scholarly journals Is Osteogenic Differentiation of Human Nucleus Pulposus Cells a Possibility for Biological Spinal Fusion?

Cartilage ◽  
2018 ◽  
Vol 11 (2) ◽  
pp. 181-191 ◽  
Author(s):  
Sharon J. Brown ◽  
Sarah A. Turner ◽  
Birender S. Balain ◽  
Neil T. Davidson ◽  
Sally Roberts
Keyword(s):  
Extracellular Matrix ◽  
Osteogenic Differentiation ◽  
Spinal Fusion ◽  
Nucleus Pulposus ◽  
Monolayer Culture ◽  
Intervertebral Discs ◽  
Nucleus Pulposus Cells ◽  
Alizarin Red ◽  
Dihydroxyvitamin D ◽  
Polymerase Chain

Objective The purpose of this study was to investigate whether a simple, biologically robust method for inducing calcification of degenerate intervertebral discs (IVD) could be developed to provide an alternative treatment for patients requiring spinal fusion. Design Nucleus pulposus (NP) cells isolated from 14 human IVDs were cultured in monolayer and exposed to osteogenic medium, 1,25-dihydroxyvitamin D3 (VitD3), parathyroid hormone (PTH), and bone morphogenic proteins (BMPs) 2/7 to determine if they could become osteogenic. Similarly explant cultures of IVDs from 11 patients were cultured in osteogenic media with and without prior exposure to VitD3 and BMP-2. Osteogenic differentiation was assessed by alkaline phosphatase activity and areas of calcification identified by alizarin red or von Kossa staining. Expression of osteogenic genes during monolayer culture was determined using polymerase chain reaction and explant tissues assessed for BMP inhibitors. Human bone marrow–derived mesenchymal stromal cells (MSCs) were used for comparison. Results Standard osteogenic media was optimum for promoting mineralization by human NP cells in monolayer. Some osteogenic differentiation was observed with 10 nM VitD3, but none following application of PTH or BMPs. Regions of calcification were detected in 2 of the eleven IVD tissue explants, one cultured in osteogenic media and one with the addition of VitD3 and BMP-2. Conclusions Human NP cells can become osteogenic in monolayer and calcification of the extracellular matrix can also occur, although not consistently. Inhibitory factors within either the cells or the extracellular matrix may hinder osteogenesis, indicating that a robust biological fusion at this time requires further optimization.

scholarly journals Effect of dexamethasone as osteogenic supplementation in in vitro osteogenic differentiation of stem cells from human exfoliated deciduous teeth

2021 ◽  
Vol 32 (1) ◽  
Author(s):  
Mariane Beatriz Sordi ◽  
Raissa Borges Curtarelli ◽  
Izabella Thaís da Silva ◽  
Gislaine Fongaro ◽  
Cesar Augusto Magalhães Benfatti ◽  
...  
Keyword(s):  
Stem Cells ◽  
Extracellular Matrix ◽  
Osteogenic Differentiation ◽  
Culture Media ◽  
Deciduous Teeth ◽  
Free Calcium ◽  
Alizarin Red ◽  
Matrix Mineralization ◽  
Media Supplementation

AbstractIn in vitro culture systems, dexamethasone (DEX) has been applied with ascorbic acid (ASC) and β-glycerophosphate (βGLY) as culture media supplementation to induce osteogenic differentiation of mesenchymal stem cells. However, there are some inconsistencies regarding the role of DEX as osteogenic media supplementation. Therefore, this study verified the influence of DEX culture media supplementation on the osteogenic differentiation, especially the capacity to mineralize the extracellular matrix of stem cells from human exfoliated deciduous teeth (SHED). Five groups were established: G1—SHED + Dulbecco’s Modified Eagles’ Medium (DMEM) + fetal bovine serum (FBS); G2—SHED + DMEM + FBS + DEX; G3—SHED + DMEM + FBS + ASC + βGLY; G4—SHED + DMEM + FBS + ASC + βGLY + DEX; G5—MC3T3-E1 + α Minimal Essential Medium (MEM) + FBS + ASC + βGLY. DNA content, alkaline phosphatase (ALP) activity, free calcium quantification in the extracellular medium, and extracellular matrix mineralization quantification through staining with von Kossa, alizarin red, and tetracycline were performed on days 7 and 21. Osteogenic media supplemented with ASC and β-GLY demonstrated similar effects on SHED in the presence or absence of DEX for DNA content (day 21) and capacity to mineralize the extracellular matrix according to alizarin red and tetracycline quantifications (day 21). In addition, the presence of DEX in the osteogenic medium promoted less ALP activity (day 7) and extracellular matrix mineralization according to the von Kossa assay (day 21), and more free calcium quantification at extracellular medium (day 21). In summary, the presence of DEX in the osteogenic media supplementation did not interfere with SHED commitment into mineral matrix depositor cells. We suggest that DEX may be omitted from culture media supplementation for SHED osteogenic differentiation in vitro studies.

scholarly journals Human Bone Marrow Mesenchymal Stromal Cell-Derived Extracellular Vesicles Promote Proliferation of Degenerated Nucleus Pulposus Cells and the Synthesis of Extracellular Matrix Through the SOX4/Wnt/β-Catenin Axis

2021 ◽  
Vol 12 ◽  
Author(s):  
Haoyu Wang ◽  
Fei Li ◽  
Wenrui Ban ◽  
Jing Zhang ◽  
Guiqi Zhang
Keyword(s):  
Extracellular Matrix ◽  
Nucleus Pulposus ◽  
Extracellular Vesicles ◽  
Target Genes ◽  
Intervertebral Disk ◽  
Human Bone ◽  
Nucleus Pulposus Cells ◽  
Synthetic Genes ◽  
Downstream Target ◽  
Transcription Factor 4

Objective: Intervertebral disk degeneration (IDD) is a major cause of pain in the back, neck, and radiculus. Mesenchymal stem cells (MSCs)-derived extracellular vesicles (EVs) are therapeutic in musculoskeletal degenerative diseases such as IDD. This study explored the effect and functional mechanism of human bone MSCs (hBMSCs)-derived EVs in proliferation and apoptosis of degenerated nucleus pulposus cells (DNPCs) and extracellular matrix (ECM) synthesis.Methods: Extracellular vesicles were isolated from hBMSCs and identified. DNPCs were induced by TNF-α. EVs were incubated with DNPCs for 24h. Internalization of EVs by DNPCs, DNPCs proliferation, apoptosis, and expressions of ECM synthetic genes, degrading genes and miR-129-5p were assessed. Downstream target genes of miR-129-5p were predicted. Target relation between miR-129-5p and SRY-box transcription factor 4 (SOX4) was verified. DNPCs proliferation, apoptosis, and ECM synthesis were measured after treatment with EVs and miR-129-5p inhibitor or SOX4 overexpression. Expressions of SOX4 and Wnt/β-catenin pathway-related proteins were determined.Results: hBMSC-EVs promoted DNPCs proliferation, inhibited apoptosis, increased expressions of ECM synthetic genes, and reduced expressions of ECM degrading genes. hBMSC-EVs carried miR-129-5p into DNPCs. Silencing miR-129-5p in EVs partially inverted the effect of EVs on DNPCs proliferation and ECM synthesis. miR-129-5p targeted SOX4. SOX4 overexpression annulled the effect of EVs on DNPCs proliferation and ECM synthesis. Expressions of Wnt1 and β-catenin were decreased in EVs-treated DNPCs, while silencing miR-129-5p in EVs promoted expressions of Wnt1 and β-catenin.Conclusion: hBMSC-EVs promoted DNPCs proliferation and ECM synthesis by carrying miR-129-5p into DNPCs to target SOX4 and deactivating the Wnt/β-catenin axis.

scholarly journals Bone Mesenchymal Stem Cells Promote Extracellular Matrix Remodeling of Degenerated Nucleus Pulposus Cells via the miR-101-3p/EIF4G2 Axis

2021 ◽  
Vol 9 ◽  
Author(s):  
Zeng Wang ◽  
Xiaolin Ding ◽  
Feifei Cao ◽  
Xishan Zhang ◽  
Jingguo Wu
Keyword(s):  
Extracellular Matrix ◽  
Nucleus Pulposus ◽  
Quantitative Polymerase Chain Reaction ◽  
Matrix Remodeling ◽  
Nucleus Pulposus Cells ◽  
Promoting Effect ◽  
Bone Mesenchymal Stem Cells ◽  
Ecm Remodeling ◽  
Lumbar Vertebral Fracture ◽  
Related Proteins

The etiology of lumbocrural pain is tightly concerned with intervertebral disk degeneration (IDD). Bone mesenchymal stem cell (BMSC)-based therapy bears potentials for IDD treatment. The properties of microRNA (miRNA)-modified BMSCs may be altered. This study investigated the role and mechanism of BMSCs promoting extracellular matrix (ECM) remodeling of degenerated nucleus pulposus cells (NPCs) via the miR-101-3p/EIF4G2 axis. NPCs were collected from patients with IDD and lumbar vertebral fracture (LVF). The expressions of miR-101-3p and ECM-related proteins, Collagen-I (Col-I) and Collagen-II (Col-II), were detected using the reverse transcription-quantitative polymerase chain reaction. The expressions of Col-I and Col-II, major non-collagenous component Aggrecan, and major catabolic factor Matrix metalloproteinase-13 (MMP-13) were detected using Western blotting. BMSCs were cocultured with degenerated NPCs from patients with IDD. Viability and apoptosis of NPCs were measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and flow cytometry. After the degenerated NPCs were transfected with the miR-101-3p inhibitor, the expressions of ECM-related proteins, cell viability, and apoptosis were detected. The targeting relationship between miR-101-3p and EIF4G2 was verified. Functional rescue experiments verified the effects of miR-101-3p and EIF4G2 on ECM remodeling of NPCs. Compared with the NPCs of patients with LVF, the degenerated NPCs of patients with IDD showed downregulated miR-101-3p, Col-II, and Aggrecan expressions and upregulated MMP-13 and Col-I expressions. BMSCs increased the expressions of miR-101-3p, Aggrecan, and Col-II, and decreased the expressions of MMP-13 and Col-I in degenerated NPCs. BMSCs enhanced NPC viability and repressed apoptosis. Downregulation of miR-101-3p suppressed the promoting effect of BMSCs on ECM remodeling. miR-101-3p targeted EIF4G2. Downregulation of EIF4G2 reversed the inhibiting effect of the miR-101-3p inhibitor on ECM remodeling. In conclusion, BMSCs increased the miR-101-3p expression in degenerated NPCs to target EIF4G2, thus promoting the ECM remodeling of NPCs.

scholarly journals Periostin: An Emerging Molecule With a Potential Role in Spinal Degenerative Diseases

2021 ◽  
Vol 8 ◽  
Author(s):  
Daxue Zhu ◽  
Wupin Zhou ◽  
Zhen Wang ◽  
Yidian Wang ◽  
Mingqiang Liu ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Matrix Protein ◽  
Bone Development ◽  
Intervertebral Discs ◽  
Extracellular Matrix Protein ◽  
Degenerative Diseases ◽  
Nucleus Pulposus Cells ◽  
Cell Proliferation And Differentiation ◽  
Potential Biomarker ◽  
Disc Cells

Periostin, an extracellular matrix protein, is widely expressed in a variety of tissues and cells. It has many biological functions and is related to many diseases: for example, it promotes cell proliferation and differentiation in osteoblasts, which are closely related to osteoporosis, and mediates cell senescence and apoptosis in chondrocytes, which are involved in osteoarthritis. Furthermore, it also plays an important role in mediating inflammation and reconstruction during bronchial asthma, as well as in promoting bone development, reconstruction, repair, and strength. Therefore, periostin has been explored as a potential biomarker for various diseases. Recently, periostin has also been found to be expressed in intervertebral disc cells as a component of the intervertebral extracellular matrix, and to play a crucial role in the maintenance and degeneration of intervertebral discs. This article reviews the biological role of periostin in bone marrow-derived mesenchymal stem cells, osteoblasts, osteoclasts, chondrocytes, and annulus fibrosus and nucleus pulposus cells, which are closely related to spinal degenerative diseases. The study of its pathophysiological effects is of great significance for the diagnosis and treatment of spinal degeneration, although additional studies are needed.

scholarly journals LncRNA GAS5 as miR-26a-5p Sponge Regulates the PTEN/PI3K/Akt Axis and Affects Extracellular Matrix Synthesis in Degenerative Nucleus Pulposus Cells in vitro

2021 ◽  
Vol 12 ◽  
Author(s):  
Liang Tan ◽  
Yifang Xie ◽  
Ye Yuan ◽  
Kai Hu
Keyword(s):  
Extracellular Matrix ◽  
Nucleus Pulposus ◽  
Intervertebral Disc Degeneration ◽  
Nucleus Pulposus Cell ◽  
Nucleus Pulposus Cells ◽  
Akt Inhibitor ◽  
Matrix Synthesis ◽  
Lncrna Gas5

The role of lncRNA growth arrest specific 5 (GAS5) in degenerative nucleus pulposus cell (NPC) apoptosis has been reported, but the mechanism of GAS5 in extracellular matrix (ECM) synthesis in intervertebral disc degeneration (IDD) remains unknown. We aimed to investigate the mechanism of GAS5 in ECM synthesis in degenerative NPCs. GAS5 expression was measured in degenerative NPCs (CP-H170) and normal NPCs (CP-H097). siRNA-mediated GAS5 knockdown was transfected to NPCs to detect cell viability and the expression of ECM-related genes (Collagen II, aggrecan, Collagen I, and MMP-3). Subcellular localization of GAS5 was analyzed. The downstream gene and pathway of GAS5 in degenerative NPCs were explored. As our results indicated, lncRNA GAS5 was upregulated in degenerative NPCs. Silencing GAS5 improved the viability of degenerative NPCs and increased ECM synthesis. GAS5 was mainly located in the cytoplasm of NPCs. LncRNA GAS5 sponged miR-26a-5p to regulate PTEN. Overexpression of miR-26a-5p promoted ECM synthesis in degenerative NPCs. Akt inhibitor LY294002 reversed the promotion of silencing GAS5 on ECM synthesis of degenerative NPCs. In conclusion, lncRNA GAS5 sponged miR-26a-5p to upregulate PTEN and inhibit the PI3K/Akt pathway, thus inhibiting ECM synthesis of degenerative NPCs.

scholarly journals Effect of Lentivirus-mediated GDF5 Transfection on Differentiation of Rabbit Nucleus Pulposus Mesenchymal Stem Cells

2020 ◽  
Author(s):  
kun zhu ◽  
Rui Zhao ◽  
Yuchen Ye ◽  
Gang Xu ◽  
Changchun Zhang
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Nucleus Pulposus ◽  
Lentiviral Vector ◽  
Intervertebral Discs ◽  
Degenerative Disease ◽  
The Other ◽  
Control Group ◽  
Nucleus Pulposus Cells ◽  
Qrt Pcr

Abstract Background: Disc degenerative disease is a common senile degenerative disease, which seriously affects the quality of life of patients.The purpose of this study is to observe the biological and cytological characteristics of rabbit nucleus pulposus mesenchymal stem cells (NPMSCs), and to determine the effect of growth differentiation factor 5(GDF5) on the differentiation of rabbit NPMSCs by lentivirus transfection.Methods: In vitro culture model of rabbit NPMSCs was established and NPMSCs cells were identified by flow cytometry (FCM)and quantitative real-time PCR(qRT-PCR). Then NPMSCs were divided into three groups: lentiviral vector carrying GDF5 was used to transfect NPMSCs, to determine the transfection rate, which was recorded as transfection group, and the NPMSCs transfected with ordinary lentiviral vector was recorded as control group, NPMSCs without processing was recorded as normal group. FCM, qRT-PCR and Western Blot(WB) were used to detected the change of NPMSCs.Results: The transfected NPMSCs by GDF5 became longer and narrower, and the cell density decreased,and the positive rate of GDF5 in the transfected group was significantly higher than that in the other two groups (P<0.05). The mRNA expression of KRT8, KRT18, KRT19 in the transfected group was significantly higher than the other two groups(P<0.05),the result of WB were the same to qRT-PCR. Conclusions: GDF5 can induce the differentiation of NPMSCs and repair degenerative intervertebral discs. Lentiviral vector carrying GDF5 can be integrated into the chromosome genome of NPMSCs and promote differentiation of NPMSCs into nucleus pulposus cells(NPCs).

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