(C-99) Rapidly Activating Cellulose-based Hemostatic Sealants for Uncontrollable Bleeding

Excessive bleeding poses a significant threat as it can lead to tissue necrosis, infection or death induced by hypothermia. Despite significant advancements in the development of hemostatic agents, there still remains a need to improve the essential properties required for urgent situations. In emergency settings, hemostatic agents should function immediately to minimize blood loss and exhibit robustness and bio-adhesiveness to withstand the blood pressure and flow in the bleeding area. Moreover, the material should be convenient to handle, ensuring effective application even in urgent situations. To meet these criteria, we introduced two phenolic moieties, catechol (CA) and pyrogallol (PG) into the system. CA and PG are known to be oxidized to form o-quinone, which can form covalent and noncovalent bonds immediately with various proteins on tissue surfaces, promoting rapid and strong tissue adhesion. To effectively harness these functional moieties, we utilized carboxymethyl cellulose (CMC) as a polymer backbone of the system. CMC, known for its high biocompatibility, mechanical strength, abundance, and cost-effectiveness, has been widely used for biomedical applications. Here, we present the development of an instant and user-friendly hemostatic agent by conjugating catechol and pyrogallol to CMC (CMC-CA, CMC-PG).

Sodium CMC was dissolved in 2-morpholinoethanesulfonic acid (MES) buffer. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were added to the CMC solution, followed by the addition of dopamine hydrochloride or 5-hydroxydopamine to synthesize CMC-CA or CMC-PG, respectively. The synthesized solution was dialyzed overnight, and the products were lyophilized.

 The mechanical properties of CMC-CA and CMC-PG were evaluated using a rotating rheometer (MCR 102) and a universal testing machine (UTM). The elasticity, adhesiveness, shear strength, and burst power of the materials were assessed.

 Human adipose-derived stem cells (hADSC) were encapsulated in CMC-CA and CMC-PG hydrogels. Cell viability was assessed using a Live/Dead assay. The immunogenicity of the materials was evaluated by quantifying TNF-α secretion from RAW 264.7 cells co-cultured with the hydrogels.

 Animal experiments were conducted using a mouse liver hemorrhaging model. CMC-CA, CMC-PG, and Fibrin glue (control) were applied to the liver lesions. Blood loss was quantified and compared among the groups. In vivo biocompatibility was assessed by implanting the materials into the subcutaneous space and retrieving them at each time point. The biocompatibility of the materials was analyzed through histological examination of the retrieved tissues.

CMC-based hemostatic hydrogels were verified to possess a robust modulus ensuring their structural integrity and ability to withstand blood pressure. Additionally, they exhibited strong adhesiveness to wet tissue. The patch form of hydrogel exhibited a 16~19-fold increase in the elastic modulus compared to the hydrogel form and a 3~4-fold increase in adhesiveness. Furthermore, in vitro cytocompatibility test provided strong evidence that CMC-based materials exhibit excellent biocompatibility and does not induce inflammatory responses. Moreover, CMC-based materials demonstrated excellent in vivo biocompatibility and enhanced hemostatic capabilities in a mouse liver hemorrhage model. In particular, the CMC-PG derivative exhibited improved mechanical properties and adhesive strength compared to CMC-CA, making it more suitable for medical applications that require stronger physical properties. Oxidant-free crosslinking via in situ autoxidation further enhanced the biocompatibility and utility of the CMC-PG derivative in urgent clinical settings. This approach offers an effective strategy for immediate bleeding control in emergency situations, even when handled by untrained personnel. Our bio-inspired hydrogels hold potential for various biomedical applications, including off-the-shelf cell therapy and drug delivery, given their robustness and tunability.