Photocurable extracellular matrix sealant for cessation of venous hemorrhage.

Hemorrhage is the second leading cause of death in patients under 46 years of age in the United States. Cessation of hemorrhage prevents hemorrhagic shock and tissue hypoxia. Controlling the bleed via direct pressure or tourniquet is often the first line of defense, but long-term care requires staples, hemostatic agents, or sealants that seal the vessel and restore blood flow. Here, we compare a new photocurable extracellular matrix sealant (pcECM) with low, medium, and high crosslink density formulations to a commercially available fibrin-based sealant, TISSEEL®. pcECM has potential uses in surgical and remote settings due to room temperature storage conditions and fast preparation time. Here, we determine if pcECM sealant can stop venous hemorrhage in a murine model, adhere to the wound site in vivo throughout the wound-healing process, and has the mechanical properties necessary for stopping hemorrhage. Adjusting pcECM crosslinking density significantly affected viscosity, swelling, burst strength, tensile strength, and elasticity of the sealant. 3-Dimensional ultrasound volume segmentations showed pcECM degrades to 17 ± 8% of its initial implant volume by day 28. Initially, local hemodynamic changes were observed, but returned close to baseline levels by day 28. Acute inflammation was observed near the puncture site in pcECM implanted mice, and we observed inflammatory markers at the 14-day explant for both sealants. pcECM and fibrin sealant successfully sealed the vessel in all cases, and consistently degraded over 14-28 days. pcECM is a durable sealant with tunable mechanical properties and possible uses in hemorrhage control and other surgical procedures.

Effect of Cross-Linkers on Mussel- and Elastin-Inspired Adhesives on Physiological Substrates.

Surgical adhesives can be useful in wound closure because they reduce the risk of infection and pain associated with sutures and staples. However, there are no commercially available surgical adhesives for soft tissue wound closure. To be effective, soft tissue adhesives must be soft and flexible, strongly cohesive and adhesive, biocompatible, and effective in a moist environment. To address these criteria, we draw inspiration from the elasticity and resilience of elastin proteins and the adhesive of marine mussels. We used an elastin-like polypeptide (ELP) for the backbone of our adhesive material due to its elasticity and biocompatibility. A mussel-inspired adhesive molecule, l-3,4-dihydroxyphenylalanine (DOPA), was incorporated into the adhesive to confer wet-setting adhesion. In this study, an ELP named YKV was designed to include tyrosine residues and lysine residues, which contain amine groups. A modified version of YKV, named mYKV, was created through enzymatic conversion of tyrosine residues into DOPA. The ELPs were combined with iron(III) nitrate, sodium periodate, and/or tris(hydroxymethyl)phosphine (THP) cross-linkers to investigate the effect of DOPA- and amine-based cross-linking on adhesion strength and cure time on porcine skin in a warm, humid environment. Incorporation of DOPA into the ELP increased adhesive strength by 2.5 times and reduced failure rates. Iron cross-linkers improved adhesion in the presence of DOPA. THP increased adhesion for all proteins tested even in the absence of DOPA. Using multiple cross-linkers in a single formulation did not significantly improve adhesion. The adhesives with the highest performance (iron nitrate mixed with mYKV and THP mixed with YKV or mYKV) on porcine skin had 10-18 times higher adhesion than a commercial sealant and reached appreciable adhesive strength within 10 min.

Cytocompatibility of a mussel-inspired poly(lactic acid)-based adhesive.

Incorporating catechols into polymers can provide strong adhesion even in moist environments, and these polymers show promise for use in several biomedical applications. Surgical adhesives must have strong bonds, be biocompatible, and function in a moist environment. Poly(lactic acid) (PLA) has a long history as a biocompatible material for hard tissue device fixation. By combining these concepts, catechol-containing poly(lactic acid) (cPLA) polymers are created that are strongly adhesive and degrade in physiological environments. Here, we evaluated the cytocompatibility of cPLA with iron(III) or periodate (IO4- ) cross-linkers. Fibroblasts cultured in cPLA leachate or on cPLA films generally had slower growth and lower metabolism compared with PLA controls but no differences in viability. These results demonstrated that cPLA was not cytotoxic but that including catechols reduced cell health. When cPLA was cross-linked with periodate, cells generally had reduced metabolism, slower cell growth, and poor actin fiber formation compared with PLA. These results are attributed to the cytotoxicity of periodate since cells cultured with periodate leachate had extremely low viability. Cells grown on the films of iron-cross-linked cPLA generally had high viability and metabolism but slower proliferation than PLA controls. These results indicate that the cPLA and iron-cross-linked cPLA systems are promising materials for biomedical adhesive applications.

pH-Sensitive Mechanical Properties of Elastin-Based Hydrogels.

Ionizable amino acids in protein-based hydrogels can confer pH-responsive behavior. Because elastin-like polypeptides (ELPs) have an established sequence and can crosslink to form hydrogels, they are an ideal system for creating pH-sensitive materials. This study examines different parameters that might affect pH-sensitive behavior and characterizes the mechanical and physical properties between pH 3 and 11 of three ELP-based crosslinked hydrogels. The first finding is that varying the amount of crosslinker affects the overall stiffness and resilience of the hydrogels but does not strongly affect water content, swelling ratio, or pH sensitivity. Second, the choice of two popular tag sequences, which vary in histidine and aspartic acid content, does not have a strong effect on pH-sensitive properties. Last, selectively blocking lysine and tyrosine residues through acetylation significantly decreases the pH-sensitive zeta potential. Acetylated hydrogels also demonstrate different behavior at low pH values with reduced swelling, reduced water content, and higher stiffness. Overall, this work demonstrates that ELP hydrogels with ionizable groups are promising materials for environmentally-responsive applications such as drug delivery, tissue engineering, and microfluidics.

Critical factors for the bulk adhesion of engineered elastomeric proteins.

Many protein-based materials, such as soy and mussel adhesive proteins, have been the subject of scientific and commercial interest. Recently, a variety of protein adhesives have been isolated from diverse sources such as insects, frogs and squid ring teeth. Many of these adhesives have similar amino acid compositions to elastomeric proteins such as elastin. Although elastin is widely investigated for a structural biomaterial, little work has been done to assess its adhesive potential. In this study, recombinant elastin-like polypeptides were created to probe the factors affecting adhesion strength. Lap shear adhesion was used to examine the effects of both extrinsic factors (pH, concentration, cross-linker, humidity, cure time and cure temperature) and intrinsic factors (protein sequence, structure and molecular weight). Of the extrinsic factors tested, only humidity, cure time and cure temperature had a significant effect on adhesion strength. As water content was reduced, adhesion strength increased. Of the intrinsic factors tested, amino acid sequence did not significantly affect adhesion strength, but less protein structure and higher molecular weights increased adhesion strength directly. The strengths of proteins in this study (greater than 2 MPa) were comparable to or higher than those of two commercially available protein-based adhesives, hide glue and a fibrin sealant. These results may provide general rules for the design of adhesives from elastomeric proteins.

Designing Smart Materials with Recombinant Proteins.

Recombinant protein design allows modular protein domains with different functionalities and responsive behaviors to be easily combined. Inclusion of these protein domains can enable recombinant proteins to have complex responses to their environment (e.g., temperature-triggered aggregation followed by enzyme-mediated cleavage for drug delivery or pH-triggered conformational change and self-assembly leading to structural stabilization by adjacent complementary residues). These "smart" behaviors can be tuned by amino acid identity and sequence, chemical modifications, and addition of other components. A wide variety of domains and peptides have smart behavior. This review focuses on protein designs for self-assembly or conformational changes due to stimuli such as shifts in temperature or pH.

Effect of localizing fruit and vegetable consumption on greenhouse gas emissions and nutrition, Santa Barbara County.

The US agrifood system is very productive, but highly centralized and resource intensive with very weak links between production and consumption. This contributes to high levels of malnutrition and greenhouse gas emissions (GHGE). A popular approach to improvement is localization-reducing direct transport (farm to retail distance, or "food miles"). We examined Santa Barbara County (SBC) California, which mirrors the high production, nutritional and environmental problems, and growing localization movement of California. SBC ranks in the top 1% of US counties in value of agricultural products, and >80% of this value is produce (fruits and vegetables). We calculated the amount of produce grown in and consumed in SBC and estimated that >99% of produce grown in SBC is exported from the county, and >95% of produce consumed in SBC is imported. If all produce consumed in SBC was grown in the county (100% localization), it would reduce GHGE from the agrifood system <1%, and not necessarily affect nutrition. While food miles capture only a portion of the environmental impact of agrifood systems, localization could be done in ways that promote synergies between improving nutrition and reducing GHGE, and many such efforts exist in SBC.