In Vivo and In Vitro Investigation of a Novel Gelatin/Sodium Polyacrylate Composite Hemostatic Sponge for Topical Bleeding.

Designing a functional and efficient blood-clotting agent is a major challenge. In this research, hemostatic scaffolds (GSp) were prepared from the superabsorbent, inter-crosslinked polymer sodium polyacrylate (Sp) bound to a natural protein gelatin (G) loaded with thrombin (Th) by a cost-effective freeze-drying method. Five compositions were grafted (GSp0.0, Gsp0.1, GSp0.2, GSp0.3, GSp0.3-Th) where the concentration of Sp varied but the ratios of G remained the same. The fundamental physical characteristics that increased the amounts of Sp with G gave synergistic effects after interacting with thrombin. Due to the presence of superabsorbent polymer (SAP) swelling capacities in GSp0.3 and GSp0.3-Th surge forward 6265% and 6948%, respectively. Pore sizes became uniform and larger (ranging ≤ 300 µm) and well-interconnected. The water-contact angle declined in GSp0.3 and GSp0.3-Th to 75.73 ± 1.097 and 75.33 ± 0.8342 degrees, respectively, thus increasing hydrophilicity. The pH difference was found to be insignificant as well. In addition, an evaluation of the scaffold in in vitro biocompatibility with the L929 cell line showed cell viability >80%, so the samples were nontoxic and produced a favorable environment for cell proliferation. The composite GSp0.3-Th revealed the lowest HR (%) (2.601%), and the in vivo blood-clotting time (s) and blood loss (gm) supported hemostasis. Overall, the results showed that a novel GSp0.3-Th scaffold can be a potential candidate as a hemostatic agent.

Papaverine loaded injectable and thermosensitive hydrogel system for improving survival of rat dorsal skin flaps.

Vasospasm during reconstructive microsurgery is a common, uncertain, and devastating phenomena concerning flap survival. Topical vasodilators as antispasmodic agents are widely used to reduce vasospasm and enhance microvascular anastomosis in reconstructive microsurgery. In this study, thermo-responsive hydrogel (CNH) was fabricated by grafting chitosan (CS) and hyaluronic acid (HA) to poly(N-isopropylacrylamide) (PNIPAM). Papaverine, an anti-spasmodic agent, was then loaded to evaluate its effect on rat skin flap survival. Post-operative flap survival area and water content of rat dorsal skin flap were measured at 7 days after intradermal application of control hydrogel (CNHP0.0) and papaverine loaded hydrogel (CNHP0.4). Tissue malondialdehyde (MDA) content and superoxide dismutase (SOD) activity was measured using enzyme linked immunosorbent assay (ELISA) to determine oxidative stress in flaps. Hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) were performed to evaluate flap angiogenesis and inflammatory markers. Results showed that CNHP0.4 hydrogel could reduce tissue edema (35.63 ± 4.01%), improve flap survival area (76.30 ± 5.39%), increase SOD activity and decrease MDA content. Consequently, it also increased mean vessel density, upregulated expression of CD34 and VEGF, decreased macrophage infiltration, and reduced CD68 and CCR7 expression based on IHC staining. Overall, these results indicate that CNHP0.4 hydrogel can enhance angiogenesis with anti-oxidative and anti-inflammatory effects and promote skin flap survival by preventing vascular spasm.

Decellularized liver extracellular matrix and thrombin loaded biodegradable TOCN/Chitosan nanocomposite for hemostasis and wound healing in rat liver hemorrhage model.

During deep noncompressible wound management, surgery, transplantation or post-surgical hemorrhage, rapid blood absorption and hemostasis are the key factors to be taken into consideration to reduce unexpected deaths from severe trauma. In this study, a novel hemostatic biodegradable nanocomposite was fabricated where decellularized liver extracellular matrix (L-ECM) was loaded with two natural polymers (oxidized cellulose and chitosan) in association with thrombin. Plant-derived oxidized cellulose nanofiber (TOCN) and Chitosan (CS) from deacylated chitin were self-assembled with each other by electrostatic interactions. ECM was prepared by the whole tissue decellularization process and incorporated into the composite as a source of collagen and other integrated growth factors to promote wound healing. Thrombin was also anchored with the polymers by freeze drying for enhanced hemostatic efficiency of the composite. This study is the first of its kind to report non-solubilized L-ECM and thrombin loaded TOCN and CS composite, CN/CS/EM-Th for faster hemostasis effect in a rat tail amputation (~71 s) and liver avulsion model (~41 s). Furthermore, excellent liver wound regeneration efficacy was observed in-vivo in comparison to the commercially available oxidized regenerated cellulose product SURGICEL gauge.

Fabrication of thrombin loaded TEMPO-oxidized cellulose nanofiber-gelatin sponges and their hemostatic behavior in rat liver hemorrhage model.

Excessive blood loss due to trauma or major surgical intervention can be life threatening which necessitates rapid hemorrhage management for the prevention of such bleeding related sufferings. Broad interest in developing new hemostatic technologies have been paid for bleeding control but none of them found completely satisfactory especially in terms of rapid clotting, absorbability, porosity, cost effectiveness and safety. To address these issues, a combination of active and passive hemostatic materials from biological sources could be a wise choice. Therefore, plant-derived TEMPO-oxidized nanocellulose (TOCN)/biopolymer gelatin (G) sponge was successfully prepared in co-operation with intrinsic blood coagulation enzyme thrombin (Th) via freeze drying method and their application as rapid hemostatic dressing was investigated. Morphological and in vitro characteristics of the samples were evaluated where uniformity, porosity, swelling, degradation behavior had direct relationship with the percent gelatin incorporation. In vitro hemocompatibility and cyto-compatibility of these sponges were confirmed as well. Among the samples, TOCN 2.5G-Th sponge exhibited excellent hemostatic effect, rapid absorbability, minimum clotting time (1.37 ± 0.152 min) and reduction of blood loss was ensured through rat liver punch biopsy model. The results demonstrated that, Th enhanced blood coagulation, platelet and red blood cell aggregation following application of biopolymer TOCN 2.5G-Th sponge compared with samples devoid of Th. In short, the functional, cost effective and nontoxic sponge developed via facile preparation could potentially be used as an absorbable biomaterial to achieve immediate hemostasis. HighlightsPlant-derived TEMPO-oxidized nanocellulose (TOCN) and biopolymer gelatin (G) was successfully used to prepare a hemostatic sponge in combination with intrinsic blood coagulation enzyme thrombin (Th).The TG sponge combines the advantages of TOCN and gelatin, exhibiting biocompatibility, biodegradability and superior blood-absorption performance.The TOCN 2.5G-Th sponge improves plasma absorption, red blood cell adhesion, aggregation, platelet adhesion and activation leading to enhanced hemostasis effect and shorter hemostasis time in vitro and in vivo.