Gene expression profiling of chondrocytes from a porcine impact injury model

Ashwell, M.S.; O’Nan, A. T.; Gonda, Michael G.; Mente, Peter L.

doi:10.1016/j.joca.2007.12.012

Cited by 25 publications

(34 citation statements)

References 63 publications

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“…CAPNS1 (calpain small subunit 1) is able to promote cell proliferation, migration, and adhesion [44]. In contrast, LEG1 (galectin-1) overexpression is thought to inhibit normal mouse fibroblast growth and increase apoptosis [45].…”

Section: Discussionmentioning

confidence: 99%

A Study of the Wound Healing Mechanism of a Traditional Chinese Medicine,Angelica sinensis, Using a Proteomic Approach

Hsiao¹,

Hung²,

Tsai

et al. 2012

Evidence-Based Complementary and Alternative Medicine

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Angelica sinensis (AS) is a traditional Chinese herbal medicine that has been formulated clinically to treat various form of skin trauma and to help wound healing. However, the mechanism by which it works remains a mystery. In this study we have established a new platform to evaluate the pharmacological effects of total AS herbal extracts as well as its major active component, ferulic acid (FA), using proteomic and biochemical analysis. Cytotoxic and proliferation-promoting concentrations of AS ethanol extracts (AS extract) and FA were tested, and then the cell extracts were subject to 2D PAGE analysis. We found 51 differentially expressed protein spots, and these were identified by mass spectrometry. Furthermore, biomolecular assays, involving collagen secretion, migration, and ROS measurements, gave results that are consistent with the proteomic analysis. In this work, we have demonstrated a whole range of pharmacological effects associated with Angelica sinensis that might be beneficial when developing a wound healing pharmaceutical formulation for the herbal medicine.

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Section: Discussionmentioning

confidence: 99%

A Study of the Wound Healing Mechanism of a Traditional Chinese Medicine,Angelica sinensis, Using a Proteomic Approach

Hsiao¹,

Hung²,

Tsai

et al. 2012

Evidence-Based Complementary and Alternative Medicine

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“…There have been a number of studies involving organ and tissue culture of different parts of the knee tissues, in which viability was not assessed (Jacoby and Jayson, 1976;Hollander et al, 1991;Fay et al, 2006;Tchetina et al, 2006;Ashwell et al, 2008;Ashwell et al, 2013). The studies utilised minimal sample numbers, and any tissue destructive method of viability assessment was not possible due to restrictive sample numbers.…”

Section: Km Elson Et Al Monitoring Of Osteochondral Organ Culturementioning

confidence: 99%

“…Organ culture has been employed in studies of meniscal tissue (Dai et al, 2013;Narita et al, 2009), articular cartilage (Jacoby andJayson, 1976;Hollander et al, 1991;Fay et al, 2006;Tchetina et al, 2006), OC explants (Amin et al, 2009a;Seol et al, 2014), and whole porcine patellae (Ashwell et al, 2008;Ashwell et al, 2013). However, organ culture is extremely challenging, as conditions in these cultures must be able to maintain tissue viability and stable matrix composition.…”

Section: Introductionmentioning

confidence: 99%

Non-destructive monitoring of viability in an ex vivo organ culture model of osteochondral tissue

Elson

Fox²,

Tipper³

et al. 2015

eCM

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Organ culture is an increasingly important tool in research, with advantages over monolayer cell culture due to the inherent natural environment of tissues. Successful organ cultures must retain cell viability. The aim of this study was to produce viable and non-viable osteochondral organ cultures, to assess the accumulation of soluble markers in the conditioned medium for predicting tissue viability. Porcine femoral osteochondral plugs were cultured for 20 days, with the addition of Triton X-100 on day 6 (to induce necrosis), camptothecin (to induce apoptosis) or no toxic additives. Tissue viability was assessed by the tissue destructive XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxyanilide tetrazolium salt) assay method and LIVE/DEAD ® staining of the cartilage at days 0, 6 and 20. Tissue structure was assessed by histological evaluation using haematoxylin & eosin and safranin O. Conditioned medium was assessed every 3-4 days for glucose depletion, and levels of lactate dehydrogenase (LDH), alkaline phosphatase (AP), glycosaminoglycans (GAGs), and matrix metalloproteinase (MMP)-2 and MMP-9. Necrotic cultures immediately showed a reduction in glucose consumption, and an immediate increase in LDH, GAG, MMP-2 and MMP-9 levels. Apoptotic cultures showed a delayed reduction in glucose consumption and delayed increase in LDH, a small rise in MMP-2 and MMP-9, but no significant effect on GAGs released into the conditioned medium. The data showed that tissue viability could be monitored by assessing the conditioned medium for the aforementioned markers, negating the need for tissue destructive assays. Physiologically relevant whole-or part-joint organ culture models, necessary for research and pre-clinical assessment of therapies, could be monitored this way, reducing the need to sacrifice tissues to determine viability, and hence reducing the sample numbers necessary.

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“…[12] with the exception of ppia (NM_214353.1). The ppia primers were designed with Beacon Designer software (Premier Biosoft Intl., Palo Alto, CA) from porcine gene sequences as previously described (F: 5’-GCAGACAAAGTTCCAAAGACAG-3’, R: 5’-AGATGCCAGGACCCGTATG-3’) [17] spanning an intron to detect genomic contamination.…”

Section: Methodsmentioning

confidence: 99%

“…We used the nine genes identified by Nygard and co-workers [12] as potential housekeeping genes as a starting point with the addition of peptidylprolyl isomerase A ( ppia ). Ppia was added because it has been used as a normalizing gene in cartilage for both OA-related [13,14] and non-OA related studies [15,16] and it exhibited no differential expression in impacted and control cartilage specimens in our previous work [17]. …”

Section: Introductionmentioning

confidence: 99%

Identification of stable normalization genes for quantitative real-time PCR in porcine articular cartilage

McCulloch

Ashwell

O’Nan

et al. 2012

J Animal Sci Biotechnol

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BackgroundExpression levels for genes of interest must be normalized with an appropriate reference, or housekeeping gene, to make accurate comparisons of quantitative real-time PCR results. The purpose of this study was to identify the most stable housekeeping genes in porcine articular cartilage subjected to a mechanical injury from a panel of 10 candidate genes.ResultsTen candidate housekeeping genes were evaluated in three different treatment groups of mechanically impacted porcine articular cartilage. The genes evaluated were: beta actin, beta-2-microglobulin, glyceraldehyde-3-phosphate dehydrogenase, hydroxymethylbilane synthase, hypoxanthine phosphoribosyl transferase, peptidylprolyl isomerase A (cyclophilin A), ribosomal protein L4, succinate dehydrogenase flavoprotein subunit A, TATA box binding protein, and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein—zeta polypeptide. The stability of the genes was measured using geNorm, BestKeeper, and NormFinder software. The four most stable genes measured via geNorm were (most to least stable) succinate dehydrogenase flavoprotein, subunit A, peptidylprolyl isomerase A, glyceraldehyde-3-phosphate dehydrogenase, beta actin; the four most stable genes measured via BestKeeper were glyceraldehyde-3-phosphate dehydrogenase, peptidylprolyl isomerase A, beta actin, succinate dehydrogenase flavoprotein, subunit A; and the four most stable genes measured via NormFinder were peptidylprolyl isomerase A, succinate dehydrogenase flavoprotein, subunit A, glyceraldehyde-3-phosphate dehydrogenase, beta actin.ConclusionsBestKeeper, geNorm, and NormFinder all generated similar results for the most stable genes in porcine articular cartilage. The use of these appropriate reference genes will facilitate accurate gene expression studies of porcine articular cartilage and suggest appropriate housekeeping genes for articular cartilage studies in other species.

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Gene expression profiling of chondrocytes from a porcine impact injury model

Cited by 25 publications

References 63 publications

A Study of the Wound Healing Mechanism of a Traditional Chinese Medicine,Angelica sinensis, Using a Proteomic Approach

A Study of the Wound Healing Mechanism of a Traditional Chinese Medicine,Angelica sinensis, Using a Proteomic Approach

Non-destructive monitoring of viability in an ex vivo organ culture model of osteochondral tissue

Identification of stable normalization genes for quantitative real-time PCR in porcine articular cartilage

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