Puff pastry-like chitosan/konjac glucomannan matrix with thrombin-occupied microporous starch particles as a composite for hemostasis
Graphical abstract
Introduction
Excessive hemorrhaging accounts for commonly observed emergent conditions both in military and civilian trauma situations (Hong et al., 2019). However, controlling serious bleeding not only depends on the hemostatic mechanisms of the body, but also requires external hemostatic intervention, lending to the need for development of novel and effective hemostatic material (Liu, Yao et al., 2018). Hence, techniques that can decrease the time required for effective control of blood loss following trauma are constantly being investigated. Recently, classical hemostatic agents such as hydrogels, porous matrices, and powders have been developed to control hemorrhage (Aydemir Sezer et al., 2018). These materials achieved hemostasis by physical shielding (Boerman et al., 2017), improving the concentration of coagulation factors (Yu et al., 2019), initiating fast-activating coagulation cascades (Liu, Yao et al., 2018), or delivering clotting agents to the wound site (Gaharwar et al., 2014). Among these materials, porous hemostats have attracted much attention (Liu, Li et al., 2018). However, these materials are limited by a relatively low efficacy for control of severe bleeding. Thus, great efforts to improve the hemostatic capacity of materials are required.
Notably, porous hemostats can promote erythrocyte and platelet aggregation by absorbing blood plasma or via electrostatic interactions, while others have been shown to activate erythrocytes and platelets at the interface to regulate hemostasis. For example, gelatin sponges concentrate blood cells and coagulation factors around the wound site by absorbing water from the blood (Yuan, Yinghui, & Sun, 2013). The amine groups of positively charged chitosan-based porous materials can aggregate with negatively charged erythrocytes via electrostatic interactions (Wang, Luo et al., 2017). Moreover, porous graphene oxide-based composites can activate platelets via Src kinase signaling to release calcium from intracellular stores (Chen, Lv et al., 2019). However, as the average diameter of blood cells (erythrocyte and platelet are approximately 7 m and 3 m, respectively) is significantly smaller than the pore size of porous hemostats, the majority of blood cells become absorbed into the interior of the material (Liu et al., 2019). Accordingly, very few accumulate at the wound-material interface, leading to poor hemostasis. Consequently, numerous studies have been carried out to develop effective physical structure hemostats to not only control severe hemorrhage, but also to allow for control of idiosyncratic wound hemorrhage. To this end, Zhang et al. produced a nanofiber sponge comprised of a layered structure through an electron-spinning method and gas-foaming technique (Zhang et al., 2019). This porous structure increased interfacial interactions between the sponge and blood cells, thereby enhancing the hemostatic capacity. Additionally, Xie et al. achieved dynamic transformation of the sponge aperture structure to one of a self-adapting bleeding wound (Chen, Carlson, Zhang, Hu, & Xie, 2018). Upon contacting blood, the compressed sponge could expand to activate hemostasis via a tamponade effect.
Apart from constructing an effective porous structure, improving the roughness of the porous interface in hemostatic materials serves to enhance the hemostatic capacity (Han, Wu, Hou, Zhao, & Xiang, 2015). For example, inorganic porous particles such as montmorillonite and kaolin have been directly embedded into hemostat sponges (Li et al., 2016; Liang et al., 2018). Unfortunately, the enhanced roughness caused by the integration of inorganic particles was suboptimal; moreover, the inorganic particles were proved cytotoxic and difficult to degrade in vivo (Jin et al., 2018). Hence, it is necessary to identify alternative materials to improve the roughness of porous interfaces. In this regard, natural bioresources may serve as ideal candidates to incorporate in hemostatic materials to enhance hemostatic performance.
Alternatively, doping materials with coagulation factors may improve the hemostatic efficiency. Specific organic coagulation factors including batroxobin, thrombin, and fibrinogen accelerate the natural coagulation cascade and control hemorrhage regardless of patient coagulation status (Mozet, Prettin, Dietze, & Dietz, 2013). These coagulation factors have, therefore, attracted attention as potential adjuncts for use to enhance the hemostatic capacity of sponge substrates. In fact, Lee and Wang doped thrombin into sponges via a freeze-drying or spray method (Li, Quan, Xu, Deng, & Wang, 2018; Shefa, Taz, Lee, & Lee, 2019), while Park developed a composite sponge incorporated with recombinant batroxobin (Seon et al., 2017). Indeed, these composite sponges exhibited superior hemostasis performance compared with regular sponges. Unfortunately, adjunctive hemostat factors generally distributed unevenly in the hosting substrate and became encased by the host substrate. These drawbacks limited the utilization efficiency and activity of coagulation factors. Thus, to avoid shielding of coagulation factors by the hosting sponge substrates, alternative assembly strategies are required to maintain their efficacy.
Herein, inspired by the porous structure of puff pastry, we designed a hemostat with a puff pastry-like hierarchical porous structure via an eco-friendly strategy. The hemostat contained three primary components: 1) a chitosan/konjac glucomannan substrate (CKS); 2) microporous starch particles (MSP); and 3) thrombin. The konjac glucomannan (KG), which consists of D-mannose, D-glucose, and acetyl groups randomly attached to the glycosyl units, is a water-soluble polysaccharide (Li, Feng et al., 2017). Owing to the abundant hydroxyl and carbonyl groups in the molecule, KG possesses excellent water absorption, which promotes the concentration of blood components (Chen, Lan et al., 2018). Chitosan (CH) is a natural material derived from chitin, which has been widely used in hemostasis and wound healing fields because of its good biodegradability, antimicrobial activity, and hemostatic properties (Liu, Yan et al., 2018). Furthermore, MSP, with high surface area and good biocompatibility, showed a negative zeta potential (Chen, Chen et al., 2019). Besides, MSP can be enzymatically disintegrated into oligosaccharides, maltose, and glucose in vivo, which are effortlessly absorbed by tissues (Hou et al., 2017).
The hemostat preparation scheme is illustrated in Fig. 1. Initially, to improve the stability and hydrophilicity, MSP was treated with sodium trimetaphosphate, which was then treated with thrombin to obtain thrombin-occupied microporous starch particles (TOMSP). Then, the TOMSP was hosted by CKS to form the hierarchical porous hemostatic system (CKS-TOMSP). We hypothesized that MSP incorporation with the CKS could improve the roughness and zeta potential in the CKS, which may enhance the interactions of the hemostatic material with the blood cells at the wound-material interface. In addition, the new loading strategy may avoid adverse shielding effects for thrombin by the hosting substrate, allowing thrombin to exert its hemostatic function effectively. To verify this hypothesis, systematic characterizations were performed to confirm the CKS-TOMSP structure, including scanning electron microscopy (SEM), flourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD) spectroscopy, and laser scanning confocal microscopy (LSCM). In addition, physical properties, biocompatibility, and in vitro blood coagulation were tested to evaluate the CKS-TOMSP. The coagulation mechanisms activated by CKS-TOMSP were also investigated from the cellular to protein levels. Furthermore, using a New Zealand white rabbit model, we studied the hemostasis performance following treatment with CKS-TOMSP. These results will provide an assessment of the hierarchical porous structure model for hemostasis.
Section snippets
Materials and chemicals
Konjac glucomannan (KG) (98 % purity) was obtained from Huaxianzi Konjac Productions Co. Ltd. (Hubei, China) and chitosan (CH) (degree of deacetylation ≥ 95.0 %) was supplied by Macklin Biochemical Co. Ltd. (Shanghai, China). Dialdehyde starch (DS) was purchased from Jinshan Modified Starch Co. Ltd. (Taian, China). Corn starch (CS) was purchased from Jinhui Biotechnology Co. Ltd. (Shanghai, China). The α-amylase (100 U/mg) and glucoamylase (100 U/mg) were obtained from Duly Biotechnology Co.
Thrombin residual ratio
The thrombin residual ratio of CKS-TOMSP was found to be remarkably higher than that of CKS-STMSP-T on the premise of identical initial thrombin concentrations (Fig. 2a). This was likely due to a more even and independent thrombin distribution in CKS-TOMSP compared to in CKS-STMSP-T. This result implies that the thrombin in CKS-TOMSP had higher blood coagulation efficiency. In addition, BCI was used to preliminarily explore the blood coagulation properties of the different substrates. CKS-TOMSP
Conclusion
In this study, we developed a new CKS-TOMSP hemostatic composite with a hierarchical porous structure system that demonstrated enhanced positive surface charge and roughness due to the hierarchical porous structure, and the even distribution of thrombin loaded on MSP. The hierarchical porous structure endowed an interception and attractive effect for blood cells to aggregate at the interface between the wound and the composite. The thrombin in the composite catalyzed the transformation of
Author contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.
Declaration of Competing Interest
The authors have no conflicts of interest to declare.
Acknowledgments
This work was supported by the National Natural Science Foundation of China (grant number 51703185), the Fundamental Research Funds for the Central Universities (grant numbers XDJK2019AC003 and SWU118125), and the Key Research and Development Program (Social Development) of Zhenjiang City (grant number SH2018001).
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These authors contributed equally.