A human synovial tendon-on-a-chip models key features of peritendinous adhesions and offers a new approach methodology for testing anti-fibrotic drugs

The authors developed a human synovial tendon-on-a-chip model that recapitulates key features of peritendinous adhesions through synovial fibroblast-driven inflammation and matrix deposition, demonstrating its utility as a new approach methodology for identifying the IL-6/JAK/STAT pathway as a therapeutic target for anti-fibrotic drugs.

The Gist

The Problem: The "Sticky Glue" of Healing

Imagine you cut your finger or injure a tendon in your hand. Your body is amazing at fixing it, but sometimes, it gets too enthusiastic. Instead of just healing the cut, the body lays down a thick, sticky layer of scar tissue that glues your tendon to the surrounding skin and muscle.

In medical terms, this is called a peritendinous adhesion. Think of it like pouring superglue between two moving parts of a machine. If your finger tendon is glued to the surrounding tissue, you can't bend your finger properly. It's stiff, weak, and painful. Currently, there are no good drugs to stop this from happening, and surgery to cut the glue often just makes more glue.

The Solution: A "Tendon-on-a-Chip"

The researchers at the University of Rochester wanted to figure out why this glue forms and how to stop it. But testing on animals (like mice) is tricky because their bodies work differently than ours. Testing on humans directly is too risky.

So, they built a Synovial Tendon-on-a-Chip (synToC).

The Analogy: Imagine a tiny, transparent Lego house built inside a microchip.

• The Bottom Room: This is the Tendon. It's filled with tendon cells and immune cells (the body's repair crew).
• The Top Room: This is the Synovial Sheath (the protective sleeve around the tendon). It's filled with special cells called Synovial Fibroblasts (FLS) and a tiny blood vessel.
• The Wall: Between the two rooms is a super-thin, porous membrane. It's like a screen door that lets cells and signals pass through but keeps the rooms separate.

This chip recreates the exact environment inside your finger where these injuries happen, but in a dish you can control.

The Discovery: The "Overzealous Foreman"

The researchers wanted to know: Who is responsible for making the glue?

They ran an experiment where they had two versions of the chip:

1. Chip A: Had the tendon and blood vessel, but the top room was empty (no synovial cells).
2. Chip B: Had the tendon, blood vessel, AND the synovial cells (FLS).

The Result:

• Chip A stayed relatively calm. The tendon healed, but didn't get stuck to the top.
• Chip B went haywire. The synovial cells (FLS) acted like overzealous foremen. Even without any extra "injury signals" added to the system, these cells started shouting orders.

They shouted two main things:

1. "Build more glue!" They started pumping out a protein called IL-6. This signal told the tendon cells to start building a thick, sticky bridge of collagen and fibronectin (the glue) that physically connected the top room to the bottom room.
2. "Call the troops!" The IL-6 signal also acted like a siren, calling immune cells (monocytes) from the blood vessel to rush into the tissue, making the inflammation worse.

The Big Surprise: Usually, scientists think you need a heavy dose of a chemical called TGF-β1 to cause this mess. But in this chip, the synovial cells caused the problem all by themselves. They were the root cause.

The Fix: Turning Down the Volume

Once they knew the synovial cells were the problem and that IL-6 was the main shout, they tried to silence them using two existing drugs already approved for other diseases (like rheumatoid arthritis):

• Tocilizumab (TCZ): Blocks the receiver so the cells can't hear the IL-6 shout.
• Tofacitinib (TOFA): Turns off the internal alarm system inside the cells.

The Result:
When they added these drugs to the chip:

• The "shouting" stopped.
• The immune troops didn't rush in.
• The sticky glue bridges never formed.
• The tendon remained smooth and free to move.

Why This Matters

This paper is a game-changer for three reasons:

1. It's Human-Specific: We finally have a model that uses human cells to see exactly how human tendons react, rather than guessing based on mice.
2. It Finds the Culprit: It proved that the cells in the "sleeve" (synovium) are the main troublemakers, not just the tendon itself.
3. It Offers a Cure: It shows that drugs we already have in our medicine cabinets could be repurposed to stop tendon adhesions before they start.

In a nutshell: The researchers built a tiny, human finger-in-a-box. They discovered that a specific type of cell acts like a noisy foreman, ordering the body to glue the tendon shut. By using existing drugs to tell that foreman to "shut up," they stopped the glue from forming. This opens the door to new, effective treatments for people who can't move their fingers after an injury.

Technical Summary

1. Problem Statement

Peritendinous adhesions are a debilitating complication of tendon injuries (particularly in Zone II flexor tendons) and surgery, affecting up to 40% of patients. These pathological scar-like connections restrict range of motion and grip strength.

• Current Limitations: Existing preclinical models (rodent and large animal) fail to fully recapitulate the complex multicellular interactions, immune trafficking, and vascular-synovial crosstalk specific to human tendon adhesion pathogenesis. Furthermore, there are no FDA-approved drugs to prevent or resolve these adhesions.
• Knowledge Gap: The specific cellular origins of the fibroblasts driving adhesions and the molecular mechanisms linking synovial inflammation to tendon fibrosis remain poorly defined, largely due to the lack of physiologically relevant human in vitro models.

2. Methodology: The Synovial Tendon-on-a-Chip (synToC)

The authors developed a human-relevant microphysiological system (MPS) called the synToC to model the intrasynovial tendon microenvironment.

• Device Architecture: The platform utilizes a modular design with two compartments separated by a 3-µm dual-scale nanomembrane (containing both nanopores and micropores).
  • Bottom Compartment (Tendon): Contains a 3D type I collagen hydrogel encapsulating primary human tendon fibroblasts and monocytes (differentiated into macrophages).
  • Top Compartment (Vascular-Synovial): Features a vascularized synovial lining. This is constructed by polymerizing a type I collagen hydrogel containing primary human fibroblast-like synoviocytes (FLS) on the basal side of the membrane, followed by seeding human umbilical vein endothelial cells (HUVECs) on the apical side to form a vascular barrier.
• Experimental Design:
  • Co-culture: The system integrates tendon fibroblasts, FLS, immune cells (monocytes/macrophages), and vascular endothelium.
  • Stimulation: Experiments were conducted with and without exogenous TGF-β1 (a known fibrotic driver) to test if FLS alone could induce pathology.
  • Drug Testing: The model was used to test two FDA-approved drugs targeting the IL-6/JAK/STAT pathway: Tocilizumab (TCZ) (anti-IL-6 receptor antibody) and Tofacitinib (TOFA) (JAK inhibitor).
• Readouts:
  • Structural: Tendon hydrogel contraction, Second Harmonic Generation (SHG) for collagen density, and adhesion contact length (fibronectin bridging).
  • Cellular: Immunofluorescence for markers of activation (α-SMA, Ki67, CD90, PDPN), immune infiltration (monocyte transmigration), and signaling (pSTAT3).
  • Molecular: Multiplex cytokine profiling of supernatants.

3. Key Contributions

• First Human Intrasynovial Model: This is the first in vitro model to integrate a vascularized synovial sheath with a tendon construct, recapitulating the "intrinsic" (tendon) and "extrinsic" (synovial) healing responses.
• Identification of FLS as Primary Drivers: The study demonstrates that synovial fibroblasts (FLS) are sufficient to drive adhesion-like pathology without exogenous TGF-β1, challenging the view that TGF-β1 is the sole initiator.
• Mechanistic Insight: The model elucidates a feed-forward loop where FLS activation leads to IL-6 secretion, monocyte recruitment, and subsequent matrix deposition.
• New Approach Methodology (NAM): The synToC serves as a predictive, human-relevant platform for screening anti-fibrotic therapies, bridging the gap between reductionist cell cultures and animal models.

4. Key Results

A. FLS Drive Adhesion Pathology Independently of TGF-β1

• Tendon Contraction: In the absence of exogenous TGF-β1, devices with FLS (+FLS) exhibited significantly greater tendon hydrogel contraction and collagen packing density compared to acellular controls (-FLS).
• Myofibroblast Differentiation: +FLS devices showed a higher proportion of α-SMA+ myofibroblasts in the tendon compartment, indicating that FLS paracrine signaling induces tendon fibrosis.
• Matrix Bridging: +FLS devices formed dense networks of fibronectin and collagen III physically bridging the synovial and tendon compartments, mimicking nascent adhesions. This occurred even without TGF-β1, whereas -FLS devices showed minimal bridging.

B. Immune Cell Recruitment and Inflammation

• Monocyte Infiltration: The presence of FLS significantly increased the transendothelial migration of monocytes from the vascular channel into the synovial and tendon compartments.
• Cytokine Profile: The model revealed a dominant inflammatory signature characterized by high levels of IL-6 and IL-8.
  • In the absence of TGF-β1, FLS drove a massive accumulation of IL-6 (100-fold increase from Day 1 to Day 5).
  • IL-6 levels correlated with monocyte infiltration and matrix deposition.

C. Pharmacological Validation

• Targeting IL-6/JAK/STAT: Treatment with Tocilizumab (TCZ) and Tofacitinib (TOFA) successfully suppressed the IL-6/JAK/STAT axis (reduced pSTAT3).
• Therapeutic Outcomes:
  • Reduced Inflammation: Both drugs significantly lowered MCP-1 and IL-8 levels and attenuated monocyte infiltration.
  • Attenuated Fibrosis: Both treatments reduced collagen III deposition in both synovial and tendon gels.
  • Adhesion Prevention: Crucially, both drugs significantly reduced the adhesion contact length (fibronectin bridging) and synovial hyperplasia.
  • Note: While TCZ blocked IL-6 signaling, it did not reduce IL-6 protein levels (consistent with receptor blockade), whereas TOFA reduced downstream signaling. TOFA uniquely reduced tendon contraction, suggesting distinct mechanisms of action on tissue remodeling.

5. Significance

• Clinical Translation: The synToC provides a mechanism-informed platform to evaluate drug candidates that would otherwise be difficult to test in animal models due to species-specific differences in immune response and tendon architecture.
• Therapeutic Target Identification: The study validates the IL-6/JAK/STAT pathway as a viable therapeutic target for preventing peritendinous adhesions. It suggests that repurposing existing drugs (TCZ, TOFA) could prevent post-surgical adhesion formation.
• Paradigm Shift: The findings shift the understanding of adhesion pathogenesis, highlighting that synovial fibroblasts act as key immune sentinels and drivers of fibrosis, rather than just passive responders to injury.
• NAM Adoption: This work supports the transition toward New Approach Methodologies (NAMs) in musculoskeletal research, offering a human-specific, high-throughput alternative to animal testing for fibrotic diseases.

In conclusion, the synToC successfully models the complex multicellular crosstalk driving peritendinous adhesions, identifies IL-6 as a critical mediator, and demonstrates the efficacy of JAK/STAT inhibition in preventing adhesion formation, paving the way for novel clinical interventions.