Semi-automatic grading system in histologic and immunohistochemistry analysis to evaluate in vitro chondrogenesis

Gutiérrez, María Lucía Lahoz; Guevara, Johana; Barrera, Luis Alejandro

doi:10.11144/javeriana.sc17-2.sags

Cited by 34 publications

(21 citation statements)

References 30 publications

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“…The following endpoints were analyzed: percent area stained for GAGs, collagen, and elastin; cell count per 20× field; and average cell size. Safranin O staining is proportional to GAG content in normal cartilage, previously confirmed with biochemical assays . Trichrome and Verhoeff's staining have been used to quantify collagen and elastin content in several tissue types …”

Section: Methodsmentioning

confidence: 82%

Age‐related histologic and biochemical changes in auricular and septal cartilage

et al. 2017

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Section: Methodsmentioning

confidence: 82%

Age‐related histologic and biochemical changes in auricular and septal cartilage

et al. 2017

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“…Micromass analysis was performed using ImageJ. Images were made binary under an RGB threshold and "Particle Analysis" was utilized to measure Alcian blue positive area and nodule number (75).…”

Section: Methodsmentioning

confidence: 99%

Domains with highest heparan sulfate–binding affinity reside at opposite ends in BMP2/4 versus BMP5/6/7: Implications for function

Billings

Yang

Mundy

et al. 2018

Journal of Biological Chemistry

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Signaling proteins, including bone morphogenetic proteins (BMPs), specifically interact with heparan sulfate (HS). These interactions regulate protein distribution and function and are largely mediated by domains rich in basic amino acids. The N-terminal region of BMP2 and BMP4 contains one such domain with a typical Cardin-Weintraub (CW) motif, but it is unclear whether the same occurs in BMP5, BMP6 and BMP7 that constitute a separate evolutionary subgroup. Peptides spanning the N-terminal domain of BMP2/4 interacted with substrate-bound HS with nanomolar affinity, but peptides spanning BMP5/6/7 N-terminal domain did not. We reexamined the entire BMP5/6/7 sequences and identified a novel CW-like motif at their Cterminus. Peptides spanning this domain displayed high affinity HS binding, but corresponding BMP2/4 C-terminal peptides did not, likely because of acidic or non-charged residue substitutions. Peptides pre-assembled into NeutrAvidin tetramers displayed the same exact binding selectivity of respective monomers, but bound HS with greater affinity. Tests of possible peptide biological activities showed that the HS-binding N-terminal BMP2/4 and C-terminal BMP5/6/7 peptides stimulated chondrogenesis in vitro, potentially by freeing endogenous BMPs. Thus, HS interactions appear largely ascribable to domains at opposite ends of BMP2/4 versus BMP5/6/7, reiterating the evolutionary distance of these BMP subgroups and possible functional diversification.

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“…It should be noted that KGN is not stable over time and thus, new batches of KGN stock were used every 2 to 3 weeks. Cultures were stained with Alcian blue (Huegel et al, 2013) and staining quantified (Gutiérrez et al, 2012) as previously described.…”

Section: Methodsmentioning

confidence: 99%

Mouse limb skeletal growth and synovial joint development are coordinately enhanced by Kartogenin

Decker

Koyama

Enomoto‐Iwamoto

et al. 2014

Developmental Biology

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Limb development requires the coordinated growth of several tissues and structures including long bones, joints and tendons, but the underlying mechanisms are not wholly clear. Recently, we identified a small drug-like molecule -we named Kartogenin (KGN)- that greatly stimulates chondrogenesis in marrow-derived mesenchymal stem cells (MSCs) and enhances cartilage repair in mouse osteoarthritis (OA) models. To determine whether limb developmental processes are regulated by KGN, we tested its activity on committed preskeletal mesenchymal cells from mouse embryo limb buds and whole limb explants. KGN did stimulate cartilage nodule formation and more strikingly, boosted digit cartilaginous anlaga elongation, synovial joint formation and interzone compaction, tendon maturation as monitored by ScxGFP, and interdigit invagination. To identify mechanisms, we carried out gene expression analyses and found that several genes, including those encoding key signaling proteins, were up-regulated by KGN. Amongst highly up-regulated genes were those encoding hedgehog and TGFβ superfamily members, particularly TFGβ1. The former response was verified by increases in Gli1-LacZ activity and Gli1 mRNA expression. Exogenous TGFβ1 stimulated cartilage nodule formation to levels similar to KGN, and KGN and TGFβ1 both greatly enhanced expression of lubricin/Prg4 in articular superficial zone cells. KGN also strongly increased the cellular levels of phospho-Smads that mediate canonical TGFβ and BMP signaling. Thus, limb development is potently and harmoniously stimulated by KGN. The growth effects of KGN appear to result from its ability to boost several key signaling pathways and in particular TGFβ signaling, working in addition to and/or in concert with the filamin A/CBFβ/RUNX1 pathway we identified previously to orchestrate overall limb development. KGN may thus represent a very powerful tool not only for OA therapy, but also limb regeneration and tissue repair strategies.

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Semi-automatic grading system in histologic and immunohistochemistry analysis to evaluate in vitro chondrogenesis

Cited by 34 publications

References 30 publications

Age‐related histologic and biochemical changes in auricular and septal cartilage

Age‐related histologic and biochemical changes in auricular and septal cartilage

Domains with highest heparan sulfate–binding affinity reside at opposite ends in BMP2/4 versus BMP5/6/7: Implications for function

Mouse limb skeletal growth and synovial joint development are coordinately enhanced by Kartogenin

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