Microchannelled alkylated chitosan sponge to treat noncompressible hemorrhages and facilitate wound healing

Xinchen Du, Le Wu, Hongyu Yan, Zhuyan Jiang, Shilin Li, Wen Li, Yanli Bai, Hongjun Wang, Zhaojun Cheng, Deling Kong, Lianyong Wang, Meifeng Zhu, Xinchen Du, Le Wu, Hongyu Yan, Zhuyan Jiang, Shilin Li, Wen Li, Yanli Bai, Hongjun Wang, Zhaojun Cheng, Deling Kong, Lianyong Wang, Meifeng Zhu

Abstract

Developing an anti-infective shape-memory hemostatic sponge able to guide in situ tissue regeneration for noncompressible hemorrhages in civilian and battlefield settings remains a challenge. Here we engineer hemostatic chitosan sponges with highly interconnective microchannels by combining 3D printed microfiber leaching, freeze-drying, and superficial active modification. We demonstrate that the microchannelled alkylated chitosan sponge (MACS) exhibits the capacity for water and blood absorption, as well as rapid shape recovery. We show that compared to clinically used gauze, gelatin sponge, CELOX™, and CELOX™-gauze, the MACS provides higher pro-coagulant and hemostatic capacities in lethally normal and heparinized rat and pig liver perforation wound models. We demonstrate its anti-infective activity against S. aureus and E. coli and its promotion of liver parenchymal cell infiltration, vascularization, and tissue integration in a rat liver defect model. Overall, the MACS demonstrates promising clinical translational potential in treating lethal noncompressible hemorrhage and facilitating wound healing.

Conflict of interest statement

The authors declare no competing interests.

© 2021. The Author(s).

Figures

Fig. 1. Fabrication and characterization of the…
Fig. 1. Fabrication and characterization of the MACSs with different porosity.
a Schematic illustration of the fabrication process of the MACSs. b Stereomicroscopic images of the PLA microfiber template, CS/PLA composite, micro channeled CS sponge, and micro channeled alkylated CS sponge. c, d Micro-CT and SEM images showing the macro and microstructure of the ACS and MACS-1/2/3. e The pore size of the ACS and MACS-1/2/3 in cross-section and longitudinal-section (pore size, n = 16). f The pore size of the ACS and MACS-1/2/3 in longitudinal-section (pore size, n = 16). g The porosity of the ACS and MACS-1/2/3 (porosity, n = 25). h, i Compressive stress-strain curves and compressive stress of the MACSs with different CS concentrations (1, 2, and 4% (w/v)) and PLA microfiber diameter (200 and 400 μm) (n = 3 independent samples). j–m Compressive stress-strain curves and compressive stress of the ACS, MCS-2, and MACS-1/2/3 before and after absorbing blood. n Mechanically reinforced folds of the ACS, MCS-2, and MACS-1/2/3 before and after absorbing blood (n = 3 independent samples). Data are expressed as mean ± SD. The significant difference was detected by one-way ANOVA with Tukey’s multiple comparisons test. The ‘ns’ indicated no significant difference, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Fig. 2. Chemical characterization of the MACSs.
Fig. 2. Chemical characterization of the MACSs.
a Modification of the CS sponge with DA in the presence of NaCNBH3 as a reducing agent. b, c Representative XPS spectra showing N1s peak of the CS and alkylated CS sponges. d The area of N1s peaks with different chemical states in the CS and alkylated CS sponges.
Fig. 3. Water/blood absorbability of the ACS…
Fig. 3. Water/blood absorbability of the ACS and MACSs.
a Macro photograph of the ACS and MACS-1/2/3 after absorbing the blood. Yellow and red arrows represented the ACS and blood, respectively. b, c Water/blood absorption capacity-time dynamic curves of the ACS and MACS-1/2/3. d–g Water/blood absorption capacity and rate of the ACS and MACS-1/2/3. h, i Macro photographs of the compressed ACS and MACS-1/2/3 before and after contact with water and blood. The yellow dotted circle represented the boundary of the ACS. j Fluid simulation images of water absorption behaviors of the ACS and MACS-1/2/3. k Total fluid speed of the ACS and MACS-1/2/3. n = 3 independent samples. Data are expressed as mean ± SD. The significant difference was detected by one-way ANOVA with Tukey’s multiple comparisons test. The ‘ns’ indicated no significant difference, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Fig. 4. Shape-memory property of the ACS…
Fig. 4. Shape-memory property of the ACS and MACSs after absorbing water and blood.
a, b Macro photographs of the water-triggered and blood-triggered shape recovery of the ACS and MACS-1/2/3. c–f Shape-recovery ratio and time of the compressed sponges. The shape-recovery time of the ACS was not shown as the compressed ACS could not restore to its original shape after absorbing the blood. n = 3 independent samples. Data are expressed as mean ± SD. the significant difference was detected by one-way ANOVA with Tukey’s multiple comparisons test. The ‘ns’ indicated no significant difference, *P < 0.05, ****P < 0.0001. g Comparison of shape-recovery time between the MACS-2 and reported hemostats. h SEM images showing the microstructure of the compressed sponges before and after absorbing water and blood. The red arrow represented the deformed microchannel.
Fig. 5. The pro-coagulant ability of the…
Fig. 5. The pro-coagulant ability of the gauze, GS, CELOXTM, CELOXTM-G, ACS, MCS-2, and MACSs.
a The BCI-time curves of various samples. b, c The percentage of adhered RBCs and platelets on various samples. n = 3 independent samples. Data are expressed as mean ± SD. The significant difference was detected by one-way ANOVA with Tukey’s multiple comparisons test. The ‘ns’ indicated no significant difference, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. d, e SEM images showing adhesion of RBCs and platelets on various samples. f Immunofluorescence staining of CD62p showing the activation of platelets on various samples. The yellow arrow represented activated platelet. g Schematic diagram illustrating the pro-coagulant mechanism of the MACSs. BCI: blood clotting index; RBCs: red blood cells.
Fig. 6. Hemostasis in the normal rat…
Fig. 6. Hemostasis in the normal rat liver perforation wound model.
a Schematic illustration of the hemostatic process of hemostats in a rat liver perforation wound model. b Photographs of the hemostatic effect of the gauze, GS, CELOXTM-G, CELOXTM, ACS, MCS-2, and MACS-2. The yellow arrow and dotted line represented the bleeding site and liver boundary, respectively. c, d Total blood loss and hemostatic time in the gauze, GS, CELOXTM-G, CELOXTM, ACS, MCS-2, and MACS-2 groups. n = 3 rats per group. Data are expressed as mean ± SD. The significant difference was detected by one-way ANOVA with Tukey’s multiple comparisons test. ****P < 0.0001.
Fig. 7. Hemostasis in a lethal pig…
Fig. 7. Hemostasis in a lethal pig liver perforation wound model.
a Schematic illustration of the hemostatic process of hemostats in a lethal pig liver perforation wound model. b Photographs of the hemostatic effect of the blank, CELOXTM, and MACS-2 groups. The yellow arrow and dotted line represented the boundary of the liver and the bleeding site, respectively. c, d Hemostatic time and total blood loss in the blank, CELOXTM, and MACS-2 groups. n = 3 pigs per group. Data are expressed as mean ± SD. The significant difference was detected by one-way ANOVA with Tukey’s multiple comparisons test. ****P < 0.0001. e Schematic diagram of hemostatic procedure and mechanism of the MACS-2.
Fig. 8. In vitro anti-infective property of…
Fig. 8. In vitro anti-infective property of the MACS-2 and other hemostats.
a, b Photographs of CFUs of S. aureus and E. coli grown on LB agar plates after contact with TCP, gauze, GS, CELOXTM-G, CELOXTM, ACS, MCS-2, and MACS-2, respectively. c, d Corresponding statistical results of the CFUs of S. aureus and E. coli. n = 3 independent samples. Data are expressed as mean ± SD. The significant difference was detected by one-way ANOVA with Tukey’s multiple comparisons test. The ‘ns’ indicated no significant difference, **P < 0.01, ***P < 0.001, ****P < 0.0001. S. aureus: staphylococcus aureus; E. coli: Escherichia coli.
Fig. 9. Liver regeneration in rat models…
Fig. 9. Liver regeneration in rat models after implantation of the ACS and MACS-2.
a DAPI staining showing cell infiltration within the ACS and MACS-2. H&E staining showing tissue ingrowth. Images of immunofluorescent staining for vWF (red) and ALB (red) indicating capillary, and LPC infiltration within the ACS and MACS-2. Yellow asterisk, pound key, and arrow represented the alkylated CS, capillary, and LPC, respectively. b–e Quantification of cell number, tissue ingrowth area, capillary number, and LPCs within the ACS and MACS-2. n = 3 independent samples. Data are expressed as mean ± SD. The significant difference was detected by one-way ANOVA with Tukey’s multiple comparisons test. The ‘ns’ indicated no significant difference, **P < 0.01, ****P < 0.0001. f PAS staining showing synthesized hepatic glycogen within the ACS and MACS-2. Images of immunofluorescent staining for HNF-4α (purple) indicating expression of key liver cytokine within the ACS and MACS-2. Yellow asterisk and arrow represented the alkylated CS and HNF-4α, respectively. g Schematic illustration of in situ liver regeneration, including the host cell infiltration and vascularization. DAPI: 4’, 6-diamidino-2-phenylindole; LPC: liver parenchymal cell; vWF: von Willebrand factor; ALB: albumin; PAS: periodic acid-schiff; HNF-4α: hepatocyte nuclear factor-4α.

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