Citrus-Derived Flavonoid Didymin Shows Promise for Healing Tendon-Bone Injuries

A natural compound extracted from a traditional Chinese medicinal herb may hold the key to one of orthopedic surgery’s most stubborn problems: the failure of tendon-bone junctions to heal properly after injury. In a new study published in Immunity, Inflammation and Disease, researchers report that didymin, the most abundant dihydroflavonoid in Clinopodium chinense (Benth.), promoted new bone formation and fibrocartilage regeneration at the tendon-bone interface in mice while simultaneously steering immune cells called macrophages away from a pro-inflammatory state and toward a repair-promoting one. The findings point to a metabolic switch inside macrophages, governed by the peroxisome proliferator-activated receptor (PPAR) signaling pathway and fatty acid oxidation, as a promising drug target for accelerating recovery from rotator cuff tears, Achilles tendon injuries, and similar damage.

Tendon-bone insertion injuries, which occur where soft tendon tissue anchors into hard bone, are among the most frequently encountered injuries in daily life and sports. Their clinical burden is growing annually, and restoring normal physiological function remains remarkably difficult. The reason lies in the anatomy: the tendon-bone junction is a graded transition zone that includes a specialized fibrocartilage layer, and unlike many tissues, it cannot regenerate itself faithfully after injury. Instead, the body fills the gap with disorganized scar tissue that has low biomechanical strength. That weakness translates directly into high postoperative re-rupture rates, making rapid, high-quality regeneration an urgent challenge in orthopedics, sports medicine, and tissue engineering.

The quality of tendon-bone healing depends heavily on how well the fibrocartilage layer regenerates at the interface, and macrophages, the versatile immune cells that flood any wound site, orchestrate much of that process. In the early phase of healing, M1-polarized macrophages dominate, secreting pro-inflammatory factors such as IL-1β and IL-6 that intensify local inflammation and recruit fibroblasts to the damaged site. Later, macrophages shift toward the M2 phenotype, releasing anti-inflammatory signals like IL-10 and Arg1 that dampen inflammation and promote tissue reconstruction. Previous work has shown that this transition from a pro-inflammatory to an anti-inflammatory microenvironment is crucial for moving from the inflammatory phase into the proliferative phase of repair, and that biologically inducing early M2 aggregation at the interface actively improves healing outcomes.

What makes the new study distinctive is its focus on macrophage metabolism. M2 macrophages rely on oxidative phosphorylation and fatty acid oxidation (FAO) for their energy supply, whereas activation of PPAR signaling and its regulatory genes increases FAO capacity. The research team, led by Xiaojun Ma and colleagues, hypothesized that if a compound could push macrophage metabolism toward FAO and oxidative phosphorylation, it would favor M2 polarization and thereby improve tendon-bone healing. Didymin was a natural candidate: earlier reports had documented its purgative, anti-inflammatory, and antioxidant properties, and prior metabolic tracing experiments had revealed that the compound enhances FAO rather than glycolysis. Crucially, didymin had already been shown to modulate the M1/M2 balance by converting pro-inflammatory M1-like macrophages into anti-inflammatory M2-like ones, without altering baseline M2 polarization.

To test the hypothesis, the researchers established a mouse model of tendon-bone healing by surgically severing the Achilles tendon near the calcaneal bone, scraping away the residual fibrocartilaginous layer, drilling a bone channel, and suturing the tendon stump back to the bone, a procedure that mimics the changes seen after rotator cuff reconstruction. Thirty-six male C57BL/6J mice were randomly assigned to six groups: a sham-operated control, an untreated model group, three didymin dose groups receiving 1, 2, or 4 milligrams per kilogram per day by oral gavage, and a positive-control group receiving the drug disulfiram at 50 milligrams per kilogram per day. Treatment began one day after surgery and continued daily for four weeks, with all protocols approved by the animal ethics committee of the People’s Hospital of Ningxia Hui Autonomous Region.

The structural results were striking. Micro-computed tomography scanning showed that high-dose didymin produced new bone volume at the interface comparable to that seen in the positive-control group, with significant increases in bone volume fraction, bone surface, trabecular number, and bone mineral density, along with significantly reduced trabecular separation compared with the untreated model group. Histological staining revealed that model-group animals suffered from disorganized collagen fibers, consolidated fibroblast nuclei, necrotic areas, and inflammatory cell infiltration at the junction, all of which were attenuated by didymin treatment. Serum biochemistry told a matching story: levels of osteocalcin, alkaline phosphatase, and calcium, all markers of bone formation activity, rose significantly in the treated animals.

Fibrocartilage regeneration, the critical determinant of healing quality, also improved. Safranin-O/fast green staining showed localized absence of the cartilage layer and abnormal chondrocyte proliferation in untreated mice, defects that didymin largely corrected. Immunofluorescence assays demonstrated that expression of the cartilage markers Sox9 and Collagen II, which dropped sharply at the injured interface, was significantly restored by the compound. The researchers also measured increased expression of TGF-β1 and TGF-β3, growth factors known to drive chondrogenesis and fibrocartilage formation, further supporting the conclusion that didymin actively promotes regeneration of the specialized transition tissue rather than merely reducing inflammation.

Flow cytometry and molecular profiling of the healing tissue revealed the immune mechanism at work. Untreated model mice showed elevated levels of F4/80-positive CD86-positive M1 macrophages and elevated IL-1β and IL-6 mRNA, while didymin administration at all three doses reduced these pro-inflammatory indicators. Medium and high doses simultaneously raised the abundance of F4/80-positive CD206-positive M2 macrophages and increased expression of Arg1 and IL-10. In parallel cell experiments using bone marrow-derived macrophages, didymin significantly reduced CD86 expression under M1-polarizing conditions and boosted levels of acetyl-CoA, the FAO enzymes Cpt1a and ACS, and the PPAR pathway proteins PPAR-γ and RXRA, indicating a metabolic reprogramming toward fatty acid oxidation.

The team then ran a decisive validation experiment using pharmacological blockers. When the PPAR-γ antagonist GW9662 or the fatty acid oxidation inhibitor etomoxir was added to didymin-treated M1 macrophages, the compound’s effects were substantially reversed: acetyl-CoA levels, CD206-positive M2 populations, and anti-inflammatory Arg1 and IL-10 expression all fell, while CD86-positive M1 populations and pro-inflammatory IL-1β and IL-6 rose again. This loss-of-function evidence strongly suggests that didymin drives macrophage polarization toward the M2 phenotype specifically by promoting the PPAR signaling-mediated FAO pathway, which in turn supports tendon-bone healing.

The authors acknowledge important limitations that temper immediate clinical translation. No biomechanical testing was performed, the antagonist and inhibitor experiments were conducted only in cell culture rather than in living animals, and all mice received local penicillin injections postoperatively, which could theoretically interact with didymin’s anti-inflammatory and antibacterial properties. Only male mice were studied, and sex is known to influence inflammatory responses, bone metabolism, and tendon healing, so verification in female animals is needed. The mouse Achilles model also cannot fully reproduce the biomechanics, vascular supply, and stress conditions of human rotator cuff attachment sites. Even so, the study lays a concrete foundation for didymin as a candidate therapy, linking a dietary flavonoid, immune cell metabolism, and musculoskeletal regeneration in a single mechanistic chain that future research can now build upon.

Subject of Research: The role of didymin in promoting tendon-bone healing through PPAR-mediated fatty acid oxidation and macrophage M2 polarization

Article Title: Didymin may Enhance Tendon‐Bone Healing Partly by Promoting PPAR‐Mediated Fatty Acid Oxidation and Macrophage M2 Polarization

Article References: Ma, X., Shen, J., Ma, J., Li, H., Li, Y., & Wan, J. (2026). Didymin may Enhance Tendon‐Bone Healing Partly by Promoting PPAR‐Mediated Fatty Acid Oxidation and Macrophage M2 Polarization. Immunity, Inflammation and Disease, 14(9), Article e70485. https://doi.org/10.1002/iid3.70485

Image Credits: AI Generated

DOI: 10.1002/iid3.70485

Keywords: didymin, tendon-bone healing, macrophage polarization, M2 macrophages, PPAR signaling, fatty acid oxidation, fibrocartilage, Achilles tendon, rotator cuff, bone regeneration, inflammation, natural compounds