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

Abstract

1Aberrant matrix turnover with elevated matrix proteolysis is a hallmark of tendon pathology. While 2 tendon disease mechanisms remain obscure, mechanical cues are central regulators. Unloading of 3 tendon explants in standard culture conditions provokes rapid cell-mediated tissue breakdown. Here we 4 show that biological response to tissue unloading depends on the mimicked physiological context. Our 5 experiments reveal that explanted tendon tissues remain functionally stable in a simulated avascular 6 niche of low temperature and oxygen, regardless of the presence of serum. This hyperthermic and 7 hyperoxic niche-dependent catabolic switch was shown by whole transcriptome analysis (RNA-seq) to 8 be a strong pathological driver of an immune-modulatory phenotype, with a stress response to reactive 9 oxygen species (ROS) and associated activation of catabolic extracellular matrix proteolysis that 10 involved lysosomal activation and transcription of a range of proteolytic enzymes. Secretomic and 11 degradomic analysis through terminal amine isotopic labeling of substrates (TAILS) confirmed that 12 proteolytic activity in unloaded tissues was strongly niche dependent. Through targeted 13 pharmacological inhibition we isolated ROS mediated oxidative stress as a major checkpoint for matrix 14 proteolysis. We conclude from these data that the tendon stromal compartment responds to traumatic 15 mechanical unloading in a manner that is highly dependent on the extrinsic niche, with oxidative stress 16 response gating the proteolytic breakdown of the functional collagen backbone. tendon fibroblasts in a collagen-rich extracellular matrix (ECM). The fact that tendons bear 20 physiologically extreme mechanical stresses is reflected by its tightly packed and highly structured 21 ECM. The load bearing tendon core is comprised by hierarchically organized type-I collagen fibrils, 22 fibres and fascicles (or fibre bundles) [1], with fascicles representing the basic functional load-bearing 23 unit of stromal tissue [2,3]. Physiological mechanical signals are understood to regulate the lifelong 24 adaption of the tissue to individual functional demands [4][5][6][7][8], with a narrow gap between beneficial and 25 detrimental mechanical loads [9][10][11][12]. This threshold is often exceeded, with tendon disorders 26 accounting for 30 -50% of all musculoskeletal clinical complaints associated with pain [13]. 27Our basic knowledge of the molecular and cellular mechanisms behind tendon physiology and 28 pathology is limited [14][15][16], yet damage and inadequate repair are considered to be central to tendon 29 disease onset and progression [17][18][19]. The stimuli that trigger tissue healing responses remain unclear, 30 potentially being a direct effect of cellular activation by overloading or by unloading after isolated fibre 31 rupture with subsequently altered cell-matrix interaction [20]. However, the mechanisms of 32 overloading and underloading may not be mutually exclusive, with tissue damage resulting in localized 33 regions...

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

“…Freshly isolated fascicles served as native control immediately after extraction. Explanted fascicles were cultured deprived from load at standard cell culture conditions (37°C, 5% CO 2 ) in high‐glucose Dulbecco's Modified Eagle's Medium (HG‐DMEM; Sigma D6429) with or without 10% fetal bovine serum (FBS) (Gibco 26140079) for 6 days (Figure 1), as previously described 17 …”

“…However, culture systems using tendon‐derived cells (TDCs) with a reduced and therefore more controllable cell environment 15 require cell isolation and expansion to minimize ethical concerns by maximizing cell yield per animal or to overcome the limited availability of human tissue for research. Expansion requires culture conditions showing similarities with tendinopathic tissue: hypercellularity, disorganized cells with loss of elongated morphology in the culture flask, 7 and serum‐rich medium resembling neovascularization 17 . Under these conditions, tenogenic cell populations lose their original healthy phenotype (“phenotypic drift”), via either transdifferentiation of cells or disproportional proliferation of subpopulations, similar to tendinopathic scenarios.…”

“…Limiting phenotypic drift of tendon cells in culture has been tried before 15 by varying biophysical (eg, impose cell morphology using anisotropic substrates 12,21‐25 or mechanical loading 20 ), biochemical (eg, partial oxygen pressure or temperature 17 ), and biological factors (eg, hormones, growth factors or cytokines 26 ), while the precise combination, dosage and time‐dependent administration of various tenogenic stimulators remains to be determined. Nonetheless, blood serum, which provides a complex, poorly defined mixture of bioactive molecules, is widely used for in vitro TDC culture to increase cell proliferation and activity.…”

Shear sensor mechano-signaling determines tendon stiffness and human jumping performance

“…To identify the responsible ion channel, we focused on candidates associated with mechanosensitive cation channel characteristics that are highly expressed in both mouse tail tendons (35) and human Achilles tendons (36) (Fig. 3b).…”

A novel, open source, low-cost bioreactor for load-controlled cyclic loading of tendon explants

“…However, recent studies have been able to identify culture conditions that reduce the drift in mechanical properties and phenotype observed in tendon explants. 51,57,59…”