Engineered Hemostatic Biomaterials for Sealing Wounds.

Hemostatic biomaterials show great promise in wound control for the treatment of uncontrolled bleeding associated with damaged tissues, traumatic wounds, and surgical incisions. A surge of interest has been directed at boosting hemostatic properties of bioactive materials via mechanisms triggering the coagulation cascade. A wide variety of biocompatible and biodegradable materials has been applied to the design of hemostatic platforms for rapid blood coagulation. Recent trends in the design of hemostatic agents emphasize chemical conjugation of charged moieties to biomacromolecules, physical incorporation of blood-coagulating agents in biomaterials systems, and superabsorbing materials in either dry (foams) or wet (hydrogel) states. In addition, tough bioadhesives are emerging for efficient and physical sealing of incisions. In this Review, we highlight the biomacromolecular design approaches adopted to develop hemostatic bioactive materials. We discuss the mechanistic pathways of hemostasis along with the current standard experimental procedures for characterization of the hemostasis efficacy. Finally, we discuss the potential for clinical translation of hemostatic technologies, future trends, and research opportunities for the development of next-generation surgical materials with hemostatic properties for wound management.

Associating growth factor secretions and transcriptomes of single cells in nanovials using SEC-seq.

Cells secrete numerous bioactive molecules that are essential for the function of healthy organisms. However, scalable methods are needed to link individual cell secretions to their transcriptional state over time. Here, by developing and using secretion-encoded single-cell sequencing (SEC-seq), which exploits hydrogel particles with subnanolitre cavities (nanovials) to capture individual cells and their secretions, we simultaneously measured the secretion of vascular endothelial growth factor A (VEGF-A) and the transcriptome for thousands of individual mesenchymal stromal cells. Our data indicate that VEGF-A secretion is heterogeneous across the cell population and is poorly correlated with the VEGFA transcript level. The highest VEGF-A secretion occurs in a subpopulation of mesenchymal stromal cells characterized by a unique gene expression signature comprising a surface marker, interleukin-13 receptor subunit alpha 2 (IL13RA2), which allowed the enrichment of this subpopulation. SEC-seq enables the identification of gene signatures linked to specific secretory states, facilitating mechanistic studies, the isolation of secretory subpopulations and the development of means to modulate cellular secretion.

Optimizing cell therapy by sorting cells with high extracellular vesicle secretion.

Critical challenges remain in clinical translation of extracellular vesicle (EV)-based therapeutics due to the absence of methods to enrich cells with high EV secretion. Current cell sorting methods are limited to surface markers that are uncorrelated to EV secretion or therapeutic potential. Here, we utilize a nanovial technology for enrichment of millions of single cells based on EV secretion. This approach is applied to select mesenchymal stem cells (MSCs) with high EV secretion as therapeutic cells for improving treatment. The selected MSCs exhibit distinct transcriptional profiles associated with EV biogenesis and vascular regeneration and maintain high levels of EV secretion after sorting and regrowth. In a mouse model of myocardial infarction, treatment with high-secreting MSCs improves heart functions compared to treatment with low-secreting MSCs. These findings highlight the therapeutic importance of EV secretion in regenerative cell therapies and suggest that selecting cells based on EV secretion could enhance therapeutic efficacy.

Secretion encoded single-cell sequencing (SEC-seq) uncovers gene expression signatures associated with high VEGF-A secretion in mesenchymal stromal cells.

Cells secrete numerous bioactive molecules essential for the function of healthy organisms. However, there are no scalable methods to link individual cell secretions to their transcriptional state. By developing and using secretion encoded single-cell sequencing (SEC-seq), which exploits hydrogel nanovials to capture individual cells and their secretions, we simultaneously measured the secretion of vascular endothelial growth factor A (VEGF-A) and the transcriptome for thousands of individual mesenchymal stromal cells (MSCs). We found that VEGF-A secretion is heterogeneous across the cell population and lowly correlated with the VEGFA transcript level. While there is a modest population-wide increase in VEGF-A secretion by hypoxic induction, highest VEGF-A secretion across normoxic and hypoxic culture conditions occurs in a subpopulation of MSCs characterized by a unique gene expression signature. Taken together, SEC-seq enables the identification of specific genes involved in the control of secretory states, which may be exploited for developing means to modulate cellular secretion for disease treatment.

Improved Humoral Immunity and Protection against Influenza Virus Infection with a 3d Porous Biomaterial Vaccine.

New vaccine platforms that activate humoral immunity and generate neutralizing antibodies are required to combat emerging pathogens, including influenza virus. A slurry of antigen-loaded hydrogel microparticles that anneal to form a porous scaffold with high surface area for antigen uptake by infiltrating immune cells as the biomaterial degrades is demonstrated to enhance humoral immunity. Antigen-loaded-microgels elicited a robust cellular humoral immune response, with increased CD4+ T follicular helper (Tfh) cells and prolonged germinal center (GC) B cells comparable to the commonly used adjuvant, aluminum hydroxide (Alum). Increasing the weight fraction of polymer material led to increased material stiffness and antigen-specific antibody titers superior to Alum. Vaccinating mice with inactivated influenza virus loaded into this more highly cross-linked formulation elicited a strong antibody response and provided protection against a high dose viral challenge. By tuning physical and chemical properties, adjuvanticity can be enhanced leading to humoral immunity and protection against a pathogen, leveraging two different types of antigenic material: individual protein antigen and inactivated virus. The flexibility of the platform may enable design of new vaccines to enhance innate and adaptive immune cell programming to generate and tune high affinity antibodies, a promising approach to generate long-lasting immunity.

Engineering a highly elastic bioadhesive for sealing soft and dynamic tissues.

Injured tissues often require immediate closure to restore the normal functionality of the organ. In most cases, injuries are associated with trauma or various physical surgeries where different adhesive hydrogel materials are applied to close the wounds. However, these materials are typically toxic, have low elasticity, and lack strong adhesion especially to the wet tissues. In this study, a stretchable composite hydrogel consisting of gelatin methacrylol catechol (GelMAC) with ferric ions, and poly(ethylene glycol) diacrylate (PEGDA) was developed. The engineered material could adhere to the wet tissue surfaces through the chemical conjugation of catechol and methacrylate groups to the gelatin backbone. Moreover, the incorporation of PEGDA enhanced the elasticity of the bioadhesives. Our results showed that the physical properties and adhesion of the hydrogels could be tuned by changing the ratio of GelMAC/PEGDA. In addition, the in vitro toxicity tests confirmed the biocompatibility of the engineered bioadhesives. Finally, using an ex vivo lung incision model, we showed the potential application of the developed bioadhesives for sealing elastic tissues.

Engineering a naturally derived hemostatic sealant for sealing internal organs.

Controlling bleeding from a raptured tissue, especially during the surgeries, is essentially important. Particularly for soft and dynamic internal organs where use of sutures, staples, or wires is limited, treatments with hemostatic adhesives have proven to be beneficial. However, major drawbacks with clinically used hemostats include lack of adhesion to wet tissue and poor mechanics. In view of these, herein, we engineered a double-crosslinked sealant which showed excellent hemostasis (comparable to existing commercial hemostat) without compromising its wet tissue adhesion. Mechanistically, the engineered hydrogel controlled the bleeding through its wound-sealing capability and inherent chemical activity. This mussel-inspired hemostatic adhesive hydrogel, named gelatin methacryloyl-catechol (GelMAC), contained covalently functionalized catechol and methacrylate moieties and showed excellent biocompatibility both in vitro and in vivo. Hemostatic property of GelMAC hydrogel was initially demonstrated with an in vitro blood clotting assay, which showed significantly reduced clotting time compared to the clinically used hemostat, Surgicel®. This was further assessed with an in vivo liver bleeding test in rats where GelMAC hydrogel closed the incision rapidly and initiated blood coagulation even faster than Surgicel®. The engineered GelMAC hydrogel-based seaalant with excellent hemostatic property and tissue adhesion can be utilized for controlling bleeding and sealing of soft internal organs.

Engineering elastic sealants based on gelatin and elastin-like polypeptides for endovascular anastomosis.

Cerebrovascular ischemia from intracranial atherosclerosis remains difficult to treat. Although current revascularization procedures, including intraluminal stents and extracranial to intracranial bypass, have shown some benefit, they suffer from perioperative and postoperative morbidity. To address these limitations, here we developed a novel approach that involves gluing of arteries and subsequent transmural anastomosis from the healthy donor into the ischemic recipient. This approach required an elastic vascular sealant with distinct mechanical properties and adhesion to facilitate anastomosis. We engineered two hydrogel-based glues: an elastic composite hydrogel based on methacryloyl elastin-like polypeptide (mELP) combined with gelatin methacryloyl (GelMA) and a stiff glue based on pure GelMA. Two formulations with distinct mechanical characteristics were necessary to achieve stable anastomosis. The elastic GelMA/mELP composite glue attained desirable mechanical properties (elastic modulus: 288 ± 19 kPa, extensibility: 34.5 ± 13.4%) and adhesion (shear strength: 26.7 ± 5.4 kPa) to the blood vessel, while the pure GelMA glue exhibited superior adhesion (shear strength: 49.4 ± 7.0 kPa) at the cost of increased stiffness (elastic modulus: 581 ± 51 kPa) and reduced extensibility (13.6 ± 2.5%). The in vitro biocompatibility tests confirmed that the glues were not cytotoxic and were biodegradable. In addition, an ex vivo porcine anastomosis model showed high arterial burst pressure resistance of 34.0 ± 7.5 kPa, which is well over normal (16 kPa), elevated (17.3 kPa), and hypertensive crisis (24 kPa) systolic blood pressures in humans. Finally, an in vivo swine model was used to assess the feasibility of using the newly developed two-glue system for an endovascular anastomosis. X-ray imaging confirmed that the anastomosis was made successfully without postoperative bleeding complications and the procedure was well tolerated. In the future, more studies are required to evaluate the performance of the developed sealants under various temperature and humidity ranges.