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Haemorrhage control is of great importance in medical care worldwide1–3. Bleeding caused by minor wounds is usually stopped by innate haemostatic mechanisms. However, uncontrolled haemorrhage following severe trauma is associated with high mortality4,5. Rapid control of bleeding is therefore imperative and has motivated the development of haemostatic materials. Although the initial goal in developing haemostatic materials was to stop bleeding, further developments have yielded mate-rials that both facilitate more efficient haemostasis and exhibit other attractive properties for wound healing, including antibacterial properties6–11, biodegradabil-ity12–15, the ability to promote angiogenesis16–19 and the ability to facilitate tissue regeneration17,20–23. Ideal hae-mostatic materials for wound healing should rapidly control bleeding, be easy to apply to different types of wounds, be biocompatible and non- cytotoxic, be easy to remove or biodegrade, be easy to process and have long- term stability during storage.

The two main factors to consider when one is devel-oping haemostatic materials are the active chemical components and the material form. In this Review, we divide the active components into four categories: natural polymers, synthetic polymers, silicon- based materi-als and metal- containing materials. The various active components used in haemostatic materials have differ-ent haemostatic functions. For example, chitosan — the most widely used haemostatic component — facilitates haemostasis by aggregating red blood cells, stimulating platelets, activating haemostasis- related pathways and promoting clot formation24–26. Silicon- based materials,

such as kaolin or zeolite, have mesoporous structures that can absorb blood and facilitate coagulation27,28. Haemostatic materials are obtained by chemically modi-fying these components or processing them into certain forms. However, developing methods of endowing the active components with more useful characteristics for wound healing without compromising their haemostatic behaviour remains a challenge.

Many forms of haemostatic materials have been devel-oped, ranging from simple to complex, from 2D to 3D, from hydrophilic to hydrophobic and from macro sized to nanosized. The form of a haemostatic material influences its functions and applications, and the most widely used forms of haemostatic materials are sponges29–31, hydro-gels1,8,10,32–38, nanofibres39–43 and particles or powders44–49. Hybrid or composite materials are attracting increasing attention because they combine useful characteristics of the different constituents while avoiding certain draw-backs of the single materials7,16,50–53. The most challenging aspect in developing a haemostatic material is designing it in a form suitable for its application. Most forms of haemostatic materials are suitable for regular- shaped wounds and with low blood flow. By contrast, it is hard to control bleeding of irregular- shaped wounds because bulk materials cannot be efficiently applied to the wound site, and high blood flow presents another challenge. Wounds at moving parts of the body require highly stretchable and adhesive haemostatic materials to avoid breakage and detachment of the dressing. Therefore, it is important to decide on the appropriate haemostatic material for specific applications.

AngiogenesisProcess leading to the formation of new blood vessels, involving sprouting and splitting from pre- existing vessels.

Haemostatic materials for wound healing applicationsBaolin Guo 1,2 ✉, Ruonan Dong 1, Yongping Liang 1 and Meng Li 1

Abstract | Wounds are one of the most common health issues, and the cost of wound care and healing has continued to increase over the past decade. The first step in wound healing is haemostasis, and the development of haemostatic materials that aid wound healing has accelerated in the past 5 years. Numerous haemostatic materials have been fabricated, composed of different active components (including natural polymers, synthetic polymers, silicon- based materials and metal- containing materials) and in various forms (including sponges, hydrogels, nanofibres and particles). In this Review, we provide an overview of haemostatic materials in wound healing, focusing on their chemical design and operation. We describe the physiological process of haemostasis to elucidate the principles that underpin the design of haemostatic wound dressings. We also highlight the advantages and limitations of the different active components and forms of haemostatic materials. The main challenges and future directions in the development of haemostatic materials for wound healing are proposed.

1State Key Laboratory for Mechanical Behavior of Materials, and Frontier Institute of Science and Technology, Xi’an Jiaotong University, Xi’an, China.2Key Laboratory of Shaanxi Province for Craniofacial Precision Medicine Research, College of Stomatology, Xi’an Jiaotong University, Xi’an, China.

✉e- mail: baoling@ mail.xjtu.edu.cn

https://doi.org/10.1038/ s41570-021-00323- z

REVIEWS

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In this Review, we explore haemostatic materials for wound healing, focusing on the chemistry of the materi-als and the processes by which they facilitate haemostasis and wound healing. We provide an overview of different haemostatic components, discuss developments made over the past 10 years in chemically modifying these components and indicate future trends. Moreover, we describe the different forms of haemostatic materi-als, highlighting their advantages and disadvantages. Current challenges in the design of haemostatic mate-rials and their application in wound healing are also discussed to inspire more in- depth research in this area.

Physiological mechanism of haemostasis and wound healingWound healing involves four overlapping (but well- defined) phases: haemostasis, inflammation, prolif-eration and remodelling54 (Box 1). Haemostasis is the process that stops bleeding by formation of a blood clot, and occurs through a mechanism with two main stages (Fig. 1). There is increasing evidence that physiological haemostasis is closely related to wound healing59–61, fur-thering research interest in the design of haemostatic materials for wound healing. The roles of the haemo-static constituents in wound healing do not end when blood flow stops. Blood clots are composed mainly of platelets and fibrin, and they represent a viable, dynamic matrix of proteins and cells that not only contributes to haemostasis but also serves as a provisional lattice for incoming inflammatory cells, fibroblasts and growth factors59. Therefore, if a haemostatic material can be removed without causing secondary damage, the remaining blood clot can further promote wound repair.

Active components of haemostatic materialsVarious active components have been used to prepare haemostatic materials for wound healing, including natural polymers, synthetic polymers, silicon- based

materials and metal- containing materials (TaBle 1). In this section, we examine how the chemical design of these active components accelerates haemostatic wound healing.

Natural polymers. Natural polymers have been widely used in biomedicine, including for haemostasis and wound healing, owing to advantageous properties, which can include biocompatibility, bioactivity, degra-dability, suitable viscoelasticity and ease of processing. Chitosan is a natural polysaccharide and the only one to have a cationic form; in blood, its amino groups are protonated, forming –NH3

+ groups. Electrostatic interac-tions with the –NH3

+ groups increase platelet adhesion62 and also facilitate rapid adhesion and aggregation of red blood cells at the damaged site, aiding the forma-tion of a thrombus3. Chitosan has therefore become the most commonly used natural polymer in haemostatic wound dressings, and chitosan- based HemCon hae-mostatic agents have been commercially available since 2002. Various strategies have been developed to modify chitosan to enhance its application in haemo-static dressings24. For example, to increase the number of positively charged centres, quaternary ammonium groups were introduced into the main chain of chi-tosan through a substitution reaction between its amino groups and glycidyltrimethylammonium chloride36,63 and, similarly, have been introduced into β- chitin through reaction of the hydroxy groups with 3- chloro-2- hydroxypropyltrimethylammonium chloride40. The introduction of quaternary ammonium groups increases blood cell adhesion36,40,63 and also enhances antimicrobial activity10,20,33, but it can also make the material toxic to normal cells64. Thiol- modified chitosan was prepared by introducing sulfhydryl groups, which interact with red blood cells to promote clotting65. Chitosan has also been modified to make it hydrophobic by Borch reduction of the amino groups with dodecyl aldehyde. The dodecyl

Fibrina highly insoluble protein polymer formed by spontaneous polymerization of fibrinogen upon excision of the a and B polypeptide chains by thrombin, and forms needle- like crystals.

Thrombusa blood clot that forms within a blood vessel or inside the heart and remains at the site of its formation.

Box 1 | the phases of wound healing

the four consecutive but overlapping phases of wound healing are haemo stasis, inflammation, proliferation and remodelling54–56 (see the figure). Haemostasis has two main stages. During the primary stage of haemostasis, platelets adhere to the injured site, where they are activated. the activated platelets further activate and aggregate free platelets to form a platelet plug. During the secondary stage of haemostasis, thrombin produced by extrinsic and intrinsic coagulation pathways converts fibrinogen into fibrin, which then forms a thrombus with platelet clots and blood cells. the important sign of the transition from haemostasis to the inflammation phase is the release of mediators and cytokines that promote the division

and proliferation of fibroblasts, thereby producing collagen. Meanwhile, inflammatory cells adhere to the fibrin net formed in the haemostatic phase and clean the wound by removing phagocytic cell debris and bacteria, and the inflammatory phase usually lasts for the first few days of wound healing. The proliferation phase occurs over the first few weeks of wound healing, during which time granulation tissue forms, new blood vessels form through angiogenesis and the epidermis regenerates. the final remodelling phase starts at 2–3 weeks after initial injury and lasts for 1 year or more. Over this period, the processes activated after injury wind down and then cease, new tissue is remodelled and the skin’s mechanical properties are restored57,58.

Primary haemostasis Secondary haemostasis Inflammation Proliferation RemodellingBleeding wound

Endothelium

PlateletRed blood cell

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tails can insert themselves into cell membranes, which aids the accumulation of red blood cells and can also kill bacteria through strong hydrophobic interactions16,20,66. Catechol- modified chitosan was prepared through ami-dation of the carboxyl groups of levodopa or dihydrocaf-feic acid with the amino groups on chitosan. The catechol groups increase the adhesion of chitosan to wet tissues, promoting the coagulation of red blood cells67,68. In addi-tion, catechol moieties can also constrict blood vessels, preventing massive bleeding69. A potential limitation of chitosan- based haemostatic agents is that they degrade slowly in vivo70, which should be taken into account when one is developing materials for wound healing.

Collagen triggers platelet activation71 in primary haemostasis and activates the expression of coagulation factors VIII, IX, XI and XII, among others, in secondary haemostasis (Fig. 1). In the process of tissue repair, col-lagen can also promote cell growth and proliferation72.

Thus, many haemostatic wound dressings containing collagen have been developed73. However, the possible allergen problems of biogenic collagen make its safety uncertain74. The production of human- like collagen through genetic engineering might avoid this issue75,76. Another way to reduce the biological safety issue of collagen is to hydrolyse it into gelatin77,78, and there are several commercial haemostatic gelatin sponges that exert haemostatic effects by increasing the con-centration of platelets and coagulation factors in the blood79. Hydrophobically modified gelatin synthe-sized by reductive amination of the amino groups with aliphatic aldehydes80 and gelatin modified with ureidopyrimidinone- hexamethylene diisocyanate32 promote haemostasis by tissue adhesion through hydro-phobic interactions80 and increased hydrogen bonding, respectively. However, facile degradation and poor stress resistance are problems for gelatin- based materials81.

XIII XIIIa

XIIX

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Va

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CrosslinkedfibrinFibrin (Ia)

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II

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X

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Xa

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KLK

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Ca2+

Ca2+

KLKPKLK

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ndar

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– – ––

––––

GPIa–IIa GPIb–IX–V

Adhesion

GPIIb–IIIa

Activation

vWF

Fig. 1 | mechanisms of primary and secondary haemostasis. During primary haemostasis, platelets quickly adhere to the injured endothelium at the bleeding site through specific receptors, namely GPIa–IIa binds to damaged subcutaneous collagen and GPIb–IX–V binds to von Willebrand factor (vWF). The platelets are then activated192, thus triggering aggregation of more locally activated platelets and the formation a platelet plug to stop bleeding188,193. The primary stage of haemostasis has inspired the development of various haemostatic materials that promote platelet enrichment and activation (such as cationic polypeptides) to promote blood coagulation85,112. In secondary haemostasis, under the synergistic effect of platelet activation, coagulation factors undergo a complex coagulation cascade to produce thrombin (also known as activated factor II (IIa)), which converts fibrinogen (also known as factor I) in adjacent plasma into fibrin (also known as factor Ia). The interwoven fibrin makes platelets clot and blood cells tangle into thrombi at the bleeding site. Thus, various haemostatic materials have been developed based on fibrinogen, fibrin and fibrin- mimetic structures (such as self- assembled peptides)147. Concurrent with thrombus formation, platelet protrusions extend into the fibrin

network, and the contraction of platelet microfilaments (actin) and myosin makes the blood clot contract, and the thrombus becomes firmer and thus can stop bleeding more effectively188,194. The coagulation cascade includes extrinsic and intrinsic pathways. The extrinsic pathway is initiated by the exposure of blood to tissue factor (TF) following tissue damage and eventually leads to the activation of factor X into factor Xa. For the intrinsic pathway, factor XII is activated when blood contacts a negatively charged surface, resulting in the downstream proteolytic activation of other coagulation factors until factor X is activated into factor Xa. Thus, haemostatic agents with negatively charged surfaces (such as anionic polysaccharides) can promote blood clotting89,91. The two pathways converge into a common pathway through factor Xa. Factor Xa cleaves prothrombin (also known as factor II) into thrombin in the presence of phospholipids and Ca2+, and thrombin converts fibrinogen into fibrin to achieve the main goal of secondary haemostasis195. Polyphosphate released from activated platelets can facilitate crosslinked fibrin clot formation and augment clot stability. HMWK, high molecular weight kininogen; KLK, kallikrein; PKLK, prekallikrein.

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Cellulose with a certain number of sugar units functionalized with carboxyl groups can combine with Fe3+ in the blood and activate coagulation fac-tor XII through the carboxyl groups on its surface,

further promoting platelet aggregation13 to realize the haemostatic effect3,77,82. Quaternized hydroxyethyl cellulose83 and rod- shaped aldehyde- functionalized cellulose nanocrystals84 showed enhanced haemostatic

Table 1 | Classification of haemostatic materials in wound healing and their haemostatic mechanism

haemostatic materiala

haemostatic mechanismb role in wound healing Limitations

Natural polymers

Chitosan Promotes adhesion and aggregation34,92 of platelets and blood cells20

Antibacterial10,20,33; accelerates infiltration of inflammatory cells; promotes granulation tissue formation10; promotes proliferation of fibroblasts120

Poor biocompatibility (quaternized chitosan); slow degradation

Collagen and gelatin

Activate platelets3; blood concentration79

Promote cell growth and proliferation72 Poor resistance to degradation; immunogenicity risk

Fibrin Blood clot formation; promotes platelet aggregation189

Promotes increase in epidermal thickness; increases the number of fibroblasts, fibrocytes46 and keratinocytes190; revascularization19

Expensive; immunogenicity risk

Cellulose Blood concentration; activates coagulation factors; promotes platelet aggregation3,13

Antibacterial84,85 Slow biodegradation

Hyaluronic acid Tissue adhesion29; blood concentration51,89,92

Keeps wound moist29; regulates TGF- β activity; promotes fibroblast migration and collagen deposition90

Expensive; hard to remove

Alginate Tissue adhesion21; blood concentration87

Keeps wound moist21; promotes granulation tissue formation and fibroblast proliferation149

Low mechanical strength and chemical stability

Dextran Promotes platelet adhesion114 Promotes cell migration and proliferation66 Unable to activate platelets and blood cells

Synthetic polymers

Polyethylene glycol Tissue adhesion16,35,36,100,105,106; blood concentration51

Good carrier for therapeutic agents52 Expensive; risk of raw material residue

Polyvinyl alcohol Blood concentration108 Good carrier for therapeutic agents109 Unable to activate platelets and red blood cells

Polyurethane Initiates coagulation and platelet aggregation105,111

Antibacterial105 Slow biodegradation; poor biocompatibility; long polymerization time (TissueGlu)

Silicon- based materials

Mesoporous silica Activates coagulation factor XII (reF.85) Antibacterial94,125 Does not degrade; hard to remove; potential metabolic toxicity

Zeolite Rapidly concentrates blood cells and releases Ca2+ to accelerate blood coagulation128

Absorbs wound exudate50 Severe exothermic reaction; does not degrade; hard to remove; potential metabolic toxicity; may cause thrombosis

Montmorillonite Blood concentration; promotes blood cell aggregation9

Good carrier for therapeutic agents171 May cause inflammation and thrombosis; does not degrade; hard to remove; potential metabolic toxicity

Kaolin Platelet activation; promotes coagulation cascade128

Revascularization128 Does not degrade; hard to remove; potential metabolic toxicity

Metal- containing materials

Ca Promotes conversion of prothrombin to thrombin136

– Do not degrade; hard to remove; toxic at high concentration

Ag Activates platelets130,148 Antibacterial

Zn Promotes blood cell aggregation138 Antibacterial138; epithelization138, promotes cell proliferation and revascularization191

Fe Stabilizes thrombin139; promotes blood cell aggregation128

Revascularization128

Ce Promotes blood cell aggregation140 Anti- inflammatory140

TGF- β, transforming growth factor- β. aThe haemostatic materials are typically based on or modified with the substances listed in this column. b‘Blood concentration’ refers to an increase in the concentration of platelets and coagulation factors in the blood.

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and antibacterial effects84,85. The quaternary ammonium groups in the former aid the aggregation of blood cells in primary haemostasis, whereas the aldehyde groups in the latter enhance tissue adhesion. However, the slow biodegradation of cellulose severely limits the applica-tion of cellulose- based materials in scenarios in which removal is not required86.

Anionic polysaccharides, such as alginate87 and hya-luronic acid51,88,89, have a high rate of water absorption, which increases the concentration of platelets and blood coagulation factors. Hyaluronic acid- based haemostatic dressings can also regulate transforming growth factor- β (TGF- β) activity and manipulate fibroblast migration and collagen deposition in subsequent repair processes90. Moreover, anionic polysaccharides can be constructed into hydrogels by crosslinking with metal ions (such as Ca2+) through their carboxyl groups14, diversifying their use in haemostatic materials. Through the nega-tive charges on their surface, anionic polysaccharides can interact with positively charged amino acids on the coagulation factor XII chain to activate the intrinsic path-way, thereby accelerating the coagulation cascade89,91. Another popular approach to modifying anionic poly-saccharides is to functionalize them with dopamine. The catechol moiety in dopamine binds to amino or sulfhy-dryl groups in tissue, endowing anionic polysaccharides with superior adhesion to wet tissue8,29 and resulting in rapid haemostasis. In addition to dopamine6,15,20,88,92,93, dihydrocaffeic acid32,68, tannic acid87,94,95 and other molecules containing ortho- polyphenol moieties have been widely used to modify anionic polysaccharides to achieve haemostasis through tissue adhesion. Catechol has also been reported to negatively charge the mate-rial, activating the intrinsic coagulation cascade and triggering coagulation factor XII, which in turn triggers a clotting cascade69. Therefore, haemostatic materials comprising compounds with ortho- polyphenol moieties show great advantages for wound healing. The enhanced adhesion can, however, be a disadvantage in situations where the haemostatic material needs to be removed.

Other natural polymers have also been used to develop haemostatic materials. For example, the alde-hyde groups in oxidized dextran can be crosslinked with an amino- containing crosslinking agent to prepare blood- absorbing sponges and hydrogels30,66. The alde-hyde groups can also crosslink with the amino groups in tissue, facilitating stronger tissue adhesion for haemosta-sis. Therefore, the design of compounds with aldehyde groups is of great significance for the development of haemostatic materials in wound healing. The silk protein fibroin does not have functional groups that promote clotting and, thus, alone does not have the potential to be a haemostatic material. However, porous films prepared by electrospinning silk fibroin can be co- processed with other synthetic or natural polymers to serve as a haemo-static material96,97. Several natural gums are used to exert a haemostatic effect by enhancing the tissue adhesion of haemostatic materials40,71,98–100. However, haemostatic materials containing natural gum generally lack strong biological mechanisms to induce blood clotting and, thus, are not as effective as the haemostatic materials discussed above. Thrombin (also known as activated

factor II or factor IIa) reacts with fibrinogen to produce fibrin, which is essential in platelet aggregation- induced haemostasis. Moreover, during wound healing, the fibrin blood clot101, which gradually dissolves, can also transport cells and growth factors into the wound102,103, promote an increase in epidermal thickness, increase the number of fibroblasts and fibrocytes and affect the behaviour of keratinocytes46. Although numerous natu-ral polymers have been used in the preparation of hae-mostatic materials for wound healing, several challenges remain in the development of natural products. These issues include large variations in properties between different batches (such as large differences in molecular weight), high prices and potential problems caused by allergenicity104.

Synthetic polymers. Synthetic polymers are increasingly used as haemostatic agents in wound healing, as they can avoid the drawbacks associated with natural polymers. Modified polyethylene glycol (PEG)- based materials are often used as physical barriers owing to their good adhesiveness to tissue, and their histocompatibility also makes them good carriers for therapeutic agents52. Furthermore, their excellent water- absorption proper-ties increase the concentration of coagulation factors and platelets within blood51 to enhance platelet aggregation in primary haemostasis105. The tissue adhesion capac-ity of PEG can be enhanced through functionalization with aldehyde groups16,36,100, N- hydroxysuccinimide106 or dopamine93 to form hydrogen bonds or Schiff base bonds with the amino groups in tissues. For example, a hyperbranched PEG- based polymer was fabricated with a hydrophobic backbone and a hydrophilic catechol side chain. Upon contact with water, the hydrophobic chains quickly self- aggregate to form a coacervate and water molecules are displaced from the adhesive surface, triggering the exposure of catechol groups to increase the adhesion of the material to tissue107. However, such modifications increase the difficulty of producing these materials on a large scale.

Polyvinyl alcohol- based haemostatic sponges expand rapidly upon water absorption and can increase the blood concentration of coagulation factors and platelets75,108–110. Such products are on the market but lack the ability to activate red blood cells and platelets108. Polyurethane foam is also widely used for haemostasis because it initi-ates the coagulation cascade and platelet aggregation105,111. The hydrophobicity of polyurethane- based materials affects their haemostatic efficacy by influencing platelet adhesion. Polyurethanes with different hydrophobicities were prepared by changing the proportion of diphenyl methyl diisocyanate, showing that the most hydro-phobic polyurethane surface had the lowest activation for coagulation but the greatest platelet adhesion112. In another example, PEG surfaces were prepared by the addition of PEG- containing amphiphilic block copoly-mers in the segmented polyurethane; long- chain PEG decreased surface hydrophobicity and reduced platelet adhesion113. Biomacromolecules such as dextran can also be incorporated to increase the adhesion of platelets to polyurethane fibres114. Polyurethane- based adhesives, such as TissueGlu, are commercially available but have

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problems such as the use of toxic isocyanates and long in vivo polymerization times (>25 min)115.

Other synthetic polymers used in the preparation of haemostatic materials in wound healing include polylactic acid6,116–118, polycaprolactone62,119, polyacrylamide6,120,121, cyanoacrylates122,123 and synthetic polypeptides85. For example, a polyacrylamide- based hydrogel matrix was functionalized with side groups that can each form three hydrogen bonds. The high density of hydrogen bonds and the ‘load- sharing’ effect enable the hydro-gels to adhere firmly to various surfaces to promote haemostasis124. However, most synthetic polymers are used as substrates to construct haemostatic materials owing to a lack of bioactivity and the ability to activate the coagulation pathway and accelerate wound healing. A few synthetic polymers (such as synthetic cationic polypeptides and cyanoacrylates) show strong haemo-static effects; thus, additional research is required for development of other synthetic polymers.

Silicon- based materials. Inorganic materials can also promote haemostasis, and most examples of inorganic haemostatic materials are silicon based. Mesoporous silica has negatively charged silanol groups on its sur-face that can interact with positively charged amino acids on the coagulation factor XII chain to activate the intrinsic coagulation pathway and accelerate the coagu-lation cascade, thus enhancing the haemostatic effect68. Incorporation of a specific ratio of mesocellular silica foam into a quaternized hydroxyethyl cellulose compos-ite hydrogel sponge accelerated the formation of thrombin83. Silver- loaded nanomesoporous silica par-ticles showed higher blood cell absorption and platelet adhesion than a commonly used haemostatic gauze. Moreover, the silica particles also notably shortened the activated partial thromboplastin time, suggesting that they have an active role in haemostasis through the intrinsic coagulation pathway125.

Another category of silicon- based haemostatic mate-rial is zeolite, which can coagulate blood by releasing Ca2+ and quickly absorb plasma and concentrate blood cells126,127. Zeolite- based QuikClot is a commercially available haemostatic material. However, its exothermic reaction upon water absorption leads to thermal damage to and necrosis of surrounding tissues, severely limiting its application. A zeolite- based material that avoids this shortcoming was fabricated by growing mesoporous single- crystal chabazite zeolite in situ on the surface of cotton fibres, achieving high procoagulant activity without undesirable thermal effects50.

Montmorillonite exerts haemostatic activity through its negatively charged surface to efficiently activate the coagulation system, although it may cause inflammation and thrombosis in the body; therefore, its commercial use is limited9. In an approach to overcome this prob-lem, montmorillonite particles were bound with polydextran aldehyde by ultrasonication, prevent-ing montmorillonite- induced peripheral vascular embolism9. QuikClot Combat Gauze, which was first marketed in 2008, is made by impregnating gauze with kaolin nanoparticles, and was approved by the US Food and Drug Administration for use in traumatic bleeding

in 2013. QuikClot Combat Gauze is a better alternative to zeolite- based QuikClot because it achieves a similar haemostatic effect without the exothermic reaction. However, QuikClot Combat Gauze has the potential to cause blood clots126. For further improvements, an Fe2O3–kaolin composite, with a large number of Fe2O3 nanoparticles anchored on a kaolin nanosheet, was pre-pared by simple precipitation and high- temperature calcination128. Similarly to kaolinite, the composite induced platelet activation and promoted the coag-ulation cascade, and Fe2O3 enhanced red blood cell aggregation. The composite also promoted vascular and immature glandular lumen formation in wound healing128.

In summary, silicon- based materials have shown good haemostatic effects, but their potential metabolic toxicity129 has restricted their development. Although approaches have been developed that have led to improvements, more work is needed to fully address their potential toxicity.

In addition to silicon- based materials, there has been widespread interest in developing graphene- modified silicon- based haemostatic agents, which have been shown to promote platelet aggregation130. The addi-tion of graphene can ameliorate the exothermic prob-lems caused by zeolite- based haemostasis through thermal conduction131. Composites of graphene with montmorillonite132 or kaolinite133 have been formed through hydrothermal reactions, and might avoid the risk of intravascular thrombosis. In addition, the electrical conductivity of graphene can also promote wound healing by enhancing the endogenous electric field, which can promote cell migration at the wound8,81. However, because graphene does not degrade, there could be metabolic and thrombogenic problems asso-ciated with the use of graphene- modified silicon- based materials in in vivo applications, and these need to be addressed in future studies134,135.

Metal- containing materials. Metal ions are also used in haemostatic wound dressings. Most commonly, Ca2+ ions are introduced to accelerate thrombosis by pro-moting the conversion of prothrombin to thrombin136. In one case, biphasic Janus self- propelling haemostatic particles were prepared by uniaxial growth of flower- like CaCO3 crystals on negatively modified microporous starch for the haemostasis of deep and non- compressible wounds. By reaction of CaCO3 with sodium bicarbonate and protonated tranexamic acid, these particles pro-duce gas to propel themselves against the bloodstream, allowing them to access deep bleeding sites, where they induce blood coagulation137. Zinc is also widely used. For example, nanoparticles comprising carboxymethyl chitosan and ZnO were prepared through in situ pro-duction of ZnO on carboxymethyl chitosan. ZnO not only enhanced the haemostatic performance of chi-tosan but also regulated each stage of wound healing138. In addition, silver nanoparticles can activate platelets130, and Fe2O3 nanoparticles stabilize thrombin and have a role in promoting blood coagulation139. The rough sur-face of hollow CeO2 nanoparticles was shown to facilitate haemostasis and also benefit electron–hole separation

Mesocellular silica foamSilicon- based nanomaterial with large- opening spherical mesopores and large pore volume; made by the reaction of tetraethyl orthosilicate under acidic conditions with the triblock copolymer Pluronic P123, mesitylene and NH4F.

Activated partial thromboplastin timeThe most commonly used sensitive screening test in clinical practice to determine the coagulation activity of the intrinsic coagulation system; measures the time taken for coagulation upon addition of activated partial thromboplastin time reagent (factor xii activator and cephalin) and Ca2+ ions to plasma at 37 °C.

ChabaziteTectosilicate mineral of the zeolite group with the formula (Ca,K2,Na2,Mg)al2Si4o12·6H2o. Chabazite- type zeolites are highly absorbent, which is beneficial for increasing the concentration of components in blood to facilitate haemostasis.

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and the production of reactive oxygen species, thereby preventing infection during the inflammation phase of wound healing140. However, few studies have focused on metal- containing haemostatic agents in wound healing, but owing to their potential haemostatic function, these materials should be given more attention.

Bio- derived or biomimetic materials. In addition to the materials mentioned so far, bio- derived haemostatic agents based on thrombin141, platelet- rich plasma- derived agents142–144 and platelet lysates with relatively low batch variability have been developed to aid wound healing84. In addition to bio- derived haemostatic materi-als, synthetic analogues that mimic native platelet func-tion have also been fabricated. For example, synthetic biomimetic platelet- like particles, containing platelet surface glycoproteins, fibrinogen or fibrinogen- derived Arg- Gly- Asp (RGD) peptides145, not only promote wound healing but also reduce the risk of biological contamination17,45,146. Platelet- like particles have also been modified with silver nanocomposites to endow them with antibacterial ability121.

In another approach, nanofibres formed from self- assembling peptides were used to coagulate blood and facilitate haemostasis. A nanofibre- based clot formed from blood components and the peptide RADA16- I through physical winding was morphologi-cally similar to the natural fibrin clot that formed in sec-ondary haemostasis147. Various peptides have also been introduced into wound dressings to promote platelet adhesion and aggregation148,149, and cationic polypep-tides can also induce blood cell aggregation and platelet activation by electrostatic attraction85. For example, the haemostatic performance of chitosan was enhanced by the addition of ε- polylysine11. However, in general, there are biosafety issues, such as exogenous pollution, that must be considered and tested for when one is develop-ing biologically derived products, and which could limit their application in haemostatic materials.

Form of haemostatic materialsFabricating compounds that promote haemostasis into suitable forms can strengthen the haemostatic effects. Multiple forms of haemostatic materials have been developed, including sponges, hydrogels, nanofibres and particles. The various forms exhibit different prop-erties and have specific application conditions. In this section, we discuss the processes by which different material forms exert haemostatic effects, including their advantages and disadvantages.

Sponges. Sponges are the most conventional form of material used in haemorrhage control, with their appli-cation dating back to 1945 (reFS150,151). Owing to their high porosity, sponges have a high blood- absorption capacity, and owing to their ability to accumulate red blood cells and platelets, they can concentrate coagula-tion factors and, thus, promote coagulation30,152. Gelatin sponges were the first to be developed and remain the most commonly used sponges for controlling haemor-rhage; these sponges are fabricated by lyophilization29,153. Although various other fabrication methods have been

developed to obtain haemostatic sponges, lyophilization still deserves attention because of its effectiveness in cre-ating porous sponges through simple steps. For example, a polydextran aldehyde- based wet- adhesive sponge that quickly absorbs blood was fabricated by simply freeze- drying the polydextran aldehyde solution30. This sponge possesses pores with sizes of ~30–50 μm and porosities of up to 97.9% (Fig. 2a). The oxidation ratio and poly-dextran aldehyde concentration can be varied to change the porosity and, thus, adjust the blood- absorbing capacity. Moreover, the residual aldehyde groups on the surface make these sponges strongly adhesive, ena-bling them to function as wound sealants. However, too many residual aldehyde groups could cause cyto-toxicity. Thus, the oxidation ratio and concentration of polydextran aldehyde must be carefully controlled. Although freeze- dried sponges possess many pores, the interconnection between the pores is low, which decreases their absorption capacity and weakens their haemostatic effect.

To increase the interconnection between pores, cryo-gel fabrication is proposed as an alternative method. Cryogels are gels that are fabricated at subzero temper-atures and have high porosity, good pore connectivity and adjustable mechanical properties. Several cryogel sponges have been fabricated using different compo-nents, including chitosan16,154, gelatin6,15, silk fibrin95 and biomolecule components11,84 (such as peptides and plate-let lysate). A series of shape- memory cryogel sponges were developed with haemostatic properties for accel-erating wound healing. To obtain the main component of the cryogel, chitosan was modified with glycidyltri-methylammonium chloride, to increase water solubil-ity and to introduce antibacterial activity, and glycidyl methacrylate through reaction with the amino groups. Diacrylate- functionalized PF127 polymer was used as a crosslinker and to recruit multiwalled carbon nano-tubes through hydrophobic interactions. The cryogel was formed at −20 °C in the presence of ammonium per-sulfate and tetramethylethylenediamine as redox initia-tors (Fig. 2b). This cryogel showed highly interconnected pores as well as good elasticity and rapid absorption and high absorption capacity. In subsequent work, dopamine was used instead of diacrylate- functionalized PF127 to crosslink gelatin at −20 °C in the presence of NaIO4, which is an oxidant that can facilitate polydopamine formation. The resulting cryogel exhibits fast and tuna-ble degradation, obviating removal after haemostasis15. Cryogels with good blood- absorption performance show much promise for haemostasis applications, but have several limitations, such as their poor mechani-cal properties (especially their poor stretchability), the long reaction times and the difficulty in removing the residual catalyst.

The formation of pores in sponges fabricated by freeze- drying and cryogel formation occurs in situ. An alternative method for sponge fabrication is to create pores by blowing gas into the material. In this way, lay-ered nanofibre sponges109 were prepared by expanding nanofibrous films (Fig. 2c). Chitosan–polyvinyl alco-hol nanofibrous films were fabricated by electrospin-ning, before immersion in a solution of NaBH4, which

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Fig. 2 | haemostatic sponges in wound healing. a | Haemostatic polydextran aldehyde (PDA) sponges formed by freeze- drying. The scanning electron microscopy images show sponges with different PDA oxidation ratios (where ‘PDA x’ indicates that the theoretical oxidation is x%)30. The pores of PDA 50 are larger than those of PDA 25, PDA 75 and PDA 100, and thus PDA 50 has the highest absorption capability. b | Fabrication of a cryogel sponge based on glycidyl methacrylate (GMA)- functionalized quaternized chitosan (QCSG)63. In the first step, chitosan is modified with glycidyltrimethylammonium chloride (GTMAC) and GMA. In a separate reaction, acryloyl chloride is used to introduce double bonds into the PF127 polymer, forming PF127- DA. Multiwalled carbon nanotubes are then dispersed in an aqueous solution of PF127- DA.

The cryogel is synthesized by co- polymerization of an aqueous solution of QCSG and the PF127- DA–carbon nanotube dispersion at −20 °C in the presence of ammonium persulfate (APS) and tetramethylethylenediamine (TEMED). c | 3D chitosan–polyvinyl alcohol (PVA) sponges formed by expanding 2D nanofibrous films109. Chitosan and PVA are mixed and then electrospun into a nanofibrous sheet. This 2D nanofibrous sheet (left) is immersed in an aqueous NaBH4 solution, and the H2 generated expands the nanofibrous sheet into a 3D nanofibrous porous sponge. The thickness of the 3D sponges increases with the immersion time (right). RT, room temperature. Part a adapted with permission from reF.30, Elsevier. Part b adapted from reF.63, CC BY 4.0 . Part c adapted with permission from reF.109, Elsevier.

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generates H2. The H2 gas flows into the nanofibrous sheets, expanding them in three dimensions to produce high- porosity sponges. The sponges have microsized pores that help absorb and concentrate blood, and the nanofibrous structures help the adhesion of other fac-tors, making their haemostatic behaviour more efficient. A concern for this fabrication method is the safety of NaBH4, and its removal would require additional steps during sponge fabrication.

A facile method to generate haemostatic sponges is to modify commercial wound dressing sponges with bioactive components. For example, gelatin sponges were coated with self- assembled nanofibres to enhance their haemostatic activity147. The sponge was coated with RADA16- I peptides and polyanions (hyaluronic acid, chondroitin sulfate and dextran sulfate) by spraying the peptide and polyanion solutions in a certain sequence at regular intervals. This layered fibrous coating is stable over a wide temperature range (−80 to 60 °C), enabling transport and storage without the need for a cold chain, and can be released when applied to wounds to help facilitate clotting by forming nanofibre- based clots. Surface modification of commercial sponges is a fast method of generating versatile sponges that exhibit favourable characteristics of both the sponge and the coating layer. It would be desirable to achieve superior integration between the coating and the sponge, such as by using covalent reactions to coat the matrix with the nanofibrous layer.

Many haemostatic sponges have been designed to have multifunctional properties, including antibacte-rial properties9,16, the ability to promote angiogenesis98 and the ability to accelerate wound healing109. However, there are challenges in applying haemostatic sponges that need to be addressed. In addition to absorption, sponges also help to stop bleeding through compres-sion owing to their expansion after swelling. However, this expansion can compress nerves. Moreover, the removal of non- degradable sponges with clots inside without causing pain and secondary bleeding remains an issue. For degradable sponges, the challenge is to control the degradation rate of the sponge to cooperate with the wound- healing timeline.

Hydrogels. Haemostatic hydrogels not only help to con-trol haemorrhaging but also keep the wound site moist, which aids wound healing155–157. A common character-istic of hydrogels used for haemorrhage control is adhe-sion, which enables the hydrogels to seal the wound and accelerate haemostasis10,14,83,107,157,158. Adhesion is mostly due to the reaction between active groups in the hydrogels and amino groups of the proteins in native tissues159–161. The Schiff base reaction is a beneficial method for fabricating hydrogels, endowing hydrogels with dynamic crosslinks and adhesive ability through the residual aldehyde groups10,36. For instance, this approach was used to develop an adhesive hydrogel from gelatin methacryloyl (GelMA) and hyaluronic acid functional-ized with N-(2- aminoethyl)−4-(4-(hydroxymethyl)−2- methoxy-5- nitrosophenoxy) butanamide (HA- NB) that forms rapidly in situ upon photoactivation1. Upon UV irradiation of a mixture of GelMA and HA- NB, the

GelMA chains crosslink through reaction of the meth-acrylic anhydride groups, forming a first crosslinked network. Furthermore, the UV irradiation of HA- NB generates aldehyde groups (Fig. 3a) that can react with amino groups from GelMA to form Schiff bases as a sec-ond crosslinked network. The photogenerated aldehyde groups also react with the amino groups from proteins or glycosaminoglycans in tissues, enabling the hydrogel to adhere to wet tissue, and the double- crosslinked network bestows high mechanical strength on the hydrogel. This hydrogel withstands pressures of up to 290 mmHg when sealed to a bleeding area, and, thus, is suitable for appli-cation in arterial bleeding during surgery. A common problem with Schiff base- induced adhesive hydrogels is the potential cytotoxicity of the numerous residual aldehyde groups. Thus, the –NH2/–CHO ratio needs to be carefully calculated to balance the adhesive function with cell viability.

In addition to Schiff base formation, polymers can be designed to adhere to tissue through other interactions. For example, a tetra- PEG- based hydrogel sealant for vis-ceral haemostasis adheres strongly to tissues through the formation of amide linkages35. Tetra- armed polyethyl-ene glycol succinimidyl succinate reacts rapidly with tetra- armed polyethylene glycol amine to form an adhe-sive hydrogel (Fig. 3b). Residual succinimidyl active ester in the hydrogel reacts with amino groups on the native tissues through the aminolysis reaction, forming stable amide bonds, resulting in efficient sealing of the wound and haemorrhage control. The hydrogel sealant shows remarkable haemostatic ability, even in anticoagulated situations, owing to its high adhesiveness (withstanding bursting pressures of up to 294 ± 27 mmHg). However, its ingredients are expensive, which is an issue for clinical application. Thus, a challenge is to generate affordable products based on this chemistry.

Nature has also provided inspiration for the design of adhesive hydrogels. For example, inspiration has been drawn from the chemistry that enables mus-sels to adhere to wet surfaces, in which catecholic amino acid on mussel foot proteins has a key role162. In one study, a double- network adhesive hydrogel was formed through physical crosslinking involving catechol–Fe3+ coordination and quadruple hydrogen bonding32 (Fig. 3c, left). The prepolymer poly(glycerol sebacate)- co- polyethylene glycol- g- catechol was reacted with ureidopyrimidinone- hexamethylene diisocyanate synthon- modified gelatin in the presence of Fe3+ to give the hydrogel. The catechol groups played the part of an adhesive reagent, forming hydrogen bonds with amido groups on the surface of tissue, participating in cationic–π interactions with amino groups on the tissue and, in some cases, reacting with the amino groups to form Schiff base bonds. The tissue adhesive strength of this hydrogel is up to 5.2 kPa, as measured by lap shear tensile testing. An adhesive hydrogel with anti-bacterial properties was fabricated8 from hyaluronic acid- g- dopamine and polydopamine- coated reduced graphene oxide (Fig. 3c, right). Crosslinking of the cat-echol groups in the two components was initiated with H2O2 and horseradish peroxidase. The tissue- adhesive properties of the hydrogel are mainly attributable to

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the interaction of the catechol and quinone groups in the hydrogel with amino and thiol groups in the proteins in native tissues, resulting in a high tissue adhesive strength of up to 6.3 ± 1.2 kPa. Mussel- inspired chem-istry is an easy and effective way to achieve adhesives. However, one concern is whether the dopamine in the hydrogel is released into the body during its application,

raising the question of how to control its release to avoid side effects.

A slug- secreted defensive mucus163 inspired the development of a tough adhesive for wet surfaces (Fig. 3d). The adhesive comprises two layers: an adhesive surface, which adheres to tissue through the formation of covalent bonds, electrostatic interactions and physical

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Fig. 3 | haemostatic hydrogels in wound healing with different adhesive mechanisms. a | Tissue adhesion through the formation of Schiff bases1. Residual aldehyde groups in the hydrogel can react with amino groups on the tissue surface, forming Schiff bases and adhering the hydrogel to the tissue. b | Tissue adhesion through the formation of amide linkages. Succinimidyl active ester in the hydrogel network reacts with amino groups on the tissue surface to form amide bonds35. c | Catechol- based adhesive hydrogels8,32. Catechol groups contribute to tissue adhesion by forming three kinds of bonds with different functional groups on the tissue surface: hydrogen bonds with amide groups, cation–π interactions with amino groups and Schiff base bonds through reaction with amino or thiol groups. d | Tissue adhesion through amide bond formation, electrostatic interactions and physical interpenetration. This adhesive comprises two layers: an adhesive surface made from a positively charged polymer and a hydrogel that acts as a dissipative matrix. The positively charged polymer with primary amino groups serves as a ‘bridging polymer’ by interacting with the tissue and the dissipative matrix. The bridging polymer absorbs on tissue through electrostatic attraction, facilitating the reaction of the amino groups on the hydrogel with carboxyl groups on the tissue surface. Moreover, when the tissue is permeable, the polymer chain can penetrate the tissue and form physical entanglements. rGO@PDA, polydopamine- coated reduced graphene oxide.

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interpenetration; and a dissipative matrix, which dissi-pates energy when stress is applied to the adhesive. The adhesive surface was formed from ‘bridging’ polymers containing primary amino groups (chitosan) that are protonated at physiological pH and interact with both the substrate and the dissipative matrix. Amino groups in the bridging polymers react with the carboxylic acid groups in the tissue surface after being absorbed by elec-trostatic attractions. Furthermore, the bridging polymer can penetrate permeable tissues, achieving physical entanglement. The dissipative matrix was formed from an alginate–polyacrylamide hydrogel; two coupling rea-gents, 1- ethyl-3-(3- dimethylaminopropyl)carbodiimide hydrochloride and N- hydroxysulfosuccinimide, facilitate the formation of covalent bonds with the bridging poly-mer and thus the integration of the adhesive layer with the dissipative matrix. This adhesive hydrogel showed remarkable adhesive properties, with an adhesive energy with regard to skin of ~1 kJ m−2 (whereas that of fibrin glues is 0.01–0.1 kJ m−2), and can strongly adhere to wet surfaces. Moreover, the hydrogel also showed a high matrix toughness of up to 10 kJ m−2, which is ~10 times higher than that of native cartilage. However, multiple coupling reagents were used in forming this hydrogel, which could lead to safety concerns.

Although adhesive ability is a beneficial characteris-tic for haemostatic hydrogels, it also leads to problems; principally, how to remove hydrogels that adhere strongly to wounds without causing secondary pain and bleeding. To address this issue, an autolytic hydro-gel was fabricated from poly(N- acryloyl-2- glycine) and hydroxyapatite (Ca10(PO4)6(OH)2) as a haemo-static adhesive164. This hydrogel has high mechanical strength, which is due to hydrogen bonding between poly(N- acryloyl-2- glycine) chains and ionic crosslinking between carboxyl moieties and Ca2+ in hydroxyapatite. The high adhesive strength is due to the carboxyl groups, which can form a large number of hydrogen bonds with different substrates. Hydroxyapatite also enhances the adhesive properties by adsorption of poly(N- acryloyl-2- glycine) chains, which exposes more carboxyl groups that can interact with tissue. The hydro-gel is autolytic in the body, as the hydrogen bonds and physical crosslinks are weakened over time by deproto-nation of the carboxyl groups in a neutral environment. When this wound dressing was planted subcutaneously in rats, its volume decreased gradually and totally dis-appeared within 14 h. Thus, one concern is that the autolysis of the dressing is too fast to achieve efficient haemostasis. Therefore, biodegradable adhesive hydro-gels could be a solution to the bleeding and pain caused by removing wound dressings, although controlling the degradability of adhesive hydrogel haemostats remains challenging.

Nanofibres. Nanofibrous matrices control haemorrhages mainly through their ability to entrap blood cells, plate-lets and other coagulation factors and the similarity of their structures to native fibrin fibres147,165. Various meth-ods are used to form nanofibrous matrices39–43,71,109,165–167, but the most conventional method is electrospinning. A portable electrospinning device designed for outdoor

use (Fig. 4a) was used to synthesize CuS composite nano-fibres that can be applied in situ to wounds to rapidly stop bleeding and to aid long- term wound healing165. The CuS nanoparticles were first generated from egg white and Cu(NO3)2·3H2O before being dispersed in a solution of polyvinylpyrrolidone and gelatin. The nanofibres formed in situ by electrospinning this solu-tion showed stronger adhesion than preformed nano-fibres and rapid haemostatic capacity. Moreover, the CuS nano particles possess photothermal antibacterial properties when triggered with near- infrared light.

Templates can also be used to assemble nanofi-brous matrices. For example, a sacrificial template was used to assemble a honeycomb- like nanofibrous mat based on chitosan34. The template was fabricated from β- cyclodextrin polyester hydrogels, which serve as a macroporous carrier and can degrade under physiolog-ical conditions. Chitosan was loaded onto the hydrogel template by immersing it in a 1- wt% chitosan solution for 7 days, and the resulting chitosan nanofibres assem-bled by imprinting the templates had honeycomb- like structures (Fig. 4b). The nanofibrous mat absorbs blood, with the honeycomb- like structure increasing the sur-face area of the mat and, thus, increasing its haemo-static efficiency. However, the formation of this chitosan mat takes a long time, limiting the application of this fabrication method.

Most nanofibrous mats have been designed to absorb blood and facilitate clot formation, for which the hydro-philicity of the materials is expected to be beneficial. However, hydrophilic fibres with rapid absorption behaviour would fill the mat with blood, and clotting in the mat can increase the difficulty of removing wound dressings39. In these cases, blood- repelling materials have been proposed as a solution168. A superhydrophobic haemostatic wound dressing coated with carbon nano-fibres (CNFs) was efficient in facilitating the formation of fibrin fibres and was easy to remove39. Exposure of blood to CNFs is proposed to induce the extrinsic coag-ulation cascade, leading to the conversion of fibrinogen to fibrin monomers. The fibrin monomers then attach to the CNFs and self- polymerize into fibrin fibres (Fig. 4c, left). The superhydrophobic surface facilitates clot for-mation but only partially adheres to the clot owing to the presence of micro air pockets at the clot–dressing interface. During clot maturation, the clot contracts, detaching CNFs from their connected fibrin and mak-ing the wound dressing easy to peel off without causing clot damage and secondary bleeding (Fig. 4c, right). However, the detailed mechanism underlying the ability of CNFs to trigger fibrin fibre formation is still unknown and requires further investigation.

Particles. Haemostatic particles and powders can be applied to a wide range of wounds, including deep and irregular- shaped wounds46,47,169,170. Mineral- based pow-ders, such as kaolin and zeolite, are the most widely used and absorb blood owing to their microporous structures50,128,171–173 A nanocomposite based on hal-loysite nanotubes (HNTs; halloysite is an aluminosil-icate clay mineral) and chitosan oligosaccharide was developed for use as a powder to accelerate haemostasis

Fibrinogena circulating glycoprotein (with a molecular mass of 340 kDa) secreted by hepatocytes and composed of two symmetrical half- molecules, each of which comprises three different polypeptide chains (aα, Bβ and γ).

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and promote wound healing173. The nanocomposite is formed by dispersing HNTs in a chitosan oligosaccharide solution and freeze- drying. Chitosan oligosaccharides have a positive zeta potential owing to their cationic amino groups, whereas the isomorphous substitution of Al3+ for Si4+ leads to negative charge on the HNTs. Electrostatic interaction leads to the deposition of

chitosan oligosaccharides on the surface of the HNTs, while the hydrogen bonding between the amine and hydroxy groups in chitosan oligosaccharides and the Si–O groups in the HNTs also contributes to nano-composite formation. Because chitosan oligosaccharide can promote coagulation, this composite was assumed to be capable of haemostasis, but additional evidence

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is needed to support this theory. This nanocomposite also promoted re- epithelization of the wound and tis-sue remodelling. Although mineral- based powders show notable promise in facilitating haemostasis, drawbacks include the difficulty of removing them completely from the injured site and the risk of causing thrombosis once the powder enters the circulation system.

These issues have motivated the search for new strat-egies to accelerate clotting by mimicking the native fac-tors involved in the haemostatic process. For example, synthetic platelet mimics were prepared by modifying deformable poly(N- isopropylacrylamide- co- acrylic acid) microgel particles with fibrin- recognition motifs174 (Fig. 5a). Biopanning assays against fibrin were used to select the recognition motifs (single- domain vari-able fragments) with the highest affinity for fibrin. The recognition motifs were subsequently coupled to the poly(N- isopropylacrylamide- co- acrylic acid) ultralow- crosslinked hydrogel by reaction with residual carboxyl groups from acrylic acid. Similarly to native platelets, the modified microgel particles showed spe-cific fibrin- recognition behaviour and high deforma-bility (Fig. 5b), enabling them to both augment clotting and induce clot retraction, thus recapitulating functions of native platelets. These platelet- like particles can also dynamically stiffen the fibrin matrices and facilitate fibroblast migration to the wound site45. Furthermore, stimulation with ultrasound increases the clot density and thickness and improves angiogenesis, which pro-mote the wound healing process17. By introducing gold44 or silver121 nanospheres, platelet- like particles can be endowed with antibacterial properties, which not only promote haemostasis but also prevent infection dur-ing wound healing. Researchers have long attempted to mimic the native haemostatic process to accelerate haemorrhage control, and platelet- like particles repre-sent a milestone in this research direction. Further mod-ifications are needed to increase the long- term stability of these platelet- like particles in blood without adversely affecting immune responses.

Other interesting particle designs for haemostatic applications and wound healing include a metal–organic framework (MOF) with embedded silver nanopar-ticles (Ag@MOF)148 (Fig. 5c). γ- Cyclodextrin MOFs (CD- MOFs), which possess spherical voids with

diameters of 1.7 nm, were fabricated by reacting γ- cyclodextrin with potassium salts. Silver nanoparti-cles were fabricated and embedded in a one- pot reaction by immersing CD- MOF particles in a AgNO3 solution. Cyclodextrin moieties in the resulting Ag@CD- MOFs were then crosslinked using diphenyl carbonate to increase the hydrophilicity of the particles, making it eas-ier to suspend them in the aqueous phase. Subsequently, the crosslinked Ag@CD- MOFs were functionalized with a fibrinogen- mimetic peptide (Gly- Arg- Gly- Asp- Ser (GRGDS)) by reacting carboxyl groups in the peptide with the surface hydroxy groups of the crosslinked Ag@CD- MOF. GRGDS enhances the haemostatic capacity of the crosslinked Ag@CD- MOFs by facilitating platelet adhesion and aggregation at the wound site. Moreover, these particles also showed promising antibacterial properties and enhanced wound healing. This demon-stration of the integration of porous MOFs with peptides and other nanoparticles to generate multifunctional and uniform particles for haemostatic applications provides insight that might lead to new research directions. For example, MOFs with nanoparticles could be designed to encapsulate growth factors or drugs to develop haemostatic materials with therapeutic properties.

Other forms. In addition to the forms discussed in the previous subsections, researchers have also developed several other forms of haemostatic materials. Here we focus on hybrid materials with haemostatic properties that can also promote wound healing. The aim of fabri-cating hybrid materials is to maintain the advantages of each constituent but to avoid their disadvantages.

One example is a hybrid haemostat based on zeolite and cotton50. Zeolite is a good coagulation agent; how-ever, zeolite powders are easily washed away from wound sites and cause thrombosis after entering the circulation system. Some commercial haemostats simply impreg-nate gauze with zeolite to form a hybrid haemostat; however, as the zeolite is not immobilized, it can easily be lost. To overcome this issue, an on- site template- free growth method was developed to bind zeolite to the surface of cotton fibres. Colloidal silica and aluminate mixtures were combined with cotton for direct hydro-thermal treatment, directly generating chabazite- type gel particles on the cotton fibres. Subsequent migration and aggregation of these chabazite- type gel particles leads to the formation of mesoporous chabazite- type zeolitic crystals (Fig. 6a). This zeolite–cotton hybrid haemostat was stable and did not leak zeolite powder when soaked in water. In addition, the clotting time of this hybrid haemostat is relatively short, and when applied in vivo in a rabbit lethal femoral artery injury model, the blood loss was small.

In another example of a hybrid haemostatic mate-rial, a composite hydrogel was made from bioglass and oxidized sodium alginate with adipic acid dihydrazide- modified γ- polyglutamic acid as the crosslinker21 (Fig. 6b). Owing to rapid ion exchange on its surface, the bioglass creates an alkaline environment, which increases the hydrogel adhesiveness to tissues by accel-erating imine bond formation. Moreover, the release of Ca2+ from the bioglass promotes the gelation of alginate.

Fig. 4 | haemostatic nanofibres in wound healing. a | In situ nanofibre formation using a handheld electrospinning device for outdoor use165. CuS nanoparticles are dispersed in a solution of polyvinylpyrrolidone (PVP) and gelatin, which is electrospun by the handheld device. The CuS nanoparticles show photothermal effects when triggered by near- infrared (NIR) irradiation, endowing the nanofibres with antibacterial properties. b | Formation of a honeycomb- structured chitosan nanofibre mat using β- cyclodextrin polyester (CDPE) as a template34. Chitosan is loaded onto the CDPE hydrogel by soaking in a chitosan solution for 7 days. Chitosan assembles into a nanofibrous structure, and sacrificing the CDPE template leaves a chitosan nanofibrous matrix with a honeycomb- like structure. c | A superhydrophobic nanofibrous gauze with carbon nanofibres (CNFs) immobilized on the surface facilitates clot formation and can be readily peeled off39. Fibrin fibres develop on the CNFs upon contact with blood. The CNFs create micro air pockets between the blood and the superhydrophobic surface, so the clot is only partially attached to the CNF surface (left). During clot maturation, contraction of the clot causes the CNFs to detach from the fibrin fibres, enabling easy removal of the clot from the CNF surface. Part a adapted with permission from reF.165, Elsevier. Part b adapted from reF.34, CC BY 4.0. Part c adapted from reF.39, CC BY 4.0.

Metal–organic framework(MoF). Class of porous materials composed of inorganic nodes (metal ions or clusters) connected by organic ligands to form 1D, 2D or 3D structures.

Bioglassa silicon- based glass–ceramic bioactive material containing calcium and phosphorus.

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This hybrid hydrogel acts as an exceptional haemostatic adhesive, facilitates vascularization by releasing Si ions and promotes tissue regeneration. However, further tests are needed to fully explore the haemostatic effects of this hybrid hydrogel. In addition, the strong tissue adhe-sion would make it hard to remove from a wound site.

Another question that needs to be addressed is whether this hydrogel can be degraded after its haemostasis function is accomplished.

A few other forms of haemostatic materials, such as membranes175 and liquid formulations176, have been reported. However, these materials did not show

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Fig. 5 | haemostatic particles in wound healing. a | Structure of platelet- like microparticles174. The inner core is formed from a poly(N- isopropylacrylamide- co- acrylic acid) (pNIPAm- AAc) microgel. Fibrin- recognition motifs are conjugated to the surface of the microgel to form the platelet- like microparticle. b | When inactive, native human platelets are round, but after activation with α- thrombin, they spread out. The morphology of the platelet- like microparticles also changes after incubation with α- thrombin: they spread out and, thus, show behaviour similar to that of native platelets174. c | Synthesis of γ- cyclodextrin metal–organic framework (CD- MOF) with embedded Ag nanoparticles (Ag@CD- MOF). CD- MOF with uniform nanopores was fabricated in advance148. CD- MOF was then impregnated with Ag+ ions by immersion in a solution of AgNO3; Ag nanoparticles (NPs) form inside CD- MOF upon reduction of Ag+. Neighbouring hydroxy groups within Ag@CD- MOF were crosslinked (CL) by forming carbonyl bonds using diphenyl carbonate (DPC) as a crosslinker and triethylamine (TEA) as a catalyst. The surface of CL- Ag@CD- MOF was subsequently functionalized with a fibrin- mimetic peptide (GRGDS). Parts a and b adapted from reF.174, Springer Nature Limited. Part c adapted with permission from reF.148, Wiley- VCH.

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impressive haemostatic ability, and problems with their application or stability cannot be ignored. Thus, these materials are not discussed further in this Review.

Comparison of different forms. Although there are many forms of haemostatic materials, we are not yet able to conclude which is the best one, as they each have their own advantages and limitations, and suitability for different applications. Sponges rapidly and effectively absorb blood once applied to the haemostatic area, and can concentrate blood and promote clot formation by aiding the aggregation of blood cells in the primary stage of haemostasis. But the good absorption also makes removal difficult. Hydrogels for haemorrhage control serve mostly as adhesives, which also function during the primary stage of haemostasis. Good adhesion helps to seal the wound and stop bleeding. The advantage of hydrogel adhesives is that they can keep the wound area moist and promote healing. But limitations include the toxicity of residual functional groups and difficulties during removal. Nanofibres mimic the native extracel-lular matrix structure and help to catch platelets and clotting factors to generate fibrin fibres and clots, con-tributing mostly to the secondary stage of haemostasis.

But nanofibres are not suitable for excessive bleeding owing to their poor absorption. Particles are suitable for deep and irregular- shaped wounds. However, their removal is a problem, and they may cause thrombosis upon entering the circulation system. Because of these advantages and disadvantages of each form of haemo-static material, hybrid materials have been developed to strengthen the pros of different forms and avoid their limitations. Although the development of hybrid hae-mostatic materials is still in the preliminary stage, more effective and multifunctional haemostatic materials can be generated in this way.

Conclusions and outlookHaemostatic materials are essential in wound treat-ment, especially in cases of severe bleeding when the inherent haemostatic system fails to stop the bleeding. Components that can facilitate haemostasis and wound healing have been well studied over the past 20 years, leading to the identification of new active components and providing insight into the mechanisms by which they stop bleeding. Among these ingredients, poly-saccharides are the most widely used, with chitosan attracting the most attention in haemorrhage control.

2 µm

a

b

Cotton

Implantable material

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CHA gel particle

Na+ OH–

Ca2+

Si

H3O+

CHA aggregate

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O

O–

NH

Si

Si Si

O

NHNH

OH

–

2

OH

2+

–

–

–

Ca

2+Ca

2+Ca

COOCOO

COO

–OH

Fig. 6 | hybrid haemostatic materials. a | Formation of zeolite- incorporated cotton by on- site template- free growth50. Chabazite- type (CHA) gel particles can be induced to form on the surface of cotton fibres. The CHA gel particles migrate on the cotton fibres and undergo aggregation into mesoporous CHA zeolitic crystals. The scanning electron microscopy image shows that the zeolite particles are well distributed on the surface of a cotton fibre. b | A hybrid adhesive hydrogel with incorporated bioglass21. The oxidized sodium alginate hydrogel is crosslinked with adipic acid dihydrazide- modified γ- polyglutamic acid. The bioglass was incorporated in the hydrogel during gel formation and has a role in tissue adhesion, material adhesion and vascularization. Rapid ion exchange on the bioglass surface creates an alkaline environment that favours imine bond formation and, thus, aids tissue adhesion. Release of Ca2+ and Si4+ ions from the bioglass promotes the gelation of alginate and accelerates vascularization, respectively. Part a adapted from reF.50, CC BY 4.0. Part b adapted from reF.21, CC BY 4.0.

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The forms of materials also affect haemorrhage control, and various forms of haemostatic materials have been developed. However, each form of haemostatic material has its own limitations, which require additional investi-gations to identify innovative solutions. In the following, we highlight some problems and future trends in haemo-static materials for wound healing applications, focusing on their chemistry, functions and applications.

Chemistry and processing. Most of the current syn-thesis routes to haemostatic materials are complicated and time- consuming. Faster and more facile chemistry would help facilitate high- volume production of mate-rials at reasonable cost. New syntheses should also be developed with consideration of the principles of green chemistry and the cost of the ingredients.

A potentially fruitful research direction would be to design haemostatic materials that participate in or pro-mote physiological haemostatic processes that have not yet been widely targeted in existing studies. For exam-ple, haemostatic materials could be designed to promote the contraction of blood clots to strengthen thrombi and thereby stopping bleeding more effectively (such as the platelet- like particles discussed earlier herein). Designing materials to mimic native haemostatic pro-cesses and components is also a future trend174. For example, the design of smart bioactive materials, such as platelet- like particles and fibrin- structured nano fibres, could provide new opportunities in haemostatic material development.

Although numerous types of haemostatic mate-rial have been developed, only a few have reached the clinical trial stage of development. Several mate-rials based on components derived from humans or animals have been applied in clinical trials, such as a lyophilized platelet- derived haemostatic product177, a fibrin sealant178 and platelet- rich fibrin179. Other mate-rials, such as chitosan- based materials180, polyurethane foam181 and a gelatin matrix182,183, have also been used in clinical studies. In general, materials comprising sim-ple components and chemicals approved by regulatory authorities are more likely to enter clinical trials.

Functions. Multifunctional materials that integrate haemostatic ability with one or more other character-istics or functions are particularly desirable for clinical applications.

For instance, materials that combine haemostatic capacity with diagnostic and/or monitoring abili-ties would have advantages over regular haemostatic materials. The pH and electrical properties of a wound site change during haemostasis and wound healing.

Materials that can monitor these physiochemical changes at the wound sites could help to determine a wound’s condition without the dressing having to be peeled off. Meanwhile, monitoring for infection would be helpful for infection control.

Further developments are also expected in materi-als that possess both therapeutic properties and hae-mostatic capacity. Haemostatic materials that promote wound healing have attracted increasing attention, and some inspired studies have been discussed herein. However, the wound healing process is complicated and most therapeutic materials are focused on skin repair184,185. Therapeutic materials that can promote the repair of internal organs are scarce but constitute an important area that requires increased research attention1,186. Some cases of bleeding are difficult to stop owing to chronic disease or chemotherapy. In such cases, haemostatic materials that provide therapeutic efficacy and haemorrhage control would be particularly power-ful. For more information on haemostatic materials for chronic wound healing, see reFS187,188.

Regardless of the form, all haemostatic materials encounter the same problem: what happens after they have completed their task? As wound healing is the final goal, the fate of haemostatic materials is to ‘disappear’, either through removal or degradation. Designing mate-rials that are easy to remove without peeling off the clot and causing secondary bleeding and pain is a major chal-lenge. In designing degradable materials, the biocom-patibility of the degradation products and the risk of an in vivo thrombo- inflammatory response should be taken into consideration. How to control the degradation rate of the haemostat is another issue, as the degradation rate should match the tissue repair process.

Applications. There are various practical properties that need to be considered in developing haemostatic materials for different applications, including their long- term stabil-ity and portability. Designing materials that can be stored for a long time even under extreme conditions, such as high or low temperatures, and that are portable is particu-larly important for military applications. Furthermore, many haemostatic materials are applied externally; thus, their aesthetics should also be considered.

In summary, future studies on haemostatic materials should focus on more than effective bleeding control. We believe that the development of haemostatic mate-rials with wound healing properties — by design of their chemistry and fabrication processes — will greatly advance their clinical application soon.

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AcknowledgementsThis work was jointly supported by the National Natural Science Foundation of China (grant no. 51973172), the Natural Science Foundation of Shaanxi Province (grant nos 2020JC-03 and 2019TD-020), the State Key Laboratory for Mechanical Behaviour of Materials, the Fundamental Research Funds for the Central Universities, the World- Class Universities (Disciplines) and the Characteristic Development Guidance Funds for the Central Universities and the Opening Project of the Key Laboratory of Shaanxi Province for Craniofacial Precision Medicine Research, College of Stomatology, Xi’an Jiaotong University (grant no. 2019LHM- KFKT008).

Author contributionsAll authors contributed to researching data for the article and writing, reviewing and editing the article before submission.

Competing interestsThe authors declare no competing interests.

Peer review informationNature Reviews Chemistry thanks W. Wang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s noteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. © Springer Nature Limited 2021

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