; and (2) HP, in which 2-day cyclic HP (0.2–0.7 MPa, 0.5 Hz) followed by 1-day constant HP (0.3 MPa) with medium replenishment at 100 µL/mL was repeated twice over 6 days (Figure 1B). Both regimens were conducted at 37 °C, 5% CO2, 3% O2, and medium osmolality at 450 mOsm/kg H2O was prepared with 4.6 g/L sodium chloride and confirmed with an osmometer (MICRO OSMETTETM).12,13 In the group without HP, the pouches were suspended within a stainless-steel mesh basket held in a 100 mL medium bottle with a stirrer to maintain sufficient mass transfer through the pouches. With HP, the pouches were placed in a culture chamber followed by installation in a pressure/perfusion culture system (TEP-2, PURPOSE, Shizuoka, Japan; Figure 1C).

Cell Viability Assay

At 6 days, the hNP cells/clusters were harvested from the pouches [n = 4 from grade 2–3: 2 males and 2 females; age, 50.5 ± 10.7 (37⁠–62); n = 4 from grade 4: 1 male and 3 females; age, 53.0±14.3 (37–67)]. The cells were incubated in calcein-AM to identify live cells and ethidium homodimer-1 to identify dead cells (LIVE/DEADTM Viability/Cytotoxicity kit) according to the manufacturer’s instructions, and images were acquired with an inverted microscope (TDM, Nikon) and a camera (D80, Nikon). Both live and dead cells were counted in four random high-power fields (×400) using the ImageJ software (https://imagej.nih.gov/ij/), and viability was expressed as the percentage of live cells.

RNA Isolation and Real-Time Reverse Transcription Polymerase Chain Reaction

At 3 and 6 days, total RNA was extracted using the RNeasy® mini kit, and 0.1 μg RNA was reverse-transcribed with random primers (High-Capacity cDNA Reverse Transcription Kit). Expression of mRNA of aggrecan core protein (ACAN), collagen types I and II (COL1A1, COL2A1), Brachyury, matrix metalloproteinase 13 (MMP13), and tissue inhibitor of metalloproteinases 2 (TIMP2) relative to glyceraldehyde 3-phosphate dehydrogenase as endogenous control14 was evaluated in quadruplicate by real-time reverse transcription polymerase chain reaction (QuantStudio 5 Real-Time PCR Systems) using TaqManTM gene expression master mix and fluorescently labeled specific probes (Supplementary Table 1, Supplemental Digital Content 1, http://links.lww.com/BRS/C29). The relative quantity of mRNA expression was analyzed by the 2−ΔΔCt method compared to the value of the sample in no HP at 3 days as 1.0 using ExpressionSuite Software v1.0.4.15

Immunohistology

At 3 and 6 days, the hNP cells/clusters were fixed in 2% paraformaldehyde/0.1 M cacodylate buffer (pH, 7.4) at 4°C, embedded in paraffin, and cut into 7 μm sections. Dewaxed sections were stained with primary antibodies against brachyury, keratan sulfate (KS), COL2, MMP13, and TRPV4. The sections were then rinsed and incubated with a biotinylated secondary antibody (VECTASTAIN® Elite ABC-HRP kit). The color was developed with 3,3’-diaminobenzidine and nickel (DAB substrate kit). Counterstaining was performed with Harris hematoxylin for KS and COL2, and with Contrast Red for MMP13 and TRPV4. Cells positive for MMP13 and TRPV4 were counted in four random high-power fields (×400) using ImageJ.

Statistical Analyses

Quantitative data were expressed as box plots in the graphs. An unpaired t test was used for the age of patients and immunohistological quantification for brachyury. Multiway repeated measures analysis of variance with Bonferroni post hoc test was used for gene expression analysis and immunohistology for MMP13 and TRPV4. Spearman rank correlation coefficient was calculated and a generalized linear model was used to assess the correlation between age and gene expression/immunopositivity, to compare the age-dependent changes between the culture conditions, and to assess the correlation between MMP13 and TIMP2 expression. P <0.05 was regarded as statistically significant using IBM SPSS Statistics 23.0 (IBM, Armonk, NY).

RESULTS

Correlation Between Pfirrmann Grade and Sulfated Glycosaminoglycan Concentration

sGAG concentration in hNP tissues classified as Pfirrmann grade 2 to 3 was significantly greater than that in grade 4 tissues (P < 0.05, Figure 1D).

Cell Viability of Human Nucleus Pulposus Cells

Cell viability was predominantly >90% in grade 2 to 3 and 4 tissues under no HP and HP at 6 days (Figure 1E).

Characteristics of Human Nucleus Pulposus Cells on Phenotypes, Metabolic Turnover, and Extracellular Matrix Production in Response to Hydrostatic Pressure

We compared cellular characteristics including age, spinal cellular origin, and grades of degeneration. There was no significant difference in the effect of age between grades 2 to 3 and 4 (P = 0.22) on gene expression of metabolic molecules: ACAN, COL2A1, MMP13, and TIMP2, and phenotype: Brachyury at 6 days in response to HP (Figure 2). In addition, hNP cells isolated from the cervical and lumbar spine also showed no significant difference in terms of metabolic molecules and phenotype genes at 6 days in response to HP (Figure 3). In contrast, hNP cells isolated from Pfirrmann grades 2 to 3 and 4 differed in gene expression of these molecules in response to HP (Figure 4A). ACAN was significantly upregulated in hNP isolated from grades 2 to 3 under HP at 6 days compared to at 3 days (P < 0.01) and hNP from grade 4 at 6 days (P < 0.01). COL2A1 exhibited a similar trend, that is, significant upregulation in hNP cells from grade 2 to 3 under HP at 6 days compared to 3 days (P < 0.01). Furthermore, COL2A1 was significantly higher in grades 2 to 3 than in grade 4 under HP at 6 days (P < 0.01). Conversely, COL2A1 was significantly upregulated in grade 4 under no HP at 6 days compared to at 3 days (P < 0.01). In terms of the fibrous matrix, COL1A1 was significantly downregulated under HP both in grades 2 to 3 and 4 compared to no HP at both time points (all, P < 0.01). As we chose a catabolic molecule, MMP13 was significantly upregulated under HP compared to no HP at both 3 and 6 days in grade 4 cells (P < 0.01 and P < 0.05, respectively); however, no changes in MMP13 were seen in grades 2 to 3. TIMP2 was upregulated under both HP and no HP in grades 2 to 3 and 4 at 6 days (P < 0.05, Figure 5A). As TIMP2 counteracts the effects of MMP13, we evaluated the correlation between MMP13 and TIMP2 in hNP cells with each grade under HP at 6 days. MMP13 and TIMP2 showed a positive correlation in grades 2 to 3 (R = 0.45), but a negative correlation in grade 4 (R = −0.30) (Figure 5B). In terms of a phenotyping marker, Brachyury was significantly upregulated without HP in grade 4 at 6 days compared to 3 days (P < 0.01) and in grades 2 to 3 at 6 days (P < 0.05) (Figure 4A).

Figure 2.

Figure 2

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The effects of HP on ECM production, phenotype, and metabolism in age-associated hNP cells. The correlations between age and gene expression of ACAN, COL2A1 and COL1A1, notochordal Brachyury, catabolic MMP13, and anticatabolic TIMP2 under no HP and HP at 6 days are shown (n = 26 for each). Linear regression analysis and Pearson analysis were used. ACAN indicates aggrecan; COL2A1 and COL1A1, collagen type II and I; ECM, extracellular matrix; hNP, human nucleus pulposus; HP, hydrostatic pressure; MMP13, matrix metalloproteinase 13; RQ, relative quantity; TIMP2, tissue inhibitor of metalloproteinases 2.

Figure 3.

Figure 3

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The effects of HP on ECM production, phenotype, and metabolism in the hNP cells from the cervical and lumbar spine. Gene expression of ACAN, COL2A1 and COL1A1, notochordal Brachyury, catabolic MMP13, and anticatabolic TIMP2 in the cervical and lumbar spine under no HP and HP at 3 and 6 days relative to no HP at 3 days are shown (n = 5 for cervical spine in grade 2–3, n = 8 for lumbar spine in grade 2–3, n = 9 for cervical spine in grade 4, and n = 4 for lumbar spine in grade 4). Data are presented with box plots. Multiway repeated-measures ANOVA with the Bonferroni post hoc test was used. ANOVA indicates analysis of variance; COL2A1 and COL1A1, collagen type II and I; ECM, extracellular matrix; hNP, human nucleus pulposus; HP, hydrostatic pressure; MMP13, matrix metalloproteinase 13; RQ, relative quantity; TIMP2, tissue inhibitor of metalloproteinases 2.

Figure 4.

Figure 4

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The effects of HP on ECM production and phenotype in hNP cells with different degrees of degeneration. A, Gene expression of ACAN, COL2A1, and COL1A1, and notochordal Brachyury in grades 2 to 3 and 4 under no HP and HP at 3 and 6 days relative to no HP at 3 days (n = 13 for each). Data are presented with box plots. Multiway repeated-measures ANOVA with the Bonferroni post hoc test was used. B, The accumulation of KS in brown counterstained with hematoxylin. C, The accumulation of COL2 in brown counterstained with hematoxylin. B and C, arrows indicate intense accumulation, each section is 7 μm thick, and the bar indicates 50 μm. ACAN indicates aggrecan; ANOVA, analysis of variance; COL2A1 and COL1A1, collagen type II and I; ECM, extracellular matrix; hNP, human nucleus pulposus; HP, hydrostatic pressure; KS, keratan sulfate; RQ, relative quantity.

Figure 5.

Figure 5

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The effects of HP on metabolic turnover (A–D) and involvement of TRPV4 in hNP cells with different degrees of degeneration (E and F). A, Gene expression of catabolic MMP13 and anticatabolic TIMP2 in grade 2 to 3 and 4 tissues under no HP and HP at 3 and 6 days relative to no HP at 3 days (n = 13 for each). Data are presented with box plots. Multiway repeated-measures ANOVA with the Bonferroni post hoc test was used. B, The correlation between MMP13 and TIMP2 expression in grades 2 to 3 and 4 under HP relative to no HP at 6 days (n = 13 for each). Linear regression analysis and Pearson analysis were used. C, The accumulation of MMP13 in black counterstained with contrast red. Arrows indicate intense accumulation, each section is 7 μm thick, and the bar indicates 50 μm. D, Positive cell percentage of MMP13 (n = 13 for each). Data are presented with box plots. Multiway repeated measures ANOVA with the Bonferroni post hoc test was used. E, The accumulation of TRPV4 in black counterstained with contrast red. Arrows indicate intense accumulation, each section is 7 μm thick, and the bar indicates 50 μm. F, Positive cell percentage of TRPV4 (n = 13 for each). Data are presented with box plots. Multiway repeated-measures ANOVA with the Bonferroni post hoc test was used. ANOVA indicates analysis of variance; hNP, human nucleus pulposus; HP, hydrostatic pressure; MMP13, matrix metalloproteinase 13; TIMP2, tissue inhibitor of metalloproteinases 2; RQ, relative quantity; TRPV4, transient receptor potential vanilloid 4.

Immunohistological Evaluation

The hNP cells from grades 2 to 3 loosely aggregated with and without HP at both days 3 and 6, whereas the cells of grade 4 were relatively densely aggregated (Figure. 4B). Immunohistological staining revealed as much accumulation of KS between cells under HP as no HP on both days. Slightly denser accumulations of COL2 were seen in grades 2 to 3 compared to grade 4 (Figure 4C). Fibrous COL1 matrices were seen in both grades 2 to 3 and 4 (data not shown). Greater accumulation of MMP13 was seen in grade 4 compared to grades 2 to 3, whereas comparable accumulation was found between no HP and HP. The number of MMP13-positive cells was significantly higher in grade 4 than in grades 2 to 3 under no HP and HP at each time point (all, P < 0.01, Figure. 5C, D).

Validation of Hydrostatic Pressure Signal Transduction Through TRPV4

We stained TRPV4 to validate whether HP was transduced through mechanosensitive ionic channels. Intense TRPV4 staining was seen at the surface of hNP cells under HP at 3 days, while it was absent without HP. TRPV4 staining was more intense in grades 2 to 3 than in grade 4. Furthermore, the ratio of TRPV4-positive cells was significantly higher under HP than no HP both in grades 2 to 3 and 4 at 3 days but diminished significantly by 6 days (all, P < 0.01, Figure 5E, F).

DISCUSSION

Clinical Relevance

It is imperative to understand the characteristics of hNP cells in degenerated IVD in order to develop successful regenerative therapeutic strategies. It is also critical to determine the indications of the degenerated IVD to be treated. However, we know little about whether hNP cells retain their viability and anabolic activity in degenerating IVD.16 Thus, we used Pfirrmann grade (designed for the lumbar spine but extended to evaluate the cervical spine) to determine the degree of ECM degeneration and evaluated cellular characteristics in hNP cells isolated from tissues of Pfirrmann grades 2 to 3 and 4.8,17 After we validated the correlation of Pfirrmann grade and sGAG density in the harvested hNP tissue, we characterized hNP cells isolated from the degenerated IVD under physiologically relevant HP in vitro. Even though current discectomy procedures include no guidelines for extracting tissue, tissue can be left within the disc space if the reherniation risk is relatively low. Thus, resident NP cells are expected to regenerate if the tissue expresses anabolic characteristics under daily spinal loading.

Regenerative Capability of Human Nucleus Pulposus Under Changes in Hydrostatic Pressure

The effects of HP on ECM synthesis have been elucidated; HP in the physiological range (0.3–1.0 MPa) increased ECM synthesis in rabbit, dog, and bovine NP cells,18–20 whereas higher HP (>2.5 MPa) induced a catabolic trend, increasing protease expression and decreasing ECM synthesis.21,22 Cadaveric and surgically obtained hNP cells from histologically nondegenerated samples exhibited an anabolic response to the physiological range of HP (0.8–1.7 MPa), whereas cells from degenerated samples either remained unaffected or showed catabolic effects under HP.23 However, these studies had limitations including relatively short culture duration under HP (30 min–2 h) and the discrepancy between histologically assessed degeneration and clinically available degeneration grading.21–23 We resolved such limitations by using our novel pressure/perfusion cell culture system with a physiologically relevant algorithm of repetitive regimens of cyclic followed by constant HP over 6 days.2

Effects of Differences in Age, Cell Origin, and Pfirrmann Grade

HP had no significant effect on the relationship between age and metabolic activity in hNP cells. In addition, differing cell origins between the cervical and lumbar spine had no effect on metabolic activity under HP. These results indicate that resident hNP cells have the potential to regenerate without regard to age or spinal level. In contrast, HP was demonstrated to enhance anabolic turnover in cells derived from grade 2 to 3 tissue relative to grade 4. Additionally, there was a positive correlation between the gene expression of MMP13 and TIMP2 in grades 2 to 3 but a negative correlation in grades 4 under HP at 6 days (Figure 5B). As catabolism in disc matrix represented by MMPs can to some degree be inhibited with TIMPs,24 these correlations revealed that hNP cells of grade 2 to 3 were significantly more capable of making anabolic molecules under HP compared to cells of grade 4. Although COL2A1 in hNP cells from grade 4 was upregulated without HP, fibrotic COL1A1 was also increased. Thus, our results indicate that hNP cells in mild-moderate degenerated IVDD have the capability to synthesize anabolic ECM under physiologically relevant HP and potential for regenerative therapy.

Validation of Mechanotransduction by Hydrostatic Pressure Through TRPV4

TRPV4 staining was more intense on hNP cell membranes from grade 2 to 3 than grade 4 under HP at 3 days and diminished in both grades by 6 days. Additionally, hNP cells accumulated newly synthesized ECM around cells over time. As our culture system increases the pure hydrostatic fluid pressure of the culture medium without changing cell shape, the newly accumulated ECM had a chance to alter membrane characteristics, reducing TRPV4 by 6 days. Other mechanosignal transduction factors (e.g., integrin) may be involved in vivo.10,11,25,26

Overall, our study suggests that regenerative therapy could be developed for mild-moderate IVDD, with the expectation that resident NP cells will express anabolic turnover under daily spinal loading and at OP equivalent to the native NP.

Limitations

First, we extended the Pfirrmann grading originally defined for lumbar IVDD to the cervical IVDD, which was validated in a prior report.27 To further support the Pfirrmann grades, we demonstrated the difference in the sGAG density between Pfirrmann grades 2 to 3 and 4. Given the clinical application, further optimization should be needed with other factors that affect the degree of degeneration.28,29 Second, hNP cells are under compressive stresses including deformation-driven shear stress, although we recapitulated HP. Further studies reproducing deviatoric stress are underway.

CONCLUSIONS

In conclusion, resident hNP cells in mild-moderate degenerated discs classified as Pfirrmann grade 2 to 3 have the potential to promote ECM production.

Key Points

  • We developed an in vitro hNP tissue model under a repetitive regimen of HP, mimicking circadian spinal loading and reproducing homeostasis within the IVD.

  • hNP cells of Pfirrmann degeneration grade 2 and 3 exhibited anabolic capability under HP and will be used for regenerative therapy as a remnant cell source.

  • The activation of transient receptor potential cation channel subfamily V member 4 (TRPV4) is a possible mechanism for disc-cell metabolic alteration under HP.

Supplementary Material

SUPPLEMENTARY MATERIAL
brs-48-728-s001.docx (37.9KB, docx)
brs-48-728-s002.docx (18.9KB, docx)
brs-48-728-s003.tiff (8.7MB, tiff)

Footnotes

Y.T.: designed the main concept of the experiments, performed all qPCR procedures and histology, analyzed statistical data, and wrote the manuscript. Y.K.: performed biochemical evaluation of discarded samples and contributed figure and table preparation. J.D.K. and S.M.: discussed all data translation and contributed to preparing the manuscript for submission.

The authors report no conflicts of interest.

Supplemental Digital Content is available for this article. Direct URL citations are provided in the HTML and PDF versions of this article on the journal’s website, www.spinejournal.com.

Contributor Information

Yoshiki Takeoka, Email: yoshiki_tkk@hotmail.com.

Yutaro Kanda, Email: ykanda1@bwh.harvard.edu.

James D. Kang, Email: jdkang@bwh.harvard.edu.

Shuichi Mizuno, Email: SMIZUNO@RICS.BWH.HARVARD.EDU.

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