Three-dimensional (3D) printing technology has become a popular tool to produce complex structures. It has great potential in the regenerative medicine field to produce customizable and reproducible scaffolds with high control of dimensions and porosity. This study was focused on the investigation of new biocompatible and biodegradable 3D-printed scaffolds with suitable mechanical properties to assist tendon and ligament regeneration. Polylactic acid (PLA) scaffolds were reinforced with 0.5 wt.% of functionalized graphite nanoplatelets decorated with silver nanoparticles ((f-EG)+Ag). The functionalization of graphene was carried out to strengthen the interface with the polymer. (f-EG)+Ag exhibited antibacterial properties against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli), an important feature for the healing process and prevention of bacterial infections. The scaffolds’ structure, biodegradation, and mechanical properties were assessed to confirm their suitability for tendon and ligamentregeneration. All scaffolds exhibited surface nanoroughness created during printing, which was increased by the filler presence. The wet state dynamic mechanical analysis proved that the incorporation of reinforcement led to an increase in the storage modulus, compared with neat PLA. The cytotoxicity assays using L929 fibroblasts showed that the scaffolds were biocompatible. The PLA+[(f-EG)+Ag] scaffolds were also loaded with human tendon-derived cells and showed their capability to maintain the tenogenic commitment with an increase in the gene expression of specific tendon/ligament-related markers. The results demonstrate the potential application of these new 3D-printed nanocomposite scaffolds for tendon and ligament regeneration.

Hyaluronic acid (HA)-based hydrogels offer a promising approach for soft tissue application due to their biocompatibility, tunable mechanical properties, ability to mimic the extracellular matrix, and capacity to support cell adhesion and proliferation. In this work, bioadhesive composite hydrogels were developed by integrating graphite derivatives (EG) into a dopamine-modified HA matrix (HA-Cat), which enhances tissue adhesion through catechol groups that mimic mussel-inspired adhesion mechanisms. The EG was functionalized via 1,3-dipolar cycloaddition reaction (f-EG), that allowed the anchoring of silver nanoparticles (f-EG-Ag) and grafting of hydrocaffeic acid (f-EG-Cat) on the functionalized EG surfaces. The hydrogels were produced by oxidative crosslinking of HA-Cat under mild basic pH conditions using sodium periodate. Indirect in vitro assays using L929 fibroblast cells showed high biocompatibility and enhanced cell proliferation at optimized composite hydrogel concentrations. These findings suggest that composite hydrogels could find an application as bioactive, adhesive scaffolds for the regeneration of soft tissues, where they can facilitate localized agent delivery and integration with the host tissue.

Aims: Protein disulfide isomerases (PDIs) are a family of chaperones resident in the endoplasmic reticulum (ER). In addition to holdase function, some members catalyze disulfide bond formation and isomerization, a crucial step for native folding and prevention of aggregation of misfolded proteins. PDIs are characterized by an arrangement of thioredoxin-like domains, with the canonical protein disulfide isomerase A1 (PDIA1) organized as four thioredoxin-like domains forming a horseshoe with two active sites, a and a ′, at the extremities. We aimed to clarify important aspects underlying the catalytic cycle of PDIA1 in the context of the full pathways of oxidative protein folding operating in the ER. Results: Using two fluorescent redox sensors, redox green fluorescent protein 2 (roGFP2) and HyPer (circularly permutated yellow fluorescent protein containing the regulatory domain of the H ₂ O ₂ -sensing protein OxyR), either unfolded or native, as client substrates, we identified the N-terminal a active site of PDIA1 as the main oxidant of thiols. From there, electrons can flow to the C-terminal a ′ active site, with the redox-dependent conformational flexibility of PDIA1 allowing the formation of an interdomain disulfide bond. The a ′ active site then acts as a crossing point to redirect electrons to ER downstream oxidases or back to client proteins to reduce scrambled disulfide bonds. Innovation and Conclusions: The two active sites of PDIA1 work cooperatively as an interdomain redox relay mechanism that explains PDIA1 oxidative activity to form native disulfides and PDIA1 reductase activity to resolve scrambled disulfides. This mechanism suggests a new rationale for shutting down oxidative protein folding under ER redox imbalance. Whether it applies to physiological substrates in cells remains to be shown.