Hydrostatic pressure mimicking diurnal spinal movements maintains anabolic turnover in bovine nucleus pulposus cells in vitro

Treatment strategies for progressive intervertebral-disc degeneration often alleviate pain and other symptoms. With the goal of developing strategies to promote the regeneration of the nucleus pulposus (NP), the present study tried to identify the biological effects of hydrostatic (HP) and osmotic pressures on NP cells. The study hypothesis was that a repetitive regimen of cyclic HP followed by constant HP in high-osmolality medium would increase anabolic molecules in NP cells. Bovine NP cells/clusters were enclosed within semi-permeable membrane pouches and incubated under a regimen of cyclic HP for 2 d followed by constant HP for 1 d, repeated 6 times over 18 d. NP cells showed a significantly increased expression of anabolic genes over time: aggrecan, chondroitin sulfate N-acetylgalactosaminyltransferase 1, hyaluronan synthase 2, collagen type 2 (p < 0.05). In addition, the expression of catabolic or degenerative genes (matrix metalloproteinase 13, collagen type 1) and cellular characteristic genes (proliferating cell nucleic antigen, E-cadherin) was suppressed. The amount of sulfated glycosaminoglycan increased significantly at day 18 compared to day 3 (p < 0.01). Immunostaining revealed deposition of extracellular-matrix molecules and localization of other specific molecules corresponding to their genetic expression. An improved understanding of how cells respond to physicochemical stresses will help to better treat the degenerating disc using either cell- or gene-based therapies as well as other potential matrix-enhancing therapies. Efforts to apply these tissue-engineering and regenerative-medicine strategies will need to consider these important physicochemical stresses that may have a major impact on the survivability of such treatments.

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

Journal of Neurosurgery Spine ◽

10.3171/2014.4.spine13870 ◽

2014 ◽

Vol 21 (2) ◽

pp. 303-309 ◽

Cited By ~ 6

Author(s):

Ganjun Feng ◽

Li Li ◽

Ying Hong ◽

Hao Liu ◽

Yueming Song ◽

...

Keyword(s):

Nucleus Pulposus ◽

Collagen Type ◽

Type Ii ◽

Collagen Type Ii ◽

Low Oxygen ◽

Nucleus Pulposus Cells ◽

Experimental Conditions ◽

3D Scaffolds

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.

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Biological Effects of β-Glucans on Osteoclastogenesis

Molecules ◽

10.3390/molecules26071982 ◽

2021 ◽

Vol 26 (7) ◽

pp. 1982

Author(s):

Wataru Ariyoshi ◽

Shiika Hara ◽

Ayaka Koga ◽

Yoshie Nagai-Yoshioka ◽

Ryota Yamasaki

Keyword(s):

Bone Resorption ◽

Bone Marrow Cells ◽

Biological Effects ◽

Osteoclast Differentiation ◽

Treatment Strategies ◽

Alcaligenes Faecalis ◽

Osteoclast Formation ◽

Osteoclast Precursors

Although the anti-tumor and anti-infective properties of β-glucans have been well-discussed, their role in bone metabolism has not been reviewed so far. This review discusses the biological effects of β-glucans on bone metabolisms, especially on bone-resorbing osteoclasts, which are differentiated from hematopoietic precursors. Multiple immunoreceptors that can recognize β-glucans were reported to be expressed in osteoclast precursors. Coordinated co-stimulatory signals mediated by these immunoreceptors are important for the regulation of osteoclastogenesis and bone remodeling. Curdlan from the bacterium Alcaligenes faecalis negatively regulates osteoclast differentiation in vitro by affecting both the osteoclast precursors and osteoclast-supporting cells. We also showed that laminarin, lichenan, and glucan from baker’s yeast, as well as β-1,3-glucan from Euglema gracilisas, inhibit the osteoclast formation in bone marrow cells. Consistent with these findings, systemic and local administration of β-glucan derived from Aureobasidium pullulans and Saccharomyces cerevisiae suppressed bone resorption in vivo. However, zymosan derived from S. cerevisiae stimulated the bone resorption activity and is widely used to induce arthritis in animal models. Additional research concerning the relationship between the molecular structure of β-glucan and its effect on osteoclastic bone resorption will be beneficial for the development of novel treatment strategies for bone-related diseases.

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Evaluation of Secretome Tenogenic Potential from Adipose Stem Cells (ACS) in Hypoxic Condition with Fresh Frozen Tendon Scaffold Using Scleraxis (Scx), Insulin-Like Growth Factor 1 (IGF-1) and Collagen Type 1

Journal of Biomimetics Biomaterials and Biomedical Engineering ◽

10.4028/www.scientific.net/jbbbe.49.111 ◽

2021 ◽

Vol 49 ◽

pp. 111-118

Author(s):

Ramadhan Ananditia Putra ◽

Heri Suroto

Keyword(s):

Cell Culture ◽

Collagen Type ◽

Hypoxic Condition ◽

Adipose Stem Cells ◽

Immunohistochemical Examination ◽

Hypoxic Conditions ◽

Fresh Frozen ◽

Collagen Type 1

Various studies have been conducted to see the scaffold that supports the regeneration of tendon. This study aims to analyze thein vitrosecretome tenogenic potential produced by ASCs culture with fresh frozen tendon scaffold in hypoxic conditions. ELISA tests for Scx and IGF-1 levels in secretome were obtained from ASC culture with fresh frozen tendon scaffold under normoxic (21%) and hypoxia (2%) conditions. The immunohistochemical examination of COL-1 was also carried out on the 2ndand 6thdays of cell culture. The secretion of Scx and IGF-1 was increased in secretome from ASC cultures using a fresh frozen tendon scaffold compared with those which did not (p <0.05). In the normoxia condition, Scx and IGF-1 in secretome with fresh frozen tendons had better results than hypoxic conditions (p <0.05). The highest Scx levels were obtained in culture on the 6thday (p <0.05), while the highest IGF-1 levels were obtained in the culture on the 2ndday (p <0.05). There was an increase in the secretion of Scx and IGF-1 from ASC cultures with fresh frozen tendon scaffold under the hypoxic condition of 2%.

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Dynamic Compression Promotes the Matrix Synthesis of Nucleus Pulposus Cells Through Up-Regulating N-CDH Expression in a Perfusion Bioreactor Culture

Cellular Physiology and Biochemistry ◽

10.1159/000488616 ◽

2018 ◽

Vol 46 (2) ◽

pp. 482-491 ◽

Cited By ~ 4

Author(s):

Yichun Xu ◽

Hui Yao ◽

Pei Li ◽

Wenbin Xu ◽

Junbin Zhang ◽

...

Keyword(s):

Nucleus Pulposus ◽

Dynamic Compression ◽

Akt Pathway ◽

Bioreactor Culture ◽

Nucleus Pulposus Cells ◽

Matrix Synthesis ◽

Static Compression ◽

Matrix Deposition ◽

The Matrix

Background/Aims: An adequate matrix production of nucleus pulposus (NP) cells is an important tissue engineering-based strategy to regenerate degenerative discs. Here, we mainly aimed to investigate the effects and mechanism of mechanical compression (i.e., static compression vs. dynamic compression) on the matrix synthesis of three-dimensional (3D) cultured NP cells in vitro. Methods: Rat NP cells seeded on small intestinal submucosa (SIS) cryogel scaffolds were cultured in the chambers of a self-developed, mechanically active bioreactor for 10 days. Meanwhile, the NP cells were subjected to compression (static compression or dynamic compression at a 10% scaffold deformation) for 6 hours once per day. Unloaded NP cells were used as controls. The cellular phenotype and matrix biosynthesis of NP cells were investigated by real-time PCR and Western blotting assays. Lentivirus-mediated N-cadherin (N-CDH) knockdown and an inhibitor, LY294002, were used to further investigate the role of N-CDH and the PI3K/Akt pathway in this process. Results: Dynamic compression better maintained the expression of cell-specific markers (keratin-19, FOXF1 and PAX1) and matrix macromolecules (aggrecan and collagen II), as well as N-CDH expression and the activity of the PI3K/Akt pathway, in the 3D-cultured NP cells compared with those expression levels and activity in the cells grown under static compression. Further analysis showed that the N-CDH knockdown significantly down-regulated the expression of NP cell-specific markers and matrix macromolecules and inhibited the activation of the PI3K/Akt pathway under dynamic compression. However, inhibition of the PI3K/Akt pathway had no effects on N-CDH expression but down-regulated the expression of NP cell-specific markers and matrix macromolecules under dynamic compression. Conclusion: Dynamic compression increases the matrix synthesis of 3D-cultured NP cells compared with that of the cells under static compression, and the N-CDH-PI3K/Akt pathway is involved in this regulatory process. This study provides a promising strategy to promote the matrix deposition of tissue-engineered NP tissue in vitro prior to clinical transplantation.

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Ligustrazine Prevents Intervertebral Disc Degeneration via Suppression of Aberrant TGFβ Activation in Nucleus Pulposus Cells

BioMed Research International ◽

10.1155/2019/5601734 ◽

2019 ◽

Vol 2019 ◽

pp. 1-9

Author(s):

Shufen Liu ◽

Yuhao Cheng ◽

Yuqi Tan ◽

Jingcheng Dong ◽

Qin Bian

Keyword(s):

Growth Factor ◽

Mouse Model ◽

Intervertebral Disc ◽

Nucleus Pulposus ◽

Disc Degeneration ◽

Intervertebral Disc Degeneration ◽

Ex Vivo ◽

Tissue Growth ◽

Nucleus Pulposus Cells

Objectives. Aberrant transforming growth factor β (TGFβ) activation is detrimental to both nucleus pulposus (NP) cells and cartilage endplates (CEPs), which can lead to intervertebral disc degeneration (IDD). Ligustrazine (LIG) reduces the expression of inflammatory factors and TGFβ1 in hypertrophic CEP to prevent IDD. In this study, we investigate the effects of LIG on NP cells and the TGFβ signaling. Design. LIG was injected to the lumbar spinal instability (LSI) mouse model. The effect of LIG was evaluated by intervertebral disc (IVD) score in the LSI mouse model. The expression of activated TGFβ was examined using immunostaining with pSmad2/3 antibody. The upright posture (UP) rat model was also treated and evaluated in the same manner to assess the effect of LIG. In ex vivo study, IVDs from four-week old mice were isolated and treated with 10−5, 10−6, and 10−7 M of LIG. We used western blot to detect activated TGFβ expression. TGFβ-treated human nucleus pulposus cells (HNPCs) were cotreated with optimized dose of LIG in vitro. Immunofluorescence staining was performed to determine pSmad2/3, connective tissue growth factor (CCN2), and aggrecan (ACAN) expression levels. Results. IVD score and the percentage of pSmad2/3+ NP cells were low in LIG-treated LSI mice in comparison with LSI mice, but close to the levels in the Sham group. Similarly, LIG reduced the overexpression of TGFβ1 in NP cells. The inhibitory effect of LIG was dose dependent. A dose of 10−5 M LIG not only strongly attenuated Smad2/3 phosphorylation in TGFβ-treated IVD ex vivo but also suppressed pSmad2/3, CCN2, and ACAN expression in TGFβ-treated NP cells in vitro. Conclusions. LIG prevents IDD via suppression of TGFβ overactivation in NP cells.

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