Mitochondrial function following rotator cuff tendon injury (RCI) influences the tendon healing. We examined the mitochondrial morphology and function under hypoxia in the shoulder tendon tissue from surgically-induced tenotomy-RCI rat model and cultured swine tenocytes. The tendon tissue was collected post-injury on 3–5 (Group-A), 10–12 (Group-B), and 22–24 (Group-C), days and the corresponding contralateral tendons were used as control for each group. There was higher protein expression of citrate synthase (P < 0.0001) [10.22 MFI (mean fluorescent intensity)] and complex-1 (P = 0.0008) (7.86 MFI) in Group-A and Group-B that decreased in Group-C [(P = 0.0201) (5.78 MFI and (P = 0.7915) (2.32 MFI), respectively] compared to control tendons. The ratio of BAX:Bcl2 (Bcl2 associated x protein:B cell lymphoma 2) in RCI tendons increased by 50.5% (Group-A) and 68.4% (Group-B) and decreased by 25.8% (Group-C) compared to normoxic controls. Hypoxia increased β-tubulin expression (P = 0067) and reduced PGC1-α (P = 0412) expression in the isolated swine tenocytes with no effect on the protein expression of Complex-1 (P = 7409) and citrate synthase (P = 0.3290). Also, the hypoxic tenocytes exhibited about 4-fold increase in mitochondrial superoxide (P < 0.0001), altered morphology and mitochondrial pore integrity, and increase in mitochondrial density compared to normoxic controls. These findings suggest the critical role of mitochondria in the RCI healing response.

Regenerative functions of exosomes rely on their contents which are influenced by pathological stimuli, including hypoxia, in rotator cuff tendon injuries (RCTI). The hypoxic environment triggers tenocytes and adjacent adipose-derived mesenchymal stem cells (ADMSCs) to release regenerative mediators to the ECM via the exosomes which elicit autocrine/paracrine responses to protect the tendon matrix from injury. We investigated the exosomal protein contents from tenocytes and subcutaneous ADMSCs from the shoulder of Yucatan microswine cultured under hypoxic conditions (2% O 2 ). The exosomal proteins were detected using high-resolution massspectrometry nano-LC-MS/MS Tribrid system and were compiled using 'Scaffold' software. Hypoxic exosomes from tenocytes and ADMSCs carried 199 and 65 proteins, respectively. The key proteins identified by mass spectrometry and associated with ECM homeostasis from hypoxic ADMSCs included MMP2, COL6A, CTSD and TN-C and those from hypoxic tenocytes were THSB1, NSEP1, ITIH4 and TN-C. These findings were confirmed at the mRNA and protein level in the hypoxic ADMSCs and tenocytes. These proteins are involved in multiple signaling pathways of ECM repair/regeneration. This warrants further investigations for their translational significance in the management of RCTI.

Replenishment of tenocytes to the injury site is an ideal strategy to improve healing response and accelerate the tendon ECM regeneration. The present study focused on the synthesis and characterization of a hybrid hydrogel scaffold system poly(propylene‐fumarate)‐alginate‐polyvinyl alcohol‐acrylic acid (PAPA) using poly(propylene‐fumarate) (PPF), alginate, polyvinyl alcohol (PVA) and acrylic acid and the in vitro investigation of bidirectional mobility of swine shoulder tenocytes (SST) for its potential application in rotator‐cuff tendon regeneration. IR analysis revealed the presence of alginate, PPF and PVA segments on the surface, SEM and AFM analyses revealed the porous and nano‐topographical features of PAPA, respectively, swelling was 712.6 ± 84.21% with the EWC (%) of 87.59 ± 1.26 having the diffusional exponent and swelling constant 0.551 and 1.8, respectively. PAPA was biodegradable, cytocompatible and supported long‐term survival of SSTs. SEM imaging revealed the adhesion, colonization, and sheet formation of SSTs within the PAPA hydrogel network. The SSTs seeded on the PAPA scaffolds were peculiar for their bidirectional migration as the anterograde movement was completed in 9 days whereas the retrograde infiltration occurred up to the depth of 198 μm. These findings suggest the promising translational potential of PAPA scaffold system in the management of rotator cuff tendon injury.