Hypoxia promotes nucleus pulposus phenotype in 3D scaffolds in vitro and in vivo

Laboratory investigation

Full access

Object

The role of oxygen in disc metabolism remains a matter of debate. Whether the effect of hypoxic priming on the nucleus pulposus phenotype can be maintained in vivo is not clear. The goal of the present study was to test the hypothesis that priming in a low oxygen tension in vitro could promote a nucleus pulposus phenotype in vivo.

Methods

Bovine nucleus pulposus cells were seeded in 3D scaffolds and subjected to varying oxygen tensions (2% and 20%) for 3 weeks. The constructs were then implanted subcutaneously for 8 weeks. Changes in the extracellular matrix were evaluated using quantitative real-time reverse transcriptase polymerase chain reaction, glycosaminoglycan (GAG) assay, DNA assay, collagen quantification, and histological and immunohistological analyses.

Results

Hypoxia resulted in greater production of sulfated glycosaminoglycan and higher levels of gene expression for collagen Type II, aggrecan, and SOX-9. Furthermore, after hypoxic priming, the subcutaneously implanted constructs maintained the nucleus pulposus phenotype, which was indicated by a significantly higher amount of glycosaminoglycan and collagen Type II.

Conclusions

Hypoxia enhanced the nucleus pulposus phenotype under experimental conditions both in vitro and in vivo. When used in combination with appropriate scaffold material, nucleus pulposus cells could be regenerated for tissue-engineering applications.

Abbreviations used in this paper:ECM = extracellular matrix; GAG = glycosaminoglycan; PBS = phosphate-buffered saline.

Abstract

Object

The role of oxygen in disc metabolism remains a matter of debate. Whether the effect of hypoxic priming on the nucleus pulposus phenotype can be maintained in vivo is not clear. The goal of the present study was to test the hypothesis that priming in a low oxygen tension in vitro could promote a nucleus pulposus phenotype in vivo.

Methods

Bovine nucleus pulposus cells were seeded in 3D scaffolds and subjected to varying oxygen tensions (2% and 20%) for 3 weeks. The constructs were then implanted subcutaneously for 8 weeks. Changes in the extracellular matrix were evaluated using quantitative real-time reverse transcriptase polymerase chain reaction, glycosaminoglycan (GAG) assay, DNA assay, collagen quantification, and histological and immunohistological analyses.

Results

Hypoxia resulted in greater production of sulfated glycosaminoglycan and higher levels of gene expression for collagen Type II, aggrecan, and SOX-9. Furthermore, after hypoxic priming, the subcutaneously implanted constructs maintained the nucleus pulposus phenotype, which was indicated by a significantly higher amount of glycosaminoglycan and collagen Type II.

Conclusions

Hypoxia enhanced the nucleus pulposus phenotype under experimental conditions both in vitro and in vivo. When used in combination with appropriate scaffold material, nucleus pulposus cells could be regenerated for tissue-engineering applications.

As many as 80% of adults experience low-back pain at some point in their lives and 5% develop chronic spinal diseases.1,14 Disc degeneration in the lumbar spine is considered as one of the key factors responsible for low-back pain.13 A variety of procedures have been developed to treat ailments related to disc failure. These procedures often involve removal of degenerative tissue or surgical stabilization, which alleviate symptoms in the short term but fail to address the underlying problem. Tissue-engineered intervertebral disc grafts have the potential to serve as an alternative source of grafts.

Strategies for engineering the intervertebral disc are also complicated by the complex fibrocartilaginous nature of the tissue. The intervertebral disc is typically composed of two distinct anatomical regions: the anulus fibrosus and the nucleus pulposus. For tissue-engineering application of the disc, either autologous or allogenic nucleus pulposus cells can be expanded in tissue culture to supply the cellular building blocks for reconstruction and improvement of the disc.7,11,16 However, like chondrocytes, nucleus pulposus cells can easily lose their phenotype characteristics in monolayer growth.8 In addition, an inappropriate in vivo environment can also alter the ultimate fate of implanted cells.12 Therefore, maintaining the nucleus pulposus phenotype remains one of the key issues in constructing a functional nucleus pulposus graft.

There are steep gradients in oxygen concentrations across the avascular disc, with pO2 falling to as low as 1% in the center of a large disc.17 A low oxygen tension environment plays a vital role in maintaining intervertebral disc physiological function, including matrix synthesis and even cell metabolism. Studies have shown that a physiological oxygen of 1% appears to promote the best nucleus pulposus phenotype for bovine intervertebral disc cells in alginate.17 In addition, Pei et al. have reported that low oxygen tension (5%) enhanced the extracellular matrix (ECM) synthesis of porcine nucleus pulposus cells in a pellet culture system.20 While culture in pellets or 3D gels such as alginate allows nucleus pulposus cells to retain their phenotype, their lack of structural integrity makes them unsuitable as a tissue-engineering scaffold when not combined with a more rigid structure. Furthermore, whether the increase in nucleus pulposus phenotype resulting from hypoxic priming in vitro can still be maintained in vivo has also not been tested.

The physiological properties of the intervertebral disc are linked to the structure of its ECM. The ECM in the healthy disc is a web of natural nanoscale fibers. As an artificial ECM, a scaffolding material often benefits from mimicking advantageous features of the natural ECM. Previously a 3D scaffold with highly interconnected macropores, a nanofibrous matrix with a fiber diameter on the scale of 100 nm (similar to that of natural collagen fibers), and a high surface-to-volume ratio was developed.24,25 These physical characteristics are desired for mimicking the native nucleus pulposus microenvironment and constructing the artificial intervertebral disc tissue. We formed a hypothesis that, when tissue is cultured on a nanofibrous scaffold, priming in a low–oxygen tension in vitro environment could promote a nucleus pulposus phenotype in vivo. To test this hypothesis, bovine nucleus pulposus cells were seeded on 3D nanofibrous scaffolds and cultured in a hypoxic environment, and the ECM production was tested both in vitro and in vivo.

Methods

Fabrication of 3D Nanofibrous Scaffolds

The nanofibrous scaffolds were prepared using a phase separation process as previously described.25 The scaffolds used in the current study have a disclike shape with a thickness of 1.5 mm, diameter of 2.5 mm, and pore size of 250–420 μm. The average fiber diameter of the nanofibrous scaffolds was between 100 and 200 nm.

Nucleus Pulposus Cell Isolation, Culture, and Seeding

Nucleus pulposus cells were isolated from the coccygeal intervertebral discs of an adult bovine tail. Caudal disc tissue was excised from the C2–4 levels in a sterile environment. Primary cultures were established as previously described.2 Briefly, the nucleus pulposus was separated through gross visual inspection. To exclude any contamination between the anulus fibrosus and nucleus pulposus region, only the innermost part of the disc was harvested as nucleus pulposus tissue. The tissue was cut into small pieces, and cells were enzymatically isolated using Type II collagenase (0.5 mg/ml; Sigma-Aldrich) for 16–18 hours under constant rotation at 37°C. After enzymatic isolation, cell suspensions were filtered through a 70-μm cell strainer and cultured in 75-cm2 flasks in Dulbecco's modified Eagle medium (Gibco) containing 10% fetal bovine serum (Gibco) and antibiotics (penicillin G, 100 U/ml; streptomycin, 0.1 mg/ml) at 37°C under 5% CO2. Cells isolated from different regions in the intervertebral disc were cultured separately for 2 passages.

To prepare scaffolds for cell seeding, 3D nanofibrous poly-l-lactide scaffolds were prewetted by soaking them in 70% ethanol for 30 minutes and washed 3 times with phosphate-buffered saline (PBS) for 30 minutes each and twice in complete medium for 2 hours each on an orbital shaker at 75 rpm. The media remaining in the scaffolds were carefully aspirated. Fifteen microliters of cell suspension (3 × 107 cells/ml) was seeded on each scaffold. After 2 hours of initial seeding, the cell-seeded scaffolds were transferred to 6-well plates with 5 ml of medium per well in a hypoxia chamber (Stemcell Technology) at 2% oxygen or at normal 20% oxygen. The medium was changed every 2 days.

Subcutaneous Implantation

The animal procedures were performed according to the protocol approved by the Sichuan University Committee of Use and Care of Laboratory Animals.

Nucleus pulposus cell–scaffold constructs were cultured under hypoxic or normoxic condition for 3 weeks before implantation. For implantation surgery, 6- to 8-week-old male nude mice were used. Surgery was performed while the mice received general inhalational anesthesia with isoflurane. To create 4 subcutaneous pockets per mouse, 2 midsagittal incisions were made on the dorsa, and 1 subcutaneous pocket was created on each side of each incision using blunt dissection. One scaffoldcell construct with or without hypoxia induction was implanted subcutaneously into each pocket at random. After placement of implants, the incisions were closed with staples. After 8 weeks, the mice were killed and the implants were harvested.

RNA Analysis

Constructs were harvested after 1 and 3 weeks of in vitro culture and homogenized with a tissue tearor. Total RNA was isolated using an RNeasy Mini Kit (Qiagen), and DNA was digested by an RNase-free DNase set (Qiagen) according to the manufacturer's protocol. The isolated RNA was then reverse-transcribed with TaqMan reverse transcription reagents (Applied Biosystems) to synthesize cDNA. Finally, real-time PCR was performed using TaqMan Universal PCR Master Mix (Applied Biosystems) and specific primers for collagen Type II (predesigned, assay ID Bt03251833_g1), aggrecan (predesigned, assay ID Bt03212190_m1), SOX-9 (primer sequences 5′-ACCATGTCCGAGGACTCTGC-3′ and 5′-AACTTGTCCTCCTCGCTCTCCTTCTT-3′), and collagen Type I (predesigned, assay ID Bt03225353_g1). All RNA samples were adjusted to yield equal amplification of GAPDH (glyceraldehyde 3-phosphate dehydrogenase predesigned; assay ID Bt03210912_g1) as an internal standard.

Histological and Immunohistological Analyses

To create histological slides, constructs were washed in PBS, fixed with 3.7% formaldehyde in PBS overnight, dehydrated through a graded series of ethanol, embedded in paraffin, and sectioned at a thickness of 5 μm. For histological observation, sections were deparaffinized, rehydrated, stained with Safranin O, and counterstained with Fast Green. For immunohistochemical staining of Type II collagen, slides were deparaffinized and pretreated with papain solution for 15 minutes. They were then incubated with Type II collagen antibody at 1:100 dilutions in 1% bovine serum albumin for 1 hour and detected by a cell and tissue staining kit (R&D Systems, Inc.) according to the manual. Lastly, immunohistochemically stained slides were counterstained with hematoxylin.

DNA and Glycosaminoglycan Assay

Constructs from the in vitro and in vivo study were harvested, washed with PBS, and digested with 200 μl papain solution (280 mg/mL in 50 mM sodium phosphate, pH 6.5, containing 5 mM N-acetyl cysteine and 50 mM EDTA) for 24 hours at 65°C. The DNA content was quantified using a fluorescence assay with Hoechst 33258 dye according to the manual (Sigma). Glycosaminoglycan (GAG) content was measured by reaction with dimethylmethylene blue. Optical density was measured at 525 nm and GAG content of each construct was calculated using shark chondroitin 4-sulfate as the standard.

Collagen Quantification

Type II collagen of in vivo samples was quantified using an indirect enzyme-linked immunosorbent assay. Briefly, samples were isolated and stored in 0.05 N acetic acid (pH 2.8) at −20°C prior to processing. All samples were treated with 500 μl of 3 M guanidine HCl and 0.05 M Tris-HCl (GuHCl) at pH 7.5 for 16 hours at 4°C with rotation. The GuHCl supernatant was removed, and the remaining samples were washed twice with sterile water and treated with 525 μl of 2 mg/ml pepsin in 0.05 N acetic acid for 48 hours rotating at 4°C. After 48 hours, pepsin was neutralized by adding 100 μl of 10× Tris-buffered saline and 25 μl of 1 N NaOH. Monoclonal antibodies to Type II collagen (1:100 dilution of supernatant) (II-II6B3; Developmental Studies Hybridoma Bank) were used. A peroxidase-based detection system using biotinylated secondary antibody (anti–rabbit IgG [H + L], Vector Laboratories) and a streptavidin-horseradish peroxidase enzyme conjugate (R&D Systems) was used. Total reacted substrate was spectrophotometrically analyzed at 450 nm using a Synergy-HT Microplate Reader (BioTek), and total protein was determined from standard curves of bovine Type II collagen (isolated from bovine nasal cartilage).

Statistical Analysis

Version 14.0 SPSS was used to perform the statistical analysis. The cell-scaffold constructs were pooled per group and used for gene analysis and GAG assay. To ensure reproducibility, 3 independent experiments were conducted using cells isolated from 3 different bovines. Data are presented as mean ± S.D. To test the significance of observed differences between the study groups, the Student t-test was applied. A value of p < 0.05 was considered to be statistically significant.

Results

To first assess the effect of hypoxia in vitro, nucleus pulposus cells were seeded on 3D scaffolds and cultured under hypoxic or normoxic condition for 3 weeks. Gene expression was quantified after 1 week and 3 weeks and revealed that expression of collagen Type II, aggrecan, and SOX-9 all increased with time under both hypoxic and normoxic conditions. After 3 weeks of in vitro culture, hypoxia significantly upregulated the gene expression levels of collagen Type II, aggrecan, and SOX-9 (Fig. 1A –C). However, the gene expression of Type I collagen was not affected by the oxygen tension (Fig. 1D). Consistent with the gene expression results, histological analysis showed more abundant GAG deposition in the scaffold (as shown by Safranin O staining), in constructs cultured for 3 weeks under hypoxia than normoxia (Fig. 2).

Fig. 1.
Fig. 1.

Results of real-time PCR analysis of gene expression for constructs in vitro: collagen Type II (A), aggrecan (B), Sox-9 (C), and collagen Type I (D). Constructs cultured for 1 week or 3 weeks under normoxic (20% oxygen) or hypoxic (2% oxygen) conditions. Statistical differences between results from hypoxic and normoxic cultures at each time point are shown. Asterisk denotes significance.

Fig. 2.
Fig. 2.

Safranin O–stained photomicrographs of nucleus pulposus cell–scaffold constructs cultured 3 weeks in vitro. More abundant GAG amounts were detected in the constructs under the hypoxic (A) than the normoxic (B) condition.

To investigate the long-term stability of engineered nucleus pulposus–like tissue, the scaffold-cell constructs were implanted into nude mice subcutaneously after in vitro culture under hypoxic or normoxic conditions for 3 weeks. After 8 weeks of implantation, the constructs were collected and subjected to histological observation. Constructs from the hypoxic group maintained their nucleus pulposus–like phenotype, indicated by strong positive staining of GAGs with Safranin O (Fig. 3A) and of Type II collagen (Fig. 3C). In contrast, constructs from the normoxic group succumbed to fibrous tissue invasion, indicated by minimal Safranin O (Fig. 3B) and Type II collagen (Fig. 3D) staining. To quantify the histological results, positively or negatively stained cells were counted in at least 4 samples per group to calculate the percentage of positive cells. The percentages of both Safranin O–positive cells and Type II collagen–positive cells from the hypoxic group were significantly higher than that from the normoxic group (Safranin O 94.3 ± 4.6% vs 21.7 ± 5.9%, respectively; Type II collagen 96.2 ± 5.6% vs 13.7 ± 4.8%, respectively).

Fig. 3.
Fig. 3.

Photomicrographs of constructs cultured in vivo for 8 weeks: Safranin O (Saf O) staining (A and B) and immunohistochemical staining for collagen Type II (Col II) (C and D). More abundant GAG and collagen Type II amounts were detected in constructs with hypoxic priming in vitro (A and C) than without hypoxic priming (B and D).

Furthermore, to quantify the synthesis of ECM in tissue-engineered nucleus pulposus, GAG and DNA content was measured after 3 weeks in vitro and after 8 weeks in vivo. The content of Type II collagen was also quantified for 8-week in vivo samples. A significantly higher GAG content was found in samples in the hypoxic group than in the normoxic group, both in vitro and in vivo (Fig. 4A). Because there was no significant difference in DNA content between the two different oxygen tensions either in vitro or in vivo, the difference in GAG content cannot be attributed to a difference in cell number (Fig. 4B). Consistent with the histological result, the Type II collagen content from the hypoxic group was also significantly higher than that of the normoxic group (Fig. 4C).

Fig. 4.
Fig. 4.

GAG and DNA content was quantified for constructs cultured 3 weeks in vitro followed by 8 weeks in vivo and Type II collagen content was also quantified for constructs cultured in vivo. Hypoxia significantly increased the accumulation of GAGs both in vitro and in vivo (A). In contrast, there was no significant difference in DNA content of nucleus pulposus cells under different oxygen tensions (B). Type II collagen content from the hypoxic group was significantly higher than that in the normoxic group (C). Asterisk denotes statistical significance.

Discussion

The goal of the present study was to investigate the effect of hypoxia on regeneration of nucleus pulposus tissue. Our results demonstrated that hypoxia could promote the nucleus pulposus phenotype in vitro, which was supported by gene expression analysis and histological results. Furthermore, exposure of nucleus pulposus cells to a low oxygen level in vitro increased ECM production and promoted maintenance of the nucleus pulposus phenotype in vivo.

The role of oxygen in disc metabolism remains a matter of debate. Ishihara et al. reported that oxygen had little effect on the GAG synthesis of bovine nucleus pulposus cells in 10%–21% oxygen, but the GAG synthesis increased significantly as the concentration fell from 10% to 5%.10 Another study showed that oxygen is necessary for GAG synthesis in nucleus pulposus cells. At 0% oxygen, nucleus pulposus cells produced very little GAG.9 The present data indicated that hypoxia is helpful for regenerating nucleus pulposus cells and maintaining their phenotype, which is consistent with recent reports.17,20 One possible reason to explain the variations among different studies might be the species from which cells are isolated and the environment in which cell metabolism takes place.

Although researchers have not yet derived a list of specific gene expression profiles that fully characterize disc cells, nucleus pulposus cells are normally indicated as “chondrocyte-like,” with characteristic markers of collagen Type II, aggrecan, and SOX-9.21,22 Aggrecan and Type II collagen are characteristic markers of chondrocytes and the primary macromolecules that make up the ECM of the nucleus pulposus, which plays a critical role in maintaining the normal physiological function of the intervertebral disc. SOX-9 has been described as having a role as a master regulator of the nucleus pulposus phenotype. SOX-9 is used as a marker indicating the success of various biological therapies for intervertebral disc degeneration. An inappropriate in vivo environment, it has been reported, can alter the ultimate fate of implanted chondrocytes.12,23 This finding raises a concern over the feasibility of using nucleus pulposus cells to repair degenerated discs, where nucleus pulposus cells might tend to form fibrous tissue after implantation. Therefore one of the key issues of constructing a functional nucleus pulposus with nucleus pulposus cells is to maintain their phenotype after implantation. Based on data from our in vitro model, we formed a hypothesis that constructs may be stably committed to the appropriate nucleus pulposus–like phenotype by priming in hypoxic condition. Our results demonstrated that abundant GAG and collagen Type II were detected in in vivo specimens with hypoxic priming. In contrast, the constructs without hypoxic priming tended to form fibrous tissue in vivo. These results indicate that hypoxic priming might serve as an efficient way to achieve a functional and stable nucleus pulposus graft for intervertebral disc regeneration, although the exact underlying mechanism needs to be further investigated.

Various studies have examined ECM production of nucleus pulposus cells associated with hypoxia in vitro.10,17,20 It has been demonstrated that nucleus pulposus cells dedifferentiated during in vitro culture and hypoxia is helpful to regenerate nucleus pulposus cells. However, there have been few reports on the in vivo performance of tissue-engineered nucleus pulposus grafts after hypoxic priming in vitro, and the question of whether the graft can maintain its phenotype in vivo has not been tested. Because the ultimate differentiation fate in vivo depends mainly on the niche where cells are implanted, an important concern is raised as to whether the regenerated nucleus pulposus graft loses its phenotype after implantation. Our study demonstrates that hypoxic priming could provide a stable nucleus pulposus phenotype after subcutaneous implantation, which indicates a potential strategy for future clinical application.

Various hydrogels have been used to provide a 3D environment for nucleus pulposus cells.4,5,15 However, hydrogel scaffolds lack structural integrity and are probably unsuitable as a tissue-engineering application when not combined with a more rigid structure. The scaffolds used in our present study were fabricated by using a phase-separation technique with a nanofibrous matrix and highly interconnected macropores. The nanofibrous matrix was developed to mimic the nanoscale fibrous morphology of collagen, a major component of ECM that is known to affect cell behavior. In our previous studies, the nanofibrous architecture was shown to provide physical cues to enhance mesenchymal stem cell differentiation into a nucleus pulposus phenotype.3 The current study proved that the low oxygen tension and the 3D nanofibrous scaffolds could mimic the physiological environment existing in the intervertebral disc to synergistically regenerate nucleus pulposus tissue.

In our present study, the nucleus pulposus cells were isolated from bovine spines. Theoretically the human lumbar intervertebral disc is the most ideal tissue for cell harvesting. However, healthy human discs suitable for in vitro synthesis experiments are difficult to obtain. Bovine intervertebral disc material has been used in previous studies as an alternative source of disc tissue. Bovine coccygeal discs are an attractive alternative because they are readily obtainable and are a cheap source of disc material. Indeed, previous studies have used this model to study disc composition, metabolism function, regulation, and biomechanics.6,18,19 Overall, the structure of the bovine and young healthy human intervertebral discs is similar. Although bovine tails sustain a lesser mechanical load than human lumbar discs, the swelling pressure of bovine coccygeal intervertebral discs has been shown to be of the same magnitude as human discs in a person resting in a prone position. Biochemically, similar in vitro proteoglycan synthesis rates and matrix synthesis responses to hydrostatic pressure have also been found in human and bovine coccygeal intervertebral discs. Both bovine and human discs also exhibit similar types and distributions of aggrecan and collagen. Therefore, bovine coccygeal intervertebral discs have been suggested as a suitable alternative to human lumbar discs for in vitro studies because of similar general properties. In the present study, the constructs were implanted in the subcutaneous pockets for 8 weeks. There are two considerations to support the adequacy of 8-week observation. The first relates to the life span of the species, in that 1 month in a rat would be equivalent to approximately 30 months in humans if one considers that the life span of a rat is about 3 years compared with approximately 90 years in humans. The other consideration relates to the biological response observed: if reactions to materials are favorable at 8 weeks, it would be unlikely that a subsequent adverse reaction would develop barring physiochemical deterioration of the material. However, an ultra–long-term observation period is still necessary to ensure the implant function in our future study.

Although the tissue generated in this study showed promise for intervertebral disc regeneration, subcutaneous implantation may not provide the same environment as that in the disc tissue. Studies on regenerative disc replacement in animal models are necessary to further test our hypothesis. The scaffold used in this study was made of poly-l-lactide, which has been widely used for biomedical applications with minimal immunogenicity. The side effect of this scaffold after long-term transplantation will be tested in our future study.

Conclusions

The experimental data demonstrate that hypoxia can enhance the nucleus pulposus phenotype under our experimental conditions both in vitro and in vivo. When used in concert with the appropriate scaffold material, hypoxia induction of nucleus pulposus cells can be applied to successfully regenerate nucleus pulposus tissue for intervertebral disc repair.

Disclosure

The authors received financial support from the National Natural Science Foundation of China (grant no. 81201430).

Author contributions to the study and manuscript preparation include the following. Conception and design: Liu, Feng. Analysis and interpretation of data: Feng, Li. Drafting the article: Feng, Li. Critically revising the article: all authors. Reviewed submitted version of manuscript: all authors. Approved the final version of the manuscript on behalf of all authors: Liu. Statistical analysis: Feng. Administrative/technical/material support: Song, Pei, Ma.

This article contains some figures that are displayed in color online but in black-and-white in the print edition.

References

  • 1

    Adogwa OCarr RKKudyba KKarikari IBagley CAGokaslan ZL: Revision lumbar surgery in elderly patients with symptomatic pseudarthrosis, adjacent-segment disease, or same-level recurrent stenosis. Part 1. Two-year outcomes and clinical efficacy. Clinical article. J Neurosurg Spine 18:1391462013

  • 2

    Chou AIReza ATNicoll SB: Distinct intervertebral disc cell populations adopt similar phenotypes in three-dimensional culture. Tissue Eng Part A 14:207920872008

  • 3

    Feng GJin XHu JMa HGupte MJLiu H: Effects of hypoxias and scaffold architecture on rabbit mesenchymal stem cell differentiation towards a nucleus pulposus-like phenotype. Biomaterials 32:818281892011

  • 4

    Francisco ATMancino RJBowles RDBrunger JMTainter DMChen YT: Injectable laminin-functionalized hydrogel for nucleus pulposus regeneration. Biomaterials 34:738173882013

  • 5

    Frith JECameron ARMenzies DJGhosh PWhitehead DLGronthos S: An injectable hydrogel incorporating mesenchymal precursor cells and pentosan polysulphate for intervertebral disc regeneration. Biomaterials 34:943094402013

  • 6

    Gahunia HKVieth RPritzker K: Novel fluorescent compound (DDP) in calf, rabbit, and human articular cartilage and synovial fluid. J Rheumatol 29:1541602002

  • 7

    Gruber HEIngram JAHanley EN Jr: Immunolocalization of thrombospondin in the human and sand rat intervertebral disc. Spine (Phila Pa 1976) 31:255625612006

  • 8

    Horner HARoberts SBielby RCMenage JEvans HUrban JP: Cells from different regions of the intervertebral disc: effect of culture system on matrix expression and cell phenotype. Spine (Phila Pa 1976) 27:101810282002

  • 9

    Horner HAUrban JP: 2001 Volvo Award Winner in Basic Science Studies. Effect of nutrient supply on the viability of cells from the nucleus pulposus of the intervertebral disc. Spine (Phila Pa 1976) 26:254325492001

  • 10

    Ishihara HUrban JP: Effects of low oxygen concentrations and metabolic inhibitors on proteoglycan and protein synthesis rates in the intervertebral disc. J Orthop Res 17:8298351999

  • 11

    Lee KIMoon SHKim HKwon UHKim HJPark SN: Tissue engineering of the intervertebral disc with cultured nucleus pulposus cells using atelocollagen scaffold and growth factors. Spine (Phila Pa 1976) 37:4524582012

  • 12

    Liu KZhou GDLiu WZhang WJCui LLiu X: The dependence of in vivo stable ectopic chondrogenesis by human mesenchymal stem cells on chondrogenic differentiation in vitro. Biomaterials 29:218321922008

  • 13

    Luoma KRiihimäki HLuukkonen RRaininko RViikari-Juntura ELamminen A: Low back pain in relation to lumbar disc degeneration. Spine (Phila Pa 1976) 25:4874922000

  • 14

    Macfarlane GJThomas ECroft PRPapageorgiou ACJayson MISilman AJ: Predictors of early improvement in low back pain amongst consulters to general practice: the influence of pre-morbid and episode-related factors. Pain 80:1131191999

  • 15

    Malhotra NRHan WMBeckstein JCloyd JChen WElliott DM: An injectable nucleus pulposus implant restores compressive range of motion in the ovine disc. Spine (Phila Pa 1976) 37:E1099E11052012

  • 16

    Moss ILGordon LWoodhouse KAWhyne CMYee AJ: A novel thiol-modified hyaluronan and elastin-like polypetide composite material for tissue engineering of the nucleus pulposus of the intervertebral disc. Spine (Phila Pa 1976) 36:102210292011

  • 17

    Mwale FCiobanu IGiannitsios DRoughley PSteffen TAntoniou J: Effect of oxygen levels on proteoglycan synthesis by intervertebral disc cells. Spine (Phila Pa 1976) 36:E131E1382011

  • 18

    Nerlich AGSchleicher EDBoos N: 1997 Volvo Award Winner in Basic Science Studies. Immunohistologic markers for age-related changes of human lumbar intervertebral discs. Spine (Phila Pa 1976) 22:278127951997

  • 19

    Ohshima HUrban JPBergel DH: Effect of static load on matrix synthesis rates in the intervertebral disc measured in vitro by a new perfusion technique. J Orthop Res 13:22291995

  • 20

    Pei MShoukry MLi JDaffner SDFrance JCEmery SE: Modulation of in vitro microenvironment facilitates synovium-derived stem cell-based nucleus pulposus tissue regeneration. Spine (Phila Pa 1976) 37:153815472012

  • 21

    Richardson SMCurran JMChen RVaughan-Thomas AHunt JAFreemont AJ: The differentiation of bone marrow mesenchymal stem cells into chondrocyte-like cells on poly-L-lactic acid (PLLA) scaffolds. Biomaterials 27:406940782006

  • 22

    Risbud MVGuttapalli AStokes DGHawkins DDanielson KGSchaer TP: Nucleus pulposus cells express HIF-1 alpha under normoxic culture conditions: a metabolic adaptation to the intervertebral disc microenvironment. J Cell Biochem 98:1521592006

  • 23

    Rutges JPDuit RAKummer JAOner FCvan Rijen MHVerbout AJ: Hypertrophic differentiation and calcification during intervertebral disc degeneration. Osteoarthritis Cartilage 18:148714952010

  • 24

    Wei GMa PX: Macroporous and nanofibrous polymer scaffolds and polymer/bone-like apatite composite scaffolds generated by sugar spheres. J Biomed Materials Res A 78:3063152006

  • 25

    Wei GMa PX: Structure and properties of nano-hydroxyapatite/polymer composite scaffolds for bone tissue engineering. Biomaterials 25:474947572004

If the inline PDF is not rendering correctly, you can download the PDF file here.

Article Information

Drs. Feng and Li contributed equally to this work.

Address correspondence to: Hao Liu, M.D., Ph.D., Department of Orthopedics, West China Hospital, Sichuan University, Chengdu, Sichuan, China. email: gjfenghx@163.com.

Please include this information when citing this paper: published online May 23, 2014; DOI: 10.3171/2014.4.SPINE13870.

© AANS, except where prohibited by US copyright law.

Headings

Figures

  • View in gallery

    Results of real-time PCR analysis of gene expression for constructs in vitro: collagen Type II (A), aggrecan (B), Sox-9 (C), and collagen Type I (D). Constructs cultured for 1 week or 3 weeks under normoxic (20% oxygen) or hypoxic (2% oxygen) conditions. Statistical differences between results from hypoxic and normoxic cultures at each time point are shown. Asterisk denotes significance.

  • View in gallery

    Safranin O–stained photomicrographs of nucleus pulposus cell–scaffold constructs cultured 3 weeks in vitro. More abundant GAG amounts were detected in the constructs under the hypoxic (A) than the normoxic (B) condition.

  • View in gallery

    Photomicrographs of constructs cultured in vivo for 8 weeks: Safranin O (Saf O) staining (A and B) and immunohistochemical staining for collagen Type II (Col II) (C and D). More abundant GAG and collagen Type II amounts were detected in constructs with hypoxic priming in vitro (A and C) than without hypoxic priming (B and D).

  • View in gallery

    GAG and DNA content was quantified for constructs cultured 3 weeks in vitro followed by 8 weeks in vivo and Type II collagen content was also quantified for constructs cultured in vivo. Hypoxia significantly increased the accumulation of GAGs both in vitro and in vivo (A). In contrast, there was no significant difference in DNA content of nucleus pulposus cells under different oxygen tensions (B). Type II collagen content from the hypoxic group was significantly higher than that in the normoxic group (C). Asterisk denotes statistical significance.

References

1

Adogwa OCarr RKKudyba KKarikari IBagley CAGokaslan ZL: Revision lumbar surgery in elderly patients with symptomatic pseudarthrosis, adjacent-segment disease, or same-level recurrent stenosis. Part 1. Two-year outcomes and clinical efficacy. Clinical article. J Neurosurg Spine 18:1391462013

2

Chou AIReza ATNicoll SB: Distinct intervertebral disc cell populations adopt similar phenotypes in three-dimensional culture. Tissue Eng Part A 14:207920872008

3

Feng GJin XHu JMa HGupte MJLiu H: Effects of hypoxias and scaffold architecture on rabbit mesenchymal stem cell differentiation towards a nucleus pulposus-like phenotype. Biomaterials 32:818281892011

4

Francisco ATMancino RJBowles RDBrunger JMTainter DMChen YT: Injectable laminin-functionalized hydrogel for nucleus pulposus regeneration. Biomaterials 34:738173882013

5

Frith JECameron ARMenzies DJGhosh PWhitehead DLGronthos S: An injectable hydrogel incorporating mesenchymal precursor cells and pentosan polysulphate for intervertebral disc regeneration. Biomaterials 34:943094402013

6

Gahunia HKVieth RPritzker K: Novel fluorescent compound (DDP) in calf, rabbit, and human articular cartilage and synovial fluid. J Rheumatol 29:1541602002

7

Gruber HEIngram JAHanley EN Jr: Immunolocalization of thrombospondin in the human and sand rat intervertebral disc. Spine (Phila Pa 1976) 31:255625612006

8

Horner HARoberts SBielby RCMenage JEvans HUrban JP: Cells from different regions of the intervertebral disc: effect of culture system on matrix expression and cell phenotype. Spine (Phila Pa 1976) 27:101810282002

9

Horner HAUrban JP: 2001 Volvo Award Winner in Basic Science Studies. Effect of nutrient supply on the viability of cells from the nucleus pulposus of the intervertebral disc. Spine (Phila Pa 1976) 26:254325492001

10

Ishihara HUrban JP: Effects of low oxygen concentrations and metabolic inhibitors on proteoglycan and protein synthesis rates in the intervertebral disc. J Orthop Res 17:8298351999

11

Lee KIMoon SHKim HKwon UHKim HJPark SN: Tissue engineering of the intervertebral disc with cultured nucleus pulposus cells using atelocollagen scaffold and growth factors. Spine (Phila Pa 1976) 37:4524582012

12

Liu KZhou GDLiu WZhang WJCui LLiu X: The dependence of in vivo stable ectopic chondrogenesis by human mesenchymal stem cells on chondrogenic differentiation in vitro. Biomaterials 29:218321922008

13

Luoma KRiihimäki HLuukkonen RRaininko RViikari-Juntura ELamminen A: Low back pain in relation to lumbar disc degeneration. Spine (Phila Pa 1976) 25:4874922000

14

Macfarlane GJThomas ECroft PRPapageorgiou ACJayson MISilman AJ: Predictors of early improvement in low back pain amongst consulters to general practice: the influence of pre-morbid and episode-related factors. Pain 80:1131191999

15

Malhotra NRHan WMBeckstein JCloyd JChen WElliott DM: An injectable nucleus pulposus implant restores compressive range of motion in the ovine disc. Spine (Phila Pa 1976) 37:E1099E11052012

16

Moss ILGordon LWoodhouse KAWhyne CMYee AJ: A novel thiol-modified hyaluronan and elastin-like polypetide composite material for tissue engineering of the nucleus pulposus of the intervertebral disc. Spine (Phila Pa 1976) 36:102210292011

17

Mwale FCiobanu IGiannitsios DRoughley PSteffen TAntoniou J: Effect of oxygen levels on proteoglycan synthesis by intervertebral disc cells. Spine (Phila Pa 1976) 36:E131E1382011

18

Nerlich AGSchleicher EDBoos N: 1997 Volvo Award Winner in Basic Science Studies. Immunohistologic markers for age-related changes of human lumbar intervertebral discs. Spine (Phila Pa 1976) 22:278127951997

19

Ohshima HUrban JPBergel DH: Effect of static load on matrix synthesis rates in the intervertebral disc measured in vitro by a new perfusion technique. J Orthop Res 13:22291995

20

Pei MShoukry MLi JDaffner SDFrance JCEmery SE: Modulation of in vitro microenvironment facilitates synovium-derived stem cell-based nucleus pulposus tissue regeneration. Spine (Phila Pa 1976) 37:153815472012

21

Richardson SMCurran JMChen RVaughan-Thomas AHunt JAFreemont AJ: The differentiation of bone marrow mesenchymal stem cells into chondrocyte-like cells on poly-L-lactic acid (PLLA) scaffolds. Biomaterials 27:406940782006

22

Risbud MVGuttapalli AStokes DGHawkins DDanielson KGSchaer TP: Nucleus pulposus cells express HIF-1 alpha under normoxic culture conditions: a metabolic adaptation to the intervertebral disc microenvironment. J Cell Biochem 98:1521592006

23

Rutges JPDuit RAKummer JAOner FCvan Rijen MHVerbout AJ: Hypertrophic differentiation and calcification during intervertebral disc degeneration. Osteoarthritis Cartilage 18:148714952010

24

Wei GMa PX: Macroporous and nanofibrous polymer scaffolds and polymer/bone-like apatite composite scaffolds generated by sugar spheres. J Biomed Materials Res A 78:3063152006

25

Wei GMa PX: Structure and properties of nano-hydroxyapatite/polymer composite scaffolds for bone tissue engineering. Biomaterials 25:474947572004

TrendMD

Metrics

Metrics

All Time Past Year Past 30 Days
Abstract Views 0 0 0
Full Text Views 99 99 29
PDF Downloads 105 105 10
EPUB Downloads 0 0 0

PubMed

Google Scholar