Tissue alignment enhances remodeling potential of tendon-derived cells - Lessons from a novel microtissue model of tendon scarring

Tendinopathy is a widespread and unresolved clinical challenge, in which associated pain and hampered mobility present a major cause for work-related disability. Tendinopathy associates with a change from a healthy tissue with aligned extracellular matrix (ECM) and highly polarized cells that are connected head-to-tail, towards a diseased tissue with a disorganized ECM and randomly distributed cells, scar-like features that are commonly attributed to poor innate regenerative capacity of the tissue. A fundamental clinical dilemma with this scarring process is whether treatment strategies should focus on healing the affected (disorganized) tissue or strengthen the remaining healthy (anisotropic) tissue. The question was thus asked whether the intrinsic remodeling capacity of tendon-derived cells depends on the organization of the 3D extracellular matrix (isotropic vs anisotropic). Progress in this field is hampered by the lack of suitable in vitro tissue platforms. We aimed at filling this critical gap by creating and exploiting a next generation tissue platform that mimics aspects of the tendon scarring process; cellular response to a gradient in tissue organization from isotropic (scarred/non-aligned) to highly anisotropic (unscarred/aligned) was studied, as was a transient change from isotropic towards highly anisotropic. Strikingly, cells residing in an 'unscarred' anisotropic tissue indicated superior remodeling capacity (increased gene expression levels of collagen, matrix metalloproteinases MMPs, tissue inhibitors of MMPs), when compared to their 'scarred' isotropic counterparts. A numerical model then supported the hypothesis that cellular remodeling capacity may correlate to cellular alignment strength. This in turn may have improved cellular communication, and could thus relate to the more pronounced connexin43 gap junctions observed in anisotropic tissues. In conclusion, increased tissue anisotropy was observed to enhance the cellular potential for functional remodeling of the matrix. This may explain the poor regenerative capacity of tenocytes in chronic tendinopathy, where the pathological process has resulted in ECM disorganization. Additionally, it lends support to treatment strategies that focus on strengthening the remaining healthy tissue, rather than regenerating scarred tissue

Tendon explant models for physiologically relevant in vitro study of tissue biology - a perspective

Background: Tendon disorders increasingly afflict our aging society but we lack the scientific understanding to clinically address them. Clinically relevant models of tendon disease are urgently needed as established small animal models of tendinopathy fail to capture essential aspects of the disease. Two-dimensional and three-dimensional cell and tissue culture models are similarly limited, lacking many physiological extracellular matrix cues required to maintain tissue homeostasis or guide matrix remodeling. These cues reflect the biochemical and biomechanical status of the tissue, and encode information regarding the mechanical and metabolic competence of the tissue. Tendon explants overcome some of these limitations and have thus emerged as a valuable tool for the discovery and study of mechanisms associated with tendon homeostasis and pathophysiology. Tendon explants retain native cell-cell and cell-matrix connections, while allowing highly reproducible experimental control over extrinsic factors like mechanical loading and nutritional availability. In this sense tendon explant models can deliver insights that are otherwise impossible to obtain from in vivo animal or in vitro cell culture models. Purpose: In this review, we aimed to provide an overview of tissue explant models used in tendon research, with a specific focus on the value of explant culture systems for the controlled study of the tendon core tissue. We discuss their advantages, limitations and potential future utility. We include suggestions and technical recommendations for the successful use of tendon explant cultures and conclude with an outlook on how explant models may be leveraged with state-of-the-art biotechnologies to propel our understanding of tendon physiology and pathology

Tendon explant models for physiologically relevant invitro study of tissue biology - a perspective

Background: Tendon disorders increasingly afflict our aging society but we lack the scientific understanding to clinically address them. Clinically relevant models of tendon disease are urgently needed as established small animal models of tendinopathy fail to capture essential aspects of the disease. Two-dimensional and three-dimensional cell and tissue culture models are similarly limited, lacking many physiological extracellular matrix cues required to maintain tissue homeostasis or guide matrix remodeling. These cues reflect the biochemical and biomechanical status of the tissue, and encode information regarding the mechanical and metabolic competence of the tissue. Tendon explants overcome some of these limitations and have thus emerged as a valuable tool for the discovery and study of mechanisms associated with tendon homeostasis and pathophysiology. Tendon explants retain native cell-cell and cell-matrix connections, while allowing highly reproducible experimental control over extrinsic factors like mechanical loading and nutritional availability. In this sense tendon explant models can deliver insights that are otherwise impossible to obtain from in vivo animal or in vitro cell culture models. Purpose: In this review, we aimed to provide an overview of tissue explant models used in tendon research, with a specific focus on the value of explant culture systems for the controlled study of the tendon core tissue. We discuss their advantages, limitations and potential future utility. We include suggestions and technical recommendations for the successful use of tendon explant cultures and conclude with an outlook on how explant models may be leveraged with state-of-the-art biotechnologies to propel our understanding of tendon physiology and pathology

Serum deprivation limits loss and promotes recovery of tenogenic phenotype in tendon cell culture systems

Current knowledge gaps on tendon tissue healing can partly be ascribed to the limited availability of physiologically relevant culture models. An unnatural extracellular matrix, high serum levels and random cell morphology in vitro mimic strong vascularization and lost cell elongation in pathology, and discord with a healthy, in vivo cell microenvironment. The thereby induced phenotypic drift in tendon-derived cells (TDCs) compromises the validity of the research model. Therefore, this research quantified the extracellular matrix (ECM)-, serum-, and cell morphology-guided phenotypic changes in tendon cells of whole tendon fascicle explants with intact ECM and TDCs cultured in a controlled microenvironmental niche. Explanted murine tail tendon fascicles were cultured in serum-rich or serum-free medium and phenotype was assessed using transcriptome analysis. Next, phenotypic marker gene expression was measured in in vitro expanded murine tail TDCs upon culture in serum-rich or serum-free medium on aligned or random collagen I patterns. Freshly isolated fascicles or TDCs served as native controls. In both systems, the majority of tendon-specific genes were similarly attenuated in serum-rich culture. Strikingly, 1-week serum-deprived culture-independent of cell morphology-converged TDC gene expression toward native levels. This study reveals a dynamic serum-responsive tendon cell phenotype. Extracting fascicles or TDCs from their native environment causes large changes in cellular phenotype, which can be limited and even reversed by serum deprivation. We conclude that serum-derived factors override matrix-integrity and cell morphology cues and that serum-deprivation stimulates a more physiological microenvironment for in vitro studies

Serum deprivation limits loss and promotes recovery of tenogenic phenotype in tendon cell culture systems

Current knowledge gaps on tendon tissue healing can partly be ascribed to the limited availability of physiologically relevant culture models. An unnatural extracellular matrix, high serum levels and random cell morphology in vitro mimic strong vascularization and lost cell elongation in pathology, and discord with a healthy, in vivo cell microenvironment. The thereby induced phenotypic drift in tendon-derived cells (TDCs) compromises the validity of the research model. Therefore, this research quantified the extracellular matrix (ECM)-, serum-, and cell morphology-guided phenotypic changes in tendon cells of whole tendon fascicle explants with intact ECM and TDCs cultured in a controlled microenvironmental niche. Explanted murine tail tendon fascicles were cultured in serum-rich or serum-free medium and phenotype was assessed using transcriptome analysis. Next, phenotypic marker gene expression was measured in in vitro expanded murine tail TDCs upon culture in serum-rich or serum-free medium on aligned or random collagen I patterns. Freshly isolated fascicles or TDCs served as native controls. In both systems, the majority of tendon-specific genes were similarly attenuated in serum-rich culture. Strikingly, 1-week serum-deprived culture—independent of cell morphology—converged TDC gene expression toward native levels. This study reveals a dynamic serum-responsive tendon cell phenotype. Extracting fascicles or TDCs from their native environment causes large changes in cellular phenotype, which can be limited and even reversed by serum deprivation. We conclude that serum-derived factors override matrix-integrity and cell morphology cues and that serum-deprivation stimulates a more physiological microenvironment for in vitro studies.ISSN:1554-527XISSN:0736-026

Minimal mechanical load and tissue culture conditions preserve native cell phenotype and morphology in tendon-a novel ex vivo mouse explant model

Appropriate mechanical load is essential for tendon homeostasis and optimal tissue function. Due to technical challenges in achieving physiological mechanical loads in experimental tendon model systems, the research community still lacks well-characterized models of tissue homeostasis and physiological relevance. Toward this urgent goal, we present and characterize a novel ex vivo murine tail tendon explant model. Mouse tail tendon fascicles were extracted and cultured for 6 days in a load-deprived environment or in a custom-designed bioreactor applying low magnitude mechanical load (intermittent cycles to 1% strain, at 1 Hz) in serum-free tissue culture. Cells remained viable, as did collagen structure and mechanical properties in all tested conditions. Cell morphology in mechanically loaded tendon explants approximated native tendon, whereas load-deprived tendons lost their native cell morphology. These losses were reflected in altered gene expression, with mechanical loading tending to maintain tendon specific and matrix remodeling genes phenotypic of native tissue. We conclude from this study that ex vivo load deprivation of murine tendon in minimal culture medium results in a degenerative-like phenotype. We further conclude that onset of tissue degeneration can be suppressed by low-magnitude mechanical loading. Thus a minimal explant culture model featuring serum-free medium with low mechanical loads seems to provide a useful foundation for further investigations. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res

Inhibition of ERK 1/2 kinases prevents tendon matrix breakdown

Tendon extracellular matrix (ECM) mechanical unloading results in tissue degradation and breakdown, with niche-dependent cellular stress directing proteolytic degradation of tendon. Here, we show that the extracellular-signal regulated kinase (ERK) pathway is central in tendon degradation of load-deprived tissue explants. We show that ERK 1/2 are highly phosphorylated in mechanically unloaded tendon fascicles in a vascular niche-dependent manner. Pharmacological inhibition of ERK 1/2 abolishes the induction of ECM catabolic gene expression (MMPs) and fully prevents loss of mechanical properties. Moreover, ERK 1/2 inhibition in unloaded tendon fascicles suppresses features of pathological tissue remodeling such as collagen type 3 matrix switch and the induction of the pro-fibrotic cytokine interleukin 11. This work demonstrates ERK signaling as a central checkpoint to trigger tendon matrix degradation and remodeling using load-deprived tissue explants.ISSN:2045-232

Inhibition of ERK 1/2 kinases prevents tendon matrix breakdown

Tendon extracellular matrix (ECM) mechanical unloading results in tissue degradation and breakdown, with niche-dependent cellular stress directing proteolytic degradation of tendon. Here, we show that the extracellular-signal regulated kinase (ERK) pathway is central in tendon degradation of load-deprived tissue explants. We show that ERK 1/2 are highly phosphorylated in mechanically unloaded tendon fascicles in a vascular niche-dependent manner. Pharmacological inhibition of ERK 1/2 abolishes the induction of ECM catabolic gene expression (MMPs) and fully prevents loss of mechanical properties. Moreover, ERK 1/2 inhibition in unloaded tendon fascicles suppresses features of pathological tissue remodeling such as collagen type 3 matrix switch and the induction of the pro-fibrotic cytokine interleukin 11. This work demonstrates ERK signaling as a central checkpoint to trigger tendon matrix degradation and remodeling using load-deprived tissue explants

Tendon response to matrix unloading is determined by the patho-physiological niche

Although the molecular mechanisms behind tendon disease remain obscure, aberrant stromal matrix turnover and tissue hypervascularity are known hallmarks of advanced tendinopathy. We harness a tendon explant model to unwind complex cross-talk between the stromal and vascular tissue compartments. We identify the hypervascular tendon niche as a state-switch that gates degenerative matrix remodeling within the tissue stroma. Here pathological conditions resembling hypervascular tendon disease provoke rapid cell-mediated tissue breakdown upon mechanical unloading, in contrast to unloaded tendons that remain functionally stable in physiological low-oxygen/-temperature niches. Analyses of the stromal tissue transcriptome and secretome reveal that a stromal niche with elevated tissue oxygenation and temperature drives a ROS mediated cellular stress response that leads to adoption of an immune-modulatory phenotype within the degrading stromal tissue. Degradomic analysis further reveals a surprisingly rich set of active matrix proteases behind the progressive loss of tissue mechanics. We conclude that the tendon stromal compartment responds to aberrant mechanical unloading in a manner that is highly dependent on the vascular niche, with ROS gating a complex proteolytic breakdown of the functional collagen backbone.ISSN:0945-053XISSN:1569-180