Ontogeny informs regeneration: explant models to investigate the role of the extracellular matrix in cartilage tissue assembly and development

McCreery, Kaitlin P.; Calve, Sarah; Neu, Corey P.

doi:10.1080/03008207.2019.1698556

Cited by 13 publications

(6 citation statements)

References 116 publications

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“…It is the relatively avascular and aneural properties of cartilage that makes it a suitable tissue choice for an ex vivo culturing system to probe factors related to cartilage degeneration and regeneration. [ 48 ] Tissue sections perpendicular to the cartilage surface were sliced using a vibratome to expose rounded, middle‐zone chondrocytes. We located live chondrocytes and the chondrocyte nuclei by staining cells with vital calcein AM and DAPI.…”

Section: Resultsmentioning

confidence: 99%

Nuclear Stiffness Decreases with Disruption of the Extracellular Matrix in Living Tissues

McCreery

Scott

et al. 2021

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Reciprocal interactions between the cell nucleus and the extracellular matrix lead to macroscale tissue phenotype changes. However, little is known about how the extracellular matrix environment affects gene expression and cellular phenotype in the native tissue environment. Here, it is hypothesized that enzymatic disruption of the tissue matrix results in a softer tissue, affecting the stiffness of embedded cell and nuclear structures. The aim is to directly measure nuclear mechanics without perturbing the native tissue structure to better understand nuclear interplay with the cell and tissue microenvironments. To accomplish this, an atomic force microscopy needle‐tip probe technique that probes nuclear stiffness in cultured cells to measure the nuclear envelope and cell membrane stiffness within native tissue is expanded. This technique is validated by imaging needle penetration and subsequent repair of the plasma and nuclear membranes of HeLa cells stably expressing the membrane repair protein CHMP4B‐GFP. In the native tissue environment ex vivo, it is found that while enzymatic degradation of viable cartilage tissues with collagenase 3 (MMP‐13) and aggrecanase‐1 (ADAMTS‐4) decreased tissue matrix stiffness, cell and nuclear membrane stiffness is also decreased. Finally, the capability for cell and nucleus elastography using the AFM needle‐tip technique is demonstrated. These results demonstrate disruption of the native tissue environment that propagates to the plasma membrane and interior nuclear envelope structures of viable cells.

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Section: Resultsmentioning

confidence: 99%

Nuclear Stiffness Decreases with Disruption of the Extracellular Matrix in Living Tissues

McCreery

Scott

et al. 2021

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“…While these methods may be used in the context of other tissues, cartilage is a suitable choice due to the ease of ex vivo culturing and ability to target the degradation with matrix-specific enzymes. It is the relatively avascular and aneural properties of cartilage that makes it a suitable tissue choice for an ex vivo culturing system to probe factors related to cartilage degeneration and regeneration 44 . Tissue sections parallel to the cartilage surface were sliced using a vibratome to expose rounded, middle-zone chondrocytes.…”

Section: Resultsmentioning

confidence: 99%

Nuclear stiffness decreases with disruption of the extracellular matrix in living tissues

McCreery¹,

Xu²,

Scott³

et al. 2020

Preprint

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Reciprocal interactions between the cell nucleus and the extracellular matrix lead to macroscale tissue phenotype changes. The extracellular environment is physically linked to the nuclear envelope and provides cues to maintain nuclear structure and cellular homeostasis regulated in part by mechanotransduction mechanisms. However, little is known about how structure and properties of the extracellular matrix in living tissues impacts nuclear mechanics, and current experimental challenges limit the ability to detect and directly measure nuclear mechanics while cells are within the native tissue environment. Here, we hypothesized that enzymatic disruption of the tissue matrix results in a softer tissue, affecting the stiffness of embedded cell and nuclear structures. We aimed to directly measure nuclear mechanics without perturbing the native tissue structure to better understand nuclear interplay with the cell and tissue microenvironments. To accomplish this, we expanded an atomic force microscopy needle-tip probe technique that probes nuclear stiffness in cultured cells to measure the nuclear envelope and cell membrane stiffness within native tissue. We validated this technique by imaging needle penetration and subsequent repair of the plasma and nuclear membranes of HeLa cells stably expressing the membrane repair protein CHMP4B-GFP. In the native tissue environment ex vivo, we found that while enzymatic degradation of viable cartilage tissues with collagenase 3 (MMP-13) and aggrecanase-1 (ADAMTS-4) decreased tissue matrix stiffness, cell and nuclear membrane stiffness is also decreased. Finally, we demonstrated the capability for cell and nucleus elastography using the AFM needle-tip technique. These results demonstrate disruption of the native tissue environment that propagates to the plasma membrane and interior nuclear envelope structures of viable cells.

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“…To quantify protein secretion, we incubated freshly harvested P3 distal humeri ex vivo for 4 days and collected and replaced the media every 2 days, an incubation period known to maintain chondrocyte viability (51,52). Notably, cartilage is suitable for ex vivo incubation studies because of its relatively avascular and aneural composition (53). During the incubation period, significantly more protein was secreted by Neo/Neo compared with ϩ/ϩ cartilage explants (p ϭ 0.0033; Fig.…”

Section: Effect Of Hspg2 On Ecm Assemblymentioning

confidence: 97%

Perlecan Knockdown Significantly Alters Extracellular Matrix Composition and Organization During Cartilage Development

Kinzer‐Ursem

et al. 2020

Molecular & Cellular Proteomics

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Perlecan is a critical proteoglycan found in the extracellular matrix (ECM) of cartilage. In healthy cartilage, perlecan regulates cartilage biomechanics and we previously demonstrated perlecan deficiency leads to reduced cellular and ECM stiffness in vivo. This change in mechanics may lead to the early onset osteoarthritis seen in disorders resulting from perlecan knockdown such as Schwartz-Jampel syndrome (SJS). To identify how perlecan knockdown affects the material properties of developing cartilage, we used imaging and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to study the ECM in a murine model of SJS, Hspg2C1532Y−Neo. Perlecan knockdown led to defective pericellular matrix formation, whereas the abundance of bulk ECM proteins, including many collagens, increased. Post-translational modifications and ultrastructure of collagens were not significantly different; however, LC-MS/MS analysis showed more protein was secreted by Hspg2C1532Y−Neo cartilage in vitro, suggesting that the incorporation of newly synthesized ECM was impaired. In addition, glycosaminoglycan deposition was atypical, which may explain the previously observed decrease in mechanics. Overall, these findings provide insight into the influence of perlecan on functional cartilage assembly and the progression of osteoarthritis in SJS.

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Ontogeny informs regeneration: explant models to investigate the role of the extracellular matrix in cartilage tissue assembly and development

Cited by 13 publications

References 116 publications

Nuclear Stiffness Decreases with Disruption of the Extracellular Matrix in Living Tissues

Nuclear Stiffness Decreases with Disruption of the Extracellular Matrix in Living Tissues

Nuclear stiffness decreases with disruption of the extracellular matrix in living tissues

Perlecan Knockdown Significantly Alters Extracellular Matrix Composition and Organization During Cartilage Development

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