Preclinical Evaluation of Colisorb: Surfactant-Free Hydrophilic Gelatin Sponge with Rapid Clotting and Enhanced Absorbency without Mechanical Compression

Abstract

Rapid hemostasis is a major challenge in surgery, especially for noncompressible wounds such as liver wounds. Conventional hemostatic products often fall short of achieving the desired level of hemostasis. The current study introduces Colisorb, a novel cross-linked gelatin-based sponge for controlling surgical bleeding. The Colisorb sponge is a superabsorbent, adhesive, biocompatible, biodegradable, mechanically stable gelatin-based sponge with superior swelling kinetics, which allows strong swelling capability up to 70× its weight, thus indicating strong capability for blood absorption. Electron microscopic examination shows that the sponge has an interconnected network structure with pore sizes of 100 to 300 μm. Colisorb sponge also exhibits higher porosity and tensile strength compared to commercial products. Additionally, the Colisorb sponge demonstrated rapid degradation in only 3 days. In vitro examination of the Colisorb sponge demonstrated that it is noncytotoxic on mouse fibroblast and human fibroblast cells. In vivo, coagulation experiments showed that Colisorb promotes hemostasis in a rat liver laceration model and significantly reduces the amount of blood loss (100 mg with Colisorb as compared to 300 mg with Cutanplast). Collectively, the findings of the current study highlight the potential of the Colisorb gelatin hemostatic sponge to drive advancements in clinical applications for managing noncompressible hemorrhage and facilitating subsequent wound closure.

1

The swelling kinetics of the Colisorb sponge as compared to the Cutanplast hemostatic sponge.

2

Scanning electron microscope (SEM) micrographs showing the internal structure of Colisorb (A) and Cutanplast (B).

3

Stress–strain curves of Colisorb and Cutanplast after repeated mechanical compression.

4

Water contact angle of Colisorb and Cutanplast.

5

In vitro degradation rate of Colisorb sponge compared to Cutanplast.

6

In vitro assessment of blood uptake and blood coagulation. (a) A photograph was taken after the blood uptake ratio test, demonstrating the structural stability of the Colisorb sponge as compared to the commercial Cutanplast. (b) Blood uptake ratio of Colisorb and Cutanplast (****P < 0.0001). The clotting time (c) of Colisorb and Cutanplast was significant compared to the untreated blood (***P < 0.001, ****P < 0.0001). (d) The blood clotting index (BCI) for both Colisorb and Cutanplast was significant compared to the control; there was no difference between Colisorb and Cutanplast. Coagulation (prothrombin time, PT) was not significantly different (e) between all groups. aPTT time (e) was significantly higher in Colisorb and Cutanplast samples compared to the control (untreated blood).

7

In vitro hemolysis assay. Blood hemolysis was minimal after Colisorb and Cutanplast. Compared to the positive control (10 μL of triton-treated blood), normal saline was used as a negative control.

8

Color observation of sponge upon RBCs and platelet adhesion test preparation for SEM imaging.

9

Scanning electron microscope (SEM) micrographs of RBC adhesion density and behavior in Colisorb and Cutanplast sponges. Multiple magnification powers were used to elucidate RBCs adhesion: (a and b) at ×240 and (c and d) at ×500 for Colisorb and Cutanplast, respectively.

10

Scanning electron microscope micrographs of platelet adhesion morphology in (a) Colisorb and (b) Cutanplast sponges.

11

Cytocompatibility of the Colisorb. Cell viability of (a) L929 cells, (b) HepG2 cells, and (c) hFB cells upon treatment with Colisorb and Cutanplast (**** P < 0.0001, *** P < 0.001).

12

The amount of blood loss from the liver in vivo (a) was significantly lower compared to that in the untreated animals. Blood Clotting Time (BCT) (b) was also significantly lower after using Colisorb and Cutanplast compared to the untreated control. Blood loss (a) and clotting time (b) were significantly lower after using Colisorb compared to Cutanplast.

13

Histological examination of the liver following laceration and hemostasis. Normal liver (a,f) showed normal histology of the hepatic lobules and hepatic cords. Nontreated control (b,g) liver samples showed dense fibrous connective tissue proliferation at the line of the incision (arrow). Gauze-treated (c,h) samples showed Colisorb-treated (d,i) samples showing fibrous tissue with vacuolar degeneration of hepatocytes and few numbers of apoptotic bodies (arrow). Cutanplast-treated samples (e,j) showed a thin layer of fibrous tissue mixed with inflammatory cells and embedded sponge material (arrow).

14

Histological assessment of skin and subcutaneous tissue. Photomicrographs of skin tissue sections showing normal (a) histological structure of epidermal and dermal layers (arrows). Colisorb-treated (b) and Cutanplast-treated (c) samples showed a normal, well-organized histological appearance of the epidermis with layers of stratified squamous epithelium covered with layers of keratinocytes. The dermis shows dense fibrous connective tissue, subcutaneous adipose tissue, and few blood capillaries without any inflammatory reaction.

15

Histological assessment of liver and kidney tissues. Assessment of the systemic toxicity following the use of Colisorb (b,e) and Cutanplast (c,f) showed that liver tissues in the Colisorb-treated group revealed mild disorganization of hepatic cords, an increased number of Kupffer cells, and narrowing of hepatic sinusoids. On the other side, Cutanplast-treated animals showed a normal histological structure similar to the normal group (a), which showed the normal histological structure of hepatic lobules and hepatic cords. The kidney tissue in all animals (d–f) exhibited a normal histological architecture, with intact glomeruli, well-defined capillary tufts, and Bowman’s capsule. Both proximal and distal convoluted tubules displayed an intact epithelial lining and a regular structural arrangement.

16

Biochemical assessment of renal and liver functions. Assessment of renal (Creatinine (a) and Urea (b)) and liver functions (ALT (c) and AST (d)) showed no significant difference between normal, Colisorb, and Cutanplast-treated animals.