Release of pro-inflammatory cytokines from muscle and bone causes tenocyte death in a novel rotator cuff in vitro explant culture model

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Secondary joint damage is the process by which a single injury can lead to detrimental changes in adjacent tissue structures, typically through the spread of inflammatory responses. We recently developed an in vitro model of secondary joint damage using a murine rotator cuff explant system, in which injuries to muscle and bone cause massive cell death in otherwise uninjured tendon. The purpose of the present study was to test the ability cytokine‐targeted and broad‐spectrum therapeutics to prevent cell death and tissue degeneration associated with secondary joint damage. We treated injured bone‐tendon‐muscle explants with either interleukin‐1 receptor antagonist, etanercept, or dexamethasone (DEX) for up to 7 days in culture. Only the low‐dose DEX treatment was able to prevent cell death and tissue degeneration. We then identified a critical window between 24 and 72 h following injury for maximal benefit of DEX treatment through timed administration experiments. Finally, we performed two tendon‐only explant studies to identify mechanistic effects on tendon health. Interestingly, DEX did not prevent cell death and degeneration in a model of cytokine‐induced damage, suggesting other targets of DEX activity. Future studies will aim to identify factors in joint inflammation that may be targeted by DEX treatment, as well as to investigate novel delivery strategies. Statement of clinical significance: Overall, this work demonstrates beneficial effects of DEX administration on preventing tenocyte death and extracellular matrix degeneration in an explant model of secondary joint damage, supporting the clinical use of low‐dose glucocorticoids for short‐term treatment of joint inflammation. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:139–149, 2020

supporting

confidence: 85%

supporting

confidence: 85%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Lose‐Dose Administration of Dexamethasone Is Beneficial in Preventing Secondary Tendon Damage in a Stress‐Deprived Joint Injury Explant Model

Connizzo

Grodzinsky

2019

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The effect of mechanical stress on enthesis homeostasis in a rat Achilles enthesis organ culture model

Saito

Nakamichi

Yoshida

et al. 2021

Tendons and ligaments are jointed to bones via an enthesis that is essential to the proper function of the muscular and skeletal structures. The aim of the study is to investigate the effect of mechanical stress on the enthesis. We used ex vivo models in organ cultures of rat Achilles tendons with calcaneus including the enthesis. The organ was attached to a mechanical stretching apparatus that can conduct cyclic tensile strain. We made the models of 1‐mm elongation (0.5 Hz, 3% elongation), 2‐mm elongation (0.5 Hz, 5% elongation), and no stress. Histological evaluation by Safranin O staining and Toluidin Blue and Picro Sirius red staining was conducted. Expression of sex‐determining region Y‐box 9 (Sox9), scleraxis (Scx), Runt‐related transcription factor 2 (Runx2), and matrix metalloproteinase 13 (Mmp13) were examined by real‐time polymerase chain reaction and immunocytochemistry. Terminal deoxynucleotidyl transferase‐mediated deoxyuridine triphosphate biotin nick end‐labeling and live/dead staining and was conducted for evaluation of the apoptosis and cell viability. The structure of the enthesis was most maintained in the model of 1‐mm elongation. The electronic microscope showed that the enthesis of the no stress model had ill‐defined borders between fibrocartilage and mineralized fibrocartilage, and that calcification of mineralized fibrocartilage occurred in the model of 2‐mm elongation. Sox9 and Scx was upregulated by 1‐mm elongation, whereas Runx2 and Mmp13 were upregulated by 2‐mm elongation. Apoptosis was inhibited by low stress. The results of this study suggested that 1‐mm elongation can maintain the structure of the enthesis, while 2‐mm elongation promotes degenerative changes.

Earlier proteoglycan turnover promotes higher efficiency matrix remodeling in MRL/MpJ tendons

Aggouras

Connizzo

2023

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While most mammalian tissue regeneration is limited, the Murphy Roths Large (MRL/MpJ) mouse has been identified to regenerate several tissues, including tendon. Recent studies have indicated that this regenerative response is innate to the tendon tissue and not reliant on a systemic inflammatory response. Therefore, we hypothesized that MRL/MpJ mice may also exhibit a more robust homeostatic regulation of tendon structure in response to mechanical loading. To assess this, MRL/MpJ and C57BL/6J flexor digitorum longus tendon explants were subjected to stress‐deprived conditions in vitro for up to 14 days. Explant tendon health (metabolism, biosynthesis, and composition), matrix metalloproteinase (MMP) activity, gene expression, and tendon biomechanics were assessed periodically. We found a more robust response to the loss of mechanical stimulus in the MRL/MpJ tendon explants, exhibiting an increase in collagen production and MMP activity consistent with previous in vivo studies. This greater collagen turnover was preceded by an early expression of small leucine‐rich proteoglycans and proteoglycan‐degrading MMP‐3, promoting efficient regulation and organization of newly synthesized collagen and allowing for more efficient overall turnover in MRL/MpJ tendons. Therefore, mechanisms of MRL/MpJ matrix homeostasis may be fundamentally different from that of B6 tendons and may indicate better recovery from mechanical microdamage in MRL/MpJ tendons. We demonstrate here the utility of the MRL/MpJ model in elucidating mechanisms of efficient matrix turnover and its potential to shed light on new targets for more effective treatments for degenerative matrix changes brought about by injury, disease, or aging.