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.
Tissues and engineered biomaterials exhibit exquisite local variation in stiffness that defines their function. Conventional elastography quantifies stiffness in soft (e.g. brain, liver) tissue, but robust quantification in stiff (e.g. musculoskeletal) tissues is challenging due to dissipation of high frequency shear waves. We describe new development of
- NSF-PAR ID:
- 10150389
- Publisher / Repository:
- Nature Publishing Group
- Date Published:
- Journal Name:
- Scientific Reports
- Volume:
- 10
- Issue:
- 1
- ISSN:
- 2045-2322
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Chemical exchange saturation transfer of glycosaminoglycans, gagCEST, is a quantitative MR technique that has potential for assessing cartilage proteoglycan content at field strengths of 7 T and higher. However, its utility at 3 T remains unclear. The objective of this work was to implement a rapid volumetric gagCEST sequence with higher gagCEST asymmetry at 3 T to evaluate its sensitivity to osteoarthritic changes in knee articular cartilage and in comparison with
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Abstract Purpose of Review Interfacial tissue exists throughout the body at cartilage-to-bone (osteochondral interface) and tendon-to-bone (enthesis) interfaces. Healing of interfacial tissues is a current challenge in regenerative approaches because the interface plays a critical role in stabilizing and distributing the mechanical stress between soft tissues (e.g., cartilage and tendon) and bone. The purpose of this review is to identify new directions in the field of interfacial tissue development and physiology that can guide future regenerative strategies for improving post-injury healing.
Recent Findings Cues from interfacial tissue development may guide regeneration including biological cues such as cell phenotype and growth factor signaling; structural cues such as extracellular matrix (ECM) deposition, ECM, and cell alignment; and mechanical cues such as compression, tension, shear, and the stiffness of the cellular microenvironment.
Summary In this review, we explore new discoveries in the field of interfacial biology related to ECM remodeling, cellular metabolism, and fate. Based on emergent findings across multiple disciplines, we lay out a framework for future innovations in the design of engineered strategies for interface regeneration. Many of the key mechanisms essential for interfacial tissue development and adaptation have high potential for improving outcomes in the clinic.
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Measuring LTGF-beta activation in live tissues in situ is a major challenge due to the short half-life of activated TGF-beta in cartilage (due to rapid receptor internalization/degradation). As such, activation assessments typically require analysis of downstream events. However, assessments of intracellular TGF-beta signaling molecules (Smad2/3 phosphorylation) yield mostly qualitative measures and reporter cell assays are not compatible with intact cartilage tissues. Alternatively, in the current project, we proposed quantifying LTGF-beta activation in situ through a novel assay that capitalizes on TGF-beta’s robust autoinduction behavior; active TGF-beta activity induces a predictable increase in synthesis of soluble LTGF-beta. The dominant fraction of newly synthesized LTGF-beta is secreted from the tissue (not retained in ECM) and stable. Accordingly, measurements of LTGF-beta secretion into culture medium allows for quantifications of TGF-beta activity in cartilage. In order to confirm that LTGF-beta secretion enhancements result from TGF-beta activity (and not other load-initiated signaling cascades), a control group can readily be utilized, consisting of TGF-beta activity inhibition from a TGF-beta-receptor specific kinase inhibitor. Using this platform, we performed the first-ever measurement of the activity of TGF-beta in cartilage explants from load-induced activation. Results demonstrate that LTGF-beta secretion rates do indeed increase with cartilage mechanical loading. Upon exposure to a TGF-beta inhibitor, LTGF-beta secretion rates return to basal control levels, thus confirming that LTGF-beta secretion enhancements can be predominantly attributed to TGF-beta activity in the tissue. Upon standard curve conversion, autoinduction assay results demonstrate that mechanical load-induced activation of ECM-bound LTGF-beta gives rise to ~0.15ng/mL of TGF-beta activity in cartilage. Importantly, this measure represents the first quantitative assessment of TGF-beta activity in articular cartilage. While these levels represent the activation of only a small fraction of the total LTGF-beta stores in the cartilage ECM (~300ng/mL), they are indeed capable of giving rise to considerable chondrocyte biosynthesis enhancements in the tissue. As such, these measurements support the mechanobiological role of load-induced LTGF-beta activation in maintaining articular cartilage integrity. The assay platform advanced in this study sets the foundation for considerable advances in our understanding of the mechanistic details and physiologic importance of load-induced LTGF-beta activation in cartilage. In the future, we plan to use this quantitative platform to assess: 1) the influence of varying loading regimens on LTGF-beta activation rates (e.g., physiologic exercise, elevated stresses, high-impact trauma), and 2) changes to load-induced LTGF-beta activation with aging or joint degeneration. An abstract on this work was presented at the 2020 ASME SB3C Conference (virtual meeting) and a full-length manuscript is currently in preparation.more » « less
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Background Healthy articular cartilage presents structural gradients defined by distinct zonal patterns through the thickness, which may be disrupted in the pathogenesis of several disorders. Analysis of textural patterns using quantitative MRI data may identify structural gradients of healthy or degenerating tissue that correlate with early osteoarthritis (OA).
Purpose To quantify spatial gradients and patterns in MRI data, and to probe new candidate biomarkers for early severity of OA.
Study Type Retrospective study.
Subjects Fourteen volunteers receiving total knee replacement surgery (eight males/two females/four unknown, average age ± standard deviation: 68.1 ± 9.6 years) and 10 patients from the OA Initiative (OAI) with radiographic OA onset (two males/eight females, average age ± standard deviation: 57.7 ± 9.4 years; initial Kellgren‐Lawrence [KL] grade: 0; final KL grade: 3 over the 10‐year study).
Field Strength/Sequence 3.0‐T and 14.1‐T, biomechanics‐based displacement‐encoded imaging, fast spin echo, multi‐slice multi‐echo
T2 mapping.Assessment We studied structure and strain in cartilage explants from volunteers receiving total knee replacement, or structure in cartilage of OAI patients with progressive OA. We calculated spatial gradients of quantitative MRI measures (eg, T2) normal to the cartilage surface to enhance zonal variations. We compared gradient values against histologically OA severity, conventional relaxometry, and/or KL grades.
Statistical Tests Multiparametric linear regression for evaluation of the relationship between residuals of the mixed effects models and histologically determined OA severity scoring, with a significance threshold at
α = 0.05.Results Gradients of individual relaxometry and biomechanics measures significantly correlated with OA severity, outperforming conventional relaxometry and strain metrics. In human explants, analysis of spatial gradients provided the strongest relationship to OA severity (
R 2 = 0.627). Spatial gradients of T2 from OAI data identified variations in radiographic (KL Grade 2) OA severity in single subjects, while conventional T2 alone did not.Data Conclusion Spatial gradients of quantitative MRI data may improve the predictive power of noninvasive imaging for early‐stage degeneration.
Evidence Level 1
Technical Efficacy Stage 1
