Mechanical gating of tendon fibrogenic transcription in systemic sclerosis

Domain: biology
Published: 2026-03-13
Editorial outcome: minor_revision

Abstract
Fibrosis arises from aberrant tissue repair in systemic sclerosis and is characterized by progressive stiffening across multiple tissues, including tendons. How altered tissue mechanics sustain fibrotic remodeling remains poorly understood, in part because of limited experimental models that capture key biophysical features of fibrosis. Here we develop a modular cantilever-based mechano-culture platform that enables tendon-like constructs to be maintained under controlled static tension. Elevated matrix tension induces fibroblast-to-myofibroblast activation and scar-like phenotypes in vitro. Analysis of preclinical and clinical models of systemic sclerosis reveals that increased three-dimensional matrix stiffness inversely correlates with transcription of major profibrotic collagens, while positively regulating genes associated with stromal–immune interactions. Co-culture with bone marrow–derived macrophages overrides tension-dependent suppression of matrix gene expression, suggesting that immune cues can supersede mechanical checkpoints. These findings demonstrate how tissue mechanics orchestrates reciprocal interactions between stromal and immune compartments to drive progressive fibrosis, and establish a reductionist platform for dissecting mechano-regulatory pathways in fibrotic diseases.

Introduction
Systemic sclerosis (SSc) is a chronic immune-mediated rheumatic disease, characterized by microvascular damage, non-resolving inflammation, and progressive fibrosis of connective tissues. (1-4) The disease represents significant clinical and socioeconomic burdens to patients, their caretakers and healthcare systems.(5-7) Globally, the prevalence of SSc is estimated to be around 10-30 cases per 100,000 population.(8) Despite the significant clinical advances in managing systemic sclerosis, musculoskeletal comorbidities contributes to the overall reduced quality of life of SSc patients.(9, 10) Tendon involvement occurs early in the development of SSc, and it is one of the strongest predictive factors of overall disease progression.(11, 12) While the aetiopathogenesis of SSc remains poorly understood, a complex picture of genetic susceptibility, small vessel damage, sustained autoimmune inflammatory and profibrotic activation and self-amplifying dysfunctional repair responses is slowly coming into focus.(9, 13) Nevertheless, relevant insights into molecular mechanisms underpinning tendon involvement are scarce, and further progress is potentially hindered by ethical concerns limiting access to tendon biopsies or scarcity in experimental models that mimic key aspects of the tendon fibrotic niche. (11, 14, 15)

The hallmark of SSc is progressive fibrosis that is dominated by persistent activation of resident stromal cells, and excessive deposition and crosslinking of the extracellular matrix (ECM). This process is thought to ultimately mediate self-amplifying activation loops accompanied by irreversible replacement of tissue stroma with a rigid, mechanically-strained connective tissue.(16-19) How extracellular matrix mechanical cues contribute to initiation or progression of fibrosis is an area of intense investigation.(19-21) In tendon, repetitive mechanical overloading disturbs the underlying homeostatic tension that is critical for maintaining matrix structure and function.(22) Injury responses are intimately linked to cell- and tissue-level mechanical homeostasis, with the extracellular matrix providing biophysical signals for appropriate remodeling and resolution responses.(22-24)

For instance, it is now evident that matrix stiffening precedes the onset of clinical fibrosis.(21, 25, 26) Whereas matrix mechanical cues are instrumental in the successful transition from activated wound healing programs to successful resolution, persistent aberrant matrix stiffening is implicated in instilling fibroblast activation and pathological tissue remodeling.(20) How these processes play out in the context of tendon involvement in SSc is unknown.

Engineered biomimetic in vitro tissue models provide an attractive avenue to explore the interplay of matrix mechanical cues and progression of fibrosis in a precise and controlled manner.(27) The most widely used technique for tuning matrix stiffness while simultaneously characterizing the magnitude of cell-generated forces involve measuring deformations of planar two-dimensional (2D) membranes or 3D hydrogel substrates as a proxy for physical forces.(28, 29) One major limitation of such approaches is the inherent assumption that more deformation translates to more traction forces, in addition to the confounding coupling of several multiple biophysical properties (e.g. stiffness and porosity). An alternative approach is combining synthetic elastic substrates of tunable stiffnesses and traction force microscopy (TFM). In this method, cells are seeded on deformable, non-degradable 2D planar surfaces that are patterned with small trackable markers, e.g. fluorescent beads or nanoparticles.(30, 31) Cells and the underlying substrate are sequentially imaged in a stressed (contractile) state, and again after cytoskeletal relaxation. The two images are analyzed to track the beads displacements and to quantify the traction forces that are needed to displace the fluorescent markers. Although the TFM methods are powerful in resolving forces with a subcellular spatial resolution, it requires significant experimental and computational resources that are complex, laborious and often not accessible to laboratories lacking engineering expertise.(32) Furthermore, cells in 2D planar in vitro models are highly polarized and often interact with the underlying substrate only on one side while in contact with cell culture medium on the other end.(33)

Hence, such reductionist approaches do not fully capture the complexity between form and function of connective tissues like tendons. In vivo, tendon stromal cells reside in highly aligned 3D niches that are rich in collagens and other proteinaceous components, such as proteoglycans, while being exposed to substantial levels of mechanical tension. Cellular tension in 3D environments can be modified by changing the intrinsic mechanical properties of the environment within which cells are encapsulated (e.g. hydrogel intrinsic stiffness), or by manipulating the extrinsic boundary stiffness by constraining the edges of isotropic fibrillar hydrogels (e.g. collagen biopolymers).(34, 35) Physically-constrained hydrogels culture models offer an attractive approach to reconstitute this complex mechano-reciprocity of tendon cell-ECM interactions in vitro.(36-40) In this approach, stromal cells are embedded in ECM-derived hydrogels that are anchored to rigid pillars to geometrically-limit isotropic contractions of the matrix, i.e. uniform compactions in all directions. As a result, cell-generated contractile forces compact the microtissue around the anchoring pillars, deflecting the free ends of each pillar inwards toward the center and driving cell-matrix alignment longitudinally along the axis of principal stresses. The shape of microtissues can be controlled by adjusting the geometry and spacing of the rigid pillars(41), while the tissue contractile forces can be quantified using beam theory or Hooke’s law.(42) Changing the compliance of the constraining pins by making it highly compliant (i.e. deflectable) or minimally compliant (i.e. rigid with minimal deflection), it is possible to tune the overall resistance of the matrix and the emergent effective stiffness.(36, 43) In turn, this resistance regulates the cellular contractility and the mechanical forces that the cells can exert on the hydrogel matrix.

Here, we hypothesize that elevated matrix tension mediates the persistence of fibrotic remodeling in SSc tendons. First, we engineered a modular mechano-culture platform that enables one to easily vary static mechanical tension of tendon-like mimetics, independently of changing bulk tissue properties, thus emulating the complex mechano-reciprocity of tendon structure-function relationship.

Next, we characterized tendons involvement in a transgenic mouse model approximating the systemic phenotypes of human SSc and used these models to explore the fibrogenic stromal-immune interactions in vitro. We conclude that mechanically-driven, feed-forward loops are likely to be a driving feature of tendon pathology in SSc.

Results
Development of mechano-culture tissue model for tendon mechanobiology
Tendons are mechanically-anchored fibrous tissues, wherein resident stromal cells are aligned longitudinally along the axis of mechanical tension.(44, 45) To recapitulate the tensional state of tendons in vitro, we engineered a modular, mechano-culture platform that supports manipulation and monitoring of cellular traction forces generated by 3D tendon-like hydrogel constructs under easily variable mechanical tension (Figure 1A and B). We constructed the device by assembling off-the-shelf components that can easily be sourced by non-engineering laboratories, thus circumventing the need for the often inaccessible methods such as photolithography or other microfabrication techniques.(46)

The device consists of two interlocked 12-well plates that are separated by a spacer. It is accessible from all sides to facilitate cell culture media replenishment and gas exchange. The upper plate contains long vertical medical-grade steel posts, which firmly lock into an optically clear bottom plate. To provide a hydrogel anchoring point, a ceramic bead is glued on top of each post. When the plates are locked together, the two vertical posts in each well are contained within an elliptical hydrogel reservoir. This design feature supports anchoring the 3D tendon-like tissues around the posts, while simultaneously maintaining tissue constructs in proximity to the floor of the bottom well for microscopic observations (Figure 1A).

In general, hydrogels stiffness can be modulated by increasing the monomer amounts or by increasing its crosslinking density. However, both approaches eventually manipulate bulk material properties; i.e. ligand density and presentation, and porosity. To decouple the effects of hydrogel rigidity from other bulk properties, we modulated the boundary rigidity of tethered collagen hydrogels by varying the base:curing agent ratios of anchoring PDMS mats (Figure 1B). This strategy facilitates tuning the hydrogel effective stiffnesse (i.e. increased resistance felts by cells), while keeping initial collagen amounts and crosslinking density constant.

First, we performed numerical simulations to explore which design parameters may have the largest impact on the steel post spring constant. Using finite element analysis (FEA) methods, we simulated the post transverse deflections in response to a 1000 µN force acting on the surface of the bead, while varying post geometries, PDMS mat properties and boundary conditions (Figure 1C). The cantilever post was modeled as a stainless steel material, whereas the bead and PDMS layer were set as linear elastic materials. The FEA model predicted that the PDMS stiffness and the active length of the post (i.e. the length from the surface of PDMS mat to the post’s free end, or in other words the PDMS thickness) have the strongest influence on the deflection of the posts (Figure 1D). Since the active length of the post is restricted by the design requirements and well dimensions, we reasoned that varying the PDMS stiffness is the best strategy to modulate boundary mechanics.

Next, we experimentally calibrated the post spring constant (k) when embedded in compliant (50:1) or rigid (30:1) PDMS mats. Using capacitive force sensors, we derived the post spring constant, which is the slop of the force-displacement linear relationship (Figure 1E and Supplementary figure A1A and B). Rigid posts were approximately 20-fold stiffer than the compliant ones, with measured spring constant of 23.5 (± 0.1) µN/µm compared to 1.34 (± 0.01) µN/µm for compliant posts. These empirically-derived values of spring constants were used to estimate the collective forces exerted by cell-populated, tensioned tissues under different mechanical boundaries.

To generate tendon-like constructs, we embedded tendon-derived stromal cells in type I collagen hydrogel. Cells compacted the polymerized hydrogel into dense fibrous tissue around the post anchoring spheres in each well (Figure 1A and F). Within two hours after seeding, tissue tension evidently increased and continued to rise by 3-fold reaching a plateauing maximum of 272 (± 60) µN at 24 h (p= 0.0014), (Figure 1G top, and Supplementary figure 1C). In that same experiment, we simultaneously tested the diffusion limits of the system by examining the rapid dynamics of tissue tension forces in response to stimulation with soluble factors.

We found that cell-mediated tissue tension was reduced after serum starvation, then increased or decreased within approximately 15 min of stimulation with the myosin activator, lysophosphatidic acid (LPA), or with non-muscle myosin II inhibitor, blebbistatin, respectively (Figure 1G and H). Next, we examined the sensitivity of the system in measuring relative differences in tissue tension forces of relatively quiescent versus activated stromal cells. We initiated the activation of stromal cells by continuously passaging rat tail-derived tendon cells on ultra-stiff tissue culture plastic which is known to induce long-term mechanical memory.(47-49) As we expected, tissue traction force significantly increased over passages by 67% at passage four (P4) and 117% at passage seven (P7) compared to passage two (P2), reaching a value of ~1353 µN at passage 14 (Figure 1I). Taken together, we established a 3D experimental platform comprising of tunable mechanical boundaries and tissue tensional states while presenting equal bulk material properties to cultured cells.

Reconstitution of fibrogenesis in tensioned tendon-like constructs
Numerous studies have implicated aberrant transforming growth factor  (TGF-) signaling in the initiation and progression of SSc.(50-55) Similarly, degenerative tendon diseases are increasingly viewed as fibro-inflammatory pathologies characterized by impaired expression of TGF superfamily and dysfunctional scarring.(56-58) TGF-β1 is considered a key profibrotic cytokine that mediates its fibrogenic effects, at least in part, by promoting the activation of “quiescent” resident stromal cells into highly-contractile myofibroblasts.(59-62) Motivated by this, we first sought to examine whether exogenous TGF-1 stimulation induces activation of naïve tendon stromal cells into a contractile phenotype within tensioned tissue constructs. Untreated stromal cells in reduced serum (i.e. 1% FBS) were used as controls (Figure 2A). Continuous treatment with TGF-1 elicited visible deflections of cantilever posts over a 72-hour period, with significant two-fold increase in contractile forces reaching a maximum value of 997 ± 141.1 µN at 48 hours. (Figure 2B and C). Whereas forces continued to rise after 48 hours under TGF-β1 treatment, tissue tension plateaued at 24 h at 511 ± 61 µN in the non-stimulated controls. However, these changes were not associated with alterations in metabolic activity, as measured by ATP assay (Figure 2D).

To correlate functional changes in cellular contractility to phenotypic activation of stromal cells, we performed immunofluorescence staining for -smooth muscle actin (-SMA); a cytoskeletal marker indictive of myofibroblasts. TGF-1-treated conditions had approximately three-fold significantly higher fluorescence intensities of -SMA than untreated controls, which is consistent with state switch to myofibroblasts (Figure 2E and F). Furthermore, quantitative analysis of cytoskeletal alignment and nuclear circularity of embedded cells revealed that TGF-1 stimulation induced higher degrees of cellular alignment and nuclear elongation along the axis of mechanical tension, reflecting active remodeling processes (Figure 2G and H). Collectively, these results underscore to the utility of the mechano-culture platform in recapitulating the hallmarks of pathological fibrosis in tensioned tendon-like tissue constructs.

Sustained mechanical tension recapitulates key aspects of pro-fibrotic activation in tendon-derived stromal cells
Progressive matrix remodeling in fibrosis results in structural changes, such as increased tissue stiffness and tension, which trigger feed-forward, self-sustaining activation loops.(13, 48, 63) We examined how naïve tendon-derived stromal cells respond to sustained mechanical tension when cultured under variable mechanical boundaries, and how such mechanical feedback can impact the phenotype of tendon-derived fibroblasts (Figure 3A).

Under rigid mechanical boundaries (i.e. high matrix tension), we observed that rat tail tendon-derived cells generated tissue tensional forces that significantly increased by approximately 25-fold after two hours, reaching a maximum value of 6904 µN (SEM ± 1113) at 24 h (Figure 3B and C). At 48h, cells embedded within hydrogels tethered to compliant boundaries (Post = 1.08 µN/µm) exerted approximately 5 nN per cell [Mean= 5.6 nN  2.6, n= 6], while cells anchored to rigid boundaries (Post = 24.9 µN/µm) had ~21-fold higher forces [Mean= 117 nN  32, n= 7] (Figure 3C). Yet despite these profound differences in cellular contractility, these changes were not associated with alterations in cellular metabolic activity, as measured by total ATP assay (Figure 3D).

Next, we investigated whether sustained mechanical tension by itself can regulate the phenotype of naïve “quiescent” tendon stromal cells. We first explored the relative impact of boundary stiffness on the activation of myofibroblasts. Rigid boundaries induced an approximately 50% increase in -SMA immune staining intensities above compliant controls (Figure 3E and F), which was consistent at the mRNA levels (Figure 3G). Moreover, analyses of cytoskeletal alignment and nuclear orientation revealed higher degree of cellular alignment and nuclear elongation along the longitudinal axis in rigidly tethered constructs (Figure 3H-I). Unexpectedly, we found that mRNA expression of pro-fibrotic matrix proteins, Col1a1 and Col3a1, were significantly downregulated, whereas decorin (Dcn) mRNA was upregulated under rigid conditions in comparison to compliant boundaries (Figure 3J - L).

Similarly, expression of markers indicative of tenogenic linage commitment of stromal cells, (Scx and Tnmd) were significantly downregulated in highly-tensioned constructs (Figure 3K). There were no differences between the rigid and compliant boundaries in the expression of ECM crosslinking genes (Lox, Loxl2, Loxl4, Tgm2) or the mechanosensitive transcription factor Mrtfa (Supplementary figure 4 A-B).

In sum, these results suggest that elevated matrix tension can feedback to independently regulate the pheno-conversion of tendon-derived stromal cells towards myofibroblasts, while appropriate tension is beneficial for tenogenic expression.

Systemic sclerosis-derived tendon cells are autonomously activated independent of matrix mechanics
Fibrotic involvement in SSc has been historically investigated in either easily accessible tissues e.g. skin, or in internal organs that are associated with high mortality rates such as lungs. Tendons are often involved early during the disease course, and tendon friction rubs have high prognostic value in predicting SSc progression over time.(64) Nonetheless, knowledge about how tendons are affected at the molecular level is scarce. This is primarily attributed to the considerable ethical concerns in obtaining research biopsies from a tissue with limited healing capacity which may further risk harming patients. Here, we give a snapshot on SSc-derived tendon tissue and stromal fibroblasts from a rare autopsy that has been donated to our laboratories after post-mortem examination (only one donor was available during the course of this study).

First, we examined the baseline gene expression in SSc tendon tissue samples using a panel of key matrisome-related genes and established markers of fibroblast activation implicated in fibro-inflammatory diseases.(18, 65-67) We compared mRNA expression levels in tendons from the SSc donor with healthy hamstring tendons and age cohort-matched diseased tendinopathic tissues from biceps and Achilles tendons (Figure 4A, Supplementary figure 7 A-B). SSc donor samples had baseline expression levels of COL1A1 transcripts that were significantly lower than age cohort-matched, non-SSc tendinopathic samples (P = 0.0265), (Figure 4A). Expression of COL1A1 mRNA in the SSc group was comparable to healthy tendons (p >0.9999), whereas VCAM1 transcripts were significantly higher in SSc samples compared to healthy hamstring controls (Supplementary figure 7 A-B). In contrast, there were no statistical differences between the SSc samples and comparison groups in the expression of COL3A1, DCN, or ACTA2 or the fibroblast activation markers (PDPN, TLR4, ICAM-1), (Supplementary figure 7 A-B).

We then explored the relative contribution of total collagen amounts and mature trivalent collagen crosslinks to tendon fibrogenesis by quantifying hydrolyzed collagen hydroxyprolines and mature hydroxyallysine-derived crosslinks: deoxypyridinoline (DPD) and pyridinoline (PYD), respectively, in tissue homogenates.

Results revealed no differences in total collagen content between the three tendon groups (Figure 4B). In contrast, SSc tendons had slightly higher amounts of PYD/DPD mature trivalent crosslinks (p> 0.069) and higher (T ) denaturation temperatures (p= 0.0552) compared to diseased onset tendinopathic tendons and healthy controls, respectively. While these differences were not statistically significant, they reflected large effect sizes (Cohen’s d) between SSc versus control tendons (Figure 4C, Supplementary figure 7C and D, Supplementary figure 8).

Having examined the baseline pro-fibrogenesis characteristics of SSc tendons, we next assessed the behavior of pathologically activated SSc tendon fibroblasts using our mechano-culture platform. We quantified tissue tensional forces of cell-populated, tendon-like constructs as a proxy measure of cellular global traction forces. For this purpose, we cultured anatomically-matched tendon fibroblasts derived from the SSc donor or healthy subjects under mechanically-compliant boundary conditions (Figure 4D and E). Over a 48-hour period of culture, we observed that SSc-derived fibroblasts showed progressive increase in tissue traction forces, akin to TGF-β1 stimulated normal tendon fibroblasts (Figure 4F, Figure 2C). At 24 hr time-point, SSc-derived tendon cells exerted on average 9 nN per cell [Mean= 8.9 nN  1.2, n= 15 tissues], while healthy controls had approximately a two-fold lower forces [Mean= 5 nN  0.8, n= 15 tissues]. To assess the generalizability of these findings to other fibroblast populations, we further extended this analysis to the extensively studied SSc-derived dermal fibroblasts. Indeed, SSc dermal fibroblasts progressively exerted tissue tensional forces over a 48-hour period that were approximately four-times higher than healthy dermal controls (Figure 4F). SSc dermal fibroblasts applied forces of approximately 23 nN per cell [Mean= 22.9 nN ± 7.5, n= 24], while healthy control cells had ~2-fold lower forces [Mean= 12.3 nN ± 7.6, n= 24]. Interestingly, in that same time frame, healthy and SSc-derived tendon cells exerted substantially less tissue traction forces compared to healthy and SSc-derived dermal fibroblasts, respectively. While the difference in the order of magnitude between SSc and healthy controls was similar in skin and tendon-derived fibroblasts, SSc dermal fibroblasts exerted higher traction forces compared to SSc tendon fibroblasts across all timepoints. This difference was more pronounced at 48 hrs; where SSc dermal cells applied approximately 5-fold higher forces compared to SSc-derived tendon cells. In comparison, healthy dermal fibroblasts exerted 3.5-fold higher forces relative to healthy tendon fibroblasts (Figure 4F).

Next, we characterized the fibro-inflammatory phenotypes of tendon and dermal resident stromal fibroblasts and examined whether SSc-derived cells respond to changes in boundary rigidity. Analysis of mRNA transcripts revealed that SSc-derived tendon cells populating the tethered constructs showed a gene expression signature typical of fibro-inflammatory activation in SSc, with altered expression of FNT(EDA), IL6, IL8, ACTA2, PDPN and TLR4 mRNA relative to healthy, non-SSc compliant controls (Figure 4 G-I). In comparison, SSc-derived dermal fibroblasts showed similar trends with upregulation of FN(EDA), IL6, IL8, ACTA2, and TLR4 transcripts. However, except for TLR4, these differences were not statistically significant in dermal fibroblasts, (Supplementary figure 8 A-D). Strikingly, we found that COL1A1 and COL3A1 mRNA were significantly downregulated in SSc-derived tendon cells compared to anatomically-matched healthy controls, irrespective of the boundary rigidity (Figure 4G). Moreover, COL1A1 mRNA was significantly downregulated in healthy controls under rigid boundaries reaching transcriptional levels comparable to SSc conditions. IL6, IL8, and ICAM1 transcripts were significantly increased in SSc-derived cells in response to dysregulated boundary mechanics. Consistent with our previous observations (in Figure 3L), COL1A1 expression in healthy tendon stromal cells showed strong dependency on boundary rigidity, which was not the case neither in diseased SSc tendon cells nor in dermal fibroblasts (Figure 4J, Supplementary figure 8). This suggests that SSc tendon fibroblasts may have reduced baseline expression of COL1A1, or that altering matrisome-related transcription in response to tensional cues is an intrinsic property of healthy tendon stromal fibroblasts. Together, these results demonstrate that SSc-derived tendon cells are intrinsically activated in a cell-autonomous manner, and may possess altered tension-sensitive transcription of matrisome and stromal-immune interactions markers.

Dysregulated mechanics characterize tendon involvement in Fosl-2(Tg) mouse model of SSc
To extend our findings from the limited clinical samples of SSc tendons, we turned to Fosl-2(Tg) transgenic mouse model. Unlike bleomycin-inducible models of fibrosis, Fosl-2(Tg) mice display spontaneous SSc-like multi-organ inflammatory and autoimmune phenotypes.(68-70) We have recently reported that Fosl-2(Tg) mice develop spontaneous systemic inflammation around the age of 13-17 weeks.(71) The disease course in these mice closely approximates what is observed in human SSc cases with regards to the disease onset and progression.(72) Whereas early stages of the disease in mice are dominated by dysregulated T cell activation, late stages are characterized by vasculopathy and extensive multiorgan inflammation and fibrosis (Figure 5A).(71)

To investigate tendon involvement in Fosl-2(Tg) mice, we focused our analyses on two cohorts: 6-8 week-old and older than 14 week-old mice; hereafter termed early and established phenotype, respectively. We first characterized the micromechanical properties of tail tendon fascicles (the functional unit of tendons) by performing ramp-to-failure tests using a custom-made horizontal uniaxial testing device (Figure 5B).(73) Structural and material properties of tendon fascicles were significantly different between Fosl-2(Tg) mice compared to wild-type (WT) littermate controls. While tendon CSA, stiffness, failure stress, failure strain, failure force and stiffness were comparable to WT during early stages of the phenotype, Fosl-2(Tg) mice with established phenotype displayed 23% significant average increase in elastic moduli relative to WT controls, [P= 0.0302] (Figure 5C, Supplementary figure 13).

Next, we sought to explore potential contributing factors to these differences in tissue mechanics. First, we profiled the mRNA expression of a focused set of key matrisome and ECM regulator genes that are known to be differentially regulated in fibrotic pathologies (Figure 5D). Relative mRNA levels were similar in Fosl-2(Tg) and WT early-stage cohorts. However, expression levels of Col1a1, Col3a1, Lox, and Loxl2 were significantly reduced in Fosl-2(Tg) tendons with established phenotypes compared to WT controls.

Moreover, Col1a1 expression showed strong and significant negative correlation with tendon E. moduli in the established phenotype group (r = 0.75, P = 0.002) (Figure 5E). In contrast, quantification of total collagen content with hydroxyproline assay revealed that Fosl-2(Tg) tendons at the established phase had significantly higher collagen content relative to the early phenotype groups: WT (P= 0.0222) and Fosl-2(Tg) (P= 0.0071) (Figure 5F). However, there were no differences in average collagen amounts between the Fosl-2(Tg) and WT groups within each phenotype cohort: Early (P= 0.7007) vs. Established (P= 0.2806) (Figure 5F).

Taking this into consideration, we reasoned that the biomechanical differences in the stiff Fosl-2(Tg) tendons could emerge from the formation of ECM crosslinks, rather than collagen accumulation. To test this hypothesis, we first analyzed the molecular thermal stability of Fosl-2(Tg) tendons using differential scanning calorimetry (DSC) endotherms. Fosl-2(Tg) tendons showed significantly higher (T ) denaturation temperatures compared to the established, age-matched WT controls (P< 0.0263), and to the early Fosl-2(Tg) phenotypes (P< 0.018), (Figure 5 G-I). This likely reflects higher degree of thermal stabilization of collagen molecules in Fosl2(Tg) tendons. Since the high thermal stability suggests that elevated collagen crosslinking may underlie the observed mechanical differences, we next quantified mature hydroxyallysine-derived crosslinks DPD and PYD in Fosl-2(Tg) tendons. We found that there were significantly higher amounts of PYD/DPD mature trivalent crosslinks in the established Fosl-2(Tg) tendons compared to WT controls (P< 0.047), (Figure 5J). Similarly, quantification of fluorescence intensities of a LOX-activatable collagen peptide-sensor revealed that Achilles tendons of Fosl-2(Tg) tendons with established fibrosis had considerably higher in-situ LOX enzymatic activity compared to WT controls (p= 0.0077), (Figure 5K and L).(74) These insights, taken together with the quantification of collagen amounts and DSC measurements, suggest that increased crosslinking in Fosl-2(Tg) tendons contributes to tissue-level changes in bulk mechanics (i.e. stiffening).

Overall, these findings support the premise of disrupted mechano-homeostasis in Fosl-2(Tg) tendons and point to potential transcriptional feedback between the expression of ECM-related genes and dysregulated tendon tissue mechanics.

Transcriptome of fibrotic Fosl-2(Tg) tendons is enriched in extracellular matrix and macrophages activation signatures
Having established the fibrotic nature of Fosl-2(Tg) tendons and that tissue stiffening is a feature of this phenotype, we next sought to explore the transcriptional landscape of tendon fibrosis in Fosl-2(Tg) mice in an unbiased fashion using bulk RNA-sequencing (Figure 6A). Principle component analysis (PCA) demonstrated that the largest source of variation between Fosl-2(Tg) transgenic and WT genotypes was in the established group (Figure 6B). Supporting this, pairwise differential expression analysis showed progressive increase in the number of differentially expressed genes (DEGs) between the Fosl-2(Tg) vs. WT comparison in the established group compared to the cohort in the early phase (Figure 6C).

We next searched for the biological pathways that are enriched in the DEGs using Enrichr. We found that the DEGs in the early and established groups were functionally enriched in processes related to Epithelial–mesenchymaCl transition (EMT), Inflammation (e.g. Inflammatory Response, Interferons response, etc.) and ECM (e.g. ECM organization, Collagen Biosynthesis, etc.) pathways (Figure 6D and E). To profile the biological processes (BP) underlying these pathways, we performed gene set enrichment analysis (Figure 6 F-I). Interestingly, ECM-related processes were positively enriched in the early cohort and negatively enriched in the established phenotype. Conversely, processes related to inflammation and immune responses were positively enriched in the established fibrotic cohort (Figure 6H-I, Supplementary figure 15 A and B). Next, we examined which genes were driving the ECM and immune signatures. We found that 12 genes of the collagen family of extracellular matrix proteins were downregulated in the Fosl-2(Tg) established fibrotic group compared to WT controls, including Col1a1, Col1a2, Col2a1, Col4a5, Col6a2, Col12a1 (Supplementary figure 15C). Eight genes surrogate of macrophage activation (e.g. Cd163, Retnla, Ccl9, Ccl7, Tlr4, Myd88) were upregulated in the Fosl-2(Tg) established fibrotic tendons (Supplementary figure 15D).

To further assess the macrophage activation signatures, we used CIBERSORT digital cytometry tool to computationally deconvolute constituent cellular fractions in the bulk RNA-sequencing dataset. We mapped the proportion of 13 cell types that were previously established to be resident in mouse tendons (Figure 6 J-L and Supplementary figure 16). Fractions of immune cells and Nerve cells 3 were significantly enriched in Fosl-2(Tg) fibrotic tendons relative to WT controls (Figure 6L).

Given that previous investigations of Fosl-2(Tg) transgenic mice have established that macrophage infiltration and polarization characterize fibrotic phenotype in these mice, we explored the generalizability of these transcriptional processes across different connective tissues.(70, 75) We performed comparative analysis of the transcriptome of Fosl-2(Tg) fibrotic tendons and lung. We used a previously published dataset that closely demonstrated lung involvement in Fosl-2(Tg) mice (Figure 6M). While there was moderate overlap of 341 genes between the DEGs of Fosl-2(Tg) tendon vs. lungs transcriptome (Figure 6N), the two datasets significantly overlapped in Gene Ontology (GO) terms related to fibrosis, macrophage activation, inflammation processes (Figure 6 O and P). Interestingly, predicted key upstream regulatory transcription factors (TFs) included Nfkb1 and Sp1 TFs, which are known regulators of inflammation and fibrosis, respectively (Figure 6Q). Similarly, we then performed a comparative analysis of the whole bulk transcriptome of the Fosl-2(Tg) tendons and capsular tissues from human patients with adhesive capsulitis.(76) We found strong and significant overlap between the two datasets (Supplementary figure 18 A-D). Of interest was that eight out of the 20 overlapped GO terms and signatures were related to matrisome, cell-ECM interaction and inflammatory processes (Supplementary figure 18 B). The TF-gene interactions predicted that only four transcription factors were enriched in the overlapped features between the two datasets. Interestingly, these TFs are known to be involved in tendon inflammation (RELA, NFKB1)(77), and in the cross-talk between fibroblasts and macrophages in fibrosis (SP1, SP3)(78), (Supplementary figure 18 C).

Collectively, this comprehensive profiling of the transcriptional landscape of Fosl-2(Tg) fibrotic tendons has concretely established that repression of ECM transcription and activation of inflammation and macrophages signatures are key features of tendon involvement in Fosl-2(Tg) model of SSc.

Dysregulated mechanics and macrophages co-culture differentially regulate stromal fibroblasts activation
Recently, we have reported that Fosl-2(Tg) mice develop multiorgan systemic inflammation phenotype which is associated with increased secretion of pro-fibrotic cytokines IL-10, IL-13, IL-5 and IL-6.(71) In the current work, we have further observed that the transcriptome of fibrotic Fosl-2(Tg) tendons is positively enriched in “macrophage activation” and “inflammatory responses” signatures, and that rigid boundary mechanics boosted mRNA expression levels of markers surrogate of stromal-immune interactions (i.e. IL6, IL8, and ICASM1) in human SSc-derived tendon stromal cells (Figure 4). Given these findings, we sought to investigate whether and how altered boundary mechanics impact the crosstalk between Fosl-2(Tg) tendon stromal fibroblasts and bone marrow-derived macrophages (BMDM), (Figure 7A).

To do so, we used our mechano-culture platform to condition tendon-derived fibroblasts from mice with established fibrosis to variable boundary rigidity, either as monocultures or in direct co-culture with bone marrow derived macrophages (BMDM) in a mix-and-match approach (Figure 7 A B and C, Supplementary figure 19). We then assessed the mRNA expression of key tendon matrisome and ECM regulator genes in Fosl-2(Tg) or wild-type (WT) cells. In monocultures, WT tendon stromal cells showed modest reduction in Col1a1 expression under rigid boundary relative to compliant controls. In contrast, total mRNA of Col1a1 in Fosl-2(Tg) fibroblasts remained unchanged in rigid boundary compared to compliant conditions (Figure 7D, Supplementary figure 20). Similarly, there was no change in Col3a1 or Dcn expression in fibroblast monocultures. Interestingly, co-culture of WT fibroblasts with BMDM increased the expression of Col1a1, which was more pronounced under rigid mechanical boundaries (P= 0.76 in compliant, P= 0.42 in rigid), and when WT fibroblasts were co-cultured with Fosl-2(Tg) BMDM (P= 0.04 in compliant, P= 0.03 in rigid), (Figure 7D, Supplementary figures 20-22). While similar trends were observed when Fosl-2(Tg)-derived fibroblasts were co-cultured with BMDM, expression of Cola1 was highly variable and statistically insignificant irrespective of the mechanical boundary conditions.

mRNA expression of Col3a1 and Lox followed similar trends to Col1a1, with one difference that Col3a1 expression was only statistically significant when WT fibroblasts were in co-culture with Fosl-2(Tg) BMDM under rigid boundaries (P= 0.03) (Figure 7D, Supplementary figures 20-22).

Similarly, co-culture with BMDM induced significant shifts in the expression of cross-linking enzyme Loxl2 in the conditions where Fosl-2(Tg) fibroblasts were in co-culture with WT or Fosl-2(Tg) BMDM under rigid boundaries (P= 0.05 and 0 .03, respectively) compared to mono-cultures (Figure 7E, Supplementary figures 23-26).

Next, we quantified how boundary rigidity and co-cultures with BMDM influence tissue tensional forces of Fosl-2(Tg) and WT tendon stromal cells in vitro (Figure 7F). Fosl-2(Tg)-derived tendon cells exerted significantly higher traction forces as early as four hours (P= 0.0006), which reached a maximum value of 640.2 µN (±104.83) at 24 hr (p <0.0001). In comparison, basal tensional forces generated by WT-derived fibroblasts were approximately four-fold lower than Fosl-2(Tg) cells, which plateaued after six hours of monoculture at 169.3 µN (±29.5) (Figure 7F, left). Direct cocultures with BMDM significantly enhanced traction forces generated by WT tendon fibroblasts, with WT-derived BMDM boosting traction forces by four folds, whereas co-culture with Fosl-2(Tg) BMDM significantly increased WT fibroblasts contractility to levels comparable to activated Fosl-2(Tg) fibroblasts at 24 hr timepoint (Figure 7F and G, Supplementary figure 27).

Collectively, these findings suggest that interaction between tendon stromal fibroblasts and macrophages may underpin the induction and progression of fibrogenesis and crosslinking-mediated stiffening in Fosl-2(Tg) mice. It underscores the powerful utility of our mechano-culture platform in unwinding the complex interplay between the mechanical boundary rigidity, evolved ECM tension and the stromal-macrophage interactions in models of tendon fibro-inflammatory diseases.

Methods
Ethical Statement: Human samples were collected with written informed donor consent with voluntary participation, in compliance with the requirements of Declaration of Helsinki, Swiss Federal Human Research Act (HRA), and Zurich Cantonal Ethics Commission (Approval numbers: 2015-0089, 2020-0119). Tissue harvesting from Wistar rats and animal experiments with the Fosl-2Tg mice were approved by Zurich Cantonal Veterinary Office (Permits: ZH007/2019 Fosl-2Tg, ZH265/14 - ZH239/17 Wistar rats).

Device assembly: The device was assembled by combining two standard 12-multiwell plates interlocked on top of each other. Plates were separated by a Poly(methyl methacrylate) spacer. The top plate contained two arrays of steel pins pairs (A20007120, inox-schrauben.de) embedded within a thick mat of PDMS. Each well in the top plate’s upper (A1-A4 wells) and lower (C1-C4 wells) arrays contains one pair of pins (i.e. 8 pairs in total per plate), while the central wells were left empty. The PDMS layer was casted on top of a of polycarbonate disc. This allows for controlling the PDMS thickness as well as the depth of pins into the bottom plates. Pins were (1 ± 0.006) mm in diameter, (20 ± 0.5) mm in length and spaced approximately 4 mm apart from each other. To provide a better gel anchoring point, a ceramic bead (116913050CF: Lysing Matrix D, MP Biomedical) with an average diameter of 1.4 mm was glued on top of each post using medical grade, low-viscosity ethyl-based instant adhesive (LOCTITE® 4011 Prism Medical CA: Ellsworth Adhesives, UK). The corresponding bottom plate encompasses a PDMS well which serve as reservoirs for casting cell-laden hydrogels. The 5mm x 8mm PDMS reservoirs wells were punched out of ~2 mm thick layer using a metal puncher and glued to a ~10 mm mat of 10:1 PDMS.

Calibration of steel pillars spring constants: Post spring constants were measured experimentally using a capacitive piezoresistive MEMS force sensor mounted on an XYZ micro-manipulating arm in a horizontal configuration (FT-RS1002 Microrobotic System: FemtoTools AG, Switzerland), as described elsewhere.(98, 99) The posts were individually calibrated by bringing the tip of the force sensor into contact with approximately the center of the glued spherical beads. Displacement force is measured while simultaneously pushing against the sphere to deflect the post. For each measurement, the post was displaced until the sensor force reached a value of 400µN or 4000µN for the compliant or rigid configurations, respectively. Hooke's law is used to calculate the spring constant ():

∆𝐹
𝑘 = (1)
∆𝛿

Quantification of traction forces: To quantify tissue traction forces, cell-populated gels were cultured under tension for the indicated time periods outlined in figures legends. Timepoint “zero” was defined as the moment the growth media is added to the polymerized collagen gels. Briefly, tissue tension was quantified by monitoring the post deflection every 2h for the first 4-6 hours and then every 24h for up to 3 days. Images of the beads capping the posts were acquired with an inverted, brightfield EVOS™ XL Core Imaging System (AMEX1000, Thermo Fisher Scientific) using a 2x or 4x objectives. These images were processed with a custom-written Matlab script (Matlab® R2018b, MathWorks, Inc.) to segment the two beads and calculate the distance between the centroids (in pixels). The deflection of the posts was determined for each time point and the distance between the two beads was rescaled to the initial inter-post distance (in µm) that was experimentally measured with the FemtoTools during the calibration spring constants. Finally, tissue traction forces were calculated with Hooke's law formula using the measured deflections (∆𝛿) and the average of spring constant of each post ().

Finite element simulation of microtissue contractility: Posts and the embedding PDMS mat model was implemented in Ansys Workbench (V. 16.2., ANSYS, Inc). The posts were modelled as stainless-steel material using the default settings of the software. Spherical beads were simulated as a linear elastic material with a density of 2400 kg/m3, a Young's modulus of 185 GPa, and the PDMS was modeled as a linear elastic material with a fixed Poisson's ratio of 0.499 and a Young's modulus of 5 kPa. A hexahedral mesh was used for the post and PDMS substrate while tetrahedral mesh was fitted for the capping bead. To save computational power, only one post was modelled, and a symmetry region was added to create the second post. For the boundary conditions, fixed constrains on all sides were chosen. As loading condition, a force acting on the surface of the beads in direction of the plane of symmetry was set. The magnitude of the force acting as loading conditions was set to 1000 µN. This force represents the force generated by the contracting cells in the collagen gel. The simulations were also done using a Neo-Hookean model for the material.

Fosl-2(Tg) mice experiments: Fosl-2(Tg) mice were generated by Sanofi-Genzyme as described elsewhere.(71) Briefly, a vector containing the murine Fosl2 gene (Exons 1 to 4, corresponding introns, and truncated UTRs) was randomly inserted into the genome under the control of the MHCI promotor H2Kb. The eGFP sequence was inserted in frame after the Fosl2 transgene as a marker with a self-cleaving peptide T2A (Thosea asigna virus 2A) inserted between the two coding sequences.(71) Mice were housed under specific pathogen-free conditions at the University of Zurich. Nontransgenic mice from the same breeding were used as controls.

Biomechanical testing of tendons: For biomechanical testing of tendons, mice were anesthetized and sacrificed according to the guidelines of the Swiss Animal Welfare Ordinance (TSchV) University of Zurich. Samples were then stored in a freezer until further analysis. On the day of mechanical testing, fascicles were extracted by holding the tail from its posterior end with surgical clamps, and gently pulling off the skin until bundles of fascicles were exposed. Each fascicle was examined under a microscope (Motic AE2000, 20x magnification) for visible signs of damage, and for measuring the external diameter. Specimens with frayed ends, visible kinks or diameters less than 90µm were discarded. Micromechanical tensile testing was performed using a custom-made horizontal uniaxial test device to generate load-displacement and load-to-failure curves (10N load cell, Lorenz Messtechnik GmbH, Germany). Briefly, two-centimeter fascicle specimens were carefully mounted and kept hydrated in PBS, as previously described (Snedeker et al., 2005). Each fascicle underwent the following protocol: pre-loading to 0.015 N (L0: 0% strain), 5 cycles of pre-conditioning to 1% L0, an additional 1% strain cycle to calculate the tangential elastic modulus, and then ramped to failure to 20% strain under a predetermined displacement rate of 1 mm/s. The load-displacement data were processed using a custom-written Matlab script (Matlab® R2018b, v. 9.5.0.944444, MathWorks, Inc.). Tangent elastic moduli were calculated from the linear region of stress-strain curves (0.5-1%). Nominal stress was estimated based on the initial cross-sectional area. Cross-sectional area was calculated from microscopic images assuming fascicles have perfect cylindrical shape.

Cell culture: For all experiments, cells were maintained in Dulbecco’s Modified Eagle’s Medium with L-glutamine, sodium pyruvate, and sodium bicarbonate (DMEM high glucose - D6429, Sigma-Aldrich), supplemented with 10% FBS, 1% Penicillin-Streptomycin, 1% MEM Non-essential Amino Acid Solution (M7145, SAFC Sigma-Aldrich) and 200 μM L-ascorbic acid phosphate magnesium salt (013-19641, FUJIFILM Wako Chemicals).

Isolation of tail tendon-derived stromal cells: Tail tendon-derived stromal fibroblasts were isolated from sexually mature 12 to 14 week-old Wistar rats, or Fosl-2(Tg) transgenic mice and their wild-type controls. Rat tail tenocytes were chosen as a model in proof-of-concept experiments because they originate from the same population of tendon somatic progenitor cells as load-bearing tendons (100), and are convenient in obtaining a large number of cells at low passages. After euthanasia, tail tendon fascicles were extracted under sterile conditions as described above in the biomechanical testing section. (101) Tissue was washed once in PBS, minced into small pieces, and digested overnight in 0.2% (wt/v) Collagenase D (11088866001, Roche) in DMEM-F12 medium supplemented with 1% (v/v) Penicillin-Streptomycin. The same isolation protocol was followed for isolating tendon-derived stromal fibroblasts from Fosl-2(Tg) mice.

Isolation and differentiation of bone marrow-derived macrophages: Bone marrow-derived macrophages were prepared as detailed elsewhere.(102) In brief, bone marrow cells were harvested by flushing femurs and tibias of sex-matched, littermate mice. Bone-marrow-derived macrophages (BMDMs) were generated in DMEM - high glucose media supplemented with 10% (v/v) heat-inactivated FBS, 1% (v/v) Penicillin-Streptomycin, and 50 ng/ml of recombinant murine M-CSF (315-02, PeproTech). Half of culture medium was replenished every 3-4 days. At day 7 post-induction, macrophages were collected and used in the co-culture experiments with tendon-derived stromal fibroblasts.

Isolation of tendon-derived stromal cells from healthy and SSc tendons: Hamstring tendons (Semitendinosus and Gracilis) were collected from otherwise healthy donors undergoing surgical autograft repair of their anterior cruciate ligaments.(103) SSc tendons were collected from a single donor postmortem at the time of autopsy. Hamstring, supraspinatus, infraspinatus, subscapularis, patellar and Achilles tendons were collected by autopsy technicians under the supervision of a senior pathologist. The patient fulfilled the American College of Rheumatology (ACR)/European League Against Rheumatism (EULAR) criteria for SSc.(104) The female patient was 76 years old, and had a disease duration of from first non-Raynaud-symptom of 21 years and was suffering from diffuse cutaneous SSc with a modified Rodnan skin score as a measure of skin fibrosis of 14/51 two days before her death. The patient was positive for high-titer anti-nuclear Abs on immunofluorescence with an AC-3 pattern. Consistently, she was positive for anti-centromere Abs on ELISA. She refused taking any disease-modifying anti-rheumatic agents including immunosuppressive drugs during the course of her disease. Donor information and demographics are summarized in (Supplementary table 1). Tendons were immediately placed in (DMEM)/F12 medium (D8437, Sigma-Aldrich) in the operating room and subsequently processed in the cell culture lab. Human tendon-derived stroma cells were isolated by collagenase digestion as described elsewhere.(105) Briefly, tendon samples were washed in PBS to remove blood or tissue debris. Surrounding fat, fascia or muscles were excised, and tendon tissue was cut into approximately 5mm x 5mm pieces followed by 6-12h digestion with Collagenase, Type I (17018029, Gibco™) at 37 °C. Isolated cells were allowed to grow in DMEM/F12 supplemented with 20% heat-inactivated fetal bovine serum (FBS - 10500, Gibco™), 1% (v/v) Penicillin-Streptomycin (P/S, P0781, Sigma-Aldrich) and 1% (v/v) Amphotericin B (15290018, Gibco™). Cells were maintained in a culture incubator set at 37 °C and 5% CO2, and fresh media were replenished every 3-4 days until cells reached 80% confluency in T75 culture flasks. Cells were routinely sub-cultured once during initial expansion and were used between passages 2 and 5 for all experiments.

Fabrication of cell-laden, tendon-like constructs: Tendon-derived cells were encapsulated in hydrogels by mixing with neutralized collagen solutions prior to gelation, and cultured for 3-5 days. The two time-points were chosen to allow for full compaction of constructs (day 3) and additional days for tissue maturation (day 5). Collagen gels were produced using rat tail collagen type I (High Concentration type I collagen - 354249, Corning®). A 2x acidic collagen solution was neutralized on ice to pH 7.2-7.4 with precooled 10× PBS (70011044, Gibco™) and 1M sodium hydroxide (S5881, Sigma-Aldrich). Next, this solution was mixed at ratio of 1:1 with a 2x concentrated cell solution to reach a final collagen concentration of 1.7 mg/ml. A 90 µl of the liquified cell-laden gel solution was added per PDMS reservoirs and was allowed to polymerize for 45 min, after assembling the plates. Plates were then carefully removed from the incubator and 1 mL of supplemented culture medium was added to each well. When indicated, the cells were treated with recombinant TGF-β1 (580702, BioLegend®), as specified in figure legends, and were cultured in growth media containing 1% FBS.

Tendon fibroblasts bone marrow-derived macrophages direct co-culture experiments:
For co-culture experiments, M-CSF pre-differentiated BMDM were pooled from 2-3 mice per genotype (WT or Fosl-2(Tg)). Pooled differentiated BMDM were mixed with WT or Fosl-2(Tg) tendon fibroblasts at 1:1 mixing ratio, in a mix-and-match fashion, and at a density of 4x106 cell/ml. Pre-mixed macrophage-fibroblast populations were encapsulated in collagen hydrogels and cultured under variable tension at a final seeding density of 1x106 cell/ml, as described above. Co-cultures were maintained in DMEM - high glucose media supplemented with 10% FBS, 1% Penicillin-Streptomycin, 1% MEM Non-essential Amino Acid Solution (M7145, SAFC Sigma-Aldrich) and 200 μM L-ascorbic acid phosphate magnesium salt. Tissue tension was quantified by monitoring the post deflection for up to 48 hours.

RNA extraction from human and Fosl-2(Tg) mice tendons: Tail fascicles were placed in 2mL Eppendorf Safe-Lock tubes, snap frozen in liquid nitrogen and stored at -80C until further assayed. On the day of isolation, tubes were placed immediately in dry ice. Samples were transferred to supercooled Spex microvial cylinder (6757C3, SPEX™ SamplePrep) containing 150µl of GENEzol™ reagent (GZR200, Geneaid). Samples were cryo-grinded in a bath of liquid nitrogen using FreezerMill (6870, SPEX™ SamplePrep) until fascicles were completely disrupted. Pulverized samples were collected by rinsing the tubes with 900µl GENEzol™, transferred to a clean 1.5 ml tube and kept in dry ice. To purify RNA, tissue homogenate was thawed and mixed well with 200µl of Chloroform (102445, Merck) at a mixing ratio of 1:5. Samples were then spun down for 15min (at 15,000g, 4°C). The upper RNA-containing aqueous phase was transferred to a clean 1.5 ml tube and mixed with one part of 70% ethanol. RNA cleanup was subsequently performed using PureLink™ RNA Micro Scale Kit (12183016, Invitrogen™), including an on-column DNA digestion step with DNase I (DNASE70, Sigma-Aldrich). For collagen gels, constructs were collected in Qiagen RLT Plus lysis buffer (1053393, Qiagen), and were snap-frozen in liquid nitrogen and stored at -80°C. Samples were disrupted by vertexing for 1 min. followed by homogenization using QIAshredder columns (79654, Qiagen). Total RNA was isolated using RNeasy Plus Micro Kit (74034, Qiagen), including a step to remove genomic DNA (Qiagen gDNA Eliminator spin columns). Cleaned RNA concentration and quality were determined by spectrophotometric determination of A260/A280 ratio. Samples were stored at -80 °C, and only samples with A260/A280 ratio of 1.8-2 were used for downstream RT-qPCR analysis.

Real-time quantitative PCR (RT-qPCR): Real-time quantitative PCR were performed using StepOnePlus Real-Time PCR System (4376600, Applied Biosystems™) and TaqMan Assays. 90 ng of total RNA were reverse transcribed to cDNA using High-Capacity RNA-to-cDNA Kits (4387406 or 4368814, Applied Biosystems™). RT-qPCR reactions were run with 2μl cDNA and 8μl of TaqMan® Mastermix (containing 5 μl Universal PCR Master Mix, 0.5 μl of TaqMan primer, 2.5 μl of ultrapure water) adding up to a total volume of 10μl. Primer details and assay IDs are listed in the online supplementary material (Supplementary Table 2). Reactions were carried out in technical duplicates. Relative expression was calculated using the comparative 2-ΔCT method.(106)

Bulk RNA sequencing: Total RNA was isolated using RNeasy Plus Micro Kit (74034, Qiagen), including a genomic DNA removal step with Qiagen gDNA Eliminator spin columns. RNA-Seq library preparation was carried out by GENEWIZ (Leipzig, Germany). Briefly, RNA quantity was measured using Qubit 4.0 Fluorometer (Life Technologies, Carlsbad, CA, USA), and its RNA integrity was checked with RNA Kit on Agilent 5300 or 5600 Fragment Analyzer (Agilent Technologies, Palo Alto, CA, USA). Ribosomal RNA (rRNA) was depleted using NEBNext rRNA Depletion Kit (Human/Mouse/Rat). RNA-seq libraries were constructed using NEBNext Ultra RNA Library Prep Kit for Illumina, following the manufacturer’s guidelines (NEB, Ipswich, MA, USA). Enriched RNAs were fragmented and first strand and second strand cDNA were subsequently synthesized. cDNA fragments were end repaired and adenylated at 3’ends, universal adapter was ligated to cDNA fragments, followed by index addition and library enrichment with limited cycle PCR. Sequencing libraries were validated using NGS Kit on the Agilent Fragment Analyzer, and quantified by using Qubit 4.0 Fluorometer. The sequencing libraries were multiplexed and loaded on the flow cell on the Illumina NovaSeq 6000 instrument (or equivalent) according to manufacturer’s instructions. Libraries were sequenced using a 2x150 Pair-End (PE) configuration v1.5. to a sequencing depth of at least 20 million reads. Image analysis and base calling were conducted by the NovaSeq Control Software v1.7 on the NovaSeq instrument. Raw sequence data (.bcl files) generated from Illumina NovaSeq was converted into fastq files and de-multiplexed using Illumina bcl2fastq program version 2.20. One mismatch was allowed for index sequence identification.

Data Preprocessing and Bioinformatics Analysis: Raw sequencing data were processed using R package ezRun, which is implemented within the SUSHI framework of the Functional Genomics Center Zurich (ETH Zurich and the University of Zurich).(107) Sequencing quality control was performed with with MultiQC (version 1.9).(108) Raw.fastq files were mapped to the Mus musculus genome reference (GENCODE GRCm39 – Release_M26-2021-04-20) with STAR aligner software.(109) Reads counts per gene were calculated using featureCounts function against the reference genome (GENCODE GRCm39).[49] Differential expression analysis was imputed by using the DESeq2 R package (version 1.36.0) with default parameters.(110) Pathway enrichment analysis was carried out using Enrichr.(111) Gene set functional enrichment was performed using eVITTA package in RStudio (112), and the gene set overrepresentation analysis (ORA) implemented within clusterProfiler package [v3.18.0].(113) Cellular deconvolution was performed using the CIBERSORTx tool (114, 115), and cell-type signature matrix of De Micheli et. al. (116). Volcano plots were visualized using VolcaNoseR package.(117) Overlayed comparison of RNAseq dataset was carried out with Metascape software.(118)

Pharmacological treatments of tissues with agonists and inhibitors: To test the dynamic contractility and temporal response to stimulation with soluble factors, cell-laden collagen hydrogels were formed and cultured in standard growth media for 24h as described above. Then constructs were serum-starved in 1% FBS for another 24h to reduce the basal levels of cytoskeletal tension. Cellular contractility was modulated by incubating tissues with Oleoyl-L-α-lysophosphatidic acid (LPA) at a final concentration of 20µM (L7260, Sigma-Aldrich), or (−)-Blebbistatin at a final concentration of 50µM (B0560, Sigma-Aldrich). Vehicle-alone controls were: PBS for LPA and DMSO for Blebbistatin in serum-reduced media.

Immunostaining and confocal fluorescence microscopy: Collagen constructs were fixed while still under tension with pre-warmed 4% neutral buffered formaldehyde solution (ROTI®Histofix 3105.2, Carl Roth) for 45 min at room temperature. Subsequently, tissues were permeabilized with 0.5% Triton X-100 (93418, Sigma) in PBS for 30 min, and blocked overnight with 3% bovine serum albumin (BSA - P6154, Biowest) in a humidified chamber at 4 °C. Samples were immersed in Image-iT FX Signal Enhancer blocking solution (I36933, Invitrogen™) for at least 30 min before overnight incubation with primary antibody solution at 4 °C. After washing, tissues were incubated with the appropriate fluorescently-conjugated, isotype-specific secondary antibody for 4 hours, and protected from light. Nuclei and actin filaments were counterstained with NucBlue and AlexaFluor-conjugated phalloidin, respectively. Samples were mounted in 120 µm Secure-Seal™ adhesive spacers and embedded in Agarose, low melting point (V3841, Promega). Immunofluorescence images were acquired with the iMic spinning disk confocal microscope, fitted with a Hamamatsu Flash 4.0 sCMOS camera and a SOLE-6 Quad laser (Omicron), using 10x (N.A. 0.4) and 20× (N.A. 0.75) objectives (Olympus UPLSAPO).

Multiphoton microscopy and second harmonic generation (SHG) imaging: Multiphoton microscopy and SHG imaging were performed on unfixed, 8 µm thick mouse Achilles tendon frozen sections. Frozen sections were equilibrated to room temperature for 10 minutes. LOX-inducible probe was added to the unfixed sections and allowed to incubate at 37 °C for 3 h in a humidified chamber.(74) Slides were washed twice in PBS for a total of 5 minutes, and subsequently fixed for 10 min in ice-cold acetone at -20 °C. Following fixation, sections were washed in PBS three times (each for 5 minutes), counterstained with DRAQ5 (62251, Thermo Scientific™) at a concentration of 1:1000. Slides were mounted in Immu-Mount medium and covered with coverslips. SHG imaging was performed on sections using a Leica TCS SP8 microscope equipped with a ×25 0.95 numerical aperture (NA) Water HCX IRAPO objective and a Mai Tai XF (Spectra-Physics) MP laser tunable from 720–950 nm. The LOX-inducible probe excitation was generated at approximately 760 nm, and detected at the emission wavelength with an external detector equipped with a filter for DAPI (460 nm). Sequentially, the SHG signal was acquired with an aligned condenser (0.55NA) using an 880 nm excitation laser, and detected around half the incident wavelength (435–485) with an opened pinhole. Laser power, photomultiplier tube (PMT) voltage, gain and offset were monitored and kept constant throughout each acquisition.(119)

Differential scanning calorimetry (DSC): Mouse tail tendons were extracted as described in the “Biomechanical testing” method section and were equilibrated in PBS during testing. Human tendon tissues were collected by cryo-sectioning from unfixed, OCT-embedded tissue blocks that were stored at -80 °C (8 – 10 sections of 20 -30 µm thickness). Tissue sections were washed extensively with PBS to remove the water-based OCT embedding medium, and were allowed to equilibrate in PBS. For DSC measurements, each mouse or human sample was blotted in Kimwipes® to remove excess PBS, and were subsequently weighed, and then hermetically sealed in a 40 µl aluminum crucible pan (30389221, Mettler-Toledo). Samples heat flow was scanned to 100 °C at 5 °C/min. After scanning, pan were punctured, dried and reweighted. Endotherms were analyzed to determine onset temperature (T ), peak temperature (T ), full-width at half-maximum (FWHM) using Origin 2023.

Quantification of mature cross-links: Tendon samples were thawed in ice and equilibrated in PBS. Tissues were reduced with 0.22 mM potassium borohydride (438472, Sigma-Aldrich) for one hour at room temperature, collected by centrifugation, washed twice with Milli-Q water, and lyophilized to dryness overnight. Next, samples were resuspended to 10 – 50 mg/ml and hydrolyzed in 6 M hydrochloric acid (258148, Sigma-Aldrich) for 20 hrs at 95 °C. One-fourth of the hydrolyzed volume was used for the collagen quantification assay. HCl was evaporated to dryness from the rest. Dry hydrolysate was reconstituted in water to a final concentration of 30 mg/ml to measure mature pyridinium cross-links (Pyridinolin PYD and Desoxypyridinolin DPD). PYD and DPD cross-links were quantified using competitive ELISA immunoassay (8010, Quidel Corporation, San Diego, USA) following the manufacturer's protocol and including the rapid vigorous washing step. Results were analyzed with 4-parameter calibration curve fitting equation using GainData® online software (Arigo Biolaboratories, https://www.arigobio.com/ELISA-calculator).

Total collagen content: Collagen content was quantified using QuickZyme total collagen assay (QZBtotcol, QuickZyme Biosciences) according to the manufacturer's protocol. Briefly, tendon fascicle hydrolysate was first diluted with Milli-Q water to a final concentration of 4M HCl (1 volume hydrolysate : 0.5 volume water). Samples were further diluted 1:100 with 4M HCl to be in the linear range of the collagen standard curve. Diluted samples were pipetted to 96-well plates and incubated with Assay Buffer and Detection Buffer as specified in the manufacturer's protocol. Colorimetric absorbance was measured at 570 nm using microplate spectrophotometer (Epoch, Biotek). Collagen content and the amount of mature cross-links were normalized to tissue dry weight (mg).

Statistics: All statistical analyses were performed using Prism 10 (Version 10.3.0, GraphPad Software) and DABEST-Matlab for the estimation statistics (Matlab® R2018b, MathWorks, Inc.). Samples were checked for normality distribution with Shapiro–Wilk test. Statistical differences were evaluated with Unpaired Student t-test with Welch's correction, Ordinary one-way or two-way ANOVA (followed by Holm-Šídák's multiple comparisons test), for normally distributed data. Kruskal-Wallis or Mann–Whitney tests were used in cases where the normal distribution criterion was not met. Non-parametric: Wilcoxon-test was used for paired data. Exact P-values and post-hoc tests are reported in the figure legends. Whenever applicable, we also report the magnitude of the effect (Cohen’s d effect size) and CIs. All figures for estimation statistics, permutation p-values, and further discussion on how we defined technical vs. biological replication units are included in the Supplementary Information "see Supplementary Discussion". We note that variability in replicate numbers (n) across some experiments is due to multiple factors. In mechano-culture experiments, this variability resulted from rupture or degradation of collagen hydrogels during the experiments. In Fosl-2(Tg) experiments, it arose from low RNA yield in Fosl-2 tendons, as tissue crosslinking makes RNA extraction particularly challenging. Additionally, Fosl-2(Tg) mice often develop a severe phenotype that necessitates euthanasia for welfare reasons, leading to a relatively high dropout rate compared with WT controls within each litter. When sample numbers were limited, we prioritized the most important readouts.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The authors have conducted an elegant study, designing a novel 3D mechano-culture system that allows culture of cells under variable static tension, to mimic fibrotic conditions. The mechanisms leading to fibrosis are poorly understood, and therefore development of physiologically relevant systems in which to investigate these mechanisms are paramount to better appreciate the interplay between ECM tension, fibroblast activation and fibrotic remodelling.\nIn this study, the authors report development and validation of a simple culture system in which stiffness experienced by cells can be altered while keeping collagen content constant. They then use this system to demonstrate increased mechanical tension induces fibrosis in tendon-derived cells, similar to the response seen when cells are treated with the pro-fibrotic mediator TGF-beta. They then compared the response of tendon cells from healthy donors with those derived from a patient with systemic sclerosis, demonstrating persistent fibrotic activation in SSc cells. Finally, they investigate factors influencing the altered tendon mechanics observed in a mouse model of systemic fibrosis, demonstrating this may be driven by alterations in collagen crosslinking, although this has not been confirmed. They also show that co-culture of fibroblasts and macrophages increased WT fibroblast traction forces, when cultured with Fra2 derived macrophages.\nWhile many aspects of this study are novel and have been investigated thoroughly, I have some concerns/comments regarding the validity and robustness of the study which need to be addressed – see comments below:\nMajor comments:\n1. A consistent, and rather unexpected finding, is that col1 gene expression is decreased in pro-fibrotic conditions, and this requires further investigation. Is it possible to measure collagen synthesis at the protein level in the hydrogels? The authors do report reduction in col1a1 gene expression in tendons from fra2 mice, providing some confidence that these results are not an artefact of the in vitro culture system, however this decrease in collagen gene expression is not associated with decreased hydroxyproline content (Fig.5f – it is not clear if this data is from the early or established group). Providing data showing expression of col1a1 in healthy and SSc human tendon samples, as well as hydroxyproline content would provide further confidence in these results.\n2. The authors present some limited evidence that the differences observed in mechanical properties in tail tendons from WT and Fra2 mice are due to alterations in collagen crosslinking, however these results need to be explored in more detail to be able to conclude that altered crosslinking drives the differences in mechanics seen. I strongly recommend that the authors measure specific crosslinks in the WT and Fra2 mice tails to establish if it is indeed altered crosslinking that is driving the differences in mechanical properties seen. If human samples are available for similar analysis that would strengthen the results further. It also seems counterintuitive that levels of cross-linking associated genes are decreased in fibrotic conditions which would imply decreased, rather than increased cross-linking. Please comment on this important point. How do the authors think that altered crosslinking may ultimately result in the pro-fibrotic activation of fibroblasts observed?\n3. It is unfortunate that fibrotic samples could only be obtained from 1 human donor, and the methods do not describe how this sample was obtained, or if donor characteristics were matched with healthy tendon samples. However, the authors have attempted to address this limitation by investigating response of fibrotic cells derived from a mouse model of systemic sclerosis. Please show representative images of hydrogel compaction comparing WT and Fra2 cells in Fig. 5. Did the authors establish if there were differences in α-SMA/F-actin and cytoskeletal alignment between cells from normal and fibrotic tendons (both in human and mouse experiments)? Were the same panel of genes assessed for both human and tendon experiments? Supplementary table 1 seems to be missing from the supplementary information.\n4. The co-culture experiments are not described in the methods, and the results of these experiments are not discussed in light of the other findings of this study, nor in regard to the relevant literature.\n5. Please check figure legends carefully – in Figure 4, panels are described that are absent from the figure itself. Please clarify if immunofluorescence was performed on these samples.\n6. Please check the manuscript carefully for typos, particularly the methods section", "reviewer_2": "Interesting study looking into how dysregulated matrix mechanics affect cell phenotype towards a pro-fibrotic phenotype using tendon-derived stromal cells and systemic sclerosis (SSc) as a model. Elegant methodology with the development of a new mechanoculture platform to support the investigation of the effect of matrix stiffness on the activation of fibroblasts, with validation of dysregulated matrix mechanics in the tendons of the Fra2 mouse model of systemic sclerosis, and use of tendon-derived stromal cells from Fra2 transgenic mice and wild type mice to identify functions affected by dysregulated matrix mechanics. The authors succeed to recreate a pro-fibrotic phenotype similar to the one induced by TGFβ through tuning of matrix stiffness and report increased matrix stiffness negatively correlate with major pro-fibrotic ECM genes expression and positively correlate with the expression of markers of stromal-immune interactions and that co-culture with bone marrow-derived macrophages can regulate gene expression of tendon-derived stromal cells independently of matrix mechanics. In addition, the developed mechanoculture platform has great potential for future studies in the mechanism of fibroblast activation through dysregulated matrix mechanics.\nMajor points\n1. How does the matrix stiffness generated by the compliant and the rigid PDMS mats relate to physiologic tendon matrix stiffness? How did you determine the rigid PDMS creates an “abnormal” or higher than physiologic matrix stiffness and the compliant substrate recreates the physiologic tendon microenvironment closer?\n2. Were technical replicates used for statistical analysis instead of biological replicates (except for the SSc where this was unavoidable)? Was a statistician consulted for the use of technical replicates instead of biological for the statistical analysis?\n3. Line 272 and Figure 5C – Since you are measuring the fascicles CSA and reporting differences in CSA between wild type and Fra2Tg fascicles why not calculate and plot failure stress instead of force to facilitate the comparison of properties between wild type and Fra2Tg fascicles? Or at least mention it in the text if there was no significant difference in failure stress measured between wild type and Fra2Tg fascicles.\n4. Line 472 – Isolation of tendon-derived stromal cells - Please include donor age and gender for SSc and healthy tissue. Were the healthy controls age-matched to the SSc donor? If not, please include in the limitations as ageing can also have an impact on cell phenotype and expression.\nMinor points\nLine 144 – Within hours after seeding, tissue tension evidently increased after two hours, and … - change to avoid repetition of within hours.\nFigure 1A legend – Sagittal section might be more accurate than cross section. A cross section view of the well could be included to show the initial set up view from the top/bottom of the well.\nFigure 1A – “PDMS matt” - mat is misspelled in the actual figure.\nFigure 1G or Line 148 – Was the increase and decrease statistically significant?\nFigure 1G – Was the provenance of these cells also rat tail tendon? Maybe add in the legend.\nLine 154 – “in in” repetition.\nFigure 1I – Is the “active forces” column representing the vehicle tissue tension after 48 h? As the magnitude does not appear to correspond to the magnitude of the data point in 1G.\nLine 177 – “consistent with” appears that part of the sentence missing.\nLine 190 – “the phenotype”\nLine 200 – Maybe better to specify immune staining intensities (rather than immune intensities).\nLine 207 – The way the sentence about the tenogenic markers is formulated it would seem Mkx is also significantly decreased which does not seem to be the case from Figure 3K – please amend accordingly.\nFigure 4C – why does line graph has n=24/group and the violin plot has 16/group – isn’t the source of the data the same presented in a different way?\nFigure 4D and E legend – The legend says immunostaining quantification and the text says mRNA expression and panels appear mislabelled/the legend includes panels that do not appear in the figure. Please amend appropriately.\nFigure 4 D-G – The x axis labelling is complex when the subcategories could just be labelled “ | compliant | rigid | ” since you have already colour-coded the healthy and SSc – just a suggestion. Same for Figure 5D.\nLine 244 – You are not specifying whether the dysregulation is observed under compliant or rigid boundary mechanics conditions which gives the impression that this dysregulation is being observed under non-dysregulated mechanics/compliant boundary mechanics conditions. This is not the case for IL6 and IL8 though which are not significantly different between healthy and SSc tendon-derived stromal cells under compliant conditions. Please specify which conditions you are referring to or remove IL6 and IL8 if they are not significantly regulated.\nLine 252 – Is it an inability of SSc cells to sense boundary rigidity or expression of COL1A1 is already lower in SSc cells so it does not get significantly lower in rigid conditions? As for other genes eg IL6, IL8, and ICAM a significant difference in expression is noted between healthy and SSc cells under rigid conditions but not compliant – appearing to show a response to boundary rigidity in SSc cells. You also mention in the next sentence SSc-derived tendon cells show stiffness-sensitivity of stromal-immune interactions markers – unless that was “and (absence of) stiffness-sensitivity of stromal-immune interactions markers”.\nLine 263 – “We have recently reported…” reference (65) is missing. Also, reference 65 – is missing a title, journal volume and pages.\nFigure 5D – Are the p values for Fn1 and Lox missing?\nLine 317 – “mirroring our observations in Fra2 tendons in vivo and SSc-derived tendon fibroblasts” but you report no significant changes in Col1a1 expression under rigid conditions in SSc cells (and Lox expression is not reported) and Lox expression in Fra2 tendons does not appear to be significantly different from WT unless there are some significant p values missing from the graphs. Please clarify sentence if needed.\nLine 334 – “Elevated rigidity” – compared to what? Physiologic matrix stiffness in tendons?\nLine 368 – As you are mentioning in line 370 the referenced paper in line 368 does not really investigate fibrosis and cell environments of increased stiffness so maybe using “This was further reported/This decreased transcription was further reported” or similar could be a better representation than “This was further confirmed”.\nLine 404 – Diameter and length details are missing.\nLine 405 – “Each well in the top plate’s upper and …”\nLine 412 – “mm” repetition\nDevice assembly paragraph (line 402) – The setup of the anchoring spheres is not included but would aid in the overall understanding of the setup of the spheres in relation to the posts.\nLine 437 – “software.” comma instead?\nLine 441 – to create\nLine 541 – 90 ng – space between number and unit\nLine 551 – “The then”\nLine 570 – Statistics\n- Two-way ANOVAs are also mentioned in the figure legends but not in the statistics paragraph.\n- (from major points) Were technical replicates used for statistical analysis instead of biological replicates (except for the SSc where this was unavoidable)? Was a statistician consulted for the use of technical replicates instead of biological for the statistical analysis?\n- Some experiments had a wide variation of sample numbers per group eg Fig 5D n=4-9. Was that due to loss of samples in the process resulting in some groups having less samples than others or sample additions where it was deemed to be needed or cell numbers/availability?", "reviewer_3": "This manuscript describes a substantial body of work that addresses mechanical factors that may be highly relevant to the tendon abnormalities which are a hallmark of systemic sclerosis patients. The authors have developed a culture technique that allows mechanics tension to be applied in vitro and using this methodology have defined effects on fibroblasts and extended previous work that shows how mechanical tension may promote the contractile phenotype and profibrotic properties of fibroblasts. A key over-arching question is the extent to which these findings may apply to a broader population of fibroblasts that those derived from tendons.\nSpecific comments:\n1. It would be helpful to explain more clearly how the mechanical forces are generated and different between cultures. Whilst the authors obviously have a very clear understanding it is less apparent to readers unfamiliar with the concepts of stiffness and elasticity and this should be explained to hep with later interpretation of the data, especially for mutant mice at different time points.\n2. More extensive comparison of SSc fibroblasts should be possible even though tendon samples may not be available routinely. It would be an important control experiment to explore whether dermal fibroblasts show the same or different responses to those observed for tendon derived cells.\n3. It is unreliable to draw conclusions from a single patient sample. It would be helpful if additional material could be obtained from a range of SSc patients and perhaps compared with other fibrosing disorders such as Dupuytren’s contracture that may be more available. Although 3 biologically independent experiments are performed it is not clear how tis was achieved with a single donor.\n4. I am somewhat confused by references to the fosl2 mutant mice as well as Fra2 and the authors should clarify this and how the different mouse strains were examined (page 29 line 449)\n5. The transcription factor MRTFA is considered critical in mechano-sensing in fibroblasts, and it would be helpful to have this included in the relevant gene expression and protein assays to explore the impact on myofibroblasts differentiation and contractility.\n6. The relative importance of motility, contractility and matricellular interaction in determining the contraction in this culture system should be considered as it is unlikely that cell contractility is a major determination of tendon biomechanics that is driven more by extrinsic skeletal muscle contraction. This may be a limitation of the current approach and should be clarified.\n7. The findings reported here concerning inflammatory cells interfering with normal tension induced down regulation of ECM may be relevant to other conditions such as adhesive capsulitis and this could be discussed."}, "author_response": "First, we thank the reviewer for their careful reading of the work. Several criLcal points were raised, and we appreciate the opportunity to elaborate on these findings.\nFig.5f in the original submission included only mice from the established group. To address this point, we have addiLonally quanLfied total collagen amounts in tendons from N= 14 mice of the early group (6-8 week-old) and added 9 mice to the established (>16 week-old) group (N= 17), [Figure 5F, RebuPal figure 1 A-B]. Confirming the observaLons in the original submission, there was no staLsLcal difference in total collagen amounts between the Fosl-2(Tg) and WT controls in the early (p= 0.7007), or established (p= 0.2806) groups. InteresLngly, tendons from the established Fosl-2(Tg) group had significantly higher collagen amounts compared to the Early groups: WT controls (p= 0.0222) and Fosl-2(Tg) (p= 0.0071). Furthermore, there was no staLsLcal difference in collagen amounts between the early and established wild-type groups (p= 0.3241), suggesLng that the observed difference in collagen amounts in Fosl-2(Tg) tendons are likely mediated by the Fosl-2 transgene, and likely not by confounding effects from the aging process. (Figure 5F, RebuPal figure 1 A-B).\nThis point is addressed in the results [Lines: 327 – 332], and discussion [Lines 550 – 561] secLons.\nNext, we performed a preliminary experiment where we isolated tendon stromal cells from the Fosl-2(Tg) and WT established phenotype and seeded these cells onto silicone mechano-variant substrates with elasLc modulus (E.) of 35 kPa. We chose this elasLcity range based on our previous work where we showed that ≈35 kPa sLffness reinstates a tendon-like transcriptomic signature and favors minimizing fibro-inflammatory acLvaLon of tendon stromal cells in vitro (in comparison with Lssue culture plasLc).(1) We reasoned that the downregulaLon of Col1a1 in established Fosl-2(Tg) tendons is not a cell-autonomous process, and that removing cells from their naLve, mechanically-stressed environment will alter Col1a1 mRNA expression. Confirming our hypothesis, we found that Fosl-2(Tg)-derived tendon stromal cells expressed significantly higher levels of Col1a1 mRNA transcripts compared to WT controls (RebuPal figure 1 C). This suggests that the suppression of collagen expression in Fosl-2(Tg) is potenLally mediated, at least in part, by the sLffening of the extracellular matrix and the associated changes in its biophysical cues (i.e. elasLcity, tension, confinement, etc..). For more discussion addressing the potenLal processes mediaLng this response, we refer the reviewer to our response to their point no. 2 below. (SecLon: Mechanical confinement and RNA polymerase II repression of transcripLon).\nNext, to get a more global overview on the expression of collagens in Fosl-2(Tg) tendons (and to exclude that the observed negaLve correlaLon between Lssue sLffening and fibrogenic transcripLon might have risen from “bias” in our selecLon of Col1a1 gene), we further performed bulk RNA-seq on tendon fascicles from 28 mice [early phenotype N= 15 (7 WT + 8 Tg) and established phenotype N= 13 (6 WT + 7 Tg)]. The findings of the RNA-seq analysis are discussed in detail elsewhere, however, here we only highlight the findings that are relevant to the reviewer’s quesLons:\nA) Over-representaLon analysis (ORA) of the differenLally expressed genes showed that tendons from the established phenotype had significant over-representaLon of Gene Ontology (GO) terms related to extracellular matrix processes (GO:0030199 collagen fibril organizaLon, GO:0030198 extracellular matrix organizaLon) in the upregulated genes of the Early cohort and in the downregulated genes of the established phenotype (RebuPal figure 1 D-E, New Figure 6 D-I, New supplementary figure 15 A-D).\nB) A closer look at the genes behind these GO terms revealed that genes related to the Collagen family of matrix proteins are mostly overrepresented in the GO terms. Specifically, Col1a1, Col1a2, Col2a1, Col4a5, Col4a4, Col8a1, Col11a1 were all significantly downregulated in Fosl-2(Tg) tendons compared to WT controls in the Established phenotype (RebuPal figure 1 F, New supplementary figure 15 C).\nOverall, this finding further strengthens our original observaLons based on the transcripLon of Col1a1.\n“Providing data showing expression of col1a1 in healthy and SSc human tendon samples, as well as hydroxyproline content would provide further confidence in these results.”\nAs suggested by the reviewer, we assessed the mRNA expression of Col1a1 and Col3a1 and quanLfied total collagen amounts in healthy hamstring tendon controls, non-SSc diseased tendinopathic (biceps and Achilles) and SSc human tendon samples (tendons of different anatomical origins from the single SSc donor). The diseased tendinopathic samples were all from a cohort group of more advanced age (65-80 year-old), in order to bePer match the age of the SSc donor.\nResults showed that SSc tendon samples had baseline expression levels of Col1a1 transcripts that are significantly lower than the age-matched, non-SSc tendinopathic samples (p= 0.0265). InteresLngly, baseline Lssue expression of Col1a1 mRNA in the SSc donor was similar to the healthy tendons group (p > 0.9999) (RebuPal figure 1 G, New Figure 4 A, New supplementary figure 7 A and B).\nNext, we measured total collagen amounts by quanLfying hydroxyprolines in hydrolyzed Lssues. Results showed no differences in collagen content between the three tendon groups (RebuPal figure 1 H, New Figure 4 B). In contrast, SSc and tendinopathic tendons had higher amounts of mature PYD/DPD trivalent crosslinks compared to healthy controls (albeit not staLsLcally significant, p >0.06), (RebuPal figure 1 I, New Figure 4 C). It is important to menLon that while the tendinopathic and SSc samples were age-matched, all SSc samples were derived from a single biological donor, and as such these results should be interpreted with cauLon.\nThis point is addressed in the results [Lines: 239 – 259], and in the discussion secLons.\nRebuttal figure 1 | Analysis of collagen expression and total amounts in Fosl-2(Tg) mice and human tendons.\n(A) Baseline Col1a1 mRNA expression in fresh frozen Fosl-2(Tg) and wild-type control. (N = 6-9 mice/group, with each data point representing ΔCt value, horizontal lines indicate the median, Two-way ANOVA (genotype, disease stage) with Holm-Šídák's multiple comparisons post-hoc test). All individual gene expression is shown normalized to Anxa5 and Gapdh reference genes. (B) Quantification of hydroxyproline content in tendons (N= 14 mice/genotype (Early), N= 16 mice/genotype (Established). Ordinary Two-way ANOVA with Holm-Šídák's multiple comparisons test.\n(C) mRNA expression of Col1a1 in freshly isolated tendon stromal cells seeded on 35 kPa PDMS mechanovariant substrates. (N= 4 replicates/group from 2 experiments), with each data point representing ΔCt, horizontal lines indicate the median. (D-E) Pathway bubble plots depicting over-representation analysis (ORA) of multiple DEGs comparison between Fosl-2(Tg) vs. WT tendons in early and established phenotypes. (D) Upregulated genes. (E) Downregulated genes. (F) Normalized expression of collagen genes in bulk RNA-sequencing of established Fosl-2(Tg) vs. wild-type tendons. (G) Baseline Col1a1 mRNA expression in snap frozen human tendons. Each data point representing ΔCt value, horizontal lines indicate the median. Kruskal–Wallis one-way ANOVA with Dunn's multiple comparisons post-hoc test). All individual gene expression is shown normalized to RPL13A and GAPDH reference genes. (H) Quantification of hydroxyproline content in human tendons. Horizontal lines represent the median. Kruskal–Wallis one-way ANOVA with Dunn's multiple comparisons post-hoc test. (I) Total amounts of mature trivalent crosslinks (PYD and DPD). Black horizontal lines indicate median values. (N= 13-15 mice/genotype (Early), N= 16 mice/genotype (Established). Ordinary Two-way ANOVA with Holm-Šídák's multiple comparisons test. For (G-I): (N = 3-6 independent donors/group, except for systemic sclerosis where different anatomical tendons were derived from a single donor.\nAbbreviations: (WT): Wild-type, (GO-BP) Gene Ontology – Biological Processes.\n2. he authors present some limited evidence that the differences observed in mechanical properLes in tail tendons from WT and Fra2 mice are due to alteraLons in collagen crosslinking, however these results need to be explored in more detail to be able to conclude that altered crosslinking drives the differences in mechanics seen. I strongly recommend that the authors measure specific crosslinks in the WT and Fra2 mice tails to establish if it is indeed altered crosslinking that is driving the differences in mechanical properLes seen. If human samples are available for similar analysis that would strengthen the results further. It also seems counterintuiLve that levels of cross-linking associated genes are decreased in fibroLc condiLons which would imply decreased, rather than increased cross-linking. Please comment on this important point. How do the authors think that altered crosslinking may ulLmately result in the pro-fibroLc acLvaLon of fibroblasts observed?\nWe thank the reviewer for raising another important point. We agree that measuring collagen cross-links provides a more direct readout of crosslinking acLvity, and thus may bePer explain the mechanical phenotype.\nTo address this point, and as requested by the reviewer, we opLmized an enzyme-linked immunosorbent assay (ELISA) to quanLfy levels of total mature trivalent pyridinium cross-links (Pyridinolin PYD and Deoxypyridinolin DPD) in Fosl-2(Tg) mice tails and human tendons. The assay opLmizes a commercial kit that provides a highly sensiLve method to quanLfy PYD and DPD crosslinks for evaluaLon Pyd in urine (as a surrogate for bone turnover) using a specific anL-PYD anLbody.(2-5) Furthermore, we directly measured the in-situ enzymaLc acLvity of lysyl-oxidase (LOX) using a fluorescent LOX-acLvatable collagen pepLde-sensor probe and two-photon confocal microscopy.(6)\nCrosslinks (mouse): We performed the pyridinium cross-links ELISA on (N= 13-15 mice/genotype (Early), N= 16 mice/genotype (Established). Results showed Fosl-2(Tg) tail tendons with established fibrosis had indeed significantly higher PYD + DPD crosslinks compared to age-matched, wild-type controls (p= 0.0471) and to Fosl-2(Tg) with early phenotype (p< 0.0001), (RebuPal figure 2A, New Figure 5J). Similarly, quanLficaLon of fluorescence intensity of the LOX-acLvatable collagen pepLde-sensor revealed that Achilles tendons of Fosl-2(Tg) tendons with established fibrosis had considerably higher in-situ LOX enzymaLc acLvity compared to WT controls (p= 0.0077), (RebuPal figure 2B, New Figure 5K and L). Taken together, these addiLonal insights, together with the quanLficaLon of collagen amounts and DSC measurements, suggest that increased crosslinking in Fosl-2(Tg) tendons contribute Lssue-level changes in bulk mechanics (i.e. sLffening).\nWe have now updated the results secLon [Lines: 340 – 351] to reflect these new insights.\n“If human samples are available for similar analysis that would strengthen the results further.”\nCrosslinks (human): Furthermore, as briefly discussed in point no. 1, we quanLfied total collagen amounts and mature crosslinks in the limited human SSc samples (4 different anatomically disLnct tendons from a single donor), and compared them to tendinopathic tendons (biceps and Achilles, N= 6) and non-diseased controls (hamstring controls, N=3), (RebuPal figure 1 I, New Figure 4 C). There was no difference in total collagen amounts between the different tendon groups. However, similar to Fosl-2(Tg) Lssues, SSc and tendinopathic tendons had higher amounts of PYD/DPD crosslinks compared to non-diseased controls (albeit not staLsLcally significant; P= 0.0697 and P= 0.0644, respecLvely).\nAddiLonally, we performed DSC on the human samples. SSc tendons showed an increase in DSC Onset temperature (Median temp= 66.17 ℃) compared to diseased (Median temp= 62.46 ℃, p= 0.0552) and non-diseased controls (Median temp= 63.67 ℃, p= 0.7701), although again these differences were not staLsLcally significant (RebuPal figure 2 G and H, New Supplementary figure 7 C and D).\nOverall, the results suggest that human SSc tendons showed trends comparable to Fosl-2(Tg) tendons with established fibrosis, however we interpret these observaLons with cauLon, also within the manuscript, given the very small sample size.\nWe have now updated the results secLon [Lines: 250 – 259] in light with these new insights.\nRebuttal figure 2 | Analysis of collagen expression and total amounts in Fosl-2(Tg) mice and human tendons.\n(A) Total amounts of mature trivalent crosslinks (PYD and DPD). Black horizontal lines indicate median values. (N= 13-15 mice/genotype (Early), N= 16 mice/genotype (Established). Ordinary Two-way ANOVA with Holm-Šídák's multiple comparisons test, Cohen’s d = 0.771 [95.0%CI -0.353, 1.94]). (B) Quantification of fluorescent intensity levels of in-situ Lox collagen peptide-sensor in Established phenotype, Grey symbols indicate values for the representative images in (B). Two-tailed Mann Whitney test. (C) Endothermic onset temperature (℃), (D) peak temperature, and (E) Full-width at half-maximum (FWHM) of thermally-denatured tendons as measured by DSC (N= 14 mice/genotype (Early), N= 16 mice/genotype (Established). Ordinary Two-way ANOVA with Holm-Šídák's multiple comparisons test. (F) Total amounts of mature trivalent crosslinks (PYD and DPD). (G) Endothermic onset temperature (℃), (H) Peak temperature of thermally-denatured human tendons as measured by DSC. Ordinary Two-way ANOVA with Holm-Šídák's multiple comparisons test. For (F-H): (N = 3-6 independent donors/group, except for systemic sclerosis where different anatomical tendons were derived from a single donor.\n“It also seems counterintuiJve that levels of cross-linking associated genes are decreased in fibroJc condiJons which would imply decreased, rather than increased cross-linking. Please comment on this important point.”\nThis is a good point. Fosl-2(Tg) mice are geneLcally engineered whereby Fosl2 transgene was randomly inserted under the control of the major histocompaLbility complex class I anLgen H2Kb (MHCI) promotor. These mice develop spontaneous phenotype (e.g. dermaLLs and skin fibrosis) with variable onset of inflammaLon that depends on the mouse sex and age.\nAgainst the backdrop of this knowledge, we speculated that the low expression of Lox and Loxl2 could be due to the inherent variability of the onset of the inflammaLon/fibrosis phenotype. To begin to address this point, we checked our bulk transcriptomic dataset (Fra2(Tg) vs. WT) for the mRNA expression of all cross-linking associated genes that are known to be involved in connecLve Lssue fibrosis and/or tendon pathologies, including: Lysyl oxidase (Lox), Lysyl oxidase-like family (Loxl1, Loxl2, Loxl3, Loxl4) and Tissue transglutaminase-2 (Tgm2).\nWe performed the sequencing experiment with tendon fascicles from 28 mice [early phenotype N= 15 (7 WT + 8 Tg) and established phenotype N= 13 (6 WT + 7 Tg)] that are different from the ones used in the RT-qPCR data in (Figure 5D). InteresLngly (and confirming the RT-qPCR data), there was no staLsLcal difference in the mRNA differenLal expression of all cross-linking associated genes between the Fosl-2(Tg) and WT controls in the early phenotype (RebuPal figure 3 A, New Supplementary figure 17). In contrast, total Loxl2 and Loxl3 transcripts were significantly downregulated in established Fosl-2(Tg) tendons compared to the WT controls. Established Fosl-2(Tg) tendons had significantly lower expression of Lox, Loxl2, Loxl3, Loxl4, Tgm2 genes compared to the Fosl-2(Tg) early phenotype (RebuPal figure 3 A, New Supplementary figure 17). This data increased our confidence that the decreased expression levels of cross-linking associated genes in fibroLc Fosl-2(Tg) is not bias from the inherent variability of the phenotype onset.\nGiven that the LOX-acLvatable collagen pepLde-sensor revealed that Fosl-2(Tg) tendons with established fibrosis had considerably higher in-situ LOX enzymaLc acLvity, it is plausible that:\n1. the peak for the expression of cross-linking associated genes was somewhere in between the two Lme-points that we examined in this study; possibly between 9-13 week.\nFurthermore, the regulaLon of collagen crosslinking is a mulL-step process involving both intracellular and extracellular phases. The iniLal steps of lysine oxidaLon to form reacLve aldehydes occur intracellularly, while subsequent crosslink formaLon happens extracellularly. This separaLon of processes adds layers of regulaLon and potenLal points of modulaLon that can dissociate gene expression from actual enzymaLc acLvity.(7)\nand/or\n2. the downregulaLon of cross-linking associated genes is part of a Lssue sLffening-mediated global transcripLonal repression of mechanosensiLve and matrisome-dependent gene programs in Fosl-2 fibroLc tendons. (Discussed in detailed below. SecLon: Mechanical confinement and RNA polymerase II repression of transcripLon).\nRebuttal figure 3 | Expression of matrix crosslinking enzymes in Fosl-2(Tg) mice.\nA) Normalized RNA-Seq expression values of matrix crosslinking enzymes in tail tendons of (Fosl-2(Tg) vs. Wild-type) mice. (Fosl-2(Tg): N = 7 mice – WT: N = 6 mice). Horizontal lines indicate the mean. ANOVA Tukey's multiple comparisons test.\n“How do the authors think that altered crosslinking may ulJmately result in the pro-fibroJc acJvaJon of fibroblasts observed?”\nWe hypothesize that elevated mechanical tension/sLffening (due to altered crosslinking) mediates the pro-fibroLc acLvaLon of fibroblasts. While we acknowledge that our study did not delve into establishing direct causal relaLonship between elevated matrix tension, pro-fibroLc acLvaLon of tendon fibroblasts, and transcripLonal repression of matrisome and cross-linking associated genes, we speculate on several possibiliLes that may explain these counterintuiLve observaLons.\n1. Extracellular matrix and mechanical acLvaLon of growth factors:\nIt is widely appreciated that the ECM plays key roles in regulaLng cellular behaviour by tuning the availability and bioacLvity of bioacLve growth factors (GFs).(8) ECM - through its organizaLon, mechanical properLes, and proteolyLc acLvity – tunes the growth factors acLvity serving as a reservoir for morphogens that orchestrate Lssue paPerning during development or wound healing responses.(9, 10) Many GFs, including TGF-β's, VEGFs, FGFs, IGFs and HGF, bind with high affinity to different ECM components.(11-14) While GFs can be liberated from the ECM through the proteolyLc degradaLon of the ECM proteins, matrix mechanical tension and cell-generated forces also contribute to the regulaLon of GFs.(15)\nSeveral studies have shown that the mechanical state of extracellular matrix primes resident fibroblasts towards fibro-inflammatory acLvaLon by controlling the acLvaLon efficacy of ECM-bound growth factors.(16, 17) Of most relevance to this work is the mechanical acLvaLon of the pro-fibroLc growth factor TGF-β1. TGF-β1 is a potent growth that drives acLvaLon of stromal cells into pro-fibrogenic myofibroblasts. It is secreted by cells in the extracellular space in a complex, inacLve form as a homodimer that is non-covalently associates with its Latency Associated Pro-pepLde (LAP).(17-19) This process serves as a “safety” mechanism that prevents an unintended TGF-β1-mediated pro-fibroLc acLvaLon of fibroblasts, thus temporally uncoupling the release of TGF-β1 from its acLvaLon. In this work, cells encapsulated within rigid boundaries exerted higher tracLon forces, and it is plausible to speculate that cell tracLon forces might have led to the dissociaLon of latent TGF-β1 from its LAP domain.\nThis might lead to a feed-forward signaling loop that propagates the α-SMA-mediated cellular contracLlity and eventually contributes to the highly tensioned matrix state.\nHowever, while this hypothesis may explain the increase in α-SMA signal secondary to mechanical rigidity in our work, it does not explain the transcripLonal repression of Col1a1 in the rigid boundary condiLons. Recent work focusing on collagen-producing stromal cells in lung fibrosis revealed a counterintuiLve duality between Acta2 (i.e. α-SMA gene) and Col1a1 transcripLon. Using single-cell RNA-seq and bleomycin-induced mouse model of lung fibrosis, Tsukui et. al. showed that cells characterized by high Acta2 expression demonstrated low Col1a1 expression.(20) AddiLonally, the authors analyzed human clinical samples from idiopathic pulmonary fibrosis and systemic sclerosis lungs and found that ACTA2 transcripts poorly correlated with COL1A1 transcripLon at single-cell level.(20) Taken together (with the findings of our study), this suggest that α-SMA expression does not exclusively idenLfy pathogenic fibroblasts that produce the highest level of collagen type I. It raises the possibility that the simultaneous incorporaLon of α-SMA and the increase in expression of Col1a1 is potenLally not a universal phenomenon in all fibroLc diseases, or may reflect a prioriLzaLon of different cellular tasks during the natural course of disease progression.(21)\n2. Mechanical confinement and RNA polymerase II repression of transcripLon:\nAnother potenLal explanaLon for the observed pro-fibroLc acLvaLon of fibroblasts and the reduced transcripLon of collagen and crosslinking-related genes is that these processes are mediated through two disLnct mechanisms, and that the spaLal confinement of cells in the sLff, highly tensioned Lssues mediates transcripLonal repression of matrisome genes.\nSeveral lines of evidence from tendon and other biological systems corroborate this hypothesis. Experiments by Zhou, Franklin and colleagues using mouse embryonic fibroblasts revealed that proliferaLon of fibroblasts and expression of matrisome-associated genes (e.g. Serpine1 and Col2a1) is significantly limited by spaLal confinement.(22) The authors of this work used the term “physical space availability”; a synonymous term for spaLal confinement. They aPributed this space availability/confinement-dependent rewiring of gene expression programs to the differenLal acLvaLon of Hippo-YAP and TGF-β signaling pathways. SpaLally confined fibroblasts (i.e. high density culture) displayed higher fluorescent intensity of cytoplasmic YAP1 and downregulaLon of Serpine1 and Col2a1 mRNA transcripLon, whereas less confined cells (i.e. low density culture) exhibited increased fluorescent intensity of nuclear YAP1 and transcripLon of ECM genes.(22)\nAddiLonally, Work by Grinstein and colleagues demonstrated that the global transcripLon of Col1a1, Col3a1, and Mki67 (proliferaLon marker) is significantly repressed when tendons transiLon from cell proliferaLve-driven embryonic growth to ECM expansion homeostasis postnatally.(23) This phase of tendon development is accompanied by significant mechanical stress with increased accumulaLon of collagen matrix and Lssue tensioning resulLng in spaLal confinement of tendon cells. As tendons increase in length and ECM density, the cellular proliferaLon rate and transcripLon of ECM genes significantly decreased between postnatal day P0 and P35.(23) Similarly, Podolsky et. al. showed that although total collagen content was significantly higher in aged and fibroLc lungs, and Col1a1 and Col1a2 transcripts were universally suppressed in comparison to young non-fibroLc controls.(24) While these studies did not mechanisLcally explain the potenLal mechanical factors driving these observaLons, we speculate that the downregulaLon of cellular proliferaLon and Col1a1, Col1a2, and Col3a1 transcripts is driven, at least in part, by the spaLal confinement of tendon cells during the ECM expansion and/or sLffening.\nIn support of this argument, Dudaryeva et. al. engineered 3D micro niches with tunable confinement volume.(25) They subsequently showed that human mesenchymal stromal cells (MSCs) demonstrate marked decrease in 1) proliferaLon rates, and 2) YAP1 nuclear acLvaLon upon 3D confinement in comparison with 2D planar cultures.(25) Similarly, encapsulaLng tendon progenitor cells within sLff, highly crosslinked alginate niches spaLally confined tendon cells in 3D and restricted the degree of cellular spreading.(26) InteresLngly, spaLal confinement of tendon cells (in sLff niches) correlated with marked repression of Col1a1 transcripts compared to less-confined cells in so}er niches. Such phenotype of Col1a1 repression was also observed when kidney-like organoids are confined in 3D matrices and compared to non-confined cultures in air–liquid interface.(27) While we think all these studies provide circumstanLal evidence, it collecLvely points to a unifying principle by which spaLal mechanical confinement might mediate the global transcripLonal repression of mechanosensiLve and matrisome-dependent gene programs.\nThe precise molecular mechanism(s) by which mechanical confinement regulates global transcripLon rates sLll need to be worked out. However, one avenue that warrants further invesLgaLons is the potenLal crosstalk between mechanical cues and the RNA Polymerase II (RNAPII) enzyme. AcLve RNAPII is a hallmark of ongoing mRNA transcripLon elongaLon. It has been previously shown that sLffness-mediated mechanical stress in models of skin aging correlates with notable reducLon in cellular proliferaLon as well as in the phosphorylaLon RNAPII-S2P.(28) ChIP–seq experiments in these studies have confirmed a reduced genome occupancy of RNAPII in aged skin compared to young controls. It is indeed challenging to decouple the effect of aging processes from the aging-mediated Lssue sLffening and increased confinement in in vivo mouse models of aging. However, supporLng evidence from alternaLve mouse models of Lssue sLffening (i.e. age-matched Collagen XIV–/– and Tnx–/–) and from 3D organoid confinement have demonstrated the essenLal role of mechanical stress in reducing RNAPII occupancy and eventually in aPenuaLng global transcripLon.(28, 29)\nCollecLvely, we contemplate that the myofibroblasLc/fibrogenic acLvaLon and the transcripLonal repression of matrisome-related genes are likely mediated by two disLnct mechanisms.\nAn abridged version of these points is included in the revised manuscript in lines [485-542].\n3. It is unfortunate that fibroLc samples could only be obtained from 1 human donor, and the methods do not describe how this sample was obtained, or if donor characterisLcs were matched with healthy tendon samples. However, the authors have aPempted to address this limitaLon by invesLgaLng response of fibroLc cells derived from a mouse model of systemic sclerosis. Please show representaLve images of hydrogel compacLon comparing WT and Fra2 cells in Fig. 5. Did the authors establish if there were differences in α-SMA/F-acLn and cytoskeletal alignment between cells from normal and fibroLc tendons (both in human and mouse experiments)? Were the same panel of genes assessed for both human and tendon experiments? Supplementary table 1 seems to be missing from the supplementary informaLon.\nHuman systemic sclerosis donor: We apologize for the oversight in documenLng the sample provenance and procedure. Further, we acknowledge that including one human donor is a limitaLon of our study. Nonetheless, we consider that these specimens are excepLonally rare, and despite our best effort, only one systemic sclerosis autopsy had been available for research at our specialized terLary center over a period of six years.\nTo avoid any overstatement, we now emphasized the confounding effect of ageing and the single donor as a limitaLon in the discussion [Line: 542-546], which reads: “We acknowledge that we prioriJzed comparing the cellular tracJon forces and gene expression of SSc-derived cells to healthy cells originated from young donors, limiJng our ability to exclude the potenJal confounding effect of ageing.”\nWe have also updated the Methods secLons to include details about the collecLon process of the SSc donor. Line [685-694] reads as following:\n“SSc tendons were collected from a single donor postmortem at the Jme of autopsy. Hamstring, supraspinatus, infraspinatus, subscapularis, patellar and Achilles tendons were collected by autopsy technicians under the supervision of a senior pathologist. The paJent fulfilled the American College of Rheumatology (ACR)/European League Against RheumaJsm (EULAR) criteria for SSc.(104) The female paJent was 76 years old, and had a disease duraJon of from first non-Raynaud-symptom of 21 years and was suffering from diffuse cutaneous SSc with a modified Rodnan skin score as a measure of skin fibrosis of 14/51 two days before her death. The paJent was posiJve for high-Jter anJ-nuclear Abs on immunofluorescence with an AC-3 pa_ern. Consistently, she was posiJve for anJ-centromere Abs on ELISA. She refused taking any disease-modifying anJ-rheumaJc agents including immunosuppressive drugs during the course of her disease.”\nWT and Fosl-2(Tg) in vitro experiments: We have now included the representaLve images of hydrogel compacLon in supplementary figure [Supplementary figure 19 A-D]. Furthermore, we aPempted to perform immunostaining on α-SMA WT and Fosl-2 cells, as suggested by the reviewer. Unfortunately, despite our best effort we were unable to obtain reliable immunofluorescence data on mouse-derived cells due to the non-specific binding of primary anLbodies.\n“Were the same panel of genes assessed for both human and tendon experiments?”\nRT-qPCR gene panels: We used a limited panel of genes which included key matrisome-related and cross-linking associated genes that are most relevant to tendon biology (i.e. Col1a1, Col3a1, Lox, Loxl2, Loxl4, Tgm2). However, depending on the goal of the experiment, we have expanded the basic panel to include: 1) known markers indicaLve of tenogenic linage commitment of stromal cells (Scx, Tnmd, Mkx) as in (Figure 3K: Rat tendon-derived cells), or 2) established markers of fibroblast acLvaLon implicated in fibro-inflammatory diseases as in (Figure 4H and I: Human SSc cells).\nTo facilitate the comparability across figures, Lssues and cell types, we have included the expression of Lox, Loxl2, Loxl4, Tgm2 genes in rat tendon-derived cells in (RebuPal figure 4A, New supplementary figure 4A). We have also ploPed the normalized expression values of matrix crosslinking enzymes and fibroblast acLvaLon markers in Fosl-2(Tg) and wild-type control mice, which is collated in (RebuPal figures 3 and 4B, New supplementary figures 17 and 18A).\nSupplementary table 1 is now included in the Supplementary informaLon file.\nRebuttal figure 4 | Expression of matrix crosslinking enzymes and fibroblast activation markers.\n(A) mRNA expression of ECM crosslinking enzymes in rat tail tendon stromal cells tethered to different mechanical rigidities. (n = 8 replicates/group from 3 independent experiments, with each data point representing a ΔCt value of 2-3 pooled tissues, horizontal lines indicate the median, Two-tailed Mann Whitney test). All individual gene expression is shown normalized to Eif4a2 and Gapdh reference genes. (B) Normalized RNA-Seq expression values of fibroblast activation markers in tail tendons of (Fosl-2(Tg) vs. Wild-type) mice. (Fosl-2(Tg): N = 7 mice – WT: N = 6 mice). Horizontal lines indicate the mean. ANOVA Tukey's multiple comparisons test.\n4. The co-culture experiments are not described in the methods, and the results of these experiments are not discussed in light of the other findings of this study, nor in regard to the relevant literature.\nThank you for the catch. We have updated the Methods secLon with the co-culture experiments, which reads:\n“Tendon fibroblasts bone marrow-derived macrophages direct co-culture experiments:\nFor co-culture experiments, M-CSF pre-differenJated BMDM were pooled from 2-3 mice per genotype (WT or Fosl-2(Tg)). Pooled differenJated BMDM were mixed with WT or Fosl-2(Tg) tendon fibroblasts at 1:1 mixing raJo, in a mix-and-match fashion, and at a density of 4x106 cell/ml. Pre-mixed macrophage-fibroblast populaJons were encapsulated in collagen hydrogels and cultured under variable tension at a final seeding density of 1x106 cell/ml, as described above. Co-cultures were maintained in DMEM - high glucose media supplemented with 10% FBS, 1% Penicillin-Streptomycin, 1% MEM Non-essenJal Amino Acid SoluJon (M7145, SAFC Sigma-Aldrich) and 200 μM L-ascorbic acid phosphate magnesium salt. Tissue tension was quanJfied by monitoring the post deflecJon for up to 48 hours.”\nThis point is now addressed in the Methods secLon Line: [718 - 726].\nTo be able to make reliable statements, we have repeated the experiments with tendon stromal cells and BMDM macrophages that are derived from addiLonal new mice to increase the number of the biological units and therefore increase the confidence in our results (RebuPal figure 5, Figure 7). We have now toned down the statements and re-interpreted the results according to the new findings.\nWe expanded the Results secLon to include the new data from the co-culture experiments. Line [419 - 434] reads:\n“In monocultures, WT tendon stromal cells showed modest reducJon in Col1a1 expression under rigid boundary relaJve to compliant controls. In contrast, total mRNA of Col1a1 in Fosl-2(Tg) fibroblasts remained unchanged in rigid boundary compared to compliant condiJons (Figure 7D). Similarly, there was no change in Col3a1 or Dcn expression in fibroblast monocultures. InteresJngly, co-culture of WT fibroblasts with BMDM increased the expression of Col1a1, which was more pronounced under rigid mechanical boundaries (p= 0.76 in compliant, p= 0.42 in rigid), and when WT fibroblasts were co-cultured with Fosl-2(Tg) BMDM (p= 0.04 in compliant, p= 0.03 in rigid), (Figure 7D). While similar trends were observed when BMDM were co-cultured with Fosl-2(Tg)-derived fibroblasts, expression of Col1a1 was highly variable and staJsJcally insignificant irrespecJve of the mechanical boundaries. mRNA expression of Col3a1 and Lox followed similar trends to Col1a1, with one difference that Col3a1 expression was only staJsJcally significant when WT fibroblasts were in co-culture with Fosl-2(Tg) BMDM under rigid boundaries (p= 0.03) (Figure 7D). Similarly, co-culture with BMDM induced significant shihs in the expression of cross-linking enzyme Loxl2 which was most pronounced in the condiJons where Fosl-2(Tg) fibroblasts were in co-culture with WT or Fosl-2(Tg) BMDM under rigid boundary condiJons (p= 0.05 and 0.03, respecJvely) compared to mono-cultures (Figure 7E).”\nAnd discussed in light of relevant literature in Lines [562- 578].\nRebuttal figure 5 | Analysis of gene expression in macrophage-fibroblast co-culture experiments\n(A) Experimental design for monocultures and (B) “mix-and-match” direct co-cultures. (C) mRNA expression of ECM-related genes, and (D) ECM crosslinking enzymes in monoculture and direct cocultures of Fosl-2(Tg) and WT cells tethered to different mechanical boundary rigidities. (Mono-cultures: n = 4 replicates/group, with cells derived from 4 different mice. Co-cultures: n = 7-8 replicates/group, with tendon cells derived from 4 different mice. BMDM cells were from two different pools of cells derived from 2 different mice and pooled together. Each data point represents fold-change value, horizontal lines indicate the median. Two-way ANOVA (Genotype, co-culture mix) with Dunnett’s post-hoc test).\nAbbreviations: (WT): Wild-type. (BMDM) Bone marrow-derived macrophages.\n5. Please check figure legends carefully – in Figure 4, panels are described that are absent from the figure itself. Please clarify if immunofluorescence was performed on these samples.\nThank you for the catch. We updated Figure 4 legend and removed the mislabeling.\n6. Please check the manuscript carefully for typos, parLcularly the methods secLon\nMulLple typos have been corrected.\nReviewer #2 (Remarks to the Author):\nInteresLng study looking into how dysregulated matrix mechanics affect cell phenotype towards a pro-fibroLc phenotype using tendon-derived stromal cells and systemic sclerosis (SSc) as a model. Elegant methodology with the development of a new mechanoculture pla~orm to support the invesLgaLon of the effect of matrix sLffness on the acLvaLon of fibroblasts, with validaLon of dysregulated matrix mechanics in the tendons of the Fra2 mouse model of systemic sclerosis, and use of tendon-derived stromal cells from Fra2 transgenic mice and wild type mice to idenLfy funcLons affected by dysregulated matrix mechanics. The authors succeed to recreate a pro-fibroLc phenotype similar to the one induced by TGFβ through tuning of matrix sLffness and report increased matrix sLffness negaLvely correlate with major pro-fibroLc ECM genes expression and posiLvely correlate with the expression of markers of stromal-immune interacLons and that co-culture with bone marrow-derived macrophages can regulate gene expression of tendon-derived stromal cells independently of matrix mechanics. In addiLon, the developed mechanoculture pla~orm has great potenLal for future studies in the mechanism of fibroblast acLvaLon through dysregulated matrix mechanics.\nMajor points\n1. How does the matrix sLffness generated by the compliant and the rigid PDMS mats relate to physiologic tendon matrix sLffness? How did you determine the rigid PDMS creates an “abnormal” or higher than physiologic matrix sLffness and the compliant substrate recreates the physiologic tendon microenvironment closer?\nSLffness of 3D hydrogels can be modified by changing the intrinsic mechanical properLes (i.e. elasLc modulus), or by manipulaLng the extrinsic boundary rigidity – i.e. by constraining the edges of isotropic fibrillar hydrogels (e.g. collagen biopolymers). Here, our strategy involved tuning the elasLcity of the PDMS mats to control the rigidity of each canLlever post. Post rigidity determines the degree to which it deflects in response to collecLve (horizontal) cellular tracLon forces of tendon cells that are encapsulated within the gel. The two posts anchor and uniaxially constrain the contracLng hydrogel, thus restricLng its deformaLon and increasing the overall resistance and effecJve sJffness of the gel.\nIndeed, the Lssue-level sLffness of natural biopolymer-based 3D gels is several orders of magnitudes so}er than the bulk sLffness of tendon Lssues.(30-33) It is noteworthy to menLon that our goal here was not to Lssue-engineer a physiologic tendon Lssue, but rather to examine the role that cell-level (matrix) mechanical tensional cues play in regulaLng cellular behavior. Despite our best efforts, and although mulL-scale mechanical tesLng is one of our laboratory’s core experLse, we could not reliably apply a measurement approach that we felt could meaningfully measure the elasLc moduli of the collagen hydrogels in tension. The methodological work to meaningfully connect cell/matrix-level to Lssue-level mechanics is substanLal, and remains an ongoing effort for our lab.\n“How did you determine the rigid PDMS creates an “abnormal” or higher than physiologic matrix sJffness and the compliant substrate recreates the physiologic tendon microenvironment closer?”\nOur raLonal for creaLng a uniaxial anisotropic (i.e. aligned) tendon-like constructs is informed by our previous work where we showed that microLssues with uniaxial constraints appear to be beneficial for tenogenic expression (Scx and Mkx).(34) Based on this, we further benchmarked the tenogenic expression rigid vs. compliant tethered constructs which revealed that compliant constructs had significantly higher expression of Scx and Tnmd genes in comparison with rigid boundary condiLons (Figure 3K).\nWe have now bePer clarified these points in the introducLon [Lines: 87-102], and results secLons [221-224].\n2. Were technical replicates used for staLsLcal analysis instead of biological replicates (except for the SSc where this was unavoidable)? Was a staLsLcian consulted for the use of technical replicates instead of biological for the staLsLcal analysis?\nYes. As background, we clarify below how we defined the “biological unit” for replicaLon.\nFor all experiments involving the molecular characterizaLon of Fosl-2(Tg) mice, i.e. gene expression and cross-links analysis, we defined single animals as the individual biological unit for replicaLon. When it comes to the “mechanical” experiments - i.e. the mechano-culture of hydrogels under tension - we find the simplisLc intuiLon behind the convenLonal dichotomy of “biological” vs. “technical” replicaLon breaks down for mulLple reasons. Individual gels compact and develop tension in a way that highly varies between gels, even when cells were originaLng from the same biological donor (whether human donors or rodents). Our experience consistently showed that the inter-gel/inter-fascicle variability is always higher than the inter-donor/inter-animal variability.(35)\nTo overcome this conceptual challenge, we followed the guiding staLsLcal principles of Lazic et. al. who argued that “the frequent and common disJncJon made between ‘biological’ and ‘technical’ replicaJon is unhelpful because they are inconsistently defined, do not capture the important characterisJcs of an experiment, and do not clarify what to replicate.”(36, 37) Lazic aPributes this shortcoming to oversimplificaLon of the ‘biological’ vs. ‘technical’ dichotomy which fails to capture the complex, mulL-level hierarchy of biological organizaLon. They further argued that it is o}en that the hypothesis being tested, the experimental intervenLons, and the technical measurements may each operate at disLnct levels of biological organizaLon, thus creaLng ambiguity regarding which level should dictate the sample size for replicaLon. In an ideal scenario, the hypothesis, intervenLons, and readouts would all align at the same level of biological organizaLon. However, more o}en than not, this is not the case as it is the case with our mechano-culture experiments.\nLazic and colleagues propose a hierarchical organizaLon of experimental design differenLaLng between biological units (BU), experimental units (EU), and observaLonal units (OU).(37) Understanding the disLncLon between these units clarifies where replicaLon should take place. They recommend there are three criteria that must be fulfilled for an experimental unit to qualify as a genuine replicate: 1) Experimental Units (EUs) must be allocated independently to each experimental condiLon or intervenLon. 2) Each experimental intervenLon must be applied independently to every EU. 3) EUs should not exert influence on each other, parLcularly concerning the measured variable.\nAgainst the backdrop of this recommendaLon, we defined the individual gels as the biological units (BU) and experimental (EU) for replicaLon upon which we aim to test the hypothesis, and the boundary rigidity as the experimental intervenLon. This way the genuine replicaLon at the level of individual gels (rather than the human/animal donors) is more relevant to the hypothesis we are tesLng which is about differences in the emergent sLffness of tensioned hydrogels as a funcLon of the boundary rigidity. That is to say, we replicated and perturbed the individual gels and boundary rigidity as a biological unit–intervenLon pair as the basis for the staLsLcal inference. We believe that this reasoning bePer captures the nuance related to tesLng our experimental hypothesis.\nFurthermore, we are aware of the importance of increasing confidence in any biological findings by tesLng cells originaLng from mulLple donors/cell lines. As such, we performed all experiments with cell from at least 3 different donors with the staLsLcal inference performed at the level of individual gels rather than the average of gels (from a single donor).\nWe acknowledge this criLcal nuance was not explained in sufficient details in the original submission. Therefore, we have taken several steps to clarify this point:\n1. We removed the reference to “biological replicaLon” which may create some confusion. Line [398]: Sentence “due to the lack of biological replicaJon” is removed.\n2. We expanded the “StaLsLcal methods” secLon in lines [851] – [860], and added this discussion in the supplementary informaLon.\n3. Line 272 and Figure 5C – Since you are measuring the fascicles CSA and reporLng differences in CSA between wild type and Fra2Tg fascicles why not calculate and plot failure stress instead of force to facilitate the comparison of properLes between wild type and Fra2Tg fascicles? Or at least menLon it in the text if there was no significant difference in failure stress measured between wild type and Fra2Tg fascicles.\nIn figure 5C, we followed our established pracLce of focusing on tendon failure force and sLffness as structural properLes that reflect biologically relevant tendon funcLon, while providing elasLc modulus as a proxy measure of \"material quality\".\n4. Line 472 – IsolaLon of tendon-derived stromal cells - Please include donor age and gender for SSc and healthy Lssue. Were the healthy controls age-matched to the SSc donor? If not, please include in the limitaLons as ageing can also have an impact on cell phenotype and expression.\nApologies for the oversight. We have now compiled this informaLon in [Supplementary Table S1].\nUnfortunately, we were unable to age-match the healthy controls to the single SSc donor from the resourced materials in our biobank. However, to avoid any overstatement, we now emphasized the confounding effect of ageing and the single donor as a limitaLon in the discussion [Line: 542-544], which reads: “We acknowledge that we prioriJzed comparing the cellular tracJon forces and gene expression of SSc-derived cells to healthy cells originated from young donors, limiJng our ability to exclude the potenJal confounding effect of ageing.”\nMinor points\nLine 144 – Within hours a}er seeding, Lssue tension evidently increased a}er two hours, and … - change to avoid repeLLon of within hours.\nLine [155] reads: “Within two hours aher seeding, Jssue tension evidently increased and conJnued to rise by 3-fold reaching a plateauing maximum of (272 ± 60) µN at 24 h (p= 0.00142), (Figure 1G top, Supplementary figure 1C).”\nFigure 1A legend – SagiPal secLon might be more accurate than cross secLon. A cross secLon view of the well could be included to show the iniLal set up view from the top/boPom of the well.\nCorrected to “Sagi_al secJon views”.\nFigure 1A – “PDMS maP” - mat is misspelled in the actual figure.\nCorrected to “mat” throughout the manuscript.\nFigure 1G or Line 148 – Was the increase and decrease staLsLcally significant?\nYes, the increase in Lssue tension was staLsLcally significant at 24 hr compared to the two-hour Lmepoint (RebuPal figure 6, New supplementary figure 1C). Line [155] reads: “Within two hours aher seeding, Jssue tension evidently increased and conJnued to rise by 3-fold reaching a plateauing maximum of (272 ± 60) µN at 24 h (p= 0.00142), (Figure 1G top, Supplementary figure 1C).”\nRebuttal figure 6 | Quantification of tissue tension of rat tail-derived tendon stromal cells in Vehicle control condition.\nKruskal-Wallis test with Dunn’s post-hoc test.\nFigure 1G – Was the provenance of these cells also rat tail tendon? Maybe add in the legend.\nYes, that is correct. Legend of Figure 1G now reads: “Time-course of cell-generated tracJon forces of rat tail-derived tendon stromal cells (Vehicle control).”\nLine 154 – “in in” repeLLon.\nCorrected\nFigure 1I – Is the “acLve forces” column represenLng the vehicle Lssue tension a}er 48 h? As the magnitude does not appear to correspond to the magnitude of the data point in 1G.\nCorrect, the “acLve forces” and “residual tension” bars represent the values at the end of BlebbistaLn treatment (~ 54 hr). Thank you for spo‚ng the inconsistency – we found incorrectly transposed values in (Figure 1H). We have updated the figure accordingly.\nLine 177 – “consistent with” appears that part of the sentence missing.\nCorrected. Line [188 - 190] reads: “TGF-b1-treated condiJons had approximately three-fold significantly higher fluorescence intensiJes of a-SMA than untreated controls, which is consistent with state switch to myofibroblasts”\nLine 190 – “the phenotype”\nCorrected.\nLine 200 – Maybe bePer to specify immune staining intensiLes (rather than immune intensiLes).\nCorrected. Line [214] reads: “Rigid boundaries induced an approximately 50% increase in a-SMA immune staining intensiJes above compliant controls”\nLine 207 – The way the sentence about the tenogenic markers is formulated it would seem Mkx is also significantly decreased which does not seem to be the case from Figure 3K – please amend accordingly.\nCorrected. Line [221] reads “Similarly, expression of markers indicaJve of tenogenic linage commitment of stromal cells, (Scx and Tnmd) were significantly downregulated in highly-tensioned constructs”\nFigure 4C – why does line graph has n=24/group and the violin plot has 16/group – isn’t the source of the data the same presented in a different way?\nWe realize that we were not sufficiently accurate in describing the panels. Although we aimed to have N=24 gels in all experiments, it o}en occurs that some gels rupture or pop off posts at the later Lmepoints. We have updated the Figure 4 legend, which now reads: “(F) QuanJtaJve analysis of Jssue tracJon forces of tendon (top) and dermal (bo_om) fibroblasts. Leh insets: EvoluJon of Jssue tracJon forces as funcJon of Jme. Tendon: Cells from 3 anatomically different tendons of the SSc donor and 3 healthy young controls (n = 15-24 Jssues/Jmepoint). Skin: Cells from 3 different SSc donors and 3 healthy controls (n = 22-24 Jssues/Jmepoint). Each data point represent the mean ± SEM. Right inset: Violin plots of forces per cell, following normalizaJon to iniJal seeding density. Horizontal lines indicate the median and interquarJle range (n = Tendon: 15-16 Jssues/group | Skin: 22-24 Jssues/group)”\nFigure 4D and E legend – The legend says immunostaining quanLficaLon and the text says mRNA expression and panels appear mislabelled/the legend includes panels that do not appear in the figure. Please amend appropriately.\nCorrected.\nFigure 4 D-G – The x axis labelling is complex when the subcategories could just be labelled “ | compliant | rigid | ” since you have already colour-coded the healthy and SSc – just a suggesLon. Same for Figure 5D.\nThank you for the suggesLon. While we agree that the double labelling is redundant in (Figures 4 G-I) and 5D, we think it is useful in conveying the message of the more complex dataset with mulLple comparisons in (Figure 7 D-E). Therefore, we wish to be consistent with this visualizaLon across the whole manuscript.\nLine 244 – You are not specifying whether the dysregulaLon is observed under compliant or rigid boundary mechanics condiLons which gives the impression that this dysregulaLon is being observed under non-dysregulated mechanics/compliant boundary mechanics condiLons. This is not the case for IL6 and IL8 though which are not significantly different between healthy and SSc tendon-derived stromal cells under compliant condiLons. Please specify which condiLons you are referring to or remove IL6 and IL8 if they are not significantly regulated.\nWe agree with the reviewer on this point. The statement is about the gene expression trend in the SSc condiLons compared to the healthy controls (and irrespecLve of the boundary mechanics). We toned down the statement about the “significant dysregulaLon”, and specified that we are comparing the healthy, non-SSc compliant controls.\nLine [283 - 286] reads: “Analysis of mRNA transcripts revealed that SSc-derived tendon cells populaJng the tethered constructs showed a gene expression signature typical of fibro-inflammatory acJvaJon in SSc, with altered expression of FN(EDA), IL6, IL8, ACTA2, PDPN and TLR4 mRNA relaJve to healthy, non-SSc compliant controls (Figure 4 G-I).”\nLine 252 – Is it an inability of SSc cells to sense boundary rigidity or expression of COL1A1 is already lower in SSc cells so it does not get significantly lower in rigid condiLons? As for other genes eg IL6, IL8, and ICAM a significant difference in expression is noted between healthy and SSc cells under rigid condiLons but not compliant – appearing to show a response to boundary rigidity in SSc cells. You also menLon in the next sentence SSc-derived tendon cells show sLffness-sensiLvity of stromal-immune interacLons markers – unless that was “and (absence of) sLffness-sensiLvity of stromal-immune interacLons markers”.\nWe agree that our original conclusion represents strong overgeneralizaLon, especially given that these SSc cells are originaLng from a single donor. As alluded by the reviewer, our data does not exclude the possibility that expression of COL1A1 might have already been lower in SSc cells and that it does not get significantly lower than this baseline.\nWe have toned down this statement by ediLng the text in the Results secLon. Line [297 - 301] now reads: “This suggests that SSc tendon fibroblasts may have reduced baseline expression of COL1A1, or that altering matrisome-related transcripJon in response to boundary rigidity is an intrinsic property of healthy tendon stromal fibroblasts. Together, these results demonstrate that SSc-derived tendon cells are intrinsically acJvated in a cell-autonomous manner, and may possess altered tension-sensiJve transcripJon of matrisome and stromal-immune interacJons markers.”\nWe thank the reviewer for the insigh~ul criLcism that helped us to align this conclusion to our data.\nLine 263 – “We have recently reported…” reference (65) is missing. Also, reference 65 – is missing a Ltle, journal volume and pages.\nFigure 5D – Are the p values for Fn1 and Lox missing?\nCorrected, thank you for spo‚ng that. We have updated the reference to the published version of the preprint. Reference (65) is now reference (71).\nThe Fn1 and Lox P-values have been highlighted in Figure 5D.\nLine 317 – “mirroring our observaLons in Fra2 tendons in vivo and SSc-derived tendon fibroblasts” but you report no significant changes in Col1a1 expression under rigid condiLons in SSc cells (and Lox expression is not reported) and Lox expression in Fra2 tendons does not appear to be significantly different from WT unless there are some significant p values missing from the graphs. Please clarify sentence if needed.\nTo be able to make reliable statements, we have now repeated these experiments with tendon stromal cells that are derived from addiLonal new mice. We have now toned down the statements and re-interpreted the results according to the new findings. Line [419 - 434] reads:\n“In monocultures, WT tendon stromal cells showed modest reducJon in Col1a1 expression under rigid boundary relaJve to compliant controls. In contrast, total mRNA of Col1a1 in Fosl-2(Tg) fibroblasts remained unchanged in rigid boundary compared to compliant condiJons (Figure 7D). Similarly, there was no change in Col3a1 or Dcn expression in fibroblast monocultures. InteresJngly, co-culture of WT fibroblasts with BMDM increased the expression of Col1a1, which was more pronounced under rigid mechanical boundaries (p= 0.76 in compliant, p= 0.42 in rigid), and when WT fibroblasts were co-cultured with Fosl-2(Tg) BMDM (p= 0.04 in compliant, p= 0.03 in rigid), (Figure 7D). While similar trends were observed when BMDM were co-cultured with Fosl-2(Tg)-derived fibroblasts, expression of Cola1 was highly variable and staJsJcally insignificant irrespecJve of the mechanical boundaries. mRNA expression of Col3a1 and Lox followed similar trends to Col1a1, with one difference that Col3a1 expression was only staJsJcally significant when WT fibroblasts were in co-culture with Fosl-2(Tg) BMDM under rigid boundaries (p= 0.03) (Figure 7D). Similarly, co-culture with BMDM induced significant shihs in the expression of cross-linking enzyme Loxl2 which was most pronounced in the condiJons where Fosl-2(Tg) fibroblasts were in co-culture with WT or Fosl-2(Tg) BMDM under rigid boundary condiJons (p= 0.05 and 0.03, respecJvely) compared to mono-cultures (Figure 7E).”\nLine 334 – “Elevated rigidity” – compared to what? Physiologic matrix sLffness in tendons?\nWe argue it is the “elevated rigidity” in comparison to the compliant boundary rigidity. As discussed in the Major point #1, we showed that the compliant constructs had significantly higher expression of Scx and Tnmd transcripts compared to the rigid counterparts (Figure 3K). Based on this (and taken together with our previous work where we showed that uniaxially constrained microLssues appear to be beneficial for tenogenic expression(34)), we set the compliant boundary as the baseline for comparison throughout the study/manuscript.\nLine 368 – As you are menLoning in line 370 the referenced paper in line 368 does not really invesLgate fibrosis and cell environments of increased sLffness so maybe using “This was further reported/This decreased transcripLon was further reported” or similar could be a bePer representaLon than “This was further confirmed”.\nThank you, we agree. Line [498 - 501] reads “This decreased transcripJon was further reported in a mulJ-omics single cell profiling of the aging lung which a_ributed the dysregulated matrix remodeling in aged lungs to the increase in transcripJonal noise and aberrant epigeneJc control.”\nLine 404 – Diameter and length details are missing.\nCorrected. Diameter and length details are in line [592], which reads: “Pins were (1 ± 0.006) mm in diameter, (20 ± 0.5) mm in length and spaced approximately 4 mm apart from each other.”\nLine 405 – “Each well in the top plate’s upper and …” Corrected.\nLine 412 – “mm” repeLLon\nDevice assembly paragraph (line 402) – The setup of the anchoring spheres is not included but would aid in the overall understanding of the setup of the spheres in relaLon to the posts.\nCorrected. Line [593] reads: “To provide a be_er gel anchoring point, a ceramic bead (116913050CF: Lysing Matrix D, MP Biomedical) with an average diameter of 1.4 mm was glued on top of each post using medical grade, low-viscosity ethyl-based instant adhesive (LOCTITE® 4011 Prism Medical CA: Ellsworth Adhesives, UK).”\nLine 437 – “so}ware.” comma instead? Corrected\nLine 441 – to create\nCorrected.\nLine 541 – 90 ng – space between number and unit\nCorrected.\nLine 551 – “The then”\nCorrected.\nLine 570 – StaLsLcs\n- Two-way ANOVAs are also menLoned in the figure legends but not in the staLsLcs paragraph.\nCorrected.\n- (from major points) Were technical replicates used for staLsLcal analysis instead of biological replicates (except for the SSc where this was unavoidable)? Was a staLsLcian consulted for the use of technical replicates instead of biological for the staLsLcal analysis?\nThis point is addressed in Major point #2\n- Some experiments had a wide variaLon of sample numbers per group eg Fig 5D n=4-9. Was that due to loss of samples in the process resulLng in some groups having less samples than others or sample addiLons where it was deemed to be needed or cell numbers/availability?\nThe variability in sample numbers is due to mulLple factors. In Fosl-2(Tg) experiments, this o}en arises from the low RNA yield in Fosl-2 tendons (Lssue crosslinking makes RNA extracLon very challenging). AddiLonally, Fosl-2(Tg) mice o}en develop strong phenotype which necessitate euthanize the animal(s) for welfare reasons – this led to relaLvely high dropout rate of Fosl-2(Tg) mice from each liPer relaLve to WT controls. Whenever we had limited number of samples, we prioriLzed the most important genes (e.g. Col1a1, Lox, and housekeeping genes).\nReviewer #3 (Remarks to the Author):\nThis manuscript describes a substanLal body of work that addresses mechanical factors that may be highly relevant to the tendon abnormaliLes which are a hallmark of systemic sclerosis paLents. The authors have developed a culture technique that allows mechanics tension to be applied in vitro and using this methodology have defined effects on fibroblasts and extended previous work that shows how mechanical tension may promote the contracLle phenotype and profibroLc properLes of fibroblasts. A key over-arching quesLon is the extent to which these findings may apply to a broader populaLon of fibroblasts that those derived from tendons.\nSpecific comments:\n1. It would be helpful to explain more clearly how the mechanical forces are generated and different between cultures. Whilst the authors obviously have a very clear understanding it is less apparent to readers unfamiliar with the concepts of sLffness and elasLcity and this should be explained to hep with later interpretaLon of the data, especially for mutant mice at different Lme points.\nWe thank the reviewer for their posiLve comments, and for the opportunity to clarify these concepts.\nTissue mechanical properLes, specifically sLffness, describe the relaLonship between applied loads and resulLng deformaLons. In other words, how the applied forces on a Lssue deform its shape. The raLo between the applied load and subsequent deformaLon defines the sLffness of the Lssues.(38) It is important to menLon that sLffness/rigidity and elasLc modulus are interrelated concepts, and someLmes used interchangeably (parLcularly when the focus of a work is biological rather than biophysical). However, sLffness/rigidity precisely describes the Lssue structural properLes (force and length relaLonships), whereas the elasLc modulus defines the properLes of the material that consLtute that structure.(39, 40)\nIn the context of this work, with a focus that is both biological and biophysical, it is useful (perhaps imperaLve) to differenLate between the intrinsic sJffness (described above), global boundary sJffness, and their reciprocal relaLonship in regulaLng the cell-generated tension and the emergent properJes of Lssue and cellular behavior.(41, 42) Cellular tension in 3D environments can be modified by changing the intrinsic mechanical properLes of the environment within which cells are encapsulated (e.g. hydrogel intrinsic sLffness), or by manipulaLng the extrinsic boundary sLffness – i.e. by constraining the edges of isotropic fibrillar hydrogels (e.g. collagen biopolymers).\nThe intrinsic sLffness of hydrogels (bulk elasLc modulus) can be tuned by increasing the polymer concentraLon or by chemically crosslink its polymer backbone. However, this typically alters the porosity (polymer mesh size) and cell binding sites (ligand density and presentaLon) which may confound any biological findings.(43, 44)\nAlternaLvely, spaLally constraining fibrous hydrogels restricts its deformaLon and increases the overall resistance within the hydrogel and its effecJve sJffness.(45-47) With two constraining pins/canLlevers, hydrogel ECM alignment is directed along the longitudinal axis of the mechanical stress while minimizing the isotropic compacLon of the gel. Consequently, cells also align along the axis of the mechanical stress. In addiLon, by changing the compliance of the constraining pins by making it highly compliant (i.e. deflectable) or minimally compliant (i.e. rigid with minimal deflecLon), it is possible to tune the overall resistance of the matrix and the emergent effecJve sJffness.(48, 49) In turn, this resistance regulates the cellular contracLlity and the mechanical forces that the cells can exert on the hydrogel matrix.\nSpaLally constraining collagen hydrogels represents an aPracLve strategy to model tendon mechanobiology for mulLple reasons: 1) it uses a purely mechanical strategy where cell-generated mechanical forces and boundary constrains guide Lssue organizaLon into highly-aligned, tendon-like structures. 2) hydrogel effecLve sLffness can be tuned independently of the potenLal confounding factors associated with altering the intrinsic properLes of the material. However, it is noteworthy to point out that cell-populated fibrous hydrogels are highly dynamic, self-organizing systems. The effecLve sLffness of the hydrogel and the cellular responses are mutually condiLoned, and emerge from reciprocal interplay between different factors over Lme and across different length-scales.(42, 50, 51) As such, it is only possible to control for the hydrogel properLes (e.g. iniLal polymer concentraLon) at the beginning of the experiment.(46)\nHow the mechanical factors in our 3D mechano-culture system relate to the observaLons in Fosl-2(Tg) mutant mice can be explained by these emergent properLes. As detailed in our response to Reviewer #1 (above), we speculate that the suppression of the matrisome-related transcripLon is potenLally mediated, at least in part, by the emergent sLffening and/or confining effect of the extracellular matrix. For more discussion addressing the potenLal processes mediaLng this response, we refer the reviewer to our detailed response to Reviewer #1. (SecLon: Mechanical confinement and RNA polymerase II repression of transcripLon).(28, 29, 52)\nWe updated the introducLon and discussion secLons, where this point is now addressed in Line [87-102] and [505 - 546].\n2. More extensive comparison of SSc fibroblasts should be possible even though tendon samples may not be available rouLnely. It would be an important control experiment to explore whether dermal fibroblasts show the same or different responses to those observed for tendon derived cells.\nWe thank the reviewer for this important criLque that helped us to strengthen our conclusions.\nBased on the reviewer’s suggesLon we performed this control experiment, and we provide addiLonal data where we extensively compared dermal fibroblasts from SSc and healthy controls. We performed these experiments using human dermal fibroblasts originaLng from the forearm skin of paLents with systemic sclerosis (SSc) and healthy control donors. Skin biopsies were obtained from paLents who fulfilled the American College of Rheumatology/European League Against RheumaLsm (ACR/EULAR) 2013 classificaLon criteria.(53) We hope the reviewer finds this extended evaluaLon saLsfactory.\nFirst, we measured Lssue tracLon forces of SSc dermal fibroblasts and compared it to healthy control donors [RebuPal figure 7A]. Over a two-day period, we observed that SSc-derived dermal fibroblasts displayed progressive increase in Lssue tracLon forces similar to SSc-derived tendon fibroblasts. At 24h, SSc-derived dermal fibroblasts exerted forces of approximately 23 nN per cell [Mean= 22.88 nN ± 7.52, n= 24], while healthy control cells had ~two-fold lower tensional forces [Mean= 12.26 nN ± 7.6, n= 24]. While the difference in the order of magnitude between SSc and healthy controls was similar in skin and tendon-derived fibroblasts, SSc dermal fibroblasts exerted higher tracLon forces compared to SSc-derived tendon fibroblasts at all Lmepoints. This difference was more pronounced at 48 hrs; where SSc dermal cells applied approximately five-fold higher forces compared to SSc-derived tendon cells. In comparison, healthy dermal fibroblasts exerted 3.5-fold higher forces relaLve to healthy tendon fibroblasts [RebuPal figure 7A].\nWe have incorporated this data to (Figure 4F), and updated the results and discussion secLons, where this point is now addressed in Line [269 - 281].\nNext, we performed gene expression analysis to characterize the fibro-inflammatory phenotypes of tensioned dermal fibroblasts, and to assess whether and how SSc-derived dermal fibroblasts respond to changes in boundary sLffness and matrix tension [RebuPal figure 7 B-E]. InteresLngly, we found that SSc dermal fibroblasts exhibited a gene expression paPern consistent with fibro-inflammatory acLvaLon with upregulaLon of FN(EDA), IL6, IL8, ACTA2, PDPN and TLR4 mRNA levels, albeit with modest differences compared to tendon-derived SSc cells [RebuPal figure 7 B and D]. Unlike tendon-derived fibroblasts, dermal fibroblasts showed significant upregulaLon of matrix crosslinking enzymes LOX, LOXL2, TGM2 transcripts [RebuPal figure 7 C]. However, there were no differences in levels of COL1A1 and COL3A1 transcripts between SSc and healthy control dermal fibroblasts, and irrespecLve of the boundary rigidity.\nWe have added this data to (New supplementary Figure 8), and updated the results secLon in Line [282 – 299] to reflect these new findings.\nRebuttal figure 7 | Mechano-culture of systemic sclerosis-derived dermal stromal fibroblasts.\n(A) Quantitative analysis of tissue traction forces of tendon (top) and dermal (bottom) fibroblasts. Left insets: Evolution of tissue traction forces as function of time. Right insets: Violin plots of forces per cell, following normalization to initial seeding density. Horizontal lines indicate the median and interquartile range. (B) mRNA expression of ECM-related genes, (C) cross-links related enzymes (D) stromal activation markers, and (E) immune/inflammatory genes in dermal fibroblasts tethered to different mechanical rigidities. (n = 8 replicates/group from 3 independent experiments, with each data point representing ΔCt, horizontal lines indicate the median, Two-way ANOVA (boundary stiffness, disease stage) with Holm-Sidak post-hoc test). All individual gene expression is shown normalized to RPL13A and GAPDH reference genes.\n3. It is unreliable to draw conclusions from a single paLent sample. It would be helpful if addiLonal material could be obtained from a range of SSc paLents and perhaps compared with other fibrosing disorders such as Dupuytren’s contracture that may be more available. Although 3 biologically independent experiments are performed it is not clear how Ls was achieved with a single donor.\nWe agree with the reviewer, and we acknowledge that including one human SSc donor is a limitaLon of our study. Nonetheless, these specimens are excepLonally rare and, despite our best effort, only one systemic sclerosis autopsy had been available for research at our specialized terLary center over a period of six years. To extend the generalizability of the SSc tendon findings, we have: (1) Provided addiLonal supporLng evidence from SSc-derived dermal fibroblasts (cells derived from 3 human SSc and control donors), as explained in our response to point no. 2. (2) Included SSc tendons and cells originaLng from different anatomical locaLons for the single SSc donor. This data is now included in (Figure 4 A-C, 4F): “Baseline characterisJcs of ECM transcripJon, total collagen content, mature collagen crosslinks in human tendons”.\nUnfortunately, and despite our best effort, we were not able to source fibroblast cells from Dupuytren’s contracture at our surgical center.\n“Although 3 biologically independent experiments are performed it is not clear how Js was achieved with a single donor.”\nApologies for the oversight. We realized we were not sufficiently clear on how we defined the “biological unit” for replicaLon.\nWhen it comes to the “mechanical” experiments - i.e. the mechano-culture of hydrogels under tension - we find the simplisLc intuiLon behind the convenLonal dichotomy of “biological” vs. “technical” replicaLon breaks down for mulLple reasons. Individual gels compact and develop tension in a way that highly varies between gels, even when cells were originaLng from the same biological donor (whether human donors or rodents).\nOur experience consistently showed that the inter-gel/inter-fascicle variability is always higher than the inter-donor/inter-animal variability.(35)\nTo overcome this conceptual challenge, we followed the guiding staLsLcal principles of Lazic et. al. who argued that “the frequent and common disJncJon made between ‘biological’ and ‘technical’ replicaLon is unhelpful because they are inconsistently defined, do not capture the important characterisJcs of an experiment, and do not clarify what to replicate.”(36, 37) Lazic aPributes this shortcoming to oversimplificaLon of the ‘biological’ vs. ‘technical’ dichotomy which fails to capture the complex, mulL-level hierarchy of biological organizaLon. They further argued that it is o}en that the hypothesis being tested, the experimental intervenLons, and the technical measurements may each operate at disLnct levels of biological organizaLon, thus creaLng ambiguity regarding which level should dictate the sample size for replicaLon. In an ideal scenario, the hypothesis, intervenLons, and readouts would all align at the same level of biological organizaLon. However, more o}en than not, this is not the case as it is the case with our mechano-culture experiments.\nLazic and colleagues propose a hierarchical organizaLon of experimental design differenLaLng between biological units (BU), experimental units (EU), and observaLonal units (OU).(37) Understanding the disLncLon between these units clarifies where replicaLon should take place. They recommend there are three criteria that must be fulfilled for an experimental unit to qualify as a genuine replicate: 1) Experimental Units (EUs) must be allocated independently to each experimental condiLon or intervenLon. 2) Each experimental intervenLon must be applied independently to every EU. 3) EUs should not exert influence on each other, parLcularly concerning the measured variable.\nAgainst the backdrop of this recommendaLon, we defined the individual gels as the biological units (BU) and experimental (EU) for replicaLon upon which we aim to test the hypothesis, and the boundary rigidity as the experimental intervenLon. This way the genuine replicaLon at the level of individual gels (rather than the human/animal donors) is more relevant to the hypothesis we are tesLng which is about differences in the emergent sLffness of tensioned hydrogels as a funcLon of the boundary rigidity. That is to say, we replicated and perturbed the individual gels and boundary rigidity as a biological unit–intervenLon pair as the basis for the staLsLcal inference. We believe that this reasoning bePer captures the nuance related to tesLng our experimental hypothesis.\nFurthermore, we are aware of the importance of increasing confidence in any biological findings by tesLng cells originaLng from mulLple donors/cell lines. As such, we performed all SSc fibroblast experiments with cell from at least 3 different donors (for dermal fibroblasts), or 3 different anatomic origins of the single SSc tendons donor (for tendon fibroblasts). The staLsLcal inference performed at the level of individual gels rather than the average of gels (from a single donor).\nWe acknowledge this criLcal nuance was not explained in sufficient details in the original submission. Therefore, we removed the reference to “biological replicaLon” which may create some confusion. Line [398]: Sentence “due to the lack of biological replicaJon” is removed.\n4. I am somewhat confused by references to the fosl2 mutant mice as well as Fra2 and the authors should clarify this and how the different mouse strains were examined (page 29 line 449)\nFosl-2 (Fos Like 2) and Fra-2 (Fos-Related AnLgen 2) are synonyms aliases for the same gene Fosl2. In this work, we used a single mouse strain that overexpresses transcripLon factor Fosl-2.\nTo avoid confusion, we updated the text and figures with one alias (Fosl-2 Tg) to ensure consistency across the whole manuscript.\n5. The transcripLon factor MRTFA is considered criLcal in mechano-sensing in fibroblasts, and it would be helpful to have this included in the relevant gene expression and protein assays to explore the impact on myofibroblasts differenLaLon and contracLlity.\nWe agree with the reviewer here. We performed RT-qPCR to probe the expression of Mr~a in tendon fibroblasts tethered to different mechanical boundaries (RebuPal figure 8A). We found no staLsLcal significance between compliant vs. rigid boundaries (P = 0.2345). Furthermore, we have also ploPed the normalized expression values of Mr~a in Fosl-2(Tg) and wild-type control mice, which is collated in (RebuPal figure 8B). InteresLngly, expression of Mr~a was significantly downregulated in Fosl-2(Tg) fibroLc tendons relaLve WT control in the Established group (P = 0.042). ComputaLonal analysis of key upstream regulatory transcripLon factors (TFs) did not predict Mr~a among the key regulatory element behind the differenLally expressed genes (RebuPal figure 8C).\nWe have added part of this data to (New supplementary Figure 4), and updated the results secLon in Line [222 – 224] to reflect these new findings.\nRebuttal figure 8 | Expression of transcription factor Mrtfa in mechano-culture and Fosl-2(Tg) models.\n(A) mRNA expression of Mrtfa in rat tail tendon stromal cells tethered to different mechanical rigidities. (n = 8 replicates/group from 3 independent experiments, with each data point representing a ΔCt value of 2-3 pooled tissues, horizontal lines indicate the median, Two-tailed Mann Whitney test). All individual gene expression is shown normalized to Eif4a2 and Gapdh reference genes. (B) Normalized RNA-Seq expression values of Mrtfa in tail tendons of (Fosl-2(Tg) vs. Wild-type) mice. (Fosl-2(Tg): N = 7 mice – WT: N = 6 mice). Horizontal lines indicate the mean. ANOVA Tukey's multiple comparisons test. (C) Top 10 significantly enriched transcriptional regulators of shared targets in Fosl-2(Tg) vs. WT tendon and lung using TRRUST module. Color code denotes −log10(p-value) of hypergeometric enrichment test.\n6. The relaLve importance of moLlity, contracLlity and matricellular interacLon in determining the contracLon in this culture system should be considered as it is unlikely that cell contracLlity is a major determinaLon of tendon biomechanics that is driven more by extrinsic skeletal muscle contracLon. This may be a limitaLon of the current approach and should be clarified.\nThe reviewer raises a good point. We agree that mulLple cellular processes likely contribute to the emergent behavior of this mechano-culture system. While this work focused on a single variable, i.e. matrix tension as a funcLon boundary rigidity, the versaLlity of the pla~orm makes it amenable to repurposing to parse out the potenLal contribuLon of myriad of factors to mechanisms of tendon pathology. For example, the pla~orm can be easily adjusted to invesLgate the role of cell migraLon in tendon healing (54, 55), and to isolate the contribuLon of deposited matricellular molecules in tendon Lssue repair.(49, 56)\nAlthough we truly appreciate the reviewer’s views on the role of cell contracLlity, work by several groups have shown that contracLlity significantly contributes to tendon biomechanics and homeostasis. The work by Schiele et. al. invesLgated the cellular contribuLon to the mechanical properLes of embryonic tendons during developmental stages.(57) They specifically focused on the effect of acLn cytoskeleton on the cell-scale elasLc modulus of early-stage embryonic tendons. DisrupLon of tenocytes cytoskeleton with blebbistaLn, a myosin II inhibitor, significantly reduced Lssue elasLc modulus by approximately 20% compared to controls.(57)\nThis suggests that cytoskeletal reinforcement of tendon cells (whether contracLlity or passive intracellular tension) acLvely contributes to the structural mechanical properLes of tendons, at least during early developmental stages. Furthermore, non-muscle myosin II (NMII)-dependent cellular contracLlity indirectly contributes to ECM secreLon and alignment in tendon. Kalson et. al. showed that assembly and transport of collagen fibrils at the plasma membrane is facilitated by the contracLle NMII actomyosin machinery, and that this process is potenLally criLcal for tendon biomechanical homeostasis by maintaining collagen fibril alignment and by transmi‚ng mechanical loads back to the cell.(58) Similarly, work by Dakota and colleagues has illuminated a potenLal role for (NMII)-dependent contracLlity in regulaLng the secreLon of newly synthesized tendon matrix proteins. InhibiLon of (NMII)-dependent cellular contracLlity with blebbistaLn significantly reduced ECM deposiLon by tendon fibroblasts.(59)\nCollecLvely, this highlights that cellular contracLlity and the associated cell-ECM interacLons are criLcal not only for tendon development during embryonic morphogenesis, but also for the assembly of mechanically robust extracellular matrix during healing and repair processes.\n7. The findings reported here concerning inflammatory cells interfering with normal tension induced down regulaLon of ECM may be relevant to other condiLons such as adhesive capsuliLs and this could be discussed.\nThis is indeed a great suggesLon, and we agree with the reviewer that adhesive capsuliLs shares many of the fibro-inflammatory features with fibroLc tendons. Like tendon fibroblasts, capsular fibroblasts are embedded in a dense, highly-aligned stroma composed primarily of collagen type-I.(60, 61) Similarly, adhesive capsuliLs pathology is characterized by increase in pro-inflammatory cytokines, infiltraLon of immune cells, fibroblasts acLvaLon, and a collagen type-I to type-III matrix switch in the synovium and joint capsule, resembling many of the molecular features of tendinopathy.(61-68)\nAs adhesive capsuliLs pathology progresses towards fibrosis, so} Lssue structures within the joint (i.e. capsule, synovium, ligaments and tendons) get thicker, sLffer and more disorganized in both human adhesive capsuliLs and animal models of joint immobilizaLon.(69-72) This results in significant changes in Lssue mechanical properLes. For instance, the sLffness of the shoulder coracohumeral ligament, supra/ Infraspinatus tendons are significantly higher in symptomaLc paLents with adhesive capsuliLs than in the unaffected shoulder or healthy subjects.(73-75)Such changes in the mechanical properLes are concomitantly accompanied by alteraLons in the underlying ECM biophysical cues that Lssue-resident cell experience.(76)\nWhether and how the altered mechanics of the fibroLc niche in adhesive capsuliLs contributes to the proporLonal differences of immune cells is unknown. However, evidence from well-controlled in vitro systems revealed that matrix biophysical cues (i.e. tension and collagen alignment) accelerate the recruitment of macrophages in models of fibrosis.(77) ContracLle fibroblasts remodel the surrounding ECM generaLng regions of tensioned and highly-aligned ECM. The acLve contracLlity of fibroblasts and the highly tensioned collagen matrix serve as far-reaching signals that aPract macrophages to the proximity of contracLle myofibroblasts.(78)\nClose interacLon between macrophages and fibroblasts, via cadherin 11, creates potent profibroLc niches where TGF-β-producing macrophages and TGF-β-acLvaLng myofibroblasts sustain the progression of fibrosis through well-described feed-forward loops.(19, 77)\nWith regards to immune cells infiltraLon, recent studies that invesLgated the fibro-inflammatory changes associated with adhesive capsuliLs idenLfied several immune cells that are enriched in capsular Lssues including macrophages, T cells and mast cells.(62, 63)\nAkbar et. al. mapped the immune landscape in adhesive capsuliLs, and discovered that the immune milieu in diseased shoulder capsules is dominated by T cells with a clear shi} from macrophages to T cells-enriched populaLons compared to the resLng control Lssues.(62) Furthermore, work by Ng and colleagues reported similar observaLons where they further revealed that T cells and macrophages populate unique microanatomical niches in the capsular Lssues of adhesive capsuliLs.(79) Adhesive capsuliLs Lssues were enriched in T cells in the sublining layer and MERTK+LYVE1+MRC1+ macrophages subpopulaLon in the lining layer. While the authors aPributed these differences to the potenLal acLvaLon of developmental cellular programs, it remains to be seen if the underlying biophysical cues of the capsular matrix contribute to this differenLal homing of macrophages and T calls in adhesive capsuliLs.(79)\nTo begin to address this point, we performed a set of bulk RNA-sequencing experiments on tail tendons from early and established phases of Fosl-2(Tg) and wild-type controls. We analyzed the data in an unbiased way with the goal of potenLally idenLfying enriched inflammatory and/or immune signatures that overlap between Fosl-2(Tg) tendons and human adhesive capsuliLs. We also performed in silico deconvoluLon of the cellular fracLons in bulk transcriptomic signature using CIBERSORT pipeline.\nFirst, we found that Fosl-2(Tg) fibroLc tendons are significantly enriched in GO terms related to inflammaLon and immune response [New Figure 6, New supplementary figure 18 A-D]. InteresLngly, in silico deconvoluLon of the cellular fracLons predicted that Fosl-2(Tg) tendons with established fibrosis contain significantly higher fracLons of immune cells and nerves cells compared to wild-type controls [New Figure 6 J-L, New supplementary figure 16].\nNext, we examined the mRNA expression of established markers of fibroblast acLvaLon in tendinopathy and adhesive capsuliLs (Cd248, Mcam, Vcam1, Pdpn, Cd34, Fap).(64, 80) We found no difference between the expression levels between the Fosl-2(Tg) and wild-type controls [RebuPal figure 9 A, New supplementary figure 18 A]. Then we performed a comparaLve analysis of the whole bulk transcriptome of the Fosl-2(Tg) tendons and capsular Lssues from human paLents with adhesive capsuliLs [RebuPal figure 9 B-C, New supplementary figure 18 B-D]. We found strong and significant overlap between the two datasets. Of interest was that 8 out of the 20 overlapped GO terms and signatures were related to matrisome, cell-ECM interacLon and inflammatory processes [RebuPal figure 9 C, New supplementary figure 18 B and C].\nThe TF-gene interacLons using TRRUST database predicted that only 4 transcripLon factors were regulaLng the overlapped features between the two datasets. InteresLngly, these TFs are known to be involved in tendon inflammaLon (RELA, NFKB1)(81) and in the cross-talk between fibroblasts and macrophages in fibrosis (SP1, SP3)(82), [RebuPal figure 9 D, New supplementary figure 18 D].\nWe have added this data to the (New supplementary Figure 18, and updated the results secLon in Line [393 – 401] and discussion [568 – 578] to reflect these new insights.\nRebuttal figure 9 | Gene list-based comparative analysis of mouse tendons with established fibrosis with human capsular tissue with adhesive capsulitis. (A) Normalized RNA-Seq expression values of selected fibroblast activation markers in tendons with established phenotype (Fosl-2(Tg) vs. Wild-type). (Fosl-2(Tg): N = 7 mice – WT: N = 6 mice). Horizontal lines indicate the median. Welch's t-test. (B) Circos plot of overlapped input genes and enrichment features between the two groups. Dark orange arc and purple lines represent the differentially expressed genes that are shared between the two groups. Blue lines connect the genes that fall under the same statistically significantly enriched ontology term, reflecting the extent of functional overlap between the two comparison groups. (C) Heatmap of top significantly enriched terms (GO, KEGG, canonical pathways, etc.) using Metascape. (D) Top 5 significantly enriched transcriptional regulators of shared targets in (C) using TRRUST database for transcription factor analysis. Color code depicts −log10(hypergeometric p-value) of statistical test.\nAbbreviations: (R) Reactome, (W) WikiPathways, (K) KEGG, (H) Hallmark, (GO) Gene Ontology, (MSigDB) Molecular Signatures Database.\nResponse to reviewer 2\nPoint 3: Line 272 and Figure 5C – Since you are measuring the fascicles CSA and reporting differences in CSA between wild type and Fra2Tg fascicles why not calculate and plot failure stress instead of force to facilitate the comparison of properties between wild type and Fra2Tg fascicles? Or at least mention it in the text if there was no significant difference in failure stress measured between wild type and Fra2Tg fascicles.\nResponse: In figure 5C, we followed our established practice of focusing on tendon failure force and stiffness as structural properties that reflect biologically relevant tendon function, while providing elastic modulus as a proxy measure of \"material quality\".\nReply: I agree that reporting failure stress here, in addition to failure force, would be valuable, and recommend that results for failure stress are either included in the text or the figure, particularly as the authors describe generating stress-strain curves in Figure 5b.\nAuthors response: Thank you for pointing this out. We have reported failure stress and failure strain values in (Supplementary figure 13 E-F). We have now referred to failure stress and failure strain in the text, and have included their plots in a way that is consistent with the main figures.\nViolin plots depict tail tendon failure stress (E) and failure strain (F). Each data point represents independent sample (i.e. fascicle). Horizontal lines indicate the median and interquartile range (n = 18 (Early WT), n = 12 (Early Fosl-2(Tg)), n = 23 (Established WT), n = 24 (Established Fosl-2(Tg)) fascicles from ≥ 6 mice/group. Two-way, ANOVA (genotype, disease stage) with Holm-Šídák's post-hoc test.\nFinal minor point: Some experiments had a wide variation of sample numbers per group eg Fig 5D n=4-9. Was that due to loss of samples in the process resulting in some groups having less samples than others or sample additions where it was deemed to be needed or cell numbers/availability?\nResponse: The variability in sample numbers is due to multiple factors. In Fosl-2(Tg) experiments, this often arises from the low RNA yield in Fosl-2 tendons (tissue crosslinking makes RNA extraction very challenging). Additionally, Fosl-2(Tg) mice often develop strong phenotype which necessitate euthanize the animal(s) for welfare reasons – this led to relatively high dropout rate of Fosl-2(Tg) mice from each litter relative to WT controls. Whenever we had limited number of samples, we prioritized the most important genes (e.g. Col1a1, Lox, and housekeeping genes).\nReply: It would be helpful to include this information in the supplementary information to allow the reader to understand the wide variation in sample numbers\nAuthors response: We agree. We have included a brief explanation in the Statistics section (Lines: 858 – 865]."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed all my comments and concerns thoroughly and I have no further comments on the revised manuscript.", "reviewer_3": "The authors have carefully and comprehensively addressed the points raised at review. This is appreciated and the rebuttal document includes significant new data and analysis. The revised paper is clearer and more complete. No further comments."}, "author_response": ""}]

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