A review: Carrier-based hydrogels containing bioactive molecules and stem cells for ischemic stroke therapy

Abstract

Ischemic stroke (IS), a cerebrovascular disease, is the leading cause of physical disability and death worldwide. Tissue plasminogen activator (tPA) and thrombectomy are limited by a narrow therapeutic time window. Although strategies such as drug therapies and cellular therapies have been used in preclinical trials, some important issues in clinical translation have not been addressed: low stem cell survival and drug delivery limited by the blood-brain barrier (BBB). Among the therapeutic options currently sought, carrier-based hydrogels hold great promise for the repair and regeneration of neural tissue in the treatment of ischemic stroke. The advantage lies in the ability to deliver drugs and cells to designated parts of the brain in an injectable manner to enhance therapeutic efficacy. Here, this article provides an overview of the use of carrier-based hydrogels in ischemic stroke therapy and focuses on the use of hydrogel scaffolds containing bioactive molecules and stem cells. In addition to this, we provide a more in-depth summary of the composition, physicochemical properties and physiological functions of the materials themselves. Finally, we also outline the prospects and challenges for clinical translation of hydrogel therapy for IS.

Graphical abstract

Here, this article provides an overview of the use of carrier-based hydrogels in ischemic stroke therapy and focuses on the use of hydrogel scaffolds containing bioactive molecules and stem cells. In addition to this, we provide a more in-depth summary of the composition, physicochemical properties and physiological functions of the materials themselves. Finally, we also outline the prospects and challenges for clinical translation of hydrogel therapy for IS.

Highlights

• •Reviewing recent advances in carrier-based hydrogels for ischemic stroke treatment.
• •Hydrogels containing bioactive molecules and stem cells in ischemic stroke.
• •Impact of carrier-based hydrogel properties on IS treatment.
• •Challenges and prospects of hydrogel for IS applications.

1. Introduction

Stroke is one of the leading causes of global disability and death, affecting nearly 800,000 people annually in the United States each year and accounting for approximately 1 in 19 deaths [1,2]. In China, from 1990 to 2019, the annual incidence of stroke rose 86 % to 276.7 cases per 100,000 population [3,4]. In addition, the mortality rate has increased by 32.3 %, which is among the highest in the world [4].

Stroke is an acute cerebrovascular disease that can be clinically classified into ischemic and hemorrhagic strokes based on the etiology [5]. While ischemic stroke accounts for 85 % of the cases, it is characterized by vascular occlusion, leading to insufficient perfusion of oxygenated blood and the formation of cerebral infarction [6]. After 5 min of ischemia in brain tissue, it can cause both nerve cell death in the brain and transmission of damage to the surrounding uninjured brain tissue, leading to impaired brain function and neurological dysfunction. And for many years, alteplase (tissue plasminogen activator, tPA) was the only drug therapy for ischemic stroke reperfusion approved by Food and Drug Administration (FDA). However, this approach has a limited therapeutic time window, typically 4.5 h after stroke, resulting in more than 90 % of patients not benefiting from it [7,8]. Meanwhile, mechanical means (endovascular thrombectomy, ET) has been a new approach to treating stroke in recent years. Still, it also faces the same problem of a narrow window of time for treatment (within 6 h after stroke) and the possibility of device- and procedure-related complications [9,10], which makes it possible to perform this treatment in only 6 % of patients [11]. As a result, ischemic strokes that are not treated in a timely manner and have a poor postoperative outcome can lead to disability, which can have a significant negative impact on the patient's life as well as on their families [12].

Subsequently, scientists have developed neuroprotective drugs for treating ischemic stroke, but this therapy has also failed to achieve clinical translation due to the limitations of the blood-brain barrier (BBB) and off-target utility [13]. Most drugs require high concentrations of systemic administration to cross the BBB, but may cause systemic toxicity due to their off-target distribution. In addition to this, preliminary clinical trials of stem cell therapies have been conducted to explore their safety, feasibility and efficacy [14,15]. However, the translation of stem cell therapies into clinical practice still faces serious challenges, including low survival rates due to immune system attack, limited long-term viability and the risk of tumor formation [16,17]. Therefore, there is a need to develop new therapeutic strategies to address the current therapeutic challenges in ischemic stroke.

As science and technology continue to advance, biomaterials are beginning to be developed for use in medicine and are providing new therapeutic approaches for nervous system regeneration [18]. To date, several new biomaterial-based strategies for the treatment of stroke have been developed to address the above challenges [[19], [20], [21]]. Different biomaterials have been used as delivery vehicles for drugs, growth factors, or cells that can compensate for the inability of systemic routes of drug delivery to achieve clinical efficacy and ensure the timing and amount of clinical response required for drugs [22]. Among the many biomaterials, hydrogel is a polymer system with a very high water content, its unique injectability, and suitable porosity that allows it to be used as a carrier for the delivery of cells, drugs, and proteins, etc., and it has become a more widely researched material [18]. Due to the high water content, the hydrogel has a suitable elastic modulus. It exhibits a 3D structure that mimics the properties of the natural extracellular matrix within the central nervous system (CNS) [23,24]. Hydrogels are ideal candidates for neural tissue engineering due to their high biocompatibility, ease of functionalization and controlled physical properties [25]. Hydrogels can deliver a wide range of bioactive molecules and constitute a tunable substrate for the axonal growth of endogenous or transplanted cells, thus promoting tissue regeneration [23]. Furthermore, hydrogels are an ideal carrier system for neuroprosthetics, which can transplant therapeutic stem cells or molecules into the target brain region with considerable precision. The high porosity and appropriate pore size of hydrogels can influence the degree of inward tissue growth, which provides excellent restoration of stroke-induced brain damage [26,27].

Carrier-based hydrogels are promising functional scaffolds for the treatment of IS. Therefore, it is necessary to summarize the application of carrier-based hydrogels in the treatment of IS and provide some insights for researchers to design hydrogels. There have been articles summarizing the application and progress of hydrogel in stroke treatment, but they mostly focus on the introduction of IS path mechanisms and hydrogel therapeutic principles, and do not provide an in-depth summary of the composition of the material itself, its physicochemical properties, physiological functions, and application design [28,29]. Therefore, this review summarizes the application of carrier-based hydrogels in the treatment of IS in a more systematic manner based on existing studies (Fig. 1). First, we briefly describe the pathophysiology of IS and the limitations of existing treatment strategies, followed by an in-depth discussion of the advantages and disadvantages of using different biomaterial preparations in hydrogel therapy for IS. The discussion then turns to the use of carrier-based hydrogels in IS therapy, with a particular focus on the use of hydrogel scaffolds containing bioactive molecules and stem cells. This is followed by an analysis of the physicochemical properties that are required of hydrogels used for IS. Finally, the limitations of hydrogels in IS therapy and the future challenges in developing more complex carrier-based hydrogels are outlined.

Fig. 1.

Schematic diagram of hydrogel-based treatment of ischemic stroke. (Inner Ring) The classification of hydrogels for ischemic stroke. Based on their sources, they can be classified into natural and synthetic hydrogels, including chitosan, hyaluronic acid, polyethylene glycol, polyvinyl alcohol, etc.; (Middle Ring) Requirements to be met by hydrogel scaffolds for ischemic stroke, including porosity and pore size, biodegradability, conductivity, and elasticity, all of which affect the ability of hydrogels to repair nerves; (Outer Ring) Carrier-based hydrogels are used in ischemic stroke therapy. They are mainly hydrogel systems containing bioactive molecules and stem cells. Among the bioactive molecules currently used in the treatment of IS are mainly BDNF, VEGF, EPO, etc., and stem cells are mainly MSCs, NSCs and hiPSCs. Finally, there are also composite and stimulus-responsive hydrogel systems.

2. Pathophysiology of IS and existing treatment strategies

2.1. Pathophysiology of IS

IS, also known as cerebral infarction, is caused by brain's blood flow disruption, leading to cerebral vascular thrombosis [30,31]. Cerebral ischemia is followed by a series of pathological cascade of events that ultimately leads to neuronal death [32]. First, disruption of cerebral blood flow depletes glucose in the brain, leading to blocked adenosine triphosphate (ATP) synthesis and decreased energy, disturbed ionic gradients, and acid-base imbalance [33]. Thus, an ischemic and hypoxic environment induces a variety of physiological, biochemical, and molecular changes [34,35]: 1) excitotoxicity mediated by the massive release of the excitatory amino acid glutamate and a surge in cytosolic calcium influx [36]; 2) when the production of free radicals such as reactive oxygen/nitrogen free radicals (ROS/RNS) exceeds the endogenous scavenging capacity of antioxidant defense systems, the mitochondrial dysfunction caused by oxidative and nitrative stress [37]; ROS also cause tissue destruction and cell death through processes such as DNA damage, protein damage, lipid peroxidation, and damage to cytoskeletal structures [38]; 3) microglia are activated to invade the infarct core and periphery, and then release pro-inflammatory cytokines, such as TNF - α, IL-1 β, and IL-6. These cytokines exacerbate the inflammatory response and irreversible neuronal damage occurs by recruiting and infiltrating neutrophils, monocytes, and T-cells to the brain lesion [39]. Damage induced by excitotoxicity, oxidative stress, and mitochondrial disorders in IS may induce multiple cell signaling cascades leading to neuronal cells undergoing programmed or non-programmed death [40]. Usually, programmed death includes apoptosis and autophagy [41], whereas non-programmed death is cell necrosis, possibly caused by external stimuli [42]. All these events occur with irreversible damage to brain tissue and ultimately lead to severe neurological dysfunction (Fig. 2).

Fig. 2.

A summary for the pathophysiology involved in ischemic stroke. (1) Excitotoxicity mediated by the massive release of the excitatory amino acid glutamate and a surge in cytosolic calcium influx. (2) microglia are activated to release pro-inflammatory cytokines, such as TNF - α, IL-1 β, and IL-6. These cytokines exacerbate the inflammatory response. (3) Cell death signaling pathways, which mainly involves autophagy, apoptosis and necroptosis in ischemic stroke. (4) Oxidative stress, which is mainly characterized by ROS/RNS production and mitochondrial dysfunction.

2.2. Existing treatment strategies

The guidelines for the early management of stroke state that ischemic stroke is treated by removing the blood clot thereby relieving ischemia and hypoxia in the brain and restoring blood flow or cerebral collateral circulation as early as possible to prevent further damage to brain tissue [43]. The extent of cerebral collateral circulation is a key clinical factor influencing the prognosis of patients with ischemic stroke. It is closely related to whether intravenous thrombolysis and endovascular therapy are more effective [44]. To date, the only FDA-approved treatment for ischemic stroke is intravenous recombinant tPA, which is used to reperfuse brain tissue by thrombolysis and recanalization of obstructed vessels [45]. Mechanical endovascular thrombectomy (ET), on the other hand, is also a newer therapeutic approach as a complementary approach to tPA therapy, which can be used alone or in combination with fibrinolytic drugs. However, both have a narrow time window for treatment, <4.5 h for tPA therapy [46], and ET is also limited to 6 h of stroke onset [47]. Less than 10 % of ischemic stroke patients can obtain and benefit from these treatments because certain hospitals are unable to perform ET [48]. Notably, both approaches carry a potential risk of hemorrhagic conversion during treatment, which may exacerbate ischemic stroke [49].

Over the past decades, an increasing number of scientists have investigated the pathophysiology and pathogenesis of ischemic stroke and have identified neuroprotective agents as a promising strategy for brain protection [50]. Since neurons are difficult to regenerate after injury, protection of neurons and restoration of neurological function are essential in the treatment of stroke to prolong the duration and effectiveness of the therapeutic window [51]. Although most neuroprotective drugs have shown good results in preclinical studies and can be used to slow down further damage to brain tissue, none of them have been successfully taken to the clinic [52]. The main reason for this is that treatment requires delivery of neuroprotective agents to the lesion site in the brain. Still, traditional oral and intravenous routes of administration are limited by the blood-brain barrier (BBB), resulting in the inability to deliver drugs, growth factors, etc. To the brain [53]. As a result, high systemic doses are required to achieve therapeutic concentrations at the site of injury, which is usually systemically cytotoxic [54]. Systemic delivery leads to off-target distribution of therapeutic molecules, which can result in undesirable side effects, such as tumors and fibrosis [55]. In addition to this, systemic delivery of vascular growth factor (VEGF) leads to further opening of the BBB, exacerbates edema, and promotes the formation of a disorganized vascular system [56]. Therefore, this method has not been investigated for appropriate drug delivery as well as dosage at this time. The failure to develop numerous neuroprotective drugs suggests that modulating a single therapeutic target may not be sufficient to achieve clinically relevant neuroprotection [57].

Other strategies currently used for treating ischemic stroke IS include stem cell therapy, biomaterials therapy [58]. Stem cell therapy is multi-targeted compared to previous treatment options, and it is considered a major regenerative medicine approach to restore neuronal function and improve treatment outcomes. Potential mechanisms of stem cell therapy include but are not limited to, direct replacement of missing, damaged, or dysfunctional cells, enhancement of neuronal survival, mobilization of endogenous stem cells, and modulation of immune responses [59]. In many preclinical studies, transplantation of stem or progenitor cells after ischemic stroke has been shown to increase neuronal survival and improve neurological function to some extent [58,60,61]. Although stem cell therapy has been proven to be effective, there are still numerous issues that need to be addressed, such as: ethical issues, safety issues, cell dose, optimal timing and route of administration. Some studies have shown that the following factors mainly limit stem cell therapy: 1) Rejection of transplanted cells due to immune system attacks. Transplanted cells enter the brain in suspension, which results in low graft survival [62]. 2) The tumorigenicity of transplanted cells needs to be considered, and stem cell transplantation into the brain may lead to tumor formation [63]. 3) Controlled treatment of transplanted exogenous neural stem cells to regulate differentiation and achieve the desired therapeutic effect remains to be explored [64]. 4) Insufficient migration of transplanted cells to the lesion site, along with ineffective integration into the injured brain tissue [65]. 5) Each cell needs to undergo safety and efficacy testing; the most effective and safest route of administration, timing of delivery, and dosage have yet to be found [66], and the optimal timing of transplantation remains a controversial topic. Hydrogel can be used as a carrier for stem cells, its good biocompatibility and similar structure to natural extracellular matrix are more conducive to the survival of stem cells after transplantation, which brings a new strategy for stem cell transplantation, and is a promising approach to promote brain repair after ischemic stroke (Fig. 3).

Fig. 3.

The treatment strategies of ischemic stroke. (1) Common treatment methods: tPA and ET. Both tPA and ET are limited by the treatment time window. (2) Neuroprotective agents as a promising strategy for brain protection, but traditional oral and intravenous routes of administration are limited by the blood-brain barrier (BBB). (3) A new strategy for treating IS: biomaterials (hydrogels and nanoparticles). Different biomaterials can be used as delivery vehicles for drugs, growth factors or cells.

3. Hydrogel ischemic stroke biomaterials

Hydrogel, a popular material for repairing ischemic stroke, has the following advantages. First, hydrogel is an ideal carrier that can be loaded with cells, drugs, growth factors, and other bioactive molecules and deliver these substances directly to the injury site. Second, it has a suitable pore size for efficient nutrient and metabolite transportation, leading to better cell migration [67,68]. Third, hydrogels have high water content and good biocompatibility, which will not harm the graft or host cells [69]. Fourth, hydrogels have adjustable mechanical strength that matches the modulus of the damaged brain tissue and can influence cell differentiation by modulating mechanical strength [70]. Finally, hydrogels used for IS repair promote neuroregeneration by supporting and controlling cell attachment, proliferation, and differentiation [69,71].

For effective use in ischemic stroke, hydrogels must be biocompatible, have an appropriate elastic modulus and be non-toxic. In contrast, crosslinking represents a pivotal aspect of hydrogel design and synthesis, and can be attained through a multitude of methodologies. This process markedly influences the characteristics of the resulting hydrogel, including its mechanical strength, biocompatibility, and responsiveness to external stimuli [72]. Various cross-linking methods including chemical cross-linking with reagents such as glutaraldehyde or EDC/NHS, physical cross-linking through freeze-thaw cycles or ionic interactions, enzymatic bio-cross-linking using e.g., transglutaminase, and photo-cross-linking using photosensitizing materials, which give a wide range of options for enhancing the structural integrity and functional properties of hydrogels [73,74]. Chemical cross-linking can provide stable and robust hydrogels for long-term release of bioactive molecules. However, the reagents/solvents used in cross-linking strategies have limited biodegradability and may be toxic. Physical cross-linking using non-covalent interactions may produce hydrogels that are responsive to internal stimuli such as changes in pH, leading to better control of drug/bioactive molecule release [75]. However, physical crosslinked hydrogels are not stable enough in physiological environments and are therefore limited in therapeutic IS. Enzymatic cross-linking offers the possibility of biocompatible and cell-friendly hydrogels, but enzymes are easily inactivated, which can lead to changes in the properties of the hydrogel [76]. Photo-crosslinked hydrogels allow for precise control of hydrogel properties and have the potential to create complex structures for customized therapeutic applications [77]. However, the synthetic monomers used to prepare photo-crosslinked hydrogels are often toxic, and removing these hazardous substances remains a challenging problem [78]. Together, these crosslinking techniques offer promising avenues for the development of hydrogels for IS, but there are still challenges to be addressed (Fig. 4).

Fig. 4.

Cross linking and composition of hydrogels. (1) Cross linking methods of hydrogels: chemical cross linking with glutaraldehyde、EDC/NHS or other reagents, physical cross linking through freeze-thaw cycle or ion interaction, enzymatic biological cross linking using transglutaminase and other enzymes, and photo cross linking using photosensitive materials. (2) Composition of hydrogels: a. Natural hydrogels include hyaluronic acid, collagen, gelatin, etc. Their main advantages include good biocompatibility; wide range of sources, easy accessibility; good biological activity can participate in processes such as cell differentiation and migration. But there are still some drawbacks, such as low mechanical strength; poor stability; performance differences between different batches, etc. b. Synthetic hydrogels include polyethylene glycol, polyethylene, etc. The main advantages include adjustable physical and chemical properties, which can be specially optimized for specific applications; stable performance; low production costs and large-scale production; controllable degradation, etc. The remaining drawbacks include poor biocompatibility; low biological activity, no existing natural cell binding sites; low compatibility between degradation rate and neural tissue regeneration, etc.

Hydrogels formed by cross-linking exhibit a diverse range of properties and applications, and are classified according to various criteria. The composition of hydrogels has a significant impact on their biocompatibility. A common approach is to differentiate between the various types of hydrogels on the basis of their composition, classifying them into two main categories: natural and synthetic hydrogels. The former are derived from natural materials, including hyaluronic acid, collagen, gelatin, and so forth, while the latter are synthesized from man-made polymers, such as polyethylene glycol and polyvinyl alcohol. Natural hydrogels are biocompatible and easy to obtain. In contrast, synthetic hydrogels are more stable and have adjustable physical and chemical properties. Natural and synthetic hydrogels are described in detail below, with examples of the advantages and disadvantages of the different materials.

3.1. Natural hydrogels

Natural hydrogels are hydrogels composed of natural biomaterials. Natural biomaterials are highly diverse and multifunctional biomaterials produced under natural conditions. They usually have excellent biocompatibility and bioactive properties and do not produce toxic side effects, making them promising materials for treating ischemic stroke [79]. However, the mechanical properties and stability of natural hydrogels are generally poor, and sometimes their degradation rate is too fast for them to have a slow-release effect [80]. Some natural hydrogels for IS as well as for neuroprosthetics will be briefly presented and discussed below.

3.1.1. Hyaluronic acid

Hyaluronic acid (HA) is a glycosaminoglycan that is highly hydrophilic and helps retain large amounts of water in connective tissues [81]. It is one of the components of the extracellular matrix and interacts with other extracellular molecules involved in cell differentiation, migration, and angiogenesis [82]. Because of its distinct molecular structure and physical-chemical properties, HA can interact with cell surface proteins to trigger signalling systems for proliferation, differentiation, and other functions [83,84]. Therefore, HA is a popular biomaterial for treating ischemic stroke due to its outstanding biocompatibility and biodegradability.

Because HA can hold large amounts of water, it is widely used in hydrogels and can serve as an excellent carrier for bioactive molecules and cells. In a previous study, HA hydrogels loaded with Nogo-66 receptor antibody were implanted into the necrotic areas of stroke rats, and the results showed that the antibodies were released from the hydrogels and diffused into the surrounding tissues and lasted up to 8 weeks [85]. In vitro studies have shown that HA hydrogels can promote the growth of neural synapses [86]. Neural synapses are protrusions arising from the cytosol of a nerve cell and are categorized into dendrites and axons. They play the role of information transmission. Meanwhile, previous studies have shown that HA hydrogel supports the survival and growth of neural precursor cells [70,87], neural stem cells [88], schwann cells [89], etc., and enables them to proliferate and differentiate.

However, hyaluronic acid is usually non-adhesive to cells, thus requiring it to bind to other ECM components or cell adhesion sequences [79]. For example, in a study by Hou et al., a laminin-modified HA hydrogel was implanted into cortical defects to repair brain injury. The results showed that the synthesized HA hydrogel induced angiogenesis promoted the extension of neural synapses, and inhibited the formation of neuroglial scarring [90]. In another study, Tian et al. developed a hyaluronic acid-poly-D-lysine (PDL) copolymer hydrogel, which was able to support cell adhesion and growth [91]. In addition, HA has been shown to modulate astrocyte proliferation, which can inhibit glial scar formation by reducing the thickness of glial proliferation [92,93]. However, since HA typically fails to adhere to cells, cell binders must be incorporated into hydrogels to facilitate cell attachment and growth. This underscores the importance of achieving an optimal ratio between the individual components. Additionally, HA is relatively expensive and degrades rapidly in vivo, which can result in a precipitous decline in mechanical strength. In conclusion, HA monomaterials continue to encounter significant challenges when synthesizing hydrogels.

3.1.2. Collagen

Collagen is the most abundant and widely distributed protein in mammals and one of the important components of the neural extracellular matrix. Like many ECM components, collagen regulates cell migration, proliferation and differentiation [94]. Currently, there are 28 different types of collagens, of which type I collagen is the most used collagen in the biomedical field. Type I collagen supports axonal growth and plays an important role in developing the nervous system [95]. FDA has approved a variety of collagen nerve conduits for clinical use in peripheral nerve injury [96]. Collagen has excellent neurotissue compatibility and is therefore a good candidate for promoting brain tissue regeneration [97].

Collagen hydrogels can serve as good carriers for bioactive molecules and stem cells [98,99]. In previous studies, it was shown that collagen hydrogels can release nerve growth factor (NGF), which is able to protect cholinergic neurons [100]. In addition to NGF, the collagen hydrogel loaded glial cell line-derived neurotrophic factor (GDNF), a scaffold that significantly increased dopaminergic neuron survival and nerve fiber growth [101]. With its superior biocompatibility and ability to mimic extracellular matrix, collagen hydrogel can be used in conjunction with stem cells to further promote neural tissue repair. Collagen hydrogels containing laminin-derived peptides can be used as carriers for neural stem cells and can improve the viability of NSCs [102]. Meanwhile, in another study, Moxon et al. doped collagen fibers into an alginate hydrogel that lacked cell-binding sequences and loaded it with human induced pluripotent stem cell (iPSC)-derived neurons, which could exhibit cell attachment, neuron maturation, and the hydrogel supported the formation of neural networks [103]. In a study by Lu et al., collagen scaffolds filled with human bone marrow mesenchymal stem cells reduced lesion volume and improved sensorimotor function in rats after injury [104].

Collagen is a natural ECM component that binds and interacts with cell surface receptors, but such interactions have not been well studied. In addition, normal brain ECM contains only a small amount of collagen and a large amount of glycosaminoglycans such as hyaluronic acid, chondroitin sulfate [105]. As a result, injecting or placing collagen hydrogels with unnaturally high concentrations of collagen in the brain may produce some undesirable side effects. At the same time, collagen degrades at too rapid a rate in vivo and needs to be chemically modified to improve stability; it also needs to be thoroughly tested for immunity/allergenicity due to its heterologous nature.

3.1.3. Gelatin

Gelatin is obtained through the partially controlled degradation of collagen and therefore shares some of the same properties as collagen while being easier to process. Gelatin-based hydrogels are used in a variety of biomedical applications, such as device coatings, drug delivery, wound dressings, etc., due to their biocompatibility and ability to promote cell adhesion and proliferation [106].

And in neurological applications, gelatin hydrogels have shown sufficient potential for application. In previous studies, it has been shown that gelatin-based hydrogels can be loaded with basic fibroblast growth factor (bFGF) and achieve a sustained release in vivo for 14 days, while also promoting nerve axon regeneration [107]. In a study by Nakaguchi et al. gelatin hydrogel microspheres containing hepatocyte growth factors were injected into the striatum, which sustained the release of growth factors and thus increased the number of new neurons migrating from the subventricular zone (SVZ) of the brain into the striatum [108]. In a more recent study it was also shown that injectable gelatin-norbornene nanofiber hydrogels (GNF) containing growth factors mimicked the natural extracellular matrix structure and significantly increased the average size of lectin-positive blood vessels in the IS brain [109]. However, gelatin has poor mechanical strength and degrades rapidly. Therefore, gelatin usually needs to be mixed with other biomaterials to enhance the properties of hydrogels.

3.1.4. Silk fibroin and sericin

Silk fibroin and Sericin are the two main proteins in silk. Silk is a unique biomaterial with good mechanical properties and controlled biodegradability. The degradation rate of silk fibroin is relatively slow, which renders them an appropriate choice for biomedical or tissue engineering applications that necessitate long-term stability [110]. Studies conducted in vitro have shown that silk fibroins are biocompatible, can support dorsal root ganglion (DRG) growth, and may facilitate the survival of ceviche cells [111]. Gu et al. developed a silk fibroin hydrogel with a high-strength and aligned microgroove morphology, which can regulate the growth of ceviche cells, promote axon growth, and guide the sprouting of neuromasts [112]. In an in vivo model, silk hydrogel is highly biocompatible and does not cause persistent inflammation [113]. The study revealed that the brain exhibited a higher stem cell content following transplantation with filipin hydrogel, in comparison to mice that had been implanted with stem cells alone [114].

Past studies have shown that sericin has neurotrophic and neuroprotective effects, promoting axonal extension and branching and preventing the death of primary neurons due to hypoxia [115]. In another study, Zhang et al. prepared chitosan-sericin-gel composite hydrogel scaffolds, which supported the growth of ceviche cells, and their degradation products also had excellent properties in promoting the proliferation of ceviche cells and inducing the differentiation of PC12 cells [116]. In addition, the degradation products of sericin proteins are rich in glycine and serine, and glycine plays a regulatory role in many neuronal circuits [117,118]. In vivo studies have demonstrated the considerable potential of sericin hydrogels. In Wang et al.'s study, an injectable silk hydrogel containing mesenchymal stem cells (MSCs) was prepared, and when injected into the IS brain the cells were able to migrate out and infiltrate into the surrounding brain tissue at an average depth of 147 μm, which contrasts with the rapid cell loss of directly transplanted cells [119].

These studies have shown that both sericin and silk fibroin proteins have good biocompatibility and excellent mechanical properties, and their slow and controlled degradation rates are favorable for long-term implantation in medical applications, so they are expected to become a major repair material for ischemic stroke in the future.

3.1.5. Chitosan

Chitosan is a polysaccharide obtained by partial deacetylation of chitin. Chitosan has many favorable properties such as biocompatibility, biodegradability, and antimicrobial activity [120]. Therefore, chitosan has also been used to repair nerve damage.

The ability of chitosan to accelerate the rate of healing in the nervous system is attributed to its ability to enhance the adhesion of neuronal cells to their membranes [121]. Recently, it was found that PDL-modified thermoresponsive chitosan/glycerophosphate (GP) hydrogels promoted cell survival and neurite growth [122]. Liu et al. prepared self-repairing hydrogels with semi-interpenetrating polymer networks by doping HA into chitosan-based hydrogels. The results showed that the unique structure of the hydrogel facilitated cell spreading, migration, proliferation, and differentiation in vitro, and it provided a relaxed microenvironment for axonal growth, which could improve brain injury [123]. Although chitosan has good antimicrobial and anti-inflammatory properties, it has some drawbacks, such as poor solubility at physiological pH and difficulty in controlling gelation time, as well as general interaction with nerve cells and inability to regulate the mechanical properties to support 3D neural protrusion extension [124].

3.1.6. Alginate

Alginate is a naturally occurring anionic polymer usually obtained from seaweed.Due to its biocompatibility, low toxicity and ease of gelation, it is now widely used in biomedical fields such as wound repair dressings, drug delivery vehicles, in vitro cell culture, tissue engineering, etc. [125] Alginate has antioxidant capacity and protects neurons from H₂O₂ damage [126,127].

Alginate hydrogels have potential in the field of neural tissue repair. In a study by Emerich et al., alginate hydrogels containing VEGF provided up to 14 days of growth factor release in vivo, whereas VEGF was undetectable after only 4–8 h by direct injection [128]. Previous studies have also shown that alginate hydrogel as a three-dimensional scaffold for culturing embryonic stem cells can effectively support neural differentiation and promote neurite growth and extension [129]. Similarly, alginate hydrogels containing mesenchymal stem cells showed maximum length of axon growth compared to controls [130]. However, alginate hydrogels still have some limitations in nerve repair. Since mammals do not produce endogenous alginase, alginate hydrogels may take months to degrade in vivo [131]. Meanwhile, alginate lacks sequences for cell adhesion and usually needs to be used in combination with other ECM components. This may limit the use of alginate hydrogels in brain tissue repair.

3.2. Synthetic hydrogels

Synthetic hydrogels are made up of synthetic polymers. These polymers, which are made by humans, can be mass-produced, have stable properties and are cheap to produce. Synthetic hydrogels are well-stabilized and have adjustable mechanical strength [132]. Some of the synthetic hydrogels used in treating IS are briefly described and discussed below.

3.2.1. Polythene glycol

The most extensively researched synthetic material is polythene glycol (PEG), a stable, non-toxic, and biocompatible polymer. PEG hydrogels are excellent biomolecular carriers that enable controlled release [133].

PEG hydrogels have been shown to promote axonal regeneration and myelin sheath regeneration in a study by Estrada et al. [134] In another study, Lampe et al. developed PEG hydrogels formed by photoinitiated polymerization for neurotrophic factor delivery. They designed a PEG hydrogel encapsulated with PLGA particles and utilized this hydrogel to deliver brain-derived neurotrophic factor (BDNF) and glial-derived neurotrophic factor (GDNF) to the brain, enabling controlled release of growth factors. BDNF was released slowly over the full 56 days, whereas GDNF was released by day 28 and was found to attenuate microglia activation and inflammatory responses after implantation [135]. Similarly, PEG hydrogels are good carriers for cells, and neural progenitor cells (NPCs) encapsulated in PEG hydrogels can survive for up to 17 days. In addition, more than half of the cells differentiated into mature neurons (Neurofilament 200 positive) [136]. However, PEG hydrogels have poor mechanical compatibility with tissues, and modifications of collagen and laminin are generally required to reduce the stiffness of the hydrogel. Currently, encapsulation of cells into PEG hydrogels is mostly based on photopolymerization, which subsequently may affect cell viability. At the same time, the lactic acid released during the PEG gelation process may affect cell function [137].

3.2.2. Methacrylate and methacrylamide

Methacrylate-based hydrogels' chemical and spatial structure can be tailored to produce mechanical properties like those of neural tissue [138]. Hydrogels based on poly (2-hydroxyethyl methacrylate) (PHEMA) and poly[N-(2-hydroxypropyl) methacrylamide] (PHPMA) are a group of highly biocompatible polymers, both used in the field of neural repair [139]. Implantation of PHEMA hydrogels enhanced axonal regeneration [140], and subsequent studies incorporated oriented polycaprolactone fibers into PHEMA hydrogels, which provide support and contact guidance for extending axons and invading cells [141]. However, PHEMA has no adhesion or attraction to neurons and needs to be modified to improve biocompatibility. For example, a super porous PHEMA hydrogel modified with a laminin-derived peptide developed by Kubinová et al. was able to improve cell adhesion properties and promote neural stem cell differentiation [142]. PHEMA still has some drawbacks in that it does not possess good biodegradability, which limits its application [143].

PHPMA is more biocompatible than PHEMA and has great potential for nerve repair [144]. It has been shown that PHPMA hydrogels supported angiogenesis and induced hemotransfusion at the injury site within 2 weeks after transplantation and retained the ability to regenerate axons up to 3 months after injury [145]. Peptide and aminosugar sequence-modified PHPMA hydrogels can increase adhesion properties to host neural tissue [146]. In a study by Woerly et al. PHPMA hydrogels containing RGD adhesion peptides showed interconnected porous structures, resulting in more inward axons growth [147]. Woerly et al. prepared a PHPMA hydrogel containing a block-modified saccharin portion of ganglioside GM3,for example, 3′-sialyllactose, is a bioactive epitope recognized by many cell surface receptors on human cells [148]. The results showed that the modified hydrogels have good neurohistocompatibility and can store endogenous self-migrating stem cells and, for a fraction of them, support their neural differentiation in non-neurogenic regions [148].

3.2.3. Polyvinyl alcohol

Polyvinyl alcohol (PVA) is a polymer formed by polymerization reaction and alcoholysis of vinyl acetate. In studies of peripheral nerve regeneration, it has been demonstrated that PVA nerve conduits can maintain the regeneration of damaged nerves and have good axonal growth density [149]. Meanwhile, in vivo experiments have shown that PVA hydrogel prevents the migration of inflammatory cells after laminectomy and subsequently reduces scar tissue formation in the spinal canal [150]. In another study, Oh et al. prepared PVA/HA hydrogels with a wide range of stiffness gradients, and stem cells could be differentiated towards different cell types by adjusting the stiffness of the hydrogels [151]. However, the hydrogel of PVA has low bioactivity, while the degradation rate is not sufficiently matched to neural tissue regeneration.

In summary, natural hydrogels have excellent biocompatibility, and some materials, such as collagen, also have bioactive sequences for cellular recognition, which may be beneficial for ischemic stroke repair. Meanwhile, the natural hydrogel has good biocompatibility and can integrate well with the host brain tissue. However, the mechanical properties of natural hydrogels tend to be poor and may vary from batch to batch of natural polymers, which limits the mass production of natural hydrogels [152]. Synthetic hydrogels are attractive because their physical and chemical properties (e.g., degradation rate, porosity, mechanical strength, etc.) can be specifically optimized for specific applications [153]. However, the biocompatibility of synthetic hydrogels poses a few challenges for their application because natural cell-binding sites do not exist on synthetic polymers. Thus, synthetic hydrogels are not sufficiently compatible with cells and tissues and may cause immune reactions (Fig. 4). Therefore, combining the advantages of natural and synthetic hydrogels and exploring the therapeutic strategy of combining the two may provide a more optimal solution for the treatment of ischemic stroke.

4. Application of carrier-based hydrogels in IS therapy

Neural recovery and regeneration after ischemic stroke are a complex process involving multiple factors and cells and limited endogenous neural restorative capacity. Therefore, the replacement of damaged tissues with exogenous cells/stem cells, and/or neuroprotection or stimulation of endogenous stem cell proliferation and differentiation with the help of bioactive molecules have become the current focus of ischemic stroke treatment. Hydrogels have an excellent performance in acting as carriers and support structures for bioactive molecules and cell delivery. Hydrogel, as an important biomaterial, is a scaffold formed by a network of crosslinked polymer units with high water content (>90 %) and has a three-dimensional structure like that of natural tissue ECM. Hydrogel, as a biocompatible carrier, can transport bioactive molecules to the site of injury in a targeted manner, thus achieving their long-term release, improving neuroinflammation and promoting nerve regeneration after IS. In addition, it mimics the structure of the ECM and the unique biophysical properties of neural tissue, enabling it to provide a suitable microenvironment for the growth of transplanted or neoplastic cells, promoting the survival, differentiation, and functional integration of the transplanted cells, as well as binding to the transplanted cells to inhibit inflammation and so on [70,154]. Meanwhile, another unique ability of hydrogels is the ability to be injectable and form gels in situ, which enables them to fill irregularly shaped lesion cavities in the brain and form structural supports [155]. Thus, carrier-based hydrogels have emerged as a potential biomaterial resource for treating ischemic stroke and are ideal for repairing brain injuries. A few new therapeutic strategies have been developed for carrier-based hydrogels, including loaded bioactive molecules, stem cells, or a combination of the two, aiming to mitigate further damage to peripheral tissues and restore some neurological functions after ischemic stroke. In this section, we will focus on the use of hydrogels containing bioactive molecules and stem cells in the treatment of ischemic stroke and provide an overview of the different therapeutic forms of carrier-based hydrogels, including but not limited to composite hydrogels, stimuli-responsive hydrogels, nanogels, microgels and hydrogel microspheres.

4.1. Hydrogels containing bioactive molecules

Although the brain initiates some repair mechanisms on its own after a stroke, their effects are far from sufficient to restore the damage caused by the stroke. Bioactive molecules can regulate and promote neural repair through corresponding receptors. Many studies have reported that various bioactive molecules can promote neuronal survival and growth, which is beneficial to the repair of the nervous system [156]. However, bioactive factors alone usually suffer from the BBB barrier, low bioavailability, poor tissue distribution, and short half-lives. At the same time, higher doses may in turn cause adverse immune responses. Whereas hydrogels contain up to 90 % water, and their three-dimensional network structure facilitates the loading of bioactive molecules, they also have elasticity and flexibility similar to natural extracellular matrix (ECM) [157]. Since ischemic stroke results in irregular cavities in the brain, injectable hydrogels have become an ideal vehicle for minimally invasive targeted delivery of bioactive molecules/cells [158]. For example, the Shoichet lab has developed hyaluronic acid-methylcellulose hydrogel (HAMC), a hydrogel that has been shown to gel quickly and can be injected, which makes it possible to fill arbitrarily irregularly shaped lesion cavities [159]. Therefore, therapeutic strategies combining bioactive molecules with scaffold materials, especially hydrogels, may bring new directions for repair after ischemic stroke. Hydrogel binds to bioactive molecules to maintain their activity for a long period of time, reducing the impact of the blood-brain barrier and bringing better therapeutic effects. Table 1 summarizes hydrogels containing bioactive molecules and their results in the treatment of IS processes(Table 1).

Table 1.

Summary of some representative hydrogel systems containing bioactive molecules for use in ischemic stroke.

Reference	Wang et al. (2012) [161]	Obermeyer et al. (2019) [163]	Cook et al. (2017) [166]	Nih et al. (2018) [176]	Yanev et al. (2022) [177]	Wang et al. (2013) [178]
Outcome	EPO delivered from HAMC poststroke resulted in attenuated inflammatory response, reduced stroke cavity size, increased number of both neurons in the peri-infarct region and migratory neuroblasts in the subventricular zone.	Local delivery of BDNF with hydrogel results in short-term recovery in forelimb function, as well as hindlimb recovery at week 7 testing. The system preserves mature neurons in the peri-lesional tissue and decreasing lesion volume.	HA hydrogel-BDNF combination achieves BDNF release over weeks and promotes motor recovery and axonal sprouting, enhances the initial migration of immature neurons to peri-infarct cortex and supports their long-term survival.	The delivery of hydrogel promotes tissue formation de novo, and results in axonal networks along thee generated blood vessels. This regenerated tissue produces functional recovery through the established axonal networks.	Results show that long-term release of VEGF and Ang1 from an intralesionally injected hydrogel reinforces post-stroke vascularization and neuronal survival in the perilesional region, ultimately resulting in improved functional outcome.	Our composite-mediated, sequential delivery of EGF-PEG and EPO attenuates the inflammatory response of brain tissue, then leads to tissue repair in a mouse stroke model and minimizes damage compared to ICV infusion.
Growth factors/Small molecules/Drugs	Erythropoietin (EPO)	Brain-derived neurotropic factor (BDNF)	BDNF	Vascular endothelial growth factor (VEGF)	VEGF and Angiopoietin 1 (Ang1)	Pegylated EGF (EGF-PEG), erythropoietin (EPO)
Hydrogel system	Hyaluronan/methyl cellulose (HAMC) hydrogel	HAMC hydrogel/PLGA nanoparticles	HA hydrogel	HA hydrogel/heparin nanoparticles (nH)	RADA16-I, pH-sensitive peptide nanofiber-based self-assembling hydrogel	HAMC hydrogel

To date, only two growth factors have been used as therapeutic agents; Erythropoietin (EPO) and Granulocyte-colony stimulating factor (G-CSF) [160]. And now Nerve Growth Factor (NGF) is also gradually embarking on clinical trials, such as the clinical trials (NCT05652751 and NCT05231694) in preparation in China (https://www.clinicaltrials.gov/), i.e., the clinical efficacy of electro-acupuncture in combination with NGF in ischemic stroke. Due to the presence of the BBB, many bioactive molecules require high concentrations to reach the brain, which may cause systemic toxicity [55]. However, by loading bioactive molecules into a scaffold of biomaterials, targeted delivery of bioactive molecules to the site of injury in the brain can be achieved. In previous studies, the Shoichet lab mediated nerve regeneration by topically releasing EPO through HAMC hydrogels to attenuate the inflammatory response and promote the survival of endogenous neural stem/progenitor cells (NSPCs) (Fig. 5A) [161]. In another study, Cook et al. also used the HAMC hydrogel system as a carrier. They dispersed epidermal growth factor (EGF) in a HAMC hydrogel for minimally invasive drug delivery through the outer cortical layer, achieving up to 2 days of EGF release in vivo with significantly higher release than the other groups. In addition, the release of EGF stimulated the proliferation of endogenous NSPCs [162]. In addition to delivering EPO and EGF, HAMC hydrogels have shown good performance in delivering brain-derived neurotrophic factor (BDNF) [163] and in combination therapy(Fig. 6A) [164].

Fig. 5.

Hydrogel systems loaded with different bioactive molecules for treating IS. (A) HAMC hydrogel loaded with EPO, locally released EPO to attenuate the inflammatory response and promotes NSPCs survival to mediate nerve regeneration. Reproduced with permission from Ref. [161]. Copyright 2012, Elsevier. (B) HyStem®-C hydrogel loaded with BDNF could deliver BDNF to the brain in a targeted manner, and the inflammatory response was reduced in treated stroke rats. Reproduced with permission from Ref. [167]. Copyright 2018, MDPI. (C) Injecting HA hydrogel loaded with VEGF directly into the stroke cavity induced the formation of vascular and neuronal structures, thereby promoting the recovery of neurological function. Reproduced with permission from Ref. [176]. Copyright 2018, Springer Nature.

Fig. 6.

Scheme illustrating the combination treatment of IS with a hydrogel system containing two bioactive molecules. (A) HAMC hydrogel loaded with EPO and CsA achieved local and sustained co-release of bioactive molecules, accelerated functional recovery and improved tissue repair. Reproduced with permission from Ref. [164]. Copyright 2020, Elsevier. (B) HA-AC hydrogels loaded with PF127/BIO and PLGA/VEGF stimulated functional recovery after stroke. Reproduced with permission from Ref. [179]. Copyright 2022, Elsevier.

Neurotrophic factors are growth factors that promote neuronal survival and regulate central nervous system development. BDNF and nerve growth factor (NGF) are currently being studied in greater depth as potential stroke treatment options [165]. BDNF is a neurotrophic protein that promotes functional recovery in several post-stroke animal models. Cook et al. utilized BDNF-loaded hyaluronic acid hydrogels to locally release BDNF in the infarct cavity of a mouse stroke model for neurorestoration in the chronic phase after stroke. In a mouse stroke model, compared with the rapid 1-week release of directly injected BDNF, hydrogel-delivered BDNF continued to diffuse from the stroke lumen into the peri-infarct tissues over a 3-week period, achieving a slow release of BDNF that facilitated the recovery of motor function. Whereas in chronic stroke in nonprimates, hydrogel-released BDNF could be detected 2 cm from the infarction [166]. In another study, Ravina et al. delivered BDNF via HyStem®-C (thiol-modified hyaluronic acid and gelatin) hydrogel, through which a 3-week continuous release of BDNF could be achieved and most of the BDNF was released from the hydrogel on day 22, which allows for long-term BDNF therapy. The experimental results also showed that positive microglia and astrocytes were significantly reduced in stroke rats treated with hydrogel + BDNF, which suggests that the use of hydrogel-targeted delivery of BDNF to the brain can alleviate ischemic brain injury by reducing neuroinflammatory responses(Fig. 5B) [167]. In addition to this, NGF was added to the laminin-modified agarose gel, and the resulting trophic factor gradient stimulated directed synapse extension in the dorsal root ganglion (DRG) in the gel [168]. Meanwhile, in Wang et al.'s study, NGF-loaded hyaluronic acid hydrogel had good injectability, biodegradability, and excellent biocompatibility, and this hydrogel was able to reduce neuroinflammation at the site of injury and accelerate the healing process of the damaged brain tissue [169]. A recent study reported a multifunctional in situ hydrogel delivery system for co-delivery of antioxidants and NGF. After hydrogel injection in the IS model of rats, labeling of microvessels in the ischemic area by Glut-1 staining at week 6 showed that the hydrogel containing NGF had a significant effect on angiogenesis. Meanwhile, the hydrogel combined with antioxidants and NGF for the treatment of IS significantly improved the neurological deficits after cerebral ischemic injury in rats [170]. However, there are still some limitations of the above studies, which have identified behavioral effects, tissue biodistribution, and neurorestorative effects of hydrogels in different stroke models as well as in rodents and primates, and have demonstrated that BDNF/NGF-containing hydrogel induces axon germination and neurogenesis after stroke, but have not established a causal mechanism for the behavioral restorative effects.

Angiogenesis and reconstruction of the functional microvascular system are important and necessary for the restoration of nervous system function. Many studies have begun to focus on vascular endothelial growth factor (VEGF) as a therapeutic agent for nerve repair [[171], [172], [173]]. VEGF is expressed after ischemic brain injury and is involved in brain repair processes by regulating angiogenesis, neurogenesis, and neural protrusion growth [174]. In a study by George et al. they utilized a protein-based composite hydrogel system to load VEGF and matrix metalloproteinase-9 (MMP-9). The results showed that this system could mimic the effect of stem cells on functional recovery after ischemia and could control the release of molecules to enhance stroke repair [175]. Nih et al. ed designed VEGF-containing hydrogel biomaterials and injected them directly into the stroke cavity, which induces the formation of blood vessels and neuronal structures, thereby promoting neurological recovery [176]. The results showed that HA gel + hcV (high aggregation density of VEGF) induced the formation of robust, mature and highly developed vascular beds within the stroke lumen and patterned axons along these vessels growing inward(Fig. 5C). Similarly, a pH-sensitive peptide nanofiber-based self-assembled hydrogel prepared by Yanev P et al. following the loading of VEGF and Ang1 enhances post-stroke vascularization and neuronal survival in the peri-lesion region, ultimately promoting functional recovery [177]. In the latest study, injection of gelatin-norbornene nanofibrous (GNF) hydrogel alone improved sensorimotor function, treatment with GNF hydrogels containing vascular endothelial growth factor (VEGF) was more effective than GNF injections alone, and VEGF released from GNF hydrogels increased the mean size of lectin-positive blood vessels in the brain [109].

In addition, due to the extreme complexity of the pathological process of stroke, the development of combination therapies targeting different phases is an effective therapeutic strategy, leading to the sequential release of bioactive molecules. For example, Wang Y et al. designed a novel delivery system consisting of a HAMC hydrogel and polymer particles containing polyethylene glycolized epidermal growth factor (EGF-PEG) and EPO. Their study achieved the sequential release of EGF-PEG and EPO and stimulated endogenous NSPCs, thereby promoting nerve regeneration [178]. Similarly, Liu Y et al. developed an HA-based hydrogel delivery system in which glycogen synthase kinase 3 beta (GSK3β) inhibitor 6-bromoindirubin-3′oxime (BIO) nanoparticles were loaded with Pluronic F127 (PF127), and poly (lactic-co-glycolic acid) (PLGA) porous microspheres loaded with vascular endothelial growth factor (VEGF) to achieve sequential release of bioactive molecules jointly(Fig. 6B). In contrast, the larger-sized, water-insoluble PLGA microspheres, which release VEGF from them slowly and chronically at a later stage, enhance angiogenesis in the stroke region and promote the maturation of neovasculature and the formation of a network, which ultimately enhances the repair of brain tissue [179].

Hydrogels, an emerging therapeutic modality for ischemic stroke, can deliver bioactive molecules to designated sites in the brain, thereby reducing neuroinflammatory responses, promoting angiogenesis and improving motor function. However, the causal mechanism by which hydrogels containing bioactive molecules improve motor function is currently unknown. Therefore, future studies should focus more on exploring the mechanisms to determine whether hydrogels improve motor function through axonal sprouting, neurogenesis, or other mechanisms. In addition to this, achieving sequential release of different bioactive molecules will be a major research direction in the future, but the release kinetics need to be better explored and the physicochemical properties of hydrogels need to be explored to achieve long term slow release of bioactive molecules. Therefore, the preparation of hydrogel scaffolds that achieve slow, ordered release of bioactive molecules is a critical step in the treatment of ischemic stroke. In addition, the execution and monitoring of biomaterial intracranial treatments can be optimized when the location and fate of the implanted material is detected by imaging techniques (e.g., magnetic resonance imaging), bridging the gap between preclinical and clinical studies [177].

4.2. Hydrogels containing stem cells

In the core region of ischemic infarction, ionic imbalance and energy crashes due to ischemia and hypoxia in the brain lead to cellular necrosis and irreversible cellular damage within a few minutes after stroke [180]. The ischemic dark zone region, on the other hand, undergoes minor pathological damage such as apoptosis and neuroinflammation. Importantly, the ischemic dark zone has been shown in many studies to have the potential for active recovery after stroke. Thus, the ischemic semi-dark zone is now considered to be an important research and clinical target for ischemic stroke treatment [181]. Therefore, rescuing neurons in the ischemic penumbra, which is highly complex in time and space, has become a major challenge in ischemic stroke treatment. While stem cells, as a class of cells with the ability of self-renewal and differentiation, differentiate into new cells that can replenish the deficits at the site of injury, although stem cell therapy is currently in its infancy, it is still expected to bring substantial benefits to patients [182]. Although few clinical stem cell trials have been registered on the U.S. FDA website, only a few stem cell treatments have been approved to go to the clinic [183]. The most significant problem lies in the complexity of central system diseases and the unfavorable microenvironment for cell survival at the site of stroke lesions, resulting in cell transplantation treatments for stroke often facing problems such as low survival rates [29].

The high-water content of hydrogels and their porous structure are ideal for encapsulating cells and/or bioactive molecules. Hydrogel is an ideal carrier system for stem cells with good biocompatibility for long-term cell transportation and release of regulatory factors at damaged sites [184]. Hydrogel scaffolds can be injected to retain stem cells at the target location, i.e., targeted delivery. At the same time, the hydrogel can act as an immunoprotective barrier to protect the transplanted cells from host inflammation, thus supporting cell survival, proliferation, etc., in vivo [185]. Therefore, hydrogels can be a good delivery vehicle for stem cells to treat ischemic stroke. Currently, the main types of stem cells used for loading hydrogels are as follows: neural stem/progenitor cells (NSPCs), mesenchymal stem cells (MSCs), human-derived induced pluripotent stem cells (hiPSCs), etc. [186], of which NSCs and MSCs are the most common cell types used for transplantation to treat IS. Table 2 summarizes hydrogels containing stem cells and illustrates their cell culture modalities and outcomes after transplantation for IS(Table 2).

Table 2.

Summary of some representative hydrogel systems containing stem cells for use in ischemic stroke.

Reference	McCrary et al. (2020) [188]	Zhong et al. (2010) [190]	Arulmoli et al. (2016) [191]	Yao et al. (2019) [195]	Fernández-García et al. (2018) [114]
Outcome	CS-A encapsulation of NPCs significantly improved angiogenesis and vascular density in the ischemic tissue, and many of these outcomes were dependent on bFGF.	The hydrogel promotes survival of NPCs and diminishes the infiltration of microglia/macrophage cells into graft.	Combination scaffolds support hNSPCs proliferation and differentiation while significantly attenuating the cell-mediated degradation seen with fibrin alone,and it increase human endothelial cell-derived vasculogenesis.	CS-HEC-HA/GP hydrogel transplantation increases the retention and survival of hUC-MSC in lesion location, but also makes a positive contribution to the survival and proliferation of endogenous neural cells.	The silk fibroin hydrogel efficiently supports the survival of intracerebrally implanted MSCs and increases functional outcomes in a model of cortical stroke that affects the forepaw sensory and motor representations.
Culture condition	‘4−/4+’ retinoic acid (RA, 1 μM) neural differentiation protocol	ES cells were plated into 0.1 % gelatin-coated tissue culture plastic in N2B27 medium with daily additions of bFGF and EGF.	Basal medium: DMEM/F12, 20 % BIT 9500, and 1 % antibiotic/antimycotic.	DMEM/F12 containing 10 % FBS, 1 % penicillin–streptomycin, and 10 ng/ml of basic fibroblast growth factor	DMEM/F12
Cell type	iPSC-NPCs	ES-NPCs	hNSPCs	hUC-MSCs	MSCs
Hydrogel system	Chondroitin-4-sulfate A (CS-A) hydrogel	Hyaluronan–Heparin–Collagen Hydrogel	Salmon fibrin/HA/laminin composite hydrogel scaffolds	CS-HEC-HA/GP hydrogel	Silk fibroin (SF)-based hydrogel

NSPCs have great potential for neural tissue repair after stroke, and they can all differentiate into neurons that can replace damaged or necrotic cells. Previous studies have shown that chondroitin sulfate-A (CS-A) promotes the maintenance of undifferentiated neural stem cells [187]. Thus, McCrary MR et al. encapsulated neural progenitor cells (NPCs) in CS-A hydrogel and had more detectable grafted cells in the hydrogel + NPCs group 2 weeks after transplantation and a 3-fold increase in the total number of differentiated neurons compared to the direct transplantation group, suggesting that CS-A hydrogel improves retention of grafted cells to promote neuronal replacement in the lesion area [188]. In a recent study, their laboratory further found that CS-A hydrogels loaded with NPCs could affect the microglia/macrophage phenotype in mice after ischemic stroke, thereby promoting regeneration(Fig. 8A) [189]. In another work, HA/heparin/collagen composite hydrogels loaded with NPCs were able to support cell survival in the infarct cavity, with a 2-fold increase in cell survival using hydrogel grafts compared to the direct cell transplantation group, and a further reduction in microglia/macrophage infiltration [190]. Similarly, Arulmoli J et al. developed a salmon fibronectin/HA/laminin composite hydrogel scaffold with human neural stem/progenitor cells (hNSPCs) (Fig. 7A). This composite scaffold supported the proliferation and differentiation of hNPSCs and salmon fibronectin stimulated the proliferation of hNPSCs more than mammalian fibronectin [191]. Previous studies have shown that transplanted NSCs survive better and exert efficacy in the presence of neurotrophic factors. In a recent study, Qi Y et al. inoculated NSCs into poly(trimethylene carbonate₁₅-F127-poly(trimethylene carbonate₁₅) (PFP) polymer hydrogel scaffolds containing three neurotrophic factors, which were BDNF, NGF and neurotrophic factor-3 (NT-3). It was found that the PFP scaffolds containing neurotrophic factors induced the differentiation of NSCs into mature neurons and significantly reduced the infarct area. Therefore, this PFP scaffold capable of stably releasing BDNF, NGF, and NT-3 can support the survival and differentiation of transplanted NSCs, thereby improving the efficacy of stroke stem cell therapy(Fig. 8B) [192]. However, a number of barriers to this treatment remain, including whether and how other types of immune cells interact with hydrogels and encapsulated NPCS, and the fact that this therapeutic strategy has only been tested in small animal models and in a single therapeutic window. Further studies on other transplanted cell types, cell type-specific alterations, injury volume effects, blood-brain barrier permeability, and cell death will help to translate the cell encapsulation strategy to treat brain injury [189].

Fig. 8.

Hydrogel systems loaded with different stem cells for treating IS. (A) Cs-A hydrogel loaded with NPCs improved vascular regeneration after IS and modulated microglia/macrophage phenotype to further promote regeneration. Reproduced with permission from Ref. [189]. Copyright 2022, Elsevier. (B) PFP polymer hydrogels loaded with neurotrophic factors and NSCs could sustain the release of growth factors, which induced the differentiation of NSCs into mature neurons and significantly reduced the infarct size. Reproduced with permission from Ref. [192]. Copyright 2022, De Gruyter. (C) In a cortical stroke model affecting sensory and motor representations of the forepaw, MSCs-loaded silk fibroin hydrogel supported MSCs survival, enhanced neuroprotection after IS, and increased functional outcomes over time. Reproduced with permission from Ref. [114]. Copyright 2018, Frontiers.

Fig. 7.

Different hydrogels can support cell growth in vitro or regulate endogenous cell proliferation in vivo, among others. (A) Combined hydrogel consisting of salmon fibronectin with HA and laminin had material properties that can polymerize with cells, mimic native brain tissue, and support hNSPC functions. Reproduced with permission from Ref. [191]. Copyright 2016, Elsevier. (B) Self assembling silk hydrogel implanted in the brain do not cause microglia/macrophages to react and modulate glial scar formation. Reproduced with permission from Ref. [196]. Copyright 2019, American Chemical Society.

Mesenchymal stem cells are likewise an important cell source for stroke therapy. MSCs have a strong proliferative capacity, multidirectional differentiation potential and immunomodulatory ability [193]. Studies have reported that MSC combined with biomaterials have also shown improvement in dysfunction in IS. For example, Yan F et al. loaded bone marrow mesenchymal stem cells (BMSCs) into chitosan-collagen porous scaffolds and showed by staining that the graft complexes were integrated with the host about 10 days after transplantation, and it was possible to observe that the cells migrated a small distance to the surrounding area, which indicated a good integration of the graft complexes into the host [194]. In another work, researchers developed a novel thermosensitive hydrogel based on chitosan, hydroxyethylcellulose, hyaluronic acid, and β-glycerophosphate (CS-HEC-HA/GP). They loaded it with human umbilical cord mesenchymal stem cells (hUC-MSCs). This hydrogel remains liquid below 25 °C and rapidly transforms into a hydrogel at a body temperature of 37 °C. Compared with the MSC-only group, the hydrogel + MSC group exhibited more MSC cells, suggesting that the scaffold effectively protected the retention of hUC-MSC in the focal area and survival was able to last up to 28 days. Meanwhile, the hydrogel + MSC group also showed more MSC cells migrating toward the hippocampal region [195]. In addition, sericin protein are also excellent materials for nerve repair, and filaments can be triggered to self-assemble to form hydrogels. Studies have shown that sericin hydrogels do not induce an inflammatory response when implanted in the brain and are able to modulate the formation of neuroglial scarring(Fig. 7B) [196]. Consequently, another work demonstrated that sericin protein hydrogels containing MSCs could promote functional recovery in mice after stroke(Fig. 8C) [114].

Hydrogel-containing stem cells therapy for ischemic stroke is a highly promising therapeutic option that reduces infarct volume, supports the survival and proliferation of transplanted stem cells, and significantly improves neuroinflammation, vascular remodeling, and sensory-motor behavioral outcomes after IS. However, the immunomodulatory mechanisms of stem cell-containing hydrogels for the treatment of IS are currently unclear. Also, the interaction between endogenous and transplanted cells will have a major impact on the treatment, which could be a major direction for future research. In addition to this, determining which type of stem cells are best for IS is crucial and further research is needed to determine the optimal timing of treatment to maximize nerve repair. Therefore, designing ideal hydrogel scaffolds that can promote stem cell survival, proliferation, and differentiation after transplantation is crucial for stroke therapy. Of course, new therapeutic scaffolds combining growth factors and stem cells are emerging as the materials continue to be upgraded, indicating that composite hydrogel scaffolds will eventually have even more promise.

4.3. Alternative forms of carrier-based hydrogel therapy for IS

Carrier-based hydrogels have been developed in a variety of forms beyond the loading of bioactive molecules or stem cells. These include, but are not limited to, composite hydrogels, stimuli-responsive hydrogels, nanogels, and hydrogel microspheres, among others. This subsection will provide a brief analysis of other forms of carrier-based hydrogels for therapeutic IS.

In recent years, neural tissue engineering, a composite therapeutic strategy that combines biologically active molecules, relevant cells, and biomaterial scaffolds, has gained increasing attention because it improves cell survival in the injured area and supports better integration of the cells with host tissues, while also achieving sustained release of bioactive molecules, thus improving the shortcomings of single treatment modalities.

Therefore, Wang et al. synthesized a new GelMA-T hydrogel by introducing taurine on the basis of methacrylated gelatin (GelMA) hydrogel. This hydrogel possesses both neuroprotective properties and the ability to promote the proliferation of neural stem cells at the same time. The in vitro results showed that GelMA-T hydrogel could be used to culture neural stem cells under 3D conditions for 7 days and could maintain the viability of neural stem cells and promote their proliferation [197]. And McCrary MR et al. prepared a CS-A hydrogel encapsulated with NPCs and bFGF based on previous studies. The results showed that the CS-A hydrogel improved transplant cell retention to facilitate the replacement of neurons in the stroke region; because bFGF has a strong angiogenic effect, CS-A acts via bFGF to potentiate NPC-mediated angiogenesis and vascular remodeling, blood flow recovery, ultimately led to the restoration of sensorimotor function in mice after IS [188]. Similarly, Zheng Y et al. developed an injectable hydrogel using imidazole group-modified gelatin methacrylate (GelMA-imid) containing stromal cell-derived factor (SDF-1α) and human amniotic membrane mesenchymal stromal cells (hAMSCs) encapsulated with polydopamine (PDA) nanoparticles. In vitro results showed that the hydrogel extract co-cultured with cells for 3 days, in which the imidazole moiety and SDF-1α acted synergistically, could promote the continuous migration of hAMSCs toward the injured area as well as their directed differentiation into neural cells. In vivo modeling results indicated that GelMA-imid/SDF-1α/hAMSCs hydrogel transplantation minimized the area of injury and was able to promote the regeneration of endogenous neuronal cells and ameliorate brain injury [198]. In addition to this, in another study, an injectable hyaluronic acid hydrogel (HT hydrogel) was synthesized using a dual enzyme cross-linking technique and used as a neural scaffold to deliver BMSC and NGF. Firstly, it was verified in vitro that HT hydrogel has good cell and blood compatibility, then it was confirmed in vivo that HT hydrogel combined with BMSC and NGF group could promote the secretion of neurotrophic factors and enhance the survival and proliferation of endogenous neuronal cells, and finally, it was confirmed by the modified Neurological Severity Score (mNSS) and the Morris Water Maze (MWM) that the combined treatment group improved the recovery of learning and memory functions in the brain-injured mice, and accelerated the healing process of damaged brain tissues [169]. And in the latest study, Zheng et al. showed that VEGF and adipose-derived stem cells (ADSCs) were co-encapsulated in a chitosan-hyaluronic acid hydrogel scaffold, and the hydrogel was subsequently injected into rats with cerebral ischemia-reperfusion injury. Hydrogel scaffolds containing VEGF enhanced the paracrine function of ADSCs, with an optimal VEGF concentration of 50 ng/ml. This combined therapeutic approach, therefore, reduced the effects of the ischemic microenvironment on the grafted stem cells and enhanced the therapeutic efficacy of ADSCs for the treatment of ischemic stroke, including a significant reduction in neuronal apoptosis and contributed to the maintenance of mitochondrial integrity and axonal morphology [199].

In addition to compounding therapeutic strategies, scientists have taken inspiration from nature to develop carrier-based hydrogels that respond to external stimuli. These hydrogels are capable of sensing and responding to external stimuli, including physical stimuli such as temperature, light and electric fields; chemical stimuli such as pH and ionic strength; and biological stimuli such as enzymes and DNA [27]. Stimuli-responsive hydrogels can then detect biochemical alterations in the diseased microenvironment and respond in a predefined manner to alleviate or improve stroke symptoms [200].

For example, Nourbakhsh et al. developed an electroactive hydrogel containing VEGF with conductivity similar to that of brain tissue, which can mimic the electrical properties of brain tissue. The results showed that this electrically conductive hydrogel could release VEGF in a sustained manner, and the infarct area of VEGF hydrogel-treated rats was reduced by more than 70 % compared to the control group [201]. In another study, a thermosensitive hydrogel containing ciliary neurotrophic factor (CNTF) was designed and developed. At a physiological pH of 37 °C, the material can undergo a sol-to-gel transition in a matter of minutes, allowing the hydrogel to serve as an effective platform for localized drug delivery. In in vitro studies, CNTF can be released continuously for 9 days [202]. In a study by Moshayedi et al., a matrix metalloproteinase (MMP)-sensitive cross-linking agent was doped into a HA hydrogel, and the results showed that more pronounced blood vessel growth was observed in the hydrogel doped with the MMP-sensitive cross-linking agent, and that this hydrogel promoted the survival of loaded human neural progenitor cells in the region of injury [203]. In another work, Jian WH et al. designed the nanohybrid hydrogel containing sulfated glycosaminoglycan-based polyelectrolyte complex nanoparticles (PCN). It can mimic the brain extracellular matrix and control the delivery of SDF-1α and bFGF in response to MMP for recruiting endogenous neural stem cells (NSC) and regulating their cellular fate. They modified MMP scavenging peptides and MMP-active peptides onto the nanoparticles and then achieved the controlled release of the loaded two growth factors by selectively cleaving the MMP scavenging peptides. The study demonstrated that MMP-responsive hydrogel was able to enhance the release of SDF-1α and bFGF, and the release of growth factors promoted the recruitment and migration of endogenous NSCs to the site of injury, thereby enhancing angiogenesis and neurogenesis [204].

Due to the complexity of the brain and the presence of the BBB, carrier-based hydrogels are typically delivered by in situ injection. This has led to increased interest in nanohydrogels or hydrogel microspheres that can somehow be injected through blood vessels to penetrate the BBB and target the site of injury. For example, in a previous study, Yang's lab synthesized hollow nanogels loaded with urokinase-type plasminogen activator (uPA) via a one-step reaction in conjunction with ultrasound for the treatment of IS. Nanogels extend in vivo circulation time of proteins compared to intravenous injection and can be sonically triggered to release proteins at a faster rate [205]. At the same time, the nanogel has excellent blood-brain barrier protection and does not increase the risk of cerebral hemorrhage [206]. In a recent study, Wilson et al. developed highly porous microgels and microgels containing heparin-norbornene nanoparticles with covalently bound SDF-1α. The results of the study showed an increase in the vasculature as early as 10 days after treatment with the microgels, in addition to an increase in neural progenitor cell recruitment, maintenance and neuronal differentiation [207]. And in a recent study, Xu et al. developed an immunomodulatory hydrogel microsphere containing interleukin-4 (IL-4). In vivo experiments, by injecting the immunomodulatory hydrogel microspheres into IS mice, an increase in the number of NeuN neurons and an increase in the expression of the anti-inflammatory factor CD206 were found after 7 and 14 days, which led to a reduction in the volume of brain atrophy and promoted neurobehavioral recovery [208].

The complexity of ischemic stroke necessitates consideration of numerous factors and targets when developing effective treatment strategies. Composite hydrogels, a form of neurotissue engineering, have emerged as a promising therapeutic approach due to their ability to address a range of needs and targets in the treatment of ischemic stroke. However, there are still many questions that need to be answered about combination therapies, such as which combination of stem cells and bioactive molecules will provide the best results; the specific functions of the different components and the mechanisms by which they exert their functions are still unclear; the interactions between bioactive molecules and stem cells are still unknown. In addition, the long-term therapeutic efficacy of hydrogels needs to be further validated in vivo, and the fate of hydrogels after they have fulfilled their purpose also needs to be considered. It is also possible to consider combining hydrogels and stimulus response to prepare smart hydrogels. In this way, the hydrogel will be able to sense the differences in the environment to respond accordingly, bringing more ideas for treatment. In addition to this, it may also be beneficial to prepare hydrogels as stimuli-responsive hydrogels, nanogels or microspheres, which could facilitate injection during surgical procedures and potentially reduce stress on the brain. Consequently, future research could focus on combination therapies and novel therapies to develop hydrogels with specific functions for multi-targeted therapies.

5. The physicochemical properties of carrier-based hydrogels for IS therapy

Ischemic stroke is usually caused by a thrombus that occludes cerebral blood vessels and is the primary type of stroke associated with long-term neurological impairment and a poor prognosis [209]. With successive ischemia, nutrient and oxygen deprivation, secondary mitochondrial dysfunction, glutamate excitotoxicity and calcium overload eventually lead to neuronal death. Despite the fact that new therapeutic strategies for IS continue to be proposed around the world, epidemiologic surveys in recent years have shown that its morbidity and mortality are still on the rise [210]. Nevertheless, there are currently no effective clinical treatments that can improve function after IS. However, hydrogels show considerable potential for treating IS, as they can be customized to mimic the properties of the brain's ECM. At the same time, the carrier-based hydrogel is capable of delivering bioactive molecules and stem cells to the site of injury, thereby modulating neuroinflammation and promoting tissue regeneration at the injury site.

The brain is a highly complex organ, and its extracellular matrix is synthesized and secreted by neurons and glial cells. These cells form highly organized extracellular structures [211]. Understanding this complexity is necessary to design and customize carrier-based hydrogels that are more suitable for IS. Therefore, it is important to consider the physicochemical properties of hydrogels, as the success of their function and the fate of the transplanted stem cells and bioactive molecules will depend on them. In this section, we will focus on the physicochemical properties (e.g., biocompatibility, biodegradability, modulus of elasticity, porosity and pore size, etc.) that need to be fulfilled by a carrier-based hydrogel for IS therapy and will elaborate on the implications of these properties for neural tissue regeneration (Fig. 9).

Fig. 9.

The Physicochemical properties of carrier hydrogels for IS therapy. (1) Biocompatibility is the most basic requirement for hydrogels, hydrogels with good biocompatibility can integrate better with the host; (2) Modulus of elasticity plays an important role in guiding the differentiation, proliferation, and growth of neural lineages; (3) Porosity and pore size are important parameters for the exchange of nutrients and waste between cells and external tissues, which also can affect the degree of inward growth of cells; (4) Injectable and self-repairing, which can make hydrogels bypass BBB to reach the stroke cavity through local injection, and fill irregular brain defects; (5) Conductivity, which can further enhance the effect of hydrogels for IS treatment.

5.1. Biocompatibility

In order to successfully deliver stem cells or bioactive molecules to the injured brain, the hydrogel should consider the biocompatibility profile at both cellular and tissue levels [212]. Specifically: histocompatibility of the implanted hydrogel with the local and/or systemic response of the host, and cytocompatibility of the hydrogel with the loaded cells, both in vivo and in vitro [213]. In a previous study, researchers injected filipin protein hydrogels into the striatum of mice to assess their biosafety. The results showed that in situ filipin protein hydrogels exhibit excellent biocompatibility and can coexist with complex neuronal circuits responsible for controlling sensorimotor functions, learning and memory - well complex mechanisms. Some chemical additions help increase the hydrogel's help increase the hydrogel's cytocompatibility [113]. For example, Arg-Gly-Asp (RGD), which is currently the most commonly used adhesion peptide, enhances cell adhesion and cell viability when added to hydrogels [214,215]. At the same time, the growth of both two- and three-dimensional neuromasts showed a concentration dependence on RGD [216]. Thus, biocompatible hydrogels transplanted into the brain integrate better with the host. For example, IKVAV-functionalized modified HA hydrogels can form permissive interfaces with tissues to facilitate inward cell growth and angiogenesis 6 weeks after brain implantation [217]. The biocompatibility of hydrogels determines their ability to serve as scaffolds for nerve growth cues in vivo.

5.2. Modulus of elasticity

The elastic modulus of a gel depends mainly on the network structure formed by the hydrogel [117]. The elastic modulus of hydrogels affects the fate of loaded cells, such as stem cell differentiation [212,[218], [219], [220]]. The modulus of elasticity (E) of typical brain tissue ranges from 0.1 to 1 kPa [221,222]. In a previous study, the elastic modulus of methacrylamide chitosan (MAC) hydrogels differentially affects the differentiation and proliferation of neural stem and progenitor cells (NSPCs). Neurons differentiated by NSPCs survive better on soft substrates, while oligodendrocytes are preferred on harder hydrogel [223]. Similarly, polyacrylamide and fibronectin hydrogels with a stiffness of approximately 250 Pa supported neurite growth with a greater abundance of neurites; whereas astrocytes had a larger spreading area on stiffer polyacrylamide and fibronectin hydrogels [224]. In a study by Massensini et al. the rheological properties of ECM hydrogels were analyzed and it was found that higher concentrations (8 mg/ml) of ECM hydrogels were more likely to be retained in the brain, with a modulus of elasticity of 500–1000 Pa, which is similar to that of brain tissue [225]. The implications of these studies suggest that fine control of the mechanical properties of hydrogels is particularly important in directing neural cell lineage differentiation, proliferation and growth.

5.3. Porosity and pore size

The porosity and pore size of hydrogel mainly depend on the concentration of hydrogel [226,227]. Pore size is particularly important when encapsulating cells in hydrogels, as cells must be able to cross hydrogel boundaries to exchange nutrients and waste with external tissues [212]. Pore size is the main factor influencing cell adhesion [228,229]. Suitable porosity and pore size create conditions for inward cell migration and can affect the extent of inward cell growth and the quality of cells within the gel [230]. It has been shown that neural stem cells in hydrogels with larger pore sizes can have higher proliferation rates, more pronounced migration, and promote their directed differentiation into neurons [217,231]. In addition to this, a strong correlation was found between the ability of sensory ganglia to extend neurons in three-dimensional gels and hydrogel pore size in the study of George P [227]. A study by Zheng et al. showed that imidazole-modified GelMA hydrogels with a mean pore size of 204.61 ± 41.41 nm were suitable for stem cell migration, proliferation and differentiation, and the in vivo experiments showed that the use of the hydrogel to treat brain injury resulted in the smallest area of damage [198]. In addition, the degree of hydrogel porosity also affects the modulus of the hydrogel, i.e. gel strength decreases with increasing porosity, so a suitable porosity is required [232].

5.4. Injectability and self-repairability

Injectable hydrogels can be injected locally to bypass the BBB and reach the stroke space, suggesting that hydrogels have shear-thinning properties and can be deformed under applied stress and injected through a syringe, where they then reassemble into an elastic gel network and protect cells from damage during injection [233]. And self-repairing properties are essential for stabilizing filled tissue defects after gelation. Self-repairing hydrogels with semi-interpenetrating polymer networks (SIPN) were prepared by doping HA into chitosan-based self-repairing hydrogels. This self-repairing hydrogel was loaded with NSCs and delivered to a zebrafish model of traumatic brain injury and a rat model of cerebral hemorrhage to investigate the therapeutic effects of the hydrogel. The results showed that the SPIN of the hydrogel provided an adaptive environment for cell spreading and migration and could be used as a scaffold to fill irregular brain defects [234]. A number of studies have highlighted the importance of shear thinning and self-repairing hydrogels in promoting cell survival, migration, proliferation and differentiation in the CNS [190,235].

5.5. Conductivity

Conductivity is used to demonstrate the ability of hydrogels to conduct electrical currents. The nervous system is a bridge for the transmission of "information", so restoration of nerve conductance is an important goal of CNS repair [236,237]. Currently, the most common method to increase the conductivity of hydrogels is to blend them with conductive polymers, e.g., polypyrrole (PPy), polyaniline (PANI), etc. [238,239] In a study by Rinoldi et al., conductive polymer hydrogels were able to support the survival of neurons and astrocytes [240]. In addition to this, the incorporation of conductive polymers was able to induce the differentiation of human neural stem cells into neurons [241]. Similarly, in vivo studies have shown that the electrical conductivity of the scaffolds further alters gene expression in the cells, and in vivo results showed that electrically pre-treated hNPC-bound scaffolds enhanced nerve repair after stroke [242].

Therefore, the rational design of hydrogels with elastic modulus comparable to host brain tissue, porous, self-repairing and biocompatible is essential to improve the efficacy of IS therapy. At the same time, hydrogels with these physicochemical properties, when used as carriers for bioactive molecules or stem cells, can better release bioactive molecules and promote the proliferation and migration of stem cells.

6. Challenges and Prospects of Hydrogel for IS applications

Hydrogel has demonstrated its unique advantages in the treatment of IS. First, hydrogel has good biocompatibility, injectability, and self-healing properties, and can fill irregularly shaped lesion cavities and provide mechanical support. Secondly, hydrogel can be used as an excellent carrier for bioactive molecules and stem cells, realizing the spatio-temporal sequential release of different molecules as well as the complex therapy of bioactive molecules and stem cells, thus greatly enhancing the therapeutic effect. Third, the hydrogel's physicochemical properties are malleable and can be designed to meet the specific microenvironmental needs after IS, which further enhances the hydrogel's ability for neural repair. However, hydrogel has some limitations in the treatment of IS, as well as many challenges in clinical translation.

First, hydrogel scaffolds must meet clinical criteria. Currently, most studies have only been validated in animal models, and there is a lack of substantial clinical trial data to validate effectiveness and safety in humans. There are currently two FDA-approved clinical trials on biomaterials for the treatment of IS (NCT02767817 and NCT04083001) (https://www.clinicaltrials.gov/). The former is to evaluate the safety and efficacy of injectable collagen scaffolds combined with mesenchymal stem cells transplantation for the treatment of patients with brain injury. The latter is to evaluate the safety of RGTA® (ReGeneraTing Agent) aka heparan sulfate analog, a polymer specifically designed to mimic endogenous heparan sulfate in the damaged extracellular matrix to ensure tissue regeneration. Most of these clinical trials remain in the validation of the biosafety of the material. Therefore, in the future, the focus should be on developing hydrogel scaffolds that are compatible with human brain tissue and have better cellular affinity and can progress toward clinical effectiveness trials as soon as possible.

Second, the human brain is extremely complex and variable, with varying tissue mechanical strength, multiple cell types, and ECM molecules. Current research is limited to small animal models, and there are many issues and challenges in translating this to large animal models with complex vascular systems, innervation, and other micro- and macro-structures, as well as to complex human biological systems. In addition to this, IS is more prevalent in the elderly and may coexist with other comorbidities in patients, but current models are limited to a single disease based on young adult rodents [28]. Therefore, there is still a large gap between this, and the actual clinical pathology faced. In the future, there is a need to develop animal models that better fit the actual clinical situation to further validate the safety and effectiveness of carrier-based hydrogels.

Third, there are some practical problems that need to be solved when hydrogels are used as carriers for drugs or bioactive molecules. Firstly, the drug loading efficiency of carrier-type hydrogels is too low, due to poor tensile strength, resulting in premature dissolution or efflux of the hydrogel from the target tissue, thus failing to achieve the required therapeutic dose [243]. Secondly, the high porosity of hydrogels and the limited control of the material properties often lead to rapid and sudden drug release, which limits the application of hydrogels for long-term drug release [244]. In the future, various properties such as elastic modulus, porosity, degradation rate, and cell adhesion should be optimized and integrated in the design of hydrogel scaffolds, so that hydrogel scaffolds with optimal functionality can be prepared.

Fourth, the lack of proper spatial and temporal control of hydrogel as a carrier for stem cells leads to poor cell penetration and uneven cell distribution, while the lack of a complex microvascular system in hydrogels in general results in poor blood vessel formation and insufficient transportation of nutrients, leading to the loss of cell viability and function at the time of transplantation [25]. In addition, stem cell transplantation also still faces some problems, such as the generation and expansion of hiPSCs is troublesome and needs to be optimized [245]; the inefficient reprogramming efficiency and immunogenicity of iPSC also challenge its clinical translational application [246]. Therefore, new hydrogel scaffolds need to meet the requirements of iPSC derivation, amplification, and differentiation, thus providing a favorable microenvironment to promote iPSC differentiation.

Apart from these, there are no clinical trials based on hydrogels for the treatment of IS, which suggests that hydrogels are still a huge translational gap away from clinical trials. There are many more challenges that hydrogels need to face in the process of translation. The first is ensuring biosafety. The short- and long-term effects of many materials when implanted in the brain are still unknown, so a major challenge is the possibility of an immune response. The second is that there is no good way to assess the properties of hydrogels in vivo and how well they function. Also, there are also various regulatory hurdles to consider, such as immunogenicity, teratoma formation, etc., when introducing cells [247]. Fourth, use for clinical applications also poses a challenge to the scalability of hydrogel production, where large-scale production must ensure consistency and reproducibility, which is critical for regulatory approval and clinical success [248]. Of course, patient-specific issues need to be considered above all else in clinical trials, and a large number of clinical trials and comprehensive evaluations are needed to ensure the efficacy and safety of hydrogels in different patient populations (Fig. 10).

Fig. 10.

Challenges and Prospects of Hydrogel for IS Applications. (1) Current challenges: lack of substantial clinical trial data; There is a significant gap between animal models and clinical practice; The efficiency of drug loaded hydrogel is too low to control the release; Immunogenicity of iPSCs; Regulatory barriers; Large scale production cannot guarantee consistency and repeatability, etc. (2) Future prospects: Optimize various performance and conduct clinical trials as early as possible; Develop animal models that are more in line with clinical conditions; Optimize the release mechanism of bioactive molecules; Customize treatment based on individual patients; Combining other therapies to promote functional recovery; Strengthening cooperation to promote clinical translation, etc.

Looking ahead, hydrogel-based studies for ischemic stroke treatment aim to explore innovative solutions and address current problems. First, we need to focus on developing hydrogels that are compatible with human brain tissues and have better cellular affinity, and adjusting their elastic modulus, porosity and other properties, so as to optimize the release mechanism of bioactive molecules and provide a better microenvironment to promote the proliferation and differentiation of stem cells. In addition, it is also necessary to develop animal models that are more suitable for clinical practice to further verify the safety and effectiveness of carrier hydrogels. Meanwhile, the use of noninvasive imaging techniques to detect and monitor hydrogels can provide important information about the functional status of implants [249]. To date, several methods have been developed that allow for the detection of hydrogels by various imaging techniques without compromising their rheological properties and biocompatibility [250,251]. In the future, non-invasive imaging techniques should be considered in conjunction with hydrogels to better visualize how they play out in vivo in real time.

Of course, personalized hydrogel therapies tailored to the specific needs of individual patients and stroke subtypes are expected to increase therapeutic efficacy and reduce side effects [252]. In addition, hydrogel therapy can be combined with other therapies, such as rehabilitation strategies, to synergistically enhance neuroprotection and functional recovery after stroke [74]. Also, increased collaboration between researchers, clinicians, and regulatory agencies is critical for the clinical translation of hydrogels, which can be accelerated for carrier-based hydrogels (Fig. 10).

In conclusion, the carrier-based hydrogels discussed in this review show great potential for nerve repair and regeneration after stroke and offer a unique approach to the treatment of IS. Despite the challenges, if we can overcome these obstacles, hydrogels could significantly improve the treatment of ischemic stroke and effectively improve patients' quality of life.

CRediT authorship contribution statement

Wenqi Yin: Writing – original draft, Conceptualization. Yuchi Jiang: Software. Guangrui Ma: Writing – review & editing, Software. Bricard Mbituyimana: Writing – review & editing, Software. Jia Xu: Writing – review & editing. Zhijun Shi: Writing – review & editing, Supervision. Guang Yang: Writing – review & editing, Supervision, Project administration, Funding acquisition. Hong Chen: Writing – review & editing, Supervision, Funding acquisition.

Ethics approval and consent to participate

This is no ethics approval and consent to participant involved in this article.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.