Preparation Strategy of Hydrogel Loaded with Natural Products and Its Research Progress in Skin Repair

After the skin is physically damaged by external factors, blood vessels rupture and bleed to flush out antigens and bacteria while triggering the first step in the skin repair process, hemostasis, to prevent excessive blood loss in the human body [1]. The process of hemostasis is physiologically manifested as the activation of platelet aggregation caused by the exposure of endothelial cells, collagen, and tissue factors, thereby releasing chemokines and growth factors to achieve hemostasis. Therefore, the hemostasis process can be roughly divided into two stages, primary coagulation (primary hemostasis) [14] and secondary coagulation (secondary hemostasis) [15], as shown in Figure 3.

Primary hemostasis is the first stage of the hemostasis process. Skin damage causes the smooth muscle of damaged blood vessels to contract and release signaling substances, reducing local blood loss and binding to platelet receptors during vascular contraction, promoting platelets to reach the site of injury, activating and releasing chemicals such as serotonin, thromboxane, and adenosine diphosphate, further enhancing vascular contraction, and ultimately forming platelet thrombi.

Secondary hemostasis is the process of forming fibrin coagulation on the site after primary hemostasis occurs. In this process, the activation of coagulation factors triggers the coagulation cascade and the production of thrombin, resulting in the conversion of plasma fibrin from soluble to insoluble and the formation of a biopolymer mesh of fibrin; eventually, together with the platelet thrombus, a thrombus is formed to immobilize the blood components. In this process, the coagulation cascade reaction, as the process of thrombospondin complex formation, can be divided into endogenous, exogenous, and common pathways [16].

Factor XII is activated into factor XIIa through contact with an external surface, which then activates factor XI to form factor XIa. Subsequently, with the participation of Ca²⁺ ions, factor XIa helps factor IX form factor IXa; finally, factor IXa combines with factor VIIIa to form the prothrombin complex. The exogenous pathway is caused by exposure to transmembrane glycoprotein tissue factor (TF) outside the bloodstream, mainly involving coagulation factors such as F, Ca²⁺, and factor VII. In the presence of Ca²⁺ ions, TF can bind to active coagulation factor VII to form the subunit VII tissue factor complex. The combination of endogenous and exogenous pathways is ultimately referred to as the common pathway, which involves the formation of prothrombin complex by Ca²⁺ ions Xa and Va factors and further activation into thrombin, promoting the conversion of fibrin and aggregation and completing blood coagulation through the synergistic action of fibrin, XIIIa factors, and blood cells [17]. This process aims to provide temporary treatment for skin wounds to prevent excessive pathogenic bacteria from entering the skin and disrupting the subsequent repair process.

One of the main stages for skin wound healing is the restoration of barrier function to prevent further injury or infection. Firstly, skin cells emit inflammatory signals, such as damage-related molecular patterns or pathogen-specific molecular patterns, which are recognized by Toll-like receptors at their site, triggering and maintaining inflammation [18]. Leukocytes, especially neutrophils, migrate with increasing gradients of chemokines until they reach the site of injury; during inflammation, neutrophils reach the site of injury and release chemokines such as interleukin 1-β (IL-1β), tumor necrosis factor-α (TNF-α), and interleukin 6 (IL-6) [19].

These chemokines amplify the immune response and attract monocytes for the ongoing healing process, which involves phagocytosis by neutrophils and monocytes. In addition, monocytes differentiate into macrophages that release transforming growth factors α and β (TGF-α and β) and insulin-like growth factor 1 (IGF-1), thereby facilitating the gradual transition from the inflammatory to the proliferative phase [20]. Activated regulatory T cells are part of the adaptive immune system. In addition to leukocytes, regulatory T cells are able to modulate tissue inflammation by attenuating interferon-gamma production and pro-inflammatory macrophage accumulation. According to relevant research, this action is mediated by the epidermal growth factor receptor pathway, which is used to promote skin wound repair [21].

The nature of the trauma may determine the length of the inflammatory phase. Acute skin wounds with large blood loss require longer hemostasis time, while chronic skin wounds that can be kept closed with simple pressure will coagulate faster [22]. Similarly, sharp injuries (e.g., incisions and laser wounds) will heal faster than wounds that produce necrotic tissue, whereas in chronic skin wounds, tissue regeneration stalls during the inflammatory phase, which can lead to pathological inflammation and failure to start wound healing at a late stage. After the inflammatory stage, the foreign cells and molecules in the skin repair part are fully removed, which clears the obstacles for the subsequent skin repair process.

This process mainly occurs during days 4 to 14 of skin wound healing, which mainly involves epithelialization, neovascularization, and granulation tissue formation [13]. Epithelialization occurs during the early stages of skin repair, during which the original epithelial cells migrate upwards in a normal pattern if the basement membrane remains intact [23]. Epidermal progenitor cells are located underneath the repaired portion of the skin, and if the epidermal cells remain intact, the normal epidermal layer will return to its normal state within two to three days; if the basement membrane has been damaged, then the epithelial cells located at the edge of the skin begin to proliferate and send out protrusions to rebuild the protective barrier [24]. The angiogenesis stimulated by TNF-α is marked by the migration of endothelial cells and the formation of capillaries, and the migration of capillaries to the wound is crucial for the normal healing of the wound, as the granulation tissue formation period and tissue deposition require nutrients provided by capillaries. If the nutrients are insufficient, it can lead to long-term non-healing of the wound; subsequently, the epithelial cells located at the edge of the skin begin to proliferate and send out protrusions to re-establish a protective barrier against loss of body fluids and further bacterial invasion [25]. Epithelialization begins shortly after trauma and is first stimulated by inflammatory cytokines, with IL-1 and TNF-α upregulating keratinocyte growth factor (KGF) gene expression in fibroblasts; in turn, fibroblasts synthesize and secrete KGF-1, KGF-2, and IL-6, which stimulate neighboring keratin-forming cells to migrate, proliferate in the wound area, and differentiate in the epidermis [26].

The final part of the proliferative phase is the formation of granulation tissue, where fibroblasts migrate from surrounding tissues to the wound site, activate, and begin to synthesize collagen and proliferate. Platelet-derived growth factor (PDGF) is the main signal of fibroblasts, which comes from platelets and macrophages; the expression of PDGF in fibroblasts is amplified by autocrine and paracrine signal transduction [27]. Fibroblasts already located at the wound site will begin to synthesize collagen and transform into myofibroblasts for wound contraction, and compared to fibroblasts from the wound site, their proliferation is lower. In this process, new granulation tissue is formed, and the structural integrity of skin is rebuilt through epithelization and angiogenesis [28]. However, the regeneration of appendages such as hair follicles, sweat glands, and neurons is a challenge in adult wound healing, and further study on this stage may be very important to promote the study of skin repair.

As angiogenesis is still occurring at the time of granulation tissue formation, this highly vascularized tissue presents clinically with a characteristic reddish coloration. Subsequently, it enters the final stage of skin repair, the remodeling stage, which mainly involves the remodeling of granulation tissue and the recombination of immature scars; this stage is also the most important stage in medical cosmetic treatment [29]. The main feature of the remodeling stage is the deposition of collagen into an organized and structured network. If there is matrix deposition at the site of skin repair, the difficulty of skin repair will be reduced, and excessive collagen synthesis will lead to hypertrophic scars or keloids [30].

This phase usually takes place two to three weeks after the start of skin repair, but the length of skin repair needs to be analyzed, with most repair processes lasting several months, and a few may last up to a year. The synthesis rate of collagen during skin repair comes not only from the increase in the number of fibroblasts but also from the net increase in collagen production in each cell. Fibroblasts begin to differentiate into muscle fibroblast contraction cells, characterized by high expression of alpha smooth muscle actin (α-SMA). MMP and other collagen types secreted by fibroblasts, macrophages, and other remaining cell types reshape the matrix, marking the formation of mature scars. The initially deposited collagen is thinner than uninjured skin, parallel to the skin. Over time, the initial collagen chains are reabsorbed, deposited thicker, and organized along the stress line [31]. These changes are also accompanied by an increase in the tensile strength of the wound, indicating a positive correlation between collagen fiber thickness, orientation, and tensile strength. As collagen recombination continues, cross-linking occurs between adjacent collagen bundles, leading to an increase in scar tensile strength. However, at the maximum value, the scar only recovers up to about 70% of its pre-injury mechanical strength [32]. Collagen found in granulation tissue is biochemically different from that of uninjured skin, with granulation tissue collagen having greater hydroxylation and glycosylation of lysine residues, and this increased glycosylation correlates with finer fiber size [33]. And researchers have shown that the collagen in scars, even after a year of maturation, is never as strong as the collagen in uninjured skin and that the wound strength never returns to 100 %, with wounds having only 3% of their final strength at 1 week; 30% at 3 weeks; and about 80% at 3 months [34].

Numerous researchers have shown that even after the remodeling phase, skin repair cannot fully restore the skin to its previously undamaged state. Faced with this problem, there is still a lot of room for skin repair. As the first physical therapy method that directly contacts the wound after disinfection, wound dressings should receive more attention and research from the medical, materials, and chemical communities. Research on wound excipients is also extremely important [35].

When the skin is subjected to physical or chemical impact, it may cause skin damage, and then the capillaries will rupture and blood will flow out. At this moment, it is very important to stop bleeding quickly. Hydrogels with remarkable hemostatic properties can significantly shorten and reduce the cell damage around the wound [45,46,47]. Generally, hemostasis involves three processes: vasoconstriction or blockage [48], formation of platelet blockage [49], and coagulation.

When bleeding occurs, blood vessels rupture and the body releases thromboxane and adrenaline, stimulating local and systemic vasoconstriction to reduce blood flow and loss [50]. Chitosan-based hemostatic hydrogels are often used for hemostasis due to their unique biocompatibility, tunable mechanical properties, injectability, ease of handling, and the ability of chitosan to aggregate blood cells and form embolisms to achieve hemostasis. Some researchers use the sol–gel method to synthesize nBG particles, which combine nano-bioglass (nBG) with silica, calcium, and phosphate ions on chitosan (Ch) hydrogel to achieve rapid coagulation. In the in vitro whole blood coagulation test and in vivo hemostasis evaluation, 2% Ch-5% nBG hydrogel stabilized a blood clot in 54 s in the case of liver bleeding and achieved hemostasis in 185 s in the case of femoral artery bleeding, which is more effective than 387 s with normal body mechanisms. Experiments have confirmed that the rapid and effective coagulation characteristics of 2% Ch-5% nBG hydrogel may be due to the synergistic effect of Ch and nBG in contact with blood [51].

In addition, the hydrogel dressing has a porous structure and rough surface, which can increase the contact area with blood, which is conducive to the rapid absorption of blood and accelerate hemostasis [52]. Shi et al. prepared a hemostatic hydrogel containing L. barbarum polysaccharide (LBP)-functionalized ultrathin MMT nanosheets (L-MMT NSs) for efficient hemostasis and wound healing, and the hydrogel showed a remarkably dispersed and porous 3D structure. They used block MMT in the hydrogel and successfully functionalized it with LBP-derived polysaccharides; in the hemostatic experiment of liver in vivo, compared with the control group, when P-L-MMT hydrogel was used, the blood loss was significantly reduced to 76 mg, which made the hydrogel show excellent hemostatic properties in the mouse model. The hydrogel prepared via this method significantly reduced the tissue damage caused by inflammation and significantly accelerated wound healing [53].

If the skin is subjected to the proliferation and invasion of bacteria and microorganisms in the process of skin healing, it may lead to chronic skin wounds, delay wound healing, and even cause systemic infection in severe cases [54]. Therefore, hydrogel wound dressings should have excellent antimicrobial properties.

Pueraria lobata is a kind of Chinese herbal medicine with food and medicine homology. Puerarin extracted from Pueraria lobata has been found to form a kind of self-assembled hydrogel of Chinese herbal medicine under the mechanical action with chitosan through recent research; in SEM, it can be seen that the number of bacteria is obviously reduced. The antibacterial rate of hydrogel to Escherichia coli is 99%, Escherichia coli and Salmonella are 96% and 96%, respectively, and it can destroy the cell membrane of bacteria and inhibit the proliferation of bacteria, showing excellent antibacterial characteristics [55]. The mechanism of hydrogel to accelerate the healing of infected wounds by inhibiting bacterial growth and inducing macrophages to polarize into the M2 phenotype is shown in Figure 5.

Some hydrogels, such as chitosan and carboxymethyl chitosan, have certain antibacterial ability, but in the face of problems such as the aggravation of bacterial infection in the wound, the ideal antibacterial effect cannot be achieved only by the hydrogel material itself, so natural substances need to be added to the hydrogel to achieve the antibacterial purpose [56]. Gaona et al. prepared a chitosan hydrogel loaded with different concentrations of carvacrol, which was used as a medical material with good biocompatibility and antibacterial properties. In the bacteriostasis experiment, the bacteriostasis rate of the hydrogel against Staphylococcus aureus was ≥95%, and the bacteriostasis rate against Escherichia coli was ≤90%. In the drug release experiment, carvacrol can release drugs in chitosan hydrogel for 48 h continuously. In addition, the in vitro hemolysis experiment showed that the hemolysis rate of all hydrogels was less than 2%, and a hemolysis rate between 0 and 2% was considered non-hemolytic and safe for medical applications. This further confirmed that the hydrogel has certain reference value for the development of wound dressings [57].

The inflammation stage is the second stage in the process of skin repair, and the regulation of inflammation on skin repair is extremely complicated. There is a risk of interruption of healing in both the early and late stages of inflammation, which is particularly obvious in chronic inflammatory wounds. In recent years, the ability of hydrogel wound dressings to mimic the function of the skin has received a great deal of attention in the field of scientific research. In view of the inflammatory stage of skin repair, we need to pay close attention to three aspects, namely the elimination of ROS (reactive oxygen species) [58], the isolation of chemokines [59], and the polarization of M1 to M2 of macrophages [60].

With regard to the removal of reactive oxygen species, Chen et al. studied a hydrogel for diabetes wounds to accelerate skin wound healing by removing ROS and other functions. In vivo results showed that H-SGPGA hydrogel significantly accelerated the process of diabetes wound repair (8.31 ± 5.54% of the wound surface on the 12th day) and promoted epidermal regeneration (79.13 ± 5.99%), collagen deposition (71.4 ± 9.1%), and angiogenesis (294.1 ± 29.6%). In vitro antioxidant testing showed that H-SG1GA (90.5 ± 0.4%) could play a synergistic antioxidant role between SGPGA and HXTL, and the ABTS clearance ability could reach 90.5 ± 0.45% within 2 h. In addition, H-SGPGA hydrogel also has an excellent DPPH clearance of 88.2 0.9%, showing excellent antioxidant performance [61].

Schirmer et al. designed a wound dressing scheme that can remove chemokines by using a validated biological hybrid poly (ethylene glycol)-glycosaminoglycan (starPEG-GAG) hydrogel. Based on the bionic reasonable design concept, this kind of hydrogel was integrated into the wound contact layer of composite fabric, and the efficient separation of CC and CXC family pro-inflammatory chemokines was achieved by regulating its local and overall charge density. In a mouse model of delayed healing, the dressing effectively alleviated the inflammatory response and promoted wound closure [62].

In the face of the above problems, the easiest way is to use hydrogel as a carrier and load natural active substances to enhance its anti-inflammatory activity. Zhou et al. developed a hyaluronic acid (HA) self-healing hydrogel (HA/SAB) containing salvinorin B (SAB) and investigated the anti-inflammatory mechanism of this hydrogel using q-PCR. They found that treatment with IL-4 significantly increased the expression of the M2 marker CD206, suggesting that it was successful in stimulating the M2 phenotype; in addition, SAB further increased the expression of CD206, suggesting a role for SAB in promoting M2 polarization and demonstrating the inhibitory effect of this hydrogel on the validation of M2 polarization by stimulating M1 in macrophages [63].

Angiogenesis is the growth of new blood vessels at the site of previously damaged blood vessels. As it has been proven that changes in their normal function can induce various diseases, the generation of new blood vessels has always been a focus of research for many scholars [64]. When the skin is damaged, inflammation stimulates blood vessels, and stimulated vascular endothelial cells will release matrix metalloproteinases (MMPs), thus decomposing the basement membrane. In this process, the activated endothelial cells will act as front-end cells, which will separate from the original position and move into the surrounding matrix, followed by proliferation of the endothelial cells to form new blood vessels [65].

Hydrogel has become a promising skin healing dressing because it can create a suitable environment for skin repair, deliver drugs, and imitate the extracellular matrix. Yang et al. developed a diabetes wound-healing hydrogel based on glycyl methacrylate gelatin (GelMA), Panax notoginseng saponin (PNS), sodium alginate microspheres, and growth factor (IGF-1) and took the expression level of CD31 as the standard. CD31 staining showed that the neovascularization of the diabetes mouse model using hydrogel was significantly greater than that of the control group. The synergistic combination of PNS and IGF-1 established a favorable microenvironment for angiogenesis and promoted efficient and complete tissue repair [66]. The mechanism of hydrogel promoting angiogenesis and accelerating wound healing in diabetes is shown in Figure 6. In addition, the hyaluronic acid (HA) self-healing hydrogel (HA/SAB) of salvianolic acid B (SAB) explored by Zhou et al. found that the HA/SAB2.5 group displayed the highest expression of CD31 and α-SMA through immunofluorescence staining, showing excellent angiogenesis-promoting ability [63].

Xu et al., in their study on tissue engineering, found that magnetic hydrogels have special angiogenic ability and some magnetic guidance for tissue repair, and they developed a curcumin magnetic nanoparticle (CMNP) using a one-pot co-precipitation method and dispersed it in hyaluronic acid (HyA) to create an angiogenic magnetic hydrogel. In the research, it was found that the porous network of HyA composite hydrogels could be used as a matrix for sustained release of curcumin, thereby improving the angiogenic properties of the composite hydrogels [67].

In the face of skin regeneration in the remodeling phase after the proliferation phase, how to restore the skin to its pre-breakage state has always been an important question in the medical community. Traditional wound dressings cannot absorb enough cell exudate and protect this part from microbial infection, which makes the skin regeneration occur as before [68]. Therefore, the development of hydrogel that can promote skin regeneration and restore the wound to the pre-injury quality is the main problem at present.

It is very important to study injectable hydrogel with high skin regeneration and healing characteristics for clinical application, Zhang et al. started with the high antioxidant properties of blueberry anthocyanin (BA) combined with carboxymethyl chitosan (CMCS) and oxidized hyaluronic acid (OHA) to explore the application of hydrogel in full skin regeneration and healing. In the wound healing experiment, 92.7% of the wounds in the experimental group were healed by the 12th day of the experiment, which significantly accelerated wound healing and promoted epithelial and tissue regeneration. In other studies, the composite hydrogel (Ag AV SF hydrogel) was prepared by introducing silver nanoparticles (AgNPs) and aloe vera (AV) as modifiers in the form of photocrosslinking, and a skin repair rat model was established for observation. It was found that the wounds of rats were resolved by wound contraction rather than re-epithelization, and the healing rate was significantly higher than that of other groups; meanwhile, the Ag-AV-SF hydrogel-treated group showed less scar tissue formation on days 7 and 21, which indicated that Ag-AV-SF hydrogel excelled in both promoting healing and inhibiting scar formation [69]. Hesperidin, as a bioflavonoid, has excellent efficacy in the treatment of wounds; however, its therapeutic use is limited due to poor water solubility and poor bioavailability. Praveen et al. attempted to develop hydrogel bandages with dendritic polymers to load hesperidin, and in vivo healing tests demonstrated that the 10% hesperidin-containing hydrogel exhibited the fastest wound closure (98.9% wound healing after 14 days) due to the properties of the hesperidin penetrating deep into the dermis with the help of the PAMAM dendritic macromolecule [70]. In addition, curcumin (Cur) can promote wound healing by affecting different stages of wound healing. Radha et al. prepared a physically cross-linked TOCN-PVA-Cur hydrogel with the addition of Cur via the freeze-thawing process and tested it in a rat model of skin repair, where H&E staining clearly showed that the epithelial cells of the experimental group were more completely repaired with this hydrogel than the control group [71].

For a long time, drug-resistant bacterial infections and biofilm formation have been challenges in biological protection, especially in the process of chronic skin repair. After the formation of skin wounds, monitoring the repair status and functional recovery during the repair process is essential. Therefore, it is necessary to have corresponding advanced materials to monitor the skin repair status at any time for disease assessment and subsequent treatment [72].

Based on the sensitivity of anthocyanins to pH changes, Zhao et al. synthesized light-curing hydrogels with EPLMA and blueberry anthocyanins (BAs) as precursors based on free radical polymerization reactions. The hydrogel is extremely sensitive to pH changes and can react quickly within 3 s, and the healing environment can be monitored by color change in response to pH changes, so using this characteristic to evaluate the change of wound pH environment plays an important role in the inflammatory stage and remodeling stage of skin repair and provides a potential treatment plan for related diseases [73]. In addition, inspired by anthocyanins, Zhang et al. performed extractions using mulberry fruits rich in anthocyanins and used their natural active substances to respond to pH and color changes. they cross-linked them with hyaluronic acid via the Schiff base reaction to prepare a “Tri Act” double-layer hydrogel dressing. The hydrogel not only has the characteristics of the natural active ingredients mentioned above but also has the ability to visually monitor infection, which simplifies the diagnosis process of skin repair. In addition, it shows therapeutic characteristics beyond conventional therapy in immunomodulation and promoting blood vessel regeneration and significantly enhances the therapeutic effect [74]. During the skin repair process, hydrogels are used as a way to maintain a moist repair environment for the skin, which promotes cellular activity and the re-epithelialization process, thus accelerating skin repair and reducing scar formation. In recent years, researchers have used turmeric extract (CLE) as a simple pH-sensitive indicator and loaded CLE into hydrogels made of hydroxyethyl cellulose grafted with green monomeric itaconic acid (IA) to prepare transparent, soft, and neutral pH-sensitive wound dressings [75]. Similarly, Ezati et al. also used curcumin in composite materials to achieve the purpose of pH response [76].

In addition, Hu et al. used the double-network hydrogel formed by covalent bonding and electrostatic interaction of chitosan and sodium alginate to load lignin Ag and quercetin–melanin (Q-Melanin) nanoparticles. Lig-Ag nanoparticles improve the conductivity and mechanization of hydrogels, which can be used for motion sensing and monitoring. Q-Melanin nanoparticles have anti-inflammatory and antioxidant effects, which, to some extent, enhance the anti-inflammatory properties of hydrogels and further improve the possibility of hydrogels acting on human monitoring, thus achieving accurate traction-assisted wound treatment [77].

Currently, many researchers focus on the degradation of hydrogels and drug release under various conditions (photodegradation, biodegradation, etc.), but there is a lack of research on the degradation of hydrogels in biological fluids, and it is impossible to determine the long-term release rate of drugs and the dynamic process of hydrogel degradation. Therefore, future research should carry out or simulate the removal rate of hydrogel in biological fluid so as to verify whether it matches the regeneration rate of wound tissue.

The data presented in this study are openly available in the article.