Engineering an extracellular matrix-functionalized, load-bearing tendon substitute for effective repair of large-to-massive tendon defects.

A significant clinical challenge in large-to-massive rotator cuff tendon injuries is the need for sustaining high mechanical demands despite limited tissue regeneration, which often results in clinical repair failure with high retear rates and long-term functional deficiencies. To address this, an innovative tendon substitute named "BioTenoForce" is engineered, which uses (i) tendon extracellular matrix (tECM)'s rich biocomplexity for tendon-specific regeneration and (ii) a mechanically robust, slow degradation polyurethane elastomer to mimic native tendon's physical attributes for sustaining long-term shoulder movement. Comprehensive assessments revealed outstanding performance of BioTenoForce, characterized by robust core-shell interfacial bonding, human rotator cuff tendon-like mechanical properties, excellent suture retention, biocompatibility, and tendon differentiation of human adipose-derived stem cells. Importantly, BioTenoForce, when used as an interpositional tendon substitute, demonstrated successful integration with regenerative tissue, exhibiting remarkable efficacy in repairing large-to-massive tendon injuries in two animal models. Noteworthy outcomes include durable repair and sustained functionality with no observed breakage/rupture, accelerated recovery of rat gait performance, and >1 cm rabbit tendon regeneration with native tendon-like biomechanical attributes. The regenerated tissues showed tendon-like, wavy, aligned matrix structure, which starkly contrasts with the typical disorganized scar tissue observed after tendon injury, and was strongly correlated with tissue stiffness. Our simple yet versatile approach offers a dual-pronged, broadly applicable strategy that overcomes the limitations of poor regeneration and stringent biomechanical requirements, particularly essential for substantial defects in tendon and other load-bearing tissues.

Non-collagenous proteins, rather than the collagens, are key biochemical factors that mediate tenogenic bioactivity of tendon extracellular matrix.

Despite the growing clinical use of extracellular matrix (ECM)-based biomaterials for tendon repair, undesired healing outcomes or complications have frequently been reported. A major scientific challenge has been the limited understanding of their functional compositions and mechanisms of action due to the complex nature of tendon ECM. Previously, we have reported a soluble ECM fraction from bovine tendons (tECM) by urea extraction, which exhibited strong, pro-tenogenic bioactivity on human adipose-derived stem cells (hASCs). In this study, to advance our previous findings and gain insights into the biochemical nature of its pro-tenogenesis activity, tECM was fractionated using (i) an enzymatic digestion approach (pepsin, hyaluronidase, and chondroitinase) to yield various enzyme-digested tECM fractions; and (ii) a gelation-based approach to yield collagen matrix-enriched (CM) and non-collagenous matrix-enriched (NCM) fractions. Their tenogenic bioactivity on hASCs was assessed. Our results collectively indicated that non-collagenous tECM proteins, rather than collagens, are likely the important biochemical factors responsible for tECM pro-tenogenesis bioactivity. Mechanistically, RNA-seq analysis revealed that tECM and its non-collagenous portion induced similar transcriptional profiles of hASCs, particularly genes associated with cell proliferation, collagen synthesis, and tenogenic differentiation, which were distinct from transcriptome induced by its collagenous portion. From an application perspective, the enhanced solubility of the non-collagenous tECM, compared to tECM, should facilitate its combination with various water-soluble biomaterials for tissue engineering protocols. Our work provides insight into the molecular characterization of native tendon ECM, which will help to effectively translate their functional components into the design of well-defined, ECM biomaterials for tendon regeneration. STATEMENT OF SIGNIFICANCE: Significant progress has been made in extracellular matrix (ECM)-based biomaterials for tendon repair. However, their effectiveness remains debated, with conflicting research and clinical findings. Understanding the functional composition and mechanisms of action of ECM is crucial for developing safe and effective bioengineered scaffolds. Expanding on our previous work with bovine tendon ECM extracts (tECM) exhibiting strong pro-tenogenesis activity, we fractionated tECM to evaluate its bioactive moieties. Our findings indicate that the non-collagenous matrix within tECM, rather than the collagenous portions, plays a major role in the pro-tenogenesis bioactivity on human adipose-derived stem cells. These insights will drive further optimization of ECM-based biomaterials, including our advanced method for preparing highly soluble, non-collagenous matrix-enriched tendon ECM for effective tendon repair.

Tendon-derived extracellular matrix induces mesenchymal stem cell tenogenesis via an integrin/transforming growth factor-ß crosstalk-mediated mechanism.

Treatment of tendon injuries is challenging. To develop means to augment tendon regeneration, we have previously prepared a soluble, low immunogenic (DNA-free), tendon extracellular matrix fraction (tECM) by urea extraction of juvenile bovine tendons, which is capable of enhancing transforming growth factor-ß (TGF-ß) mediated tenogenesis in human adipose-derived stem cells (hASCs). Here, we aimed to elucidate the mechanism of tECM-driven hASC tenogenic differentiation in vitro, focusing on the integrin and TGF-ß/SMAD pathways. Our results showed that tECM promoted hASC proliferation and tenogenic differentiation in vitro based on tenogenesis-associated markers. tECM also induced higher expression of several integrin subunits and TGF-ß receptors, and nuclear translocation of p-SMAD2 in hASCs. Pharmacological inhibition of integrin-ECM binding, focal adhesion kinase (FAK) signaling, or TGF-ß signaling independently led to compromised pro-tenogenic effects of tECM and actin fiber polymerization. Additionally, integrin blockade inhibited tECM-driven TGFBR2 expression, while inhibiting TGF-ß signaling decreased tECM-mediated expression of integrin α1, α2, and ß1 in hASCs. Together, these findings suggest that the strong pro-tenogenic bioactivity of tECM is regulated via integrin/TGF-ß signaling crosstalk. Understanding how integrins interact with signaling by TGF-ß and/or other growth factors (GFs) within the tendon ECM microenvironment will provide a rational basis for an ECM-based approach for tendon repair.