Dual green hemostatic sponges constructed by collagen fibers disintegrated from Halocynthia roretzi by a shortcut method

Abstract

Recently, biomacromolecules have received considerable attention in hemostatic materials. Collagen, an ideal candidate for hemostatic sponges due to its involvement in the clotting process, has been facing challenges in extraction from raw materials, which is time-consuming, expensive, and limited by cultural and religious restrictions associated with traditional livestock and poultry sources. To address these issues, this study explored a new shortcut method that using wild Halocynthia roretzi (HR), a marine fouling organism, as a raw material for developing HR collagen fiber sponge (HRCFs), which employed urea to disrupt hydrogen bonds between collagen fiber aggregates. This method simplifies traditional complex manufacturing processes while utilized marine waste, thus achieving dual green in terms of raw materials and manufacturing processes. FTIR results confirmed that the natural triple-helical structure of collagen was preserved. HRCFs exhibit a blood absorption ratio of 2000-3500 %, attributed to their microporous structure, as demonstrated by kinetic studies following a capillary model. Remarkably, the cytotoxicity and hemolysis ratio of HRCFs are negligible. Furthermore, during in vivo hemostasis tests using rabbit ear and kidney models, HRCFs significantly reduce blood loss and shorten hemostasis time compared to commercial gelatin sponge and gauze, benefiting from the capillary effect and collagen's coagulation activity. This study provides new insights into the design of collagen-based hemostatic biomaterials, especially in terms of both raw material and green manufacturing processes.

Keywords: Collagen fiber; Halocynthia roretzi; Hemostatic sponge.

Graphical abstract

Fig. 1

(a) Dissociation rationale of fibers. (b) Fabrication process of HRCFs.

Fig. 2

FTIR spectra of HR15(u0), HR15(u1), HR15(u3), and HR15(u5) at wavenumbers from (a) 4000-400 cm⁻¹, (b) 2000-400 cm⁻¹, (c) FESEM images of HR15(u1), HR15(u3), HR15(u5), and Gelatin at different magnifications.

Fig. 3

(a) Compressive stress-compressive strain curves of HR50(u1), HR50(u3) and HR50(u5). (b) Compressive stress-compressive strain curves of HR15(u3)-HR50(u3).

Fig. 4

(a) Porosity of HR15(u3)-HR50(u3). (b) Water absorption ratio of HR15(u3)-HR50(u3). (c) Blood absorption ratio of HR15(u3)-HR50(u3). (d) Photos of HR15(u3)-HR50(u3) after blood absorption. (e) Photos of HR50(u3) at initial, water absorption, and blood absorption. (f) Contact angle of HR50(u3) and its variation. Different letters in the bar chart indicate significant differences (p < 0.05) between samples.

Fig. 5

The absorption height and time of HR50(u3) (a) in DI water, (b) in blood. (c) Fitted curves depicting the relationship between the height of absorbed DI water and blood over time. (d) Schematic of liquid-absorbing capacity for HR50(u3) and Gelatin.

Fig. 6

Blood clotting stability of HR15(u3)-HR50(u3). (b) BCI of HR15(u3)-HR50(u3). Different letters in the bar chart indicate significant differences (p < 0.05) between samples. (c) Coagulation effects of HRCFs observed during BCI. (d) Interactions of HR50(u3) and Gelatin with blood cells.

Fig. 7

(a) Cell viability study using L929 fibroblast cells via CCK-8 assay on days 1, 3, and 5 (*p < 0.05). (b) Representative live/dead staining images of L929 fibroblast cells on days 1, 3, and 5. (c) Hemolysis ratio of HRCFs. (d) Hemolysis effects of HRCFs observed during hemolysis test.

Fig. 8

Schematic diagram illustrating the (a1) rabbit ear puncture hemostatic model, (a2) rabbit ear scratch hemostatic model. Hemostatic effects of control and sample groups observed during (b1) rabbit ear puncture experiment, (b2) rabbit ear scratch experiment. Blood loss and hemostasis time of control and sample groups in (c1) rabbit ear puncture experiment, (c2) rabbit ear scratch experiment. Different letters in the bar chart indicate significant differences (p < 0.05) between samples.

Fig. 9

(a) Schematic diagram illustrating the rabbit kidney scratch hemostatic model. (b) Hemostatic effects of HR50(u3) observed during rabbit kidney scratch experiment. (c) Blood loss and hemostasis time of control and sample groups in rabbit kidney scratch experiment; different letters in the bar chart indicate significant differences (p < 0.05) between samples. (d) Hemostatic mechanisms of HRCFs: (I) the porous structure possesses the ability to rapidly absorb blood, (II) the porous structure creates an optimal environment for the adhesion and accumulation of blood cells, (III) collagen stimulates platelets to release ADP and promotes platelets activation and aggregation, (IV) collagen plays a role in aiding and accelerating the endogenous coagulation process.