Controlled Stiffness of Direct-Write, Near-Field Electrospun Gelatin Fibers Generates Differences in Tenocyte Morphology and Gene Expression.

Tendinopathy is a leading cause of mobility issues. Currently, the cell-matrix interactions involved in the development of tendinopathy are not fully understood. In vitro tendon models provide a unique tool for addressing this knowledge gap as they permit fine control over biochemical, micromechanical, and structural aspects of the local environment to explore cell-matrix interactions. In this study, direct-write, near-field electrospinning of gelatin solution was implemented to fabricate micron-scale fibrous scaffolds that mimic native collagen fiber size and orientation. The stiffness of these fibrous scaffolds was found to be controllable between 1 MPa and 8 MPa using different crosslinking methods (EDC, DHT, DHT+EDC) or through altering the duration of crosslinking with EDC (1 h to 24 h). EDC crosslinking provided the greatest fiber stability, surviving up to 3 weeks in vitro. Differences in stiffness resulted in phenotypic changes for equine tenocytes with low stiffness fibers (∼1 MPa) promoting an elongated nuclear aspect ratio while those on high stiffness fibers (∼8 MPa) were rounded. High stiffness fibers resulted in the upregulation of matrix metalloproteinase (MMPs) and proteoglycans (possible indicators for tendinopathy) relative to low stiffness fibers. These results demonstrate the feasibility of direct-written gelatin scaffolds as tendon in vitro models and provide evidence that matrix mechanical properties may be crucial factors in cell-matrix interactions during tendinopathy formation.

Clot-Targeted Nanogels for Dual-Delivery of AntithrombinIII and Tissue Plasminogen Activator to Mitigate Disseminated Intravascular Coagulation Complications.

Disseminated intravascular coagulation (DIC) is a pathologic state that follows systemic injury and other diseases. Often a complication of sepsis or trauma, DIC causes coagulopathy associated with paradoxical thrombosis and hemorrhage. DIC upregulates the thrombotic pathways while simultaneously downregulating the fibrinolytic pathways that cause excessive fibrin deposition, microcirculatory thrombosis, multiorgan dysfunction, and consumptive coagulopathy with excessive bleeding. Given these opposing disease phenotypes, DIC management is challenging and includes treating the underlying disease and managing the coagulopathy. Currently, no therapies are approved for DIC. We have developed clot-targeted therapeutics that inhibit clot polymerization and activate clot fibrinolysis to manage DIC. We hypothesize that delivering both an anticoagulant and a fibrinolytic agent directly to clots will inhibit active clot polymerization while also breaking up pre-existing clots; therefore, reversing consumptive coagulopathy and restoring hemostatic balance. To test this hypothesis, we single- and dual-loaded fibrin-specific nanogels (FSNs) with antithrombinIII (ATIII) and/or tissue plasminogen activator (tPA) and evaluated their clot preventing and clot lysing abilities in vitro and in a rodent model of DIC. In vivo, single-loaded ATIII-FSNs decreased fibrin deposits in DIC organs and reduced blood loss when DIC rodents were injured. We also observed that the addition of tPA in dual-loaded ATIII-tPA-FSNs intensified the antithrombotic and fibrinolytic mechanisms, which proved advantageous for clot lysis and restoring platelet counts. However, the addition of tPA may have hindered wound healing capabilities when an injury was introduced. Our data supports the benefits of delivering both anticoagulants and fibrinolytic agents directly to clots to reduce the fibrin load and restore hemostatic balance in DIC.

Fighting fibrin with fibrin: Vancomycin delivery into coagulase-mediated Staphylococcus aureus biofilms via fibrin-based nanoparticle binding.

Staphylococcus aureus skin and soft tissue infection is a common ailment placing a large burden upon global healthcare infrastructure. These bacteria are growing increasingly recalcitrant to frontline antimicrobial therapeutics like vancomycin due to the prevalence of variant populations such as methicillin-resistant and vancomycin-resistant strains, and there is currently a dearth of novel antibiotics in production. Additionally, S. aureus has the capacity to hijack the host clotting machinery to generate fibrin-based biofilms that confer protection from host antimicrobial mechanisms and antibiotic-based therapies, enabling immune system evasion and significantly reducing antimicrobial efficacy. Emphasis is being placed on improving the effectiveness of therapeutics that are already commercially available through various means. Fibrin-based nanoparticles (FBNs) were developed and found to interact with S. aureus through the clumping factor A (ClfA) fibrinogen receptor and directly integrate into the biofilm matrix. FBNs loaded with antimicrobials such as vancomycin enabled a targeted and sustained release of antibiotic that increased drug contact time and reduced the therapeutic dose required for eradicating the bacteria, both in vitro and in vivo. Collectively, these findings suggest that FBN-antibiotic delivery may be a novel and potent therapeutic tool for the treatment of S. aureus biofilm infections.

Microparticle Hydrogel Material Properties Emerge from Mixing-Induced Homogenization in a Poly(ethylene glycol) and Dextran Aqueous Two-Phase System.

Polymer-polymer aqueous two-phase systems (ATPSs) are attractive for microgel synthesis, but given the complexity of phase separation, predicting microgel material properties from ATPS formulations is not trivial. The objective of this study was to determine how the phase diagram of a poly(ethylene glycol) (PEG) and dextran ATPS is related to the material properties of PEG microgel products. PEG-dextran ATPSs were prepared from four-arm 20 kDa PEG-norbornene and 40 kDa dextran in phosphate buffered saline (PBS), and the phase diagram was constructed. PEG microgels were synthesized from five ATPS formulations using an oligopeptide cross-linker and thiol-norbornene photochemistry. Thermogravimetric analysis (TGA) revealed that the polymer concentration of microgel pellets linearly correlates with the average concentration of PEG in the ATPS rather than the separated phase compositions, as determined from the phase diagram. Atomic force microscopy (AFM) and bulk rheology studies demonstrated that the mechanical properties of microgels rely on both the average concentration of PEG in the ATPS and the ATPS volume ratio as determined from the phase diagram. These findings suggest that PEG-dextran ATPSs undergo homogenization upon mixing, which principally determines the material properties of the microgels upon gelation.