Tendinopathy is the most frequent musculoskeletal disease that requires medical attention. Mechanical overload has been considered as a key driver of its pathology. However, the underline mechanism on how overload induces tendinopathy and inflammation is unclear. Extracellular mitochondria (EM) are newly identified as cell-to-cell communicators. The aim of this study is to elucidate the role of mitochondria in overload-induced inflammation.

We performed three-dimensional uniaxial stretching to mouse tendon organoid in bioreactors. Cyclic strain of uniaxial loadings included underload, normal load, and overload, according to previous work. We then harvested microvesicles including EM, from the bioreactor by differential centrifugation and evaluated their characteristics by flow cytometry and super-resolution confocal microscopy. Raw 264.7 mouse macrophage cell line was used for chemotaxis assay in a Boyden Chamber System with Magnetic-Activated Cell Sorting Technology. EM induced cytokines secretion by macrophages was analyzed by a bead-based multiplex assay panel. N-Acetyl-L-cysteine (NAC) was used as the antioxidant to tendon organoid to regulate mitochondrial fitness.

We showed mechanical load induced tendon organoid to release microvesicles including mitochondria. The size of microvesicles is mainly in the range from 220nm to 880nm. More than 75% of microvesicles could be stained by PKH26, confirming they were with lipophilic membrane. Super-resolution confocal microscopy identified two forms of mitochondria, including mitochondria encapsulated in vesicles and free mitochondria. Overload led to the degeneration of the organoid and induced microvesicles release containing most EM. Chemotaxis assay showed that EM from overloaded tendon organoid induced macrophages chemotaxis. In addition, microvesicles extracted from overloaded tendon organoid induced the production of proinflammatory cytokines including IL-6, KC (Keratinocyte-Derived Chemokine) and IL-18. NAC treatment to tendon cells could attenuate overload-induced macrophage chemotaxis.

Overload induces EM releasing from tendon cells, which leads to chemotaxis of macrophages toward tendon, resulting in induction of inflammation.

Nerve transfer is an emerging treatment to restore upper limb function in people with tetraplegia. The objective of this study is to examine if a flexible collage sheet (FCS) can act as epineurial-like substitute to promote nerve repair in nerve transfer.

A preclinical study using FCS was conducted in a rat model of sciatic nerve transection. A prospective case series study of nerve transfer was conducted in patients with C5-C8 tetraplegia who received nerve transfer to restore upper limb function. Motor function in the upper limb was assessed pre-treatment, and at 6-,12-, and 24-months post-treatment.

Macroscopic assessment in preclinical model showed nerve healing by FCS without encapsulation or adhesions. Microscopic examination revealed that a new, vascularised epineurium-like layer was observed at the FCS treatment sites, with no evidence of inflammatory reaction or nerve compression. Treatment with FCS resulted in well-organised nerve fibres with dense neurofilaments distal to the coaptation site. Axon counts performed proximal and distal to the coaptation site showed that 97% of proximal axon count of myelinated axons regenerated across the coaptation site after treatment with CND. In the proof of concept clinical study 17 nerve transfers were performed in five patients. Nerve transfers included procedures to restore triceps function (N=4), wrist/finger/thumb extension (N=6) and finger flexion (N=7). Functional motor recovery (MRC ≥3) was achieved in 76% and 88% of transfers at 12 and 24 months, respectively.

The preclinical study showed that FCS mimics epineurium and enable to repair nerve resembled to normal nerve tissue. Clinical study showed that patients received nerve transfer with FCS experienced consistent and early return of motor function in target muscles. These results provide proof of concept evidence that CND functions as an epineurial substitute and is promising for use in nerve transfer surgery.