Ligament/Tendon Culture under Hypoxic Conditions: A Systematic Review

The hypoxic environment is a substantial factor in maintenance, proliferation, and differentiation of the cell cultures. Low oxygen is known as a potent chondrogenesis stimulus in stem cells that is important for clinical application and engineering of functional cartilage. Hypoxia can potentially induce angiogenesis process by secretion of cytokines. This systematic review goal is to discover the effect of hypoxic condition on tendon/ ligament culture and the best oxygen level of hypoxia for in vitro and in vivo studies. We included 21 articles. A comprehensive review of this database confirms that the hypoxic condition is a substantial factor in the maintenance, proliferation, and differentiation of ligament/tendon cultures. Cell proliferation in the severe hypoxic (oxygen concentration of 1%) group at 24 h postcultivation was considered significant, but cell proliferation was markedly inhibited in the severe hypoxic group after 48 h.

In vitro Attempting to elucidate the specificity of pathways from environmental stress to cellular outcomes mediated by mitogen-activated protein kinase (MAPK) activation.

5%
Examine the responsiveness of cultured hPDL cells to epidermal growth factor (EGF), hypoxia, and mechanical stress, in terms of cell proliferation, differentiation, and the associated activation of three different MAPKs Cell proliferation was induced in the presence of 10 ng/mL EGF or hypoxic conditions (5% O2) Cell proliferation was inhibited by cyclic stretch (9% strain, 6 cycles/min) Alkaline phosphatase activity was increased by cyclic stretch but decreased by EGF and hypoxia.

Achilles tendons from rats
In vitro Achilles tendons were dissected from rats, primary cultures of rat tenocytes were challenged with different stimuli (hypoxia and PDGF) and VEGF secretion was measured.
The combination of cytokines and hypoxia increased VEGF production 5-fold.

Sprague-Dawley rat PDL
In vitro In the hypoxia group, cells were incubated with 2% O 2 for 1-3 d. In the reoxygenation group, cells were first incubated under the same conditions as the hypoxia group for 24 h and then returned to normoxic conditions and cultured for 1-2 additional days.

20% vs 2%
-Level of expression of the bone sialoprotein and vascular endothelial growth factor mRNAs -Alkaline phosphatase activity Significantly higher proliferation rates were observed in both the hypoxia and re oxygenation groups than in the control group.
Alkaline phosphatase activity was significantly higher in the hypoxia group.
The bone sialoprotein mRNA was expressed at significantly higher levels in the hypoxia group.
The vascular endothelial growth factor mRNA was expressed at significantly higher levels in the hypoxia group.
S u p p l e m e n t a r y Levels of vascular endothelial growth factor (VEGF), interleukin (IL)-6, IL-1b, tumor necrosis factor-alpha (TNF-), and prostaglandin E2 (PGE2) were determined using ELISAs.
Expression of the corresponding mRNAs was detected using reverse transcription-PCR Significantly higher extracellular concentrations of VEGF and IL-6 were detected in the hypoxia group.
The levels of the IL-1b mRNA and protein were only increased in the hypoxia group.
Neither TNF- nor PGE2 was detectable in samples from either group, PGE2 , VEGF, IL-6 and IL-1b production was detected after reoxygenation.
The levels of the secreted VEGF, IL-6 and IL-1b proteins and mRNA also tended to increase after reoxygenation.

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Kitase et al 33 Human PDL In vitro Gene expression in cultured human PDL cells induced by hypoxia was analyzed Less than 1% Gene expression in cultured human PDL cells induced by hypoxia was analyzed using a cDNA array, followed by RT-PCR analysis.
The expression of the chemokine receptor CCR2 was increased in PDL cells exposed to hypoxia.
Hypoxic conditions elicit the expression of proapoptotic genes.  In vitro The compressive force was adjusted by adding metal slices to the cylinder. PDLFs were subjected to 0.5, 1.0, 2.0 or 3.0 g/cm 2 of compressive force for the indicated periods.
PDLFs were transferred to a GasPak Pouch, where the total oxygen concentration was reduced to less than 1%, to induce hypoxia.

Less than 1%
RT-PCR analysis ELISA Luciferase reporter assay A hypoxic treatment for 24 h increased the levels of the IL-1β, IL-6 and IL-8 mRNAs.
Mechanical compression and hypoxia significantly increased the expression of IL-1β, IL-6, IL-8, TNF-α and VEGF in PDLFs. Increased osteogenic differentiation of co cultured PDLSCs was observed, as evidenced by markedly increased alkaline phosphatase (ALP) activity and PGE2 levels, vascular endothelial growth factor (VEGF) release, levels of the runt-related transcription factor 2 (Runx2) and Sp7 mRNAs and proteins and mineralized nodule formation.
ERK1/2 was phosphorylated in a rapid but transient manner, whereas p38 MAPK was activated in a slow and sustained manner in cells exposed to hypoxia. The effect of hypoxia upon tenocyte biology was measured ex vivo using quantitative real-time PCR, multiplex cytokine assays, apoptotic proteomic profiling, immunohistochemistry and -Hypoxic tenocytes exhibited increased production of proinflammatory cytokines (P<0.001), altered matrix regulation (P <0.01) and increased production of collagen type III through a S u p p l e m e n t a r y derived primary cells were isolated from hamstring tendon tissue obtained during hamstring tendon anterior cruciate ligament reconstruction.
annexin V staining followed by fluorescence-activated cell sorting.
-Hypoxia increased the expression of several mediators of apoptosis and thereby promoted tenocyte apoptosis. Cell proliferation and ALP activities -Cell proliferation was significantly increased in the severe hypoxia group at 24 h post-cultivation (P<0.05).
-Cell proliferation was markedly reduced at 72 h post-cultivation (P<0.05), and the difference was more marked in the severe hypoxia group (P<0.05).
-ALP activity decreased at each time point as the level of hypoxia increased. No marked difference was observed between the hypoxic and control groups after 12 h. In vitro HPDLSCs used in these experiments underwent 3 passages. Next, HPDLSCs (passage 3) were exposed to normal oxygen (21% O2) or hypoxic (2% O2) conditions and cell proliferation was evaluated.

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The proliferation rate was increased, and an increase in the osteogenic differentiation potential was increased, compared to control cells.
-Twelve weeks of transplantation, hypoxia-treated HPDLSCs differentiated into osteoblast-like cells that formed bone-like structures.
In vivo After 7 d of exposure to hypoxic and normal conditions, each cell group was transplanted subcutaneously into the back of immunocompromised mice to determine differences in osteogenic function in vivo.
The mean optical density of newly formed bone in animals injected with hypoxia-treated HPDLSCs was significantly higher than the control group (P < 0.05).
S u p p l e m e n t a r y Quantitative PCR, immunoblots, immunostaining, and a specific ROS assay were performed to determine the levels of NOX4, ROS, and several redox systems.
NOX4 and redox systems were evaluated using immunohistochemistry.
Significantly increased NOX4 levels were observed in PDL cells after hypoxic or inflammatory stimulation.
Significant upregulation of ROS and catalase.
The interaction of NOX4 and redox systems is crucial for ROS formation, which plays a pivotal role in oral diseases. 1) Cell proliferation was analyzed using flow cytometry.
2) Cell ultrastructure was analyzed using transmission electron microscopy.
Hypoxia regulates CTS-responsive changes in the proliferation and osteogenic differentiation of S u p p l e m e n t a r y factor Sp7 [SP7] were analyzed using RT-PCR and Western blotting.
4) The involvement of mitogen-activated protein kinase (MAPK) signaling pathways was investigated by treating cells with specific inhibitors and performing Western blotting. hPDLCs by modulating MAPK pathways. Hypoxia-treated hPDLCs may represent an in vitro model to explore the molecular mechanisms of periodontitis.

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Li Inhibitors of p38/MAPK or ERK were applied to PDLSCs to observe their effects on clone formation and proliferation.
The proliferation of PDLSCs cultured under hypoxic conditions was higher than the control group (P < 0.001).
A marked increase in p38 and ERK1/2 phosphorylation was observed in hypoxic PDLSCs compared to the control group (P < 0.05).
Hypoxia increased PDLSC clone formation and proliferation by activating the p38/MAPK and ERK/MAPK signaling pathways.

Chen et al 18 Human PDL
In vitro Cells passaged to P 4 were used in the experiments.
The UCSC Genome Bioinformatics database was used to locate the HIF-1, HIF1A-AS1 and HIF1A-AS2 Genes and to obtain the sequences of the HIF-1, HIF1A-AS1 and HIF1A-AS2 mRNAs. Mice were anesthetized after EAE was induced in mice using myelin oligodendrocyte glycoprotein peptide (MOG).

20% vs 3%
-Immunofluorescence staining of mouse spinal cord tissues Cells were subsequently subjected to injury by creating manual scratches with a 1-ml pipette tip. After injury, the medium was removed, and cells were incubated with fresh serum-free medium for 24 h. Neuronal cells without scratch injury were also included as a control. Human PDL In vitro The properties of periodontal ligament-derived MSCs (PDLSCs) cultivated in serum-free and serumcontaining media under hypoxic and normoxic conditions were characterized.

21% vs 3%
Cell growth, gene and protein expression, cytodifferentiation potential, genomic stability, cytotoxic response, and hard tissue generation by PDLSCs in vivo were examined.
Hypoxia did not alter the growth of PDLSCs under serum-free conditions but inhibited their osteogenic and adipogenic differentiation.
PDLSCs cultured in serum-free culture media were more susceptible to damage following exposure to extrinsic cytotoxic stimuli than cells cultured in medium supplemented with serum.
1% and 5% vs 21% The optical density values were detected, and the growth curve was constructed.
A wound healing assay was performed to observe the migration of hPDLCs.
RT-qPCR was conducted to detect the expression of cementum-related genes and Wnt signaling pathway-related genes.
RT-qPCR, Western blot, and immunofluorescence staining were performed to detect HIF levels.
The growth rate of hPDLCs decreased as the O2 content decreased, and the morphology of hPDLCs changed in the presence of different O2 concentrations.
The expression of cementum-related genes and Wnt signaling pathway-related genes increased in cells cultured under hypoxic conditions.
The reduction in the O2 concentration increased the levels of the HIF messenger RNA and protein, and HIF was gradually transported from the cytoplasm into the nucleus in cells cultured with a 1% O2 concentration. In vitro hPDLF (immortalized fibroblasts derived from human deciduous teeth) and hEMBF (immortalized fibroblasts obtained from a human embryo) were used in this study.

Western blotting
Cytokine array analysis Levels of the ANG and VEGF mRNAs were significantly increased in PDL fibroblasts cultured under hypoxic conditions, but not in embryonic fibroblasts.
Hypoxia increased the productions of ANG and VEGF proteins in PDL fibroblasts.
The expression of the HIF-1α mRNA was not affected by hypoxia in either fibroblast line, although the level of the HIF-1α protein was increased after exposure to hypoxia.
Under hypoxic conditions, HIF-1α upregulates synthesis of ANG and VEGF in PDL fibroblasts and promotes angiogenesis.

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Liu et al 25 Human PDL In vitro Hypoxia was induced by exposing cells to a tri-gas incubator with 3% O 2 , cobalt chloride (CoCl2) was used to induce the stabilization of HIF-1α protein.
After treating PDLSCs with hypoxia (3% O2) over different time periods (0, 12, 24 and 48 h), qPCR results revealed that the transcription of TGF"1 declined after 24 h and continued to decline after 48 h.

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Yan et al 28 Human PDL In vitro hPDLCs were extracted, cultured, and used between passages 3 and 6.

2%
-Real time PCR and Western blotting were performed to detect relative mRNA and protein levels.
Cells cultured in the presence of 2% O2 exhibited decreased A20 expression and an increased RANKL/OPG (R/O) ratio.
S u p p l e m e n t a r y hPDLCs were transfected with lentivirus A20 for overexpression or silencing studies. Twenty-four hours after transfection, 2.5 μg/mL puromycin was added and the cells were cultured for 24 h to select for positive cells.
-The formation of autophagosomes was measured using TEM. -Osteoclastic differentiation was assessed using TRAP staining and the hydroxyapatite resorption assay. The interactions between different proteins were observed using co-IP.