Exploration of biomaterial and stem cell-based strategies for promoting neuronal regeneration and creating engineered 3D in-vitro disease models

Abstract

The adult brain can produce only a relatively small number of new neurons, and neurogenesis occurs only in specific brain areas. Post-neuronal injury self-repair is very slow and sometimes does not occur. Therefore, advancements in regenerative therapies play an essential role in recovery from damage. This review focuses on stem cell-based neuronal repair by using biomaterials. We discuss neural tissue damage and the associated mechanisms in neuronal repair, highlighting the role of B cells along with VGLUT1/VGLUT2 (vesicular glutamate transporter 1/2) terminals and glutamate AMPA receptors, IL-1R1 signalling, and ERK/Stat6/MERTK Signalling. Furthermore, it focuses on the types of biomaterials, their characteristics, and mechanisms to overcome neuronal damage repair challenges using modern biomaterial-dependent neuronal tissue engineering techniques. Axonal regeneration can be enhanced by mixing many components (biomaterials, cells, and chemicals) to recover from neural nerve illnesses. Polymers, such as collagen, gelatin, chitosan, alginate, hyaluronan, silk fibroin, poly(L-lactic acid), poly(glycolic acid), polycaprolactone, polyphosphoester, and polyurethane, have been used to aid nerve cell growth. These polymers can be either natural or synthetic. Biomaterials that conduct electricity, such as polypyrrole, polythiophene, and polyaniline, can help neurites grow and make cells more active because they carry electrical impulses that help nerve signal travel. The primary goal of this review is to examine the current methods and uses of brain tissue engineering techniques, which include aspects of stem cell-based 3D in vitro and in vivo models, translational efforts, and challenges in clinical applications.

Introduction

Chronic neurodegenerative diseases including Parkinson’s disease (PD), Huntington’s disease, Alzheimer’s disease, and amyotrophic lateral sclerosis, as well as acute insults like ischemic and hemorrhagic stroke, are becoming extreamly prevalent as the population ages. Each of these conditions is characterized by cognitive, sensory, and/or motor impairments that are caused by loss of neuronal subpopulations [1, 2]. Neurons possess a delicate structure, which makes them highly sensitive. Minor injuries or diseases can disrupt the intricate network of dendrites, axons, and synapses. This sensitivity extends beyond physical damage, including biochemical imbalance, oxidative stress, and inflammatory responses [3, 4]. Severe physical injuries and neurodegenerative diseases are associated with irreversible damage and lack of function of the central nervous system (CNS). The inability of mature mammalian central nervous system neurons to regenerate has been observed in vertebrates. In early development, the CNS can repair itself to a limited extent, but its capacity for spontaneous regeneration is significantly diminished after childbirth [5]. Regenerative medicine uses tissue engineering and neural stem cell transplantation to promote native tissue regeneration and restore functional loss due to illness or injury. Stem cells can self-renew, increase, and specialize in several cell lineages; therefore, they have emerged as a possible treatment option for neurodegenerative illnesses, including mesenchymal stem cells (MSCs), progenitor cells, and embryonic stem cells (ESCs), which promote tissue regeneration by accelerating tissue homeostasis, metabolism, growth, and repair [6, 7]. Mesenchymal stem cells (MSCs) are the most suitable stem cell lines because of their low immunogenicity, accessibility, and reasonable quantity. Typically, the bone marrow (BMSCs), umbilical cord (UC-MSCs), and adipose tissue (ASCs) are used for their production. They are generally obtained from bone marrow (BMSCs), umbilical cord (UC-MSCs), and adipose tissue (ASCs). There are several constraints to the medicinal use of MSCs, despite numerous research and clinical trials on MSCs for various neurodegenerative disorders (NDs). The complexity of the nerve tissues impedes the future potential of many stems cell-based treatment techniques. The most significant problem is the need for correlation between stem cell activity in vitro and in vivo, as their differentiation depends critically on their microenvironment. The use of biomaterials in both in vitro and in vivo investigations has helped overcome this difficulty. Developments in tissue engineering, cell transplantation, and regenerative medicine have enabled the use of biomaterials for brain repair. Stem cells, biomaterials, and neurotrophic factors derived from specific nerve grafts are more efficient, and can significantly enhance the regenerative process. Neurotrophic factors, such as nerve growth factor, brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor, and neurotrophin-3, have been used extensively [8, 9].

Natural polymers, such as chitin, chitosan, collagen, alginate, and gelatin, and synthetic non-degradable polymers, such as silicone, synthetic biodegradable polymers, and conducting polymers, are used in different neuroregeneration procedures. One of the exclusive uses of polymers (natural and synthetic) in neurodegeneration is to construct a nerve conduit, which should ideally be thin, flexible, porous, biocompatible, biodegradable, compliant, neuroinductive, neuroconductive, and have suitable surface and mechanical characteristics [10]. As all first- and second-generation biomaterials have disadvantages, a surface-eroding polymeric scaffold is intended to provide more consistent contact-guiding signals for nerve regeneration. This is supported by a recent study using polyglycerol sebacate (PGS) as a nerve guide material because of its surface erodible and elastomeric qualities [11]. Mechanical stability and scaffold compatibility are critical qualities required for effective transplant absorption by host tissues. In nerve tissue engineering, the ideal scaffold should be malleable, safe for the tissue microenvironment, structurally sound, and falling, which might cause necrosis and inflammation after implantation. Many methods have been proposed for modifying the characteristics of popular biomaterials to make them acceptable for neural tissue engineering. A flexible poly(lactic-co-glycolic acid) PLGA scaffold was created using a micro-braiding approach to increase flexibility and porosity [12]. Studies have shown that thermal treatment can be used to join fibers to enhance the biomechanical characteristics of electrospun poly(ɛ-caprolactone) (PCL) scaffolds. At temperatures between 54 and 60 °C, thermal fiber bonding was carried out in Pluronic F127 solution. The shape, fiber diameter, pore area, tensile characteristics, suture retention strength, burst pressure strength, and compliance variations of thermally bonded electrospun PCL scaffolds have been examined [13].

In recent years, the merging of biomaterial science and stem cell technology has opened new avenues for neural regeneration and in-vitro disease modeling. In this review, we aim to explore the innovative aspects of biomaterial and stem cell-based strategies in neural repair. We will first examine the essential characteristics of biomaterials that make them suitable for stem cell-based neuronal regeneration, with a focus on naturally occurring biomaterials, synthetic biomaterials and conductive polymers. These materials will be assessed for their potential in facilitating neural repair. Furthermore, we will delve into the novel use of polymeric scaffolds as extracellular matrix analogues in neuronal repair, which play a crucial role in supporting stem cell attachment, growth, and differentiation within neural tissues. The review will also explore combinative strategies that enhance stem cell-mediated neural repair by integrating biomaterials with other therapeutic approaches, such as drug delivery systems, to improve the efficacy of stem cell therapies. Finally, we will discuss how biomaterial-based vascular support systems can be optimized to promote stem cell and neuronal repair, highlighting the emerging role of biomaterials in creating vascular networks that ensure the survival and integration of transplanted stem cells into damaged neural tissues. Through these discussions, this review seeks to provide a comprehensive overview of the current state of biomaterial and stem cell-based strategies in neural regeneration and in-vitro disease modeling, identifying key areas for future research and development (Fig. 1).

Fig. 1.

Factors contributing for neuronal injury. Neuronal damage may be induced by increased oxidative stress, triggering neuroinflammation. Additionally, neurodegenerative diseases, especially Alzheimer’s and Parkinson’s disease may lead to morphological changes in the brain. Metabolic diseases such as diabetes, cardiac conditions, obesity, and cancer can disrupt the normal functioning of the nervous system. The harmful effects of neurotoxins like botulinum toxin (BoNT), tetrodotoxin (TTX), and tetanospasmin (TeNT) result in damage to neurons, axons, and glia, leading to demyelination and subsequent neuronal injury. Environmental factors are significantly associated with neuronal complications, while Mechanical trauma, whether internal or external, can inflict physical injury, ultimately damaging the neurons. Genetic factors also play a role in neuronal development, contributing to the overall impact on neuronal damage

Neural tissue damage and physiological repair mechanisms

Neurons are susceptible to injured by a variety of injuries or insults, including stroke, ischemia, neurological disorders, optic nerve injury, spinal cord injury, and traumatic brain injury (TBI). To keep the adult nervous system functioning, it takes a lifetime to learn how to react to these assaults and fix damage. Neurons have a range of coping mechanisms depending on the kinds and severity of shocks. New synapses are formed, neurites grow again, and regenerated neurites re-associate with their synaptic partners as part of the neurite healing process. Neurite repair is challenging due to the restrictive environment in the adult nervous system, particularly in the central nervous system (CNS), which often limits the formation of differentiated neurons. Neurite repair can be inhibited by both cell-autonomous and non-cell-autonomous causes. For instance, glia cells and other nearby cells emit non-cell autonomous chemicals that prevent regenerated neurite from innervating the appropriate destinations. Inactivating developmental growth programs prevents neurite renewal in adult neurons as part of the cell-autonomous controls [14]. Direct injury triggers a cascade of cellular and molecular events. The reasons for these events can be chemical, biological, or physical. Typical causes include infectious diseases, toxins, strokes, neurodegenerative diseases, and traumatic brain injuries (TBIs) [15]. Secondary injuries can lead to neuronal damage and dysfunction. This damage is caused by apoptosis, excitotoxicity, mitochondrial damage, oxidative stress, and neuroinflammation [16, 17]. The ability of neural tissues to regenerate is comparatively lower than that of other tissues. However, the central nervous system (CNS) can only heal to a limited extent. Healing mechanisms include neuroplasticity, neurogenesis, remyelination, axonal sprouting, angiogenesis, and immunological responses. In reaction to damage, neurons have to shift from the inhibitory state to active growth in order to get around limitations on neurite repair [15, 17].

Transmembrane ion channels enable ions to flow through cell membranes.In addition to being engaged in numerous elements of developmental processes, such as neurite outgrowth and plasticity, they are crucial for regulating the excitability of neurons. Does neurite repair also include ion channels. The fundamental mechanism for how control of ion flow may affect neurite healing in response to injuries is revealed by recent research on TRPV1, Piezo, voltage gated calcium channels (VGCCs), and potassium channels. Whereas one of the most novel discovered ion channels such as mechanosensitive ion channels (MIC) are responsible for the process of mechanotransduction, which is the conversion of both external and internal physical inputs into biochemical signals [2, 3]. Vertebrates can feel touch, hear auditory cues, move their bodies, and feel pain because MIC is present in their cell membrane along with that It also helps to control bone and muscle. Eukaryotic cells also include several forms of MIC, such as transient receptor potential (TRP) channels, epithelial sodium channels/Degenerins family (ENaC/DEG), and two-pore domain potassium (K2 P) channels, which serve as sensors for organs subjected to mechanical forces. ENaC (epithelium sodium channel), DEG (degenerins), ASIC (acid sensitive ion channel), and FaNaC (FMRF amide gated sodium channel) are the four classes that make up the MIC/DEG/ENaC superfamily [18]. A new mechanical channel called Piezo was found in 2010 by Ardem Patapoutian labs, especially Coste. The word “Piezo” is derived from the Greek word (piezi), which means pressure. Regarding taxonomy (piezophiles - organisms that flourish under high pressure) and electronics (piezoelectric), the references are connected. Human Piezo proteins, which are essential for mechanosensation, are represented by the letters Piezo1 and Piezo2. At first, it wasn’t apparent if the Piezo channels needed other mechanisms to sense mechanical stress or if they were constitutively active. However, Syeda et al. subsequently clarified that the Piezo channels are intrinsically active, responding to mechanical perturbations in the cell membrane [19, 20]. The Piezo1 and Piezo2 channels found in neurons are responsive to both extracellular and intracellular mechanical stresses. The Piezo1 channel detects shear stress and the mechanical forces in the neurons’ immediate surroundings, whereas the Piezo2 channel is touch and proprioceptive-sensitive. These channels serve as cellular gatekeepers that convert mechanical forces into biochemical signals, including calcium influx, which regulate key processes such as proliferation, differentiation, migration, and inflammation [21]. To exploit this mechanobiological machinery therapeutically, biomaterial scaffolds can be engineered to modulate the physical microenvironment of cells, thereby influencing Piezo channel activation and downstream signaling cascades [22].

Piezo channel activation is primarily directed by variations in membrane tension, which can be externally controlled through the mechanical properties of the underlying scaffold. One of the most critical parameters is substrate stiffness. Cells adhere to and probe their environment via focal adhesions and actomyosin contractility; a stiffer substrate resists these forces, increasing membrane tension and triggering the opening of Piezo channels. This mechanical stress resulting from injury or changes in extracellular matrix (ECM) stiffness, activates the mechanosensitive ion channel Piezo1 in neurons and neural stem cells (NSCs). Upon activation, Piezo1 facilitates the influx of calcium ions (Ca^{2 +}) into the cell. This rise in intracellular calcium levels initiates a cascade of downstream signaling events that play a critical role in regulating neuronal differentiation, synaptic plasticity, and tissue regeneration. Through this mechanotransduction pathway, Piezo1 serves as a key sensor and mediator of mechanical cues that influence neuronal repair and functional recovery [23].

Biomaterial platforms play a critical role in modulating this mechanotransduction process by mimicking the mechanical properties of the native ECM, thereby providing optimal stiffness and topographical cues to activate Piezo1 [24]. Engineered scaffolds made from materials like collagen, gelatin, or synthetic polymers such as PLGA and PCL can be tailored to direct stem cell fate through controlled mechanical stimulation [25]. Additionally, stem cell-based strategies complement this process by supplying exogenous neural stem cells or progenitors capable of responding to mechanosensory cues, thereby enhancing neuroregeneration. Together, biomaterials and stem cell therapies synergize to promote Piezo1-mediated calcium signaling and downstream regenerative processes essential for neural repair [26].

Song, Yuanquan, and colleagues investigated how the piezo channel affected axonal regeneration after axonal damage. The Drosophila dendritic arborization (da) sensory neuron damage model was employed to investigate axon regeneration. The class III da neuron of the wild type (WT DmPiezo) did not regenerate axons, while the same class III da neuron demonstrated improved axon regeneration following Piezo channel knockdown (DmPiezoKO) and RNAi knockdown. This outcome was comparable in human and mouse Piezo1 channels. Additionally, they concentrated on class IV da neurons and discovered that whereas overexpression of mutant Piezo1 (Piezo 1-TriM) gain of function variation inhibits axon regeneration, overexpression of WT DmPiezo did not alter regeneration in class IV da neurons. They next looked at how class III neurons’ dendritic regrowth was affected by WT DmPiezo and DmPiezoKO. They noticed that compared to significant regeneration following baldness, dendritic regrowth following the removal of a single dendrite was more restricted. However, the differences between the WT and DmPiezoKO were not very noticeable. Then, using GFP-DmPiezo, they demonstrated that whereas GFP-DmPiezo is diffusely distributed in the axon and cell body of an uninjured class III da neuron, it is shown to be enriched in the development of the cone tip following axonal damage. Following damage, DmPiezo knockdown in class III da neurons shows that axon regrowth is increased when DmPiezo is removed. All of this results from a shift in membrane tension brought on by axonal damage, which further activates the MIC Piezo1 and causes calcium influx. Additionally, they discovered that following DmPiezoKO, the calcium decreased when they performed calcium imaging utilizing membrane targeting myr-GCaMP6(s). Thus, calcium influx following Piezo channel activation triggers the NO signaling pathway via CamKll. After the Piezo channel is activated, calcium influx triggers the NO signaling pathway through CamKll, which in turn suppresses cGMP kinase foraging or PKG, which inherently prevents axon regeneration. Piezo, in summary, prevents axon renewal while leaving dendrite regeneration unaffected [27]. Nociceptive ion channels play a key role in pain perception by transducing unpleasant stimuli. By increasing intracellular calcium and activating the PKA pathway in nociceptive neurons, inhibition of the nociceptive ion channel TRPV1 has been shown to restart the development of axons in sensory neurons. Axon regeneration after sciatic nerve compression did not significantly improve, according to the scientists. Given that only a small fraction of sensory neurons express TRPV1, it is possible that neurons without TRPV1 expression that also transmit their axons into the sciatic nerve might obscure the benefits of TRPV1 manipulation in axon regeneration. Receptors implicated in nociceptive stimulation are also involved in neurite healing, which is interesting. It’s interesting to note that neurite healing is influenced by receptors engaged in nociceptive stimulation. According to reports, axon regeneration is decreased or increased by activating or inhibiting the nociception receptor ORL1 via a ROCK-dependent process [28, 29]. On the other hand, in cultured DRG neurons, the voltage-gated calcium channel (VGCC) subunit Alpha2delta was found to be a negative regulator of axon development and injury-dependent regeneration. Dorsal column axon regeneration after SCI may be enhanced by systemic treatment of Pregabalin (PGB), a gabapentinoid that specifically binds to VGCC Alpha2delta1/2 subunits and inhibits calcium influx; the earlier PGB is administered, the better the axon regeneration [30].

On the other hand, along with the mechanistic channels intrinsic growth factors are also essential. It has been shown that a number of signaling pathways, such as the mammalian target of rapamycin (mTOR) pathway, encourage neurons that are not easily regenerative to return to the active growth stage. According to a recent study, injury signals may cause local regulation of mTOR translation, which in turn regulates the production of retrograde injury signaling molecules crucial for intracellular communication between axons and soma. We can learn more about the functions of mTOR in neurite healing by investigating how local RNA, including mTOR mRNA, is targeted to the damage sites and determining the targets of mTOR for local translation [31].

IL-1R1 signalling can also reduce the resolution of inflammation, promote tissue repair, and improve neurogenesis and synaptic plasticity during the chronic phase of brain injury. Considering the dual nature of IL-1R1 signalling, it has emerged as a potential therapeutic target for brain injury. This study, conducted using an IL-1R1 knockout mouse model, confirmed previous research findings regarding the role of the IL-1R1/IL-1RAcP pathway in inflammation and cognitive impairment after controlled cortical impact (CHI). This observation shows the significance of understanding the mechanism of IL-1 R/IL-1RAcPb and implies that IL-1/IL-1R1 might impact recovery and affect outcomes, besides being an essential injury biomarker. The IL-1 receptor (IL-1 R) signalling pathway interacts closely with several other critical signalling pathways essential for maintaining neurological health and responding to neuronal injury, including the JAK/STAT, PI3K/Akt, and Wnt/β-catenin pathways. When activated, the IL-1 R pathway triggers a cascade of events that affects various aspects of neuronal function and repair mechanisms. Through its connections with JAK/STAT, the pathway regulates the expression of genes crucial for inflammation regulation and tissue repair, promoting microglial polarization towards a reparative phenotype. Simultaneously, IL-1 R signalling engages the PI3K/Akt pathway, fostering cellular survival and growth, thereby supporting microglial function and preserving neuronal viability. Furthermore, the IL-1 R pathway interacts with the Wnt/β-catenin pathway, which is pivotal in processes such as neurite outgrowth, axonal repair, and synaptic plasticity. By influencing Wnt/β-catenin signalling, IL-1 R activation facilitates the brain’s ability to adapt to and recover from neuronal injuries, promoting synaptic remodeling and functional recovery. Together, these intricately linked signalling pathways form a complex network that controls the actions that maintain neuronal integrity and support processes for neuronal repair [32, 33].

Extracellular signal-regulated kinases (ERK) are signal transducers and activators of transcription 6 (Stat6) and MERTK (MER Proto-oncogene Tyrosine Kinase), which are integral to different signalling pathways governing various cellular processes, including proliferation, differentiation, survival, and inflammation. The ERK pathway, which is activated by various extracellular stimuli, adapts to downstream effects within the MAPK pathway. Stat6, a transcription factor activated by cytokines, controls downstream effects of the JAK/STAT pathway. MERTK, a receptor tyrosine kinase, is activated by its ligands Gas6 and protein S, and directs downstream effects within the TAM pathway. Each of these proteins plays a unique role in cellular regulation, contributing to the complexity and diversity of cellular responses to external stimuli [34, 35]. ERK, Stat6, and MERTK signalling can interact with each other and modulate the function of immune cells, specifically monocytes, macrophages, and microglia, after brain injury. ERK and Stat6 also regulate the expression and activation of MERTK, the primary receptor involved in efferocytosis, and remove apoptotic cells and debris. Efferocytosis is essential for resolving inflammation and recovering tissue homeostasis after brain injury [35, 36] (Figs. 2, 3, 4).

Fig. 2.

(A) The role of monocytes, macrophages, and B cells in the immune response resulting in neuronal damage. In response to neuronal injury, a protective mechanisms are triggered, leading to the release of proinflammatory cytokines. Monocytes and macrophages become activated to protect neurons. Subsequently, B lymphocytes play a role in the repair process by producing antibodies and controlling the over activation of macrophages. The use of CD45+ as a cell surface marker is essential for distinguishing these cells from microglia. This intricate interplay shows the involvement of various immune cells in the brain’s response to injury, aiming to protect and repair neuronal damage. (B) Glutamate, AMPA receptors and microglia along with Ca+ in neuronal repair. Neuronal injury triggers the release of glutamate and the formation of debris. These substances activate microglia, the immune cells of the nervous system, by binding to AMPA receptors. Microglia then engulf excess debris, secreting neurotrophic factors that facilitate neuronal healing. The interplay between glutamate transporters VGLUT1 and VGLUT2, along with the presence of the anti-inflammatory protein C1q on microglia, modulates this process. The balance among these factors decides the extent and speed of neuronal regeneration after injury. (NMDAR - NMDA receptor and AMPAR – AMPA receptor). (C) IL-R1 signalling in terminal brain injury: a phenomenon between damage and repair. This figure shows the complex dynamics of IL-R1 signalling in terminal brain injury, exploring both the damaging and reparative phases. In this highlighting the serious damage caused by the first inflammatory storm and the following anticipation of a potent recovery response via microglial reformation. (D) The collaboration of ERK, Stat6, and MERTK in neuronal repair is synergistic. ERK promotes neuronal survival and growth, Stat6 contributes to an anti-inflammatory environment and neuroprotection, while MERTK ensures efficient clearance of debris, allowing for a cleaner slate for repair and regeneration

Fig. 3.

Stem cell biomaterial interface in neural tissue repair: mechanisms of scaffold integration and regeneration. The image shows how stem cell-loaded biomaterial scaffolds regenerate neurones after CNS traumas including spinal cord and brain injury. Stem cells are mixed with natural or synthetic biomaterials to provide a therapeutic platform that mimics the original ECM. At the damage site, the biomaterial scaffold supports tissue repair structurally and biochemically. The scaffold is physically implanted into the host tissue to provide a 3D matrix for cellular interactions. Host-derived proteins immediately adsorb onto the scaffold surface after implantation, altering its biointerface and starting molecular signalling. After that, innate and adaptive immune cells infiltrate to respond to foreign material and aid healing. These immune cells release cytokines and chemokines to recruit endogenous progenitor and stem cells to the damage site. These cells deposit nascent ECM proteins to modify the microenvironment and make the scaffold physiologically active and regenerative. In the last stage, this remodelled environment promotes neuronal regeneration and host vasculature infiltration, which restore neural tissue function. This integrated approach shows how biomaterials and stem cell methods together activate host responses and restore the wounded CNS

Fig. 4.

Biomaterial-based modulation of injury response: from neural damage to regenerative signaling and scaffold integration. This picture shows how nerve, brain, and spinal cord injuries launch a complex sequence of defensive cellular processes and signalling cascades that biomaterial-based therapy techniques can control. After damage, macrophages (polarising into pro-inflammatory M1 or anti-inflammatory M2 phenotypes), astrocytes, microglia, T cells, and neutrophils activate. Whether secondary harm or repair occurs depends on these cellular responses. The central panel shows key signalling pathways involved in inflammation, apoptosis/survival, regeneration, and stem cell homing (NF-κB, IL-1 R, NLRP3 inflammasome, PI3K, Akt, ERK, MAPK). These pathways are important molecular targets for tissue healing and cell fate in regenerative medicine. Many biomaterials assist and modify these processes. These include natural biomaterials (agarose, alginate, collagen, gelatin, hyaluronic acid, and silk fibroin), semi-synthetic materials (PLGA, PLA, PEG), and synthetic polymers (polypyrrole, polyaniline, PEDOT) with unique chemical, mechanical, and biological properties. These biomaterials can be created into organoids, microporous scaffolds, hydrogels, soft hydrogels, and fibrous scaffolds to mimic the neuronal microenvironment and aid regeneration

Characteristics of biomaterials for stem cell-based neuronal regeneration

Biomaterials are natural or synthetic substances engineered to interact with biological systems for therapeutic or diagnostic purposes [37]. Their application spans a wide range of medical fields, including regenerative medicine, drug delivery, prosthetics, and implantable devices. A key characteristic of biomaterials is their ability to mimic or support biological functions, often without eliciting adverse immune responses [38, 39].

In recent decades, the field of biomaterials has evolved from passive materials used for structural support to bioactive and smart materials capable of responding to physiological stimuli, promoting tissue regeneration, or delivering therapeutic agents in a controlled manner [40].

Naturally occurring biomaterials

Naturally occurring biomaterials have emerged as promising candidates for neural regeneration due to their inherent biocompatibility, biodegradability, low immunogenicity, and ability to mimic the extracellular matrix [41, 42]. These materials, derived from natural sources such as seaweed, silk, or animal tissues, support critical cellular processes like adhesion, proliferation, and differentiation, making them ideal for nerve repair and tissue engineering applications [43, 44]. Naturally derived biomaterials like agarose, alginate, collagen, gelatin, hyaluronic acid (HA), and silk fibroin offer promising solutions for neural regeneration [43]. Together, these biomaterials hold great potential for nerve repair and brain regeneration therapies.

Agarose

Seaweeds contain agarose, a naturally occurring polysaccharide. Owing to its outstanding biocompatibility, physiochemical characteristics, thermo-reversible gelation behavior, rapid water absorption, and nontoxic nature, 3D neurite growth is encouraged in vivo. This has also been frequently studied in the context of neuronal regeneration. Agarose scaffolds offer significant potential for the stimulation of growth, neurotrophic factors, and neurogenesis [45]. Agarose gel poses challenges in tissue engineering and wound dressing applications owing to its brittleness and contractility. To overcome this, agarose has been prepared in conjunction with other polymers such as fibrin, chitosan, and gelatin. For instance, the potential of human olfactory ecto-mesenchymal stem cells (OE-MSCs) as a novel source for training motor neuron-like cells was examined using tissue culture plates (TCP) and conductive hydrogels. Biodegradable agarose has been created to treat neurological problems like spinal cord injury, traumatic brain injury, stroke models and etc [46]. In a recent study, the efficacy of a new fibrin-agarose bio-artificial nerve replacement (Nano) was examined in vivo in laboratory rats with a 10-mm sciatic nerve lesion. NeuraGen R collagen type I conduits (Coll-Nano) were used either alone or in conjunction with Nano. Rats with a 10-mm nerve gap were treated with these biomaterials either alone or in conjunction with adipose-derived mesenchymal stem cells (ADMSCs) as intraluminal fillers of biodegradable NeuraGen R conduits. These investigations showed that the addition of acellular fibrinogarose hydrogel hydrogels (FAH) and, more specifically, FAH-containing autologous dermal microstromal cells (ADMSCs) created an appropriate regenerative microenvironment that markedly improved the clinical, functional, electromyographic, and histological profiles [47].

Alginate

Alginate is a naturally occurring polysaccharide extracted from brown seaweeds such as Laminaria hyperborea, Laminaria japonica, and Macrocystis pyrifera [48]. Its extraction typically involves the treatment of algal biomass with aqueous alkaline solutions, followed by filtration and precipitation using calcium or sodium chloride. This yields alginate salts, which can subsequently be converted into alginic acid through acidification with hydrochloric acid (HCl). Further conversion and purification steps result in the production of sodium alginate powder [49, 50].

Alginate has shown promising results in neuronal repair applications due to its biocompatibility and versatility. In spinal cord injury treatment, a 3D bioprinted scaffold made of sodium alginate and gelatin, loaded with neural stem cells and oligodendrocytes, significantly improved hindlimb motor function and promoted nerve regeneration in rats with completely transected spinal cords [51]. The nanofibrous architecture and controlled release of NGF from the microspheres induced neurite extension of PC12 cells, suggesting potential applications in brain tissue regeneration. Additionally, alginate-based scaffolds have been explored for peripheral nerve repair, where they can be combined with conductive polymers like polypyrrole (PPy) or polyaniline (PANI) to enhance nerve regeneration through electrical stimulation [52].

A recent study showed that mouse neural stem cells (NSCs) can be effectively expanded in vitro using 3D calcium alginate beads (Ca-Alg-Bs) formed by gelling 1.5% sodium alginate with 3.5% CaCl₂. Optimal growth occurred at 0.8 × 10⁵ cells/mL, with cells doubling during the culture. Beads were dissolved using 55 mM sodium citrate, yielding over 88.5% cell recovery [53, 53].

Collagen

Collagen is the most abundant protein in mammals and makes up over one-third of the total protein makeup of the human body. It is also biocompatible and bioactive [54]. The biodegradability of collagen in the human body is excellent. However, there are major obstacles to its utilization in biomedical applications due to its inferior mechanical qualities and quick rate of deterioration. Collagen must thus be blended with other biomaterials to enhance its physiochemical properties. About 80–90% of the body’s collagen is made up of fibrils and neatly ordered fibers known as collagen types I, II, and III [55]. Hoban et al. explored the use of type I collagen hydrogels as a supportive matrix for transplanting GDNF-expressing mesenchymal stem cells (MSCs) into the rat brain. Their in vitro findings indicated that the hydrogel did not adversely affect the secretory function of GDNF-MSCs. Notably, it attenuated the brain’s immune reaction by suppressing astrocytic and microglial activation. Compared to other biomaterials, type I collagen hydrogel elicited a significantly lower immune response at the implantation site [56]. Baicalein-functionalized collagen scaffolds (BFCSs) have been developed to enhance neuronal differentiation and improve motor functional recovery in SCI models. These scaffolds facilitate neurogenesis and axon extension, further supporting their use in CNS injury treatment [57]. Additionally, collagen contributes to CNS stability, axon guidance, and synaptogenesis. Repairing degraded collagen with mimetic peptides can further enhance neural survival [58]. Despite its potential, a better understanding of collagen’s cellular connections and type-specific functions is necessary to maximize its therapeutic use.

Gelatin

Gelatin’s non-cytotoxicity, affordability, and compatibility with natural and synthetic polymers make it ideal for brain tissue engineering. Its chemically modified structure enhances cell adhesion and proliferation. In traumatic brain injury treatment, collagen and elastin were combined with gelatin methacryloyl to form 3D-printable neuromimicking hydrogels, cross-linked using Irgacure or Irgacure with Genipin. These scaffolds showed good thermal stability and maintained peptide structure, while Genipin also provided antibacterial properties to help prevent postsurgical brain infections [59]. In spinal cord injury (SCI), the persistent administration of neurotrophic factors is essential for establishing an environment that supports cell survival and nerve regeneration. [60]. It was reported that the design and engineering of biodegradable gelatin-based scaffolds and 3D synthetic scaffolds based on polyethylene glycol (PEG), created using a free-form manufacturing process, yields accurate interior geometry and elastic stiffness. Using confocal and electron imaging, it was demonstrated that human neurons generated from human embryonic stem (hESC) cells can attach to these scaffolds and create organoid structures that extend in three dimensions. Future improvements in the size, shape, and surface chemistry of scaffolds may make it easier to conduct long-term studies and create bioassays with practical applications [61]. Neuronal differentiation, uniform distribution, and well-defined architecture have been achieved by combining graphene nanoplatelets with methacrylamide/gelatin hydrogels [62, 63].

Hyaluronic acid (HA)

The extracellular matrix (ECM) is comprised of mucopolysaccharides, which are involved in cell migration, differentiation, angiogenesis, and tissue repair. It is widely used in neural tissue culture to promote neurite outgrowth, proliferation, and differentiation on various substratum [64, 65]. Given its abundance in neural tissues, hyaluronic acid (HA) serves as an excellent material for constructing scaffolds in neuroregenerative applications. As a major component of the extracellular matrix (ECM) in the central nervous system and neural stem cell (NSC) niches, HA supports the survival and development of NSCs. Notably, encapsulating human embryonic stem cell-derived NSCs (hESC-NS) in HA-based hydrogels has been shown to promote their differentiation into oligodendrocytes and enhance their motility, as indicated by cc1 marker expression [66]. Neural stem cell (NSC) implantation into artificial scaffolds for peripheral nerve injury is gaining considerable interest. Rabbit facial nerve transsection ends were implanted with BrdU-labeled NSCs conduit after NSCs were grown ex vivo in a hyaluronic acid (HA)-collagen composite with neurotrophin-3 [67]. Hyaluronic acid (HA) supports neuroregeneration by reducing inflammation, glial scarring, and GFAP expression due to its excellent biocompatibility. Despite its poor cell adhesion, this limitation can be addressed by blending HA with materials like silk fibroin or chitosan. These composites enhance cell binding, mechanical strength, and biodegradability, showing promising results in neural repair and peripheral nerve regeneration models [68, 69].

Silk fibroin

Regenerated silk fibroin (RSF) from Bombyx mori has shown promising results in peripheral nerve regeneration due to its support for cell viability and proliferation. Studies have also explored spider silk and regenerated spidroin (RSP) in nerve repair. In one trial, a 6 cm tibial nerve lesion in sheep was treated using a decellularized vein graft filled with spider silk strands, which supported Schwann cell migration, axonal regeneration, and myelination. Electrophysiological results matched those of the control, indicating successful nerve recovery [70]. In the context of drug delivery, SF serves as an excellent carrier due to its biocompatibility, biodegradability, and mechanical robustness, enabling sustained and controlled release of therapeutics, protection of drugs from degradation, and targeted delivery to specific neural tissues. These properties position SF as a promising material for improving treatment efficacy in disorders such as Alzheimer’s disease, Parkinson’s disease, and stroke [71]. Complementing this therapeutic potential, recombinant silk microfibers have also been effectively used as scaffolds for brain organoid development from pluripotent stem cells. These silk scaffolds address key limitations in traditional organoid cultures, such as heterogeneity and limited maturation, by promoting uniform neuroectoderm formation and enhancing oxygen and nutrient diffusion. This leads to better neuronal differentiation and structural organization, yielding organoids that are more functionally mature and suitable for neurodevelopmental studies and disease modeling. Collectively, these studies underscore the emerging role of silk-based biomaterials in both regenerative medicine and neuroscience research [72].

Silk fibroin (SF) exhibits excellent biocompatibility, low immunogenicity, and strong mechanical properties, making it a promising material for brain and nerve regeneration. SF hydrogels and nanofibers have been used to support axonal growth and bridge nerve gaps. In rats, SF combined with poly(L-lactide-co-ε-caprolactone) improved nerve repair. To enhance neural regeneration in small nerve gaps, SF-based neural conduits (SF NCs) were developed using aligned and non-aligned nanofibers loaded with GDNF and NGF. These conduits promoted increased axonal outgrowth in spinal cord and dorsal root ganglion neurons from chicken embryos, offering an effective alternative to autologous grafts [73]. Additionally, SF-based gels have the potential to be used as biomaterials in 3D bioprinting, allowing for precise structural control. However, very little silk was produced during this process. Through effective nerve functional recovery, Silk Bridge TM, which is composed of three layers, has demonstrated a balance between its physical and biological properties. However, the results of current clinical trials are positive [74, 75].

Synthetic polymeric scaffolds for neural tissue regeneration

Applications of natural biomaterials are limited because of their instability or fast rate of disintegration despite their excellent biocompatibility and capacity to enhance cell activities. To obtain better mechanical qualities, biocompatibility, and immunogenicity, researchers studying tissue engineering have preferred synthetic polymers. Neural regeneration is either non-biodegradable or biodegradable. While biomaterials containing methacrylate are not biodegradable, examples of biodegradable polymers include polyglycolic acid (PGA), polyethylene glycol (PEG), poly lactic acid (PLA), and their copolymer PLGA. Using the electrospinning method, researchers created nanofibrous PLA scaffolds with the same shape and architecture as the natural extracellular matrix. They mimic the composition and biological functions of natural ECM. Neural devices with physiochemical and physical characteristics similar to those of wounded nerve tissue have been made possible by recent developments in biomaterial technologies. They allow for modifications in the architecture and chemistry based on specific requirements. However, these artificial polymers have several drawbacks including the possibility of residual harmful monomers. Thus, novel assays for these polymers are urgently required to make them accessible and appropriate for clinical applications [76, 77].

Polylactic acid (PLA)

Polylactic acid (PLA) is a biocompatible, biodegradable synthetic polymer widely used in biomedical applications. In neural engineering, PLA scaffolds support artery formation and Schwann cell-driven axon growth. However, poor hydrophilicity limits cell adhesion. To overcome this, PLA scaffolds have been modified with NGF-loaded heparin/collagen layers to enhance hydrophilicity and support Schwann cell proliferation. Despite its benefits, PLA may suffer from structural instability, leading to breakage and crumpling [78]. Patterned PLA-based microchamber arrays offer a flexible, biocompatible system for site-specific drug delivery. Recent studies show they support neuronal cell growth and allow localized release of nerve growth factor (NGF), guiding neurite outgrowth in N2A cells toward open microchambers. This system enables controlled, on-demand stimulation of neuronal responses [79].

Regenerated nerves often show reduced nerve conduction velocity (NCV), prompting interest in electroconductive scaffolds for improved recovery. Polypyrrole/polylactic acid (PPy/PLA) is a promising conductive biomaterial for nerve tissue engineering. Bone marrow stromal cells (BMSCs), known for their low immunogenicity and multipotency, support tissue repair and can differentiate into neural cell types. A recent study showed that PPy/PLA nanofibrous scaffolds combined with BMSCs enhanced functional recovery in spinal cord injury models [80].

Poly (lactic-co-glycolic acid) (PLGA)

PLGA scaffolds have shown potential in supporting brain repair by promoting mesenchymal stem cell (MSC) and neuron adhesion, growth, and migration both in vitro and in vivo. In a recent study, MSCs and neurons tagged with GFP were seeded onto PLGA scaffolds. MSCs retained normal proliferation, adhered well, and showed neural differentiation marked by MAP2 expression and neuron-like morphology. SEM and immunohistochemistry confirmed cell attachment and migration, indicating that PLGA scaffolds support neural regeneration without hindering MSC function [81]. Polylactic glycolic acid (PLGA), an FDA-approved polymer, is widely used in brain regeneration due to its biodegradability, permeability, and suitable degradation rate. However, its hydrophobicity and lack of functional groups limit its application. These issues are addressed by adjusting PLA:PGA ratios and blending with natural polymers. A 75:25 PLA to PLGA ratio showed ideal degradation (9–12%) and stability in bodily fluids, supporting axonal regeneration in rats with spinal cord transection injuries [82]. Multichannel PLGA scaffolds seeded with Schwann cells aid brain regeneration, but their limited electrical conductivity is a drawback. This issue can be addressed by coating PLGA electrospun nanofibers with polypyrrole (PPy). Under 10 mV/cm electrical stimulation, PPy–PLGA scaffolds significantly enhanced hippocampal neuron response, promoting 40–50% longer neurites and 40–90% greater neurite production [83, 84].

Polyethylene glycol (PEG)

PEG, a water-soluble, FDA-approved synthetic polymer with low toxicity, shows strong potential in nerve regeneration. Though not bioactive, it can be combined with other polymers like PLGA. Advances in neural stem cell research and nanotechnology have boosted hope for brain repair after neurological disorders. This study explored the in vitro proliferation and neurogenic effects of PLGA and PLGA–PEG nanofibers on human SH-SY5Y cells. [85]. PEGs high biocompatibility of PEG makes it suitable for preparing hydrogels for neural tissue applications, and these hydrogels can be used to manage CNS insults. Neuronal cell growth on PEG substrates supports neuronal cell proliferation, differentiation, and survival. Culturing of NSCS on rhpo hydrogels (made of PEG and poly (L-lysine) (PLL)) revealed that specific gel blends encouraged NSC movement on polyurethane (PU) in some locations and NSC differentiation in other places [84]. Poly(ethylene glycol) diacrylate (PEGDA) hydrogels are soft, hydrated, and tissue-like, making them ideal for biomedical use. Their properties can be tuned using heat, light, or cross-linkers. Studies show that PEGDA scaffolds with log-pile and hexagonal structures support neurosphere survival, proliferation, and 3D neuronal growth. Tuj1 labeling confirms that neurons form 3D networks on both scaffold types, with larger neurospheres unable to penetrate the scaffold interior. [61] (Fig. 5).

Fig. 5.

Multimodal characterization of biomaterial scaffolds and their applications in neural tissue engineering. i) SEM images of PEG scaffolds: log-pile architecture (a, b): these images depict the log-pile internal structure of PEG scaffolds, supporting the survival and differentiation of neurospheres into neuronal morphology. The neurospheres spread from adherent cell clusters, forming both 2D and 3D intercellular projections.Hexagonal architecture (c–f): the hexagonal PEGDA scaffolds shown here demonstrate even penetration and adhesion of neurospheres, with larger neurospheres mostly excluded from the scaffold’s interior. 3D lattices (g–i): neurons form 3D lattices on both log-pile and hexagonal PEGDA scaffolds, as indicated by Tuj1 labeling of neuronal connections. The white arrow in (h) points to a cell cluster adhered to the side of the log-pile scaffold with neural connections extending along the struts. Scale: 100 μm. ii) confocal images of stiff PEGDA scaffold: hexagonal and log-pile architecture (a–f): confocal images of stiff PEGDA scaffolds, featuring hexagonal (a–c) and log-pile (d–f) structures, show the formation of 3D neural connections, labeled with Tuj1, a marker of early neurons. The white arrows in (f) highlight the log-pile structure. Scale: 100 μm iii) confocal images of soft PEGDA scaffold: hexagonal geometry (a–c): confocal images of the soft PEGDA scaffold with hexagonal geometry from selected z-sections, labeled with Tuj1 (green), MAP2 (red), and nucleus (DAPI-blue). 3D reconstruction (d): a 3D reconstruction of the hexagonal scaffold. High-resolution images (e–g): high-resolution images of the soft PEGDA scaffolds show clusters of neural cells forming 3D connections between two hexagonal geometries, with white arrows in (e) indicating the hexagonal structure. SEM images (h–k): SEM images of the soft PEGDA scaffold are provided. Scale: A–I 100 μm; J, K 10 μm. iv) scanning electron microscopy images of rat NSCs cultured on PES films: PES films coated with Laminin (a–c): images show rat NSCs cultured on PES films coated with laminin. Randomly oriented PES nanofibers (d–l): rat NSCs are cultured on randomly oriented PES nanofibers with diameters of 280 nm, 750 nm, and 1450 nm. 280 nm nanofibers (d–f): cells highlighted in yellow in (e) and (f). 750 nm nanofibers (g–j): images of NSCs on 750 nm nanofibers. 1450 nm nanofibers (k, l): images of NSCs on 1450 nm nanofibers. Scale bars indicate 10 μm (a, b, d, g, i, k), 5 μm (c, e, f), and 2 μm (h, j, l). Copyright 2009. Adapted with permission from elsevier. v) effect of NSCs transduced with GFP on PCL and PCL-immobilized GDNF (Pcl_igdf), transplanted into the rat brain parenchyma: cell ratios (A–D): the ratios of neural stem cells (A, nestin expressing cells), neurons (B, Tuj1 expressing cells), oligodendrocytes (C, NG2 expressing cells), and astrocytes (D, GFAP expressing cells) in rat striatal grafts. Immunohistochemical staining (E–J): staining of nestin (E–G, red) and GFAP (H–J) in grafts where NSCs (E, H), NSCs on PCL (F, I), and NSCs on PCL-immobilized GDNF (G, J) were transplanted into rat brain parenchyma. NSCs were transduced with GFP beforehand [86]. Copyright 2012. Adapted with permission from elsevier. vi) effects of PLLA nanofibers on axonal growth in ESC-derived neural cells: nanofiber structures (A, B): scanning electron microscopy images of random (A) and aligned nanofibers (B).Immunohistochemical staining (C–E): staining of TUBB3 in ESC-derived neural cells on a tissue culture plate (C), random nanofiber (D), and aligned nanofiber (E, horizontal direction). Axonal length and angle (F, G): axonal length on tissue culture plate [C], random nanofiber [R], and aligned nanofiber [A], and the angles of axons on tissue culture plate (control), random nanofibers, and aligned nanofibers. Scale bars indicate 50 μm [75]. Copyright 2010. Adapted with permission from Mary Ann Liebert

PEG alone has limited effectiveness in neural repair due to poor biocompatibility and inability to mimic spinal cord structure. However, PEG hydrogels treated with polylysine improve nerve cell adhesion, integration, axon regeneration, and remyelination. Despite its drawbacks, PEG is incorporated into new materials for added hydrophilicity and flexibility. By modifying PEGs end groups (e.g., aldehydes, amino, carboxyl), its applications extend to drug delivery, controlled release, chemical synthesis, and stem cell transplantation [87].

Polyurethane (PU)

High-performance synthetic polymers, such as polyurethane (PU), have attracted considerable interest for biological applications. PU have gained much traction in brain regeneration because of their unique mechanical, physical, and biodegradable qualities and biocompatibility. A thermoresponsive water-based biodegradable PU scaffold was produced by adding NSCs, using innovative 3D bioprinting techniques. Its mixing with other polymers enhances the physicochemical attributes of polyurethane for use in nerve tissue applications [88]. In a recent study, iron oxide nanoparticles (Fe2O3 NPs) were added to a polyurethane [PU]-based cell transport platform to facilitate neural induction in human induced pluripotent stem cells (hiPSCs). Forskolin, IBMX, and various FBS ratios were used to stimulate neurogenesis in hiPSCs. [89]. In addition to its benefits, it has several drawbacks such as limited mechanical strength, weak cell adherence, and sluggish disintegration. Therefore, it is necessary to design new PU systems based on innovative polyols with fewer negative consequences [90].

Poly-caprolactone (PCL)

Polycaprolactone (PCL) is a cost-effective, FDA-approved polyester known for its high crystallinity, mechanical strength, and bioabsorbability, making it suitable for nerve tissue engineering and drug delivery. Electrospun PCL scaffolds reinforced with GDNF support cortical cell growth, migration, and neurite extension. While PCL promotes cell proliferation and passage, its slow degradation and high crystallinity limit its use—issues that can be addressed through aminolysis and hydrolysis modifications [91, 92]. An in vitro study showed that Schwann cells (SCs) cultured on PCL and PLLA membranes promoted the expression of basal lamina components. SCs from neonatal rat sciatic nerves were seeded on polymer membranes, and immunocytochemistry confirmed the presence of laminin I/II and type IV collagen. These biodegradable polymers, especially PLLA, supported SC adhesion, proliferation, and ECM production, indicating their potential to enhance peripheral nerve regeneration in vivo [93]. A recent study developed antioxidant PCL/lignin nanofibers to support nerve regeneration, leveraging lignin’s potent antioxidant properties. Using solvent-free ring-opening polymerization, lignin-PCL copolymers were synthesized and blended with PCL to create nanofibrous scaffolds. These scaffolds enhanced mechanical strength and showed high free radical inhibition (up to 98.3% in 4 hours). Compared to pure PCL, the lignin-PCL nanofibers improved cell viability under oxidative stress, promoted DRG neurite outgrowth, boosted Schwann cell myelin protein expression, and increased BMSC and Schwann cell proliferation. This suggests strong potential for their use in nerve tissue engineering [94]. PCL is a widely studied biodegradable polymer for tissue engineering due to its non-immunogenicity, versatility in fabrication, and compatibility with other polymers. However, its slow degradation and hydrophobicity limit cell adhesion, infiltration, and overall biocompatibility. These issues can be addressed by blending with other materials or modifying surface properties. Despite limitations in cell ingrowth and seeding efficiency, PCL remains a promising scaffold material, offering structural flexibility and potential for innovative regenerative medicine applications [95].

Polyvinyl alcohol

Polyvinyl alcohol (PVA) is an FDA-approved polymer widely used in brain tissue engineering due to its water retention, biocompatibility, mechanical strength, and non-toxicity. PVA-based electrospun nanofibers meet key requirements for nerve regeneration, such as alignment, porosity, and 3D structure. However, its degradation into acidic byproducts and poor cell adhesion limit its use. These issues can be mitigated by incorporating bioactive compounds. For instance, conductive PVA/PEDOT scaffolds with electrical stimulation enhance cellular responses, offering strong potential for brain regeneration [96, 97]. To enhance stability in aqueous environments, PVA fibers were cross-linked with glyoxal and loaded with quercetin, an anti-inflammatory neuroprotectant. The fibers showed a maximum quercetin release of 56 ± 6% and were characterized using IR spectroscopy. Studies on fiber formation and diameter confirmed the impact of quercetin loading and cross-linking. Cytotoxicity tests with PC12Adh cells indicated the fibers were non-toxic. These quercetin-loaded, glyoxal-crosslinked PVA fibers show potential for use in nerve guides to reduce scar tissue and fibrosis [98]. Polyvinyl alcohol (PVA) is an FDA-approved polymer widely used in neural tissue engineering due to its high water retention, mechanical strength, biocompatibility, and stability. PVA-based electrospun nanofibers meet key criteria for nerve regeneration, including alignment, porosity, and 3D structure. However, its degradation into acidic byproducts and poor cell adhesion limit its effectiveness. Incorporating bioactive compounds, such as PEDOT, and applying electrical stimulation can enhance cellular responses, making PVA/PEDOT scaffolds promising for brain regeneration [99]. PVOH polymers and copolymers possess desirable properties like water retention, film formation, strength, and swelling, making them useful in pharmaceuticals and biomedical applications. Their non-toxicity and favorable mechanical behavior in water-swollen states support their use. However, large-scale production is limited due to strict regulations, and key concerns remain, including chemical cross-linker residue safety, stability of physical and ion-cross-linked structures, drug-hydrogel interactions, and long-term hydrogel stability [99, 100].

Hydrogels

Hydrogels function as regenerative platforms for axonal regrowth both with and without transplanted cells because of their excellent biocompatibility, chemical characteristics, and structural resemblance to the extracellular matrix. Compared with other tissues, the extracellular matrix (ECM) of the brain has a higher concentration of linear polysaccharides, which gives it a unique structure [1]. The brain’s extracellular matrix (ECM) is a soft network with an elastic modulus of ~0.1–1.5 kPa, much lower than that of other tissues. It mainly consists of proteoglycans formed through interactions with hyaluronic acid (HA), where the carboxylic acid group is key for protein binding. Instead of using native brain ECM, studies often employ hydrogels made from natural polysaccharides or polypeptides to mimic the ECMs mechanical and structural properties. These materials are cost-effective and support neural cell growth while maintaining cell viability [101].

Structural similarities to hyaluronic acid in guuronic and mannuronic acid residues. Because the polymer chain is negatively charged, alginate solutions can form polysaccharide hydrogels through covalent crosslinking between the guluronic acid residues and divalent cations. The encapsulation process can be performed while maintaining cell viability because gelation can occur quickly and under physiological conditions [24]. The mechanical characteristics of alginate hydrogels can be readily adjusted by adjusting the concentration of the crosslinker or the polymer. Therefore, it is possible to modify alginate hydrogels to mimic the stiffness of the brain. To encourage brain development, alginate must be chemically altered or combined with cell-adhesive substances because the polysaccharide chain lacks cell-adhesion motifs [102].

Mouse primary cortical neurons were bioprinted with a gellan gum-based bio-ink functionalized with the RDG peptide to form layered, brain-like 3D structures. Various hydrogels, using materials like hyaluronic acid, alginate, collagen, and gelatin, are explored for neural regeneration. These hydrogels, alone or loaded with neural stem cells (NSCs), can be implanted into brain tissue. In animal models, NSC-loaded hydrogels improved stem cell survival and cognitive function [103]. Cerebral organoids are key for brain research, often grown using Matrigel, a mouse tumor-derived hydrogel that lacks brain-specific complexity. A recent study used decellularized adult pig brain extracellular matrix (B-ECM) as a scaffold for human embryonic stem cell-derived brain organoids. B-ECM was processed using detergent and enzyme methods, and analyzed for structure, antigens, DNA, proteins, and mechanics. Despite the loss of some brain-specific proteins, B-ECM retained various collagen types and formed stable hydrogels at ≥ 5 mg/mL. Compared to Matrigel, B-ECM offered a more brain-relevant environment for organoid development [104].

The neurovascular unit (NVU), consisting of neurons, glial cells, vascular cells, and the surrounding ECM, regulates the blood–brain barrier (BBB). NVU disruption contributes to neurological diseases like Alzheimer’s and stroke. Using iPSCs to generate NVU cell types enables the creation of human NVU models for studying BBB function. Incorporating ECM provides essential 3D biochemical and mechanical cues. Methods such as hydrogels, transwells, 3D bioprinting, microfluidics, and organoids help model the NVU. These models are valuable for studying disease mechanisms, testing drug permeability, identifying therapies, and developing cell-based treatments [105].

Conductive polymers

Conductive polymers are extensively employed in nerve tissue scaffolds owing to their exceptional durability, ability to transmit electrical impulses, and capacity to encapsulate and deliver molecules. Both neural regeneration and the differentiation of stem cells into nerve cells are aided by electrical stimulation. The ability of conductive materials to replicate the microenvironment of stem cells and control the orientation, proliferation, and differentiation of neural stem cells makes them one of the top options for nerve regeneration. Numerous studies have demonstrated the use of conductive biomaterials such as graphene, graphite oxide, polypyrrole, polyaniline, poly(3,4-ethylenedioxythiophene), and single- and multi-walled carbon nanotubes to regulate the activities of neural stem cells with regard to neuronal differentiation and proliferation [82].

Polypyrrole (PPy): Owing to its reduced toxicity; outstanding mechanical, environmental, and thermal stability features; and changeable electrical characteristics, PPy has drawn much interest. PPy shows promise for creating electrically controlled carriers for targeted medication administration to the central nervous system and for long-term chronically implanted neuroprosthetic devices. PPy blended with other biodegradable polymers such as PCL and PLGA improves neurite adherence and development. It has been documented that the PPy-PDLLA poly (D, Lactic acid) scaffold may restore sciatic nerve deficits in rats to the level of a gold-standard autograft [106].

Polyaniline (PANI): PANi has gained popularity as a conductive polymer because of its high conductivity, affordability, ease of use, and straightforward manufacturing process. PANI scaffolds are intended for auxiliary use in neural stem cell differentiation and nerve regeneration. PANI/PLA-based electroactive electrospun fibers are efficient graft materials for PC12 cells enhanced by NGF-induced neurite outgrowth. PANI has also been proposed as a potential biosensor for electronic patches intended for therapeutic stimulation and recording [107].

Polypyrrole (PPy), polyaniline (PANI), and poly(3,4-ethylenedioxy-thiophene) (PEDOT) are electrically conductive polymers with similar chemical structures. These conductive polymers allow unrestricted electron mobility between atoms owing to a pattern of overlapping π-bonds and alternating single and double bonds. Polymers can be made more oxidative and their backbones broken by adding a dopant, which enables electrons to pass through when an electrical potential is applied, and the material characteristics of conducive polymers may be dramatically changed throughout the polymerization process by varying the types and quantities of doping agents. For instance, when chondroitin sulfate was added, the chondroitin sulfate-infused PPy became rougher. Compared to hyaluronic acid-doped PPy, polystyrene sulfonate-doped PPy is more flexible. PPy is the most researched electrically conductive polymer and has shown great promise as a biomaterial that can sustain respectable conductivity (1–75 S/m) under physiological conditions. The biocompatibility of PPy was demonstrated both in vitro and in vivo. Additionally, PPy can be readily synthesized using oxidants and electrochemical procedures because it dissolves in a variety of solvents, including water [108]. Traditionally, PPy films have been used to cultivate PC12 cells to investigate the effects of electrical stimulation on brain function. Electrical stimulation increased the length of neurite outgrowth in PC12 cells with unfunctionalized PPy by > 90%. Myelinated nerve fibers were shown to regrow four weeks after the implantation of PPy nerve conduits in the rat sciatic nerve model, and PPy was further combined with other polymers (such as poly(D, L-lactide-co-epsilon-caprolactone) and poly(lactic-co-glycolic acid)) to increase the mechanical strength and cytocompatibility of the polymer and to better control PC12 cell adherence and proliferation [109].

Biomaterials in stem cell based neuronal repair: an advanced approach

Cell therapy and tissue transplantation have long been popular targets for neurodegenerative diseases. But treating it has turned out to be more challenging than anticipated. For instance, mesencephalic transplantation alone will not be sufficient to treat Parkinson’s disease (PD), which involves more than just dopaminergic degradation in the substantia nigra. Significant issues also arise from side effects, such as the dyskinesias documented in two randomized, controlled trials [110, 111]. Teratoma development is another issue with transplanting such undifferentiated cells. Moreover, there is an unbreakable relationship between stem cells and cancer. There are habitats that draw these implanted stem cells and then trigger their proliferation and differentiation. It is also taken under consideration that there may be signals that guide the stem cells’ differentiation and subsequent migration to the appropriate niche following transplantation. They might develop into a tumor if their growth is unchecked, which could be more harmful than the initial illness [112].

Neural tissue engineering and regenerative medicine are now strongly dependent on the development of innovative and smart regenerative therapies to treat central nervous system degeneration. In order to promote regeneration outcomes, tissue engineering scaffolds have been created using both organic and inorganic materials that have been designed to interface with certain biological systems (depending on the application). It is hoped that biomaterials will help to overcome or lessen many of the obstacles that are now preventing the development of cell-based treatments for neuronal repair. These bioengineered scaffolds are logically made to give cells physical support (during differentiation and/or implantation), repair tissue architecture when necessary, and convey pertinent trophic signals to both new and remaining host cells with spatial and temporal control. Both host-derived and graft-derived neural cells can have their survival, proliferation, differentiation, and/or integration synergistically directed and controlled by neural tissue engineering, which takes use of scaffold design, material selection, and scaffold morphology. Mainly two strategies such as stimulating endogenous neurogenesis and cell transplantation were popular for brain repair and have therapeutic potential, but they also pose persistent problems for the field, such as sufficient cell numbers and tissue availability, survival, directed differentiation, and proper integration into the host tissue. Although studies have demonstrated that exposing stem cells to the right physical and chemical conditions enhances destiny determination and cellular integration, it can be challenging to provide these signals in vivo in a way that is optimal for both time and space. Bioengineered scaffolds have been suggested as a way to enhance cell-based treatments for brain healing. Researchers are looking at how biomaterials could improve cell survival and differentiation, restore tissue architecture, and encourage the integration and flexibility of both indigenous and transplanted stem cells.

These materials manage the present restrictions on stem cell treatment and have the potential to promote brain tissue regeneration and repair, which are crucial tools in regeneration and medical devices. Biomaterial-based scaffolds offer remarkable properties, such as adaptable physical and dimensional delivery, and are suitable for both in vitro and in vivo applications [83]. Owing to their unparalleled adaptability, biocompatibility, degradability, and mechanical qualities, polymeric biomaterials have garnered significant interest for neurodegeneration and neuroprotection. Biomaterial scaffolds or nanoparticles boost cell division and regeneration of nerve cells. These biomaterials produce a three-dimensional extracellular matrix that mimics several characteristics of the ECM in the living cell microenvironment [113]. Neural regeneration includes both synthetic and natural biopolymers, such as poly (L-lactic acid), poly (glycolic acid), silk fibroin, polyurethane, alginate, hyaluronan, collagen, and chitosan gelatin. Neurodegenerative medicine for transplantation and 3D in vitro neural models for drug screening have improved owing to the technological advances in biomimetic nanofibrous scaffolds. Hydrogels, carbon-based nanomaterials, and nanofibers have demonstrated the best quality in the field of brain regeneration.

Mechanisms of biomaterial-based tissue engineering and stem cells in neural repair

Polymeric scaffolds as extracellular matrix analogues: a novel approach to neuronal repair

Cells are supported by a temporary framework known as scaffolding while tissues are being built or repaired. Scaffold materials’ surface chemistry is thought to be the most important component in tissue engineering [114]. In biological systems, the extracellular matrix (ECM) serves as an interface for cell migration and interaction as well as a means of holding cells together. Therefore, it is ideal for the synthetic scaffold to stimulate cell adhesion, proliferation, and differentiation both in vitro and in vivo by imitating the ECM. As the name implies, two-dimensional tissue cultures only provide a monolayer of cells, unlike the three-dimensional nature of tissues in living things [115]. As a result, they cannot accurately simulate intricate biological interactions and are prone to hydrodynamic damage in bioreactors. Because of their vital cell-cell contact, increased surface area per unit volume, superior hydrodynamic protection, and enhanced regeneration of wounded tissue, three-dimensional tissue cultures have drawn more attention than two-dimensional cultures [116]. The transplantation of stem cells on polymeric scaffolds has been shown to result in better functional recovery and the development of neural networks to bridge the gap following spinal cord damage compared to transplantation of stem cells alone.

Combinative tactics to support stem cell-mediated repair

The regeneration capabilities of biomaterial-based technologies have significantly improved and are now widely used in practically every aspect of tissue engineering. They frequently operate as therapeutic delivery platforms with highly adjustable qualities, which are helpful for managing the local release of medications and growth hormones. This review showed that they can also act as platforms for delivering stem cells by performing various protective functions that boost survival. In light of this, therapeutic and cell-delivery biomaterial platforms are frequently viewed as two distinct approaches. Both show promise in achieving the therapeutic objectives when used alone. However, combining them with additional approaches may yield better synergistic benefits to meet the demands of increased transplant survival and regeneration. Combining seemingly unrelated methods into a single procedure could produce a robust stem cell delivery platform with potential applications beyond CNS restoration.

Biomaterial-based vascular support for stem cell and neuronal repair

The local microenvironment, sometimes referred to as the stem cell niche, significantly influences the development and activity of stem cells. High-density vascular networks are frequently observed in stem cell niches throughout the body because they transport the oxygen and nutrients required for stem cell differentiation and proliferation. Endothelial cells (ECs) are the primary cell type and crucial structural elements of blood arteries. These proteins are essential for angiogenesis. One area of the CNS where NSCs and ECs interact closely is the subventricular zone (SVZ). It has been demonstrated that SVZ NSC neurogenesis is closely correlated with how far away they are from the local vascular bed. [117] Furthermore, NSC self-renewal and neurogenesis are increased when ECs and cerebral cortex NSCs from embryonic days 10–11 (E10–11) are co-cultured in vitro in two-dimensional (2D) transwell plates. Previous in vitro models have demonstrated that signalling molecules produced by cerebral microvascular ECs further boost E10–11 and adult SVZ NSC development. These models aim to replicate the NSC niche using static or fluid flow 2D cell culture settings. NSCs and ECs have a strong association, as demonstrated by the protective function of NSCs in ECs in co-cultures against ischemic conditions through HIF-1α and VEGF signalling. Furthermore, it has been demonstrated that co-transplantation of adult NSCs produced by cortex-derived ischemia with bovine pulmonary microvascular ECs increases NSC survival at 28 and 5 d [118]. Approximately 3% of the surviving NSCs and their offspring developed NeuN+ neurons, a minimal amount, which was much higher than that of NSCs transplanted alone. Improving NSC-mediated regeneration by hydrogel-based cell transplantation may be possible by utilizing the benefits of niche modelling, in vitro co-culture, and early co-transplantation investigations. Although not much research has been conducted on in vivo co-transplantations in hydrogel networks, it is possible to implant human mesenchymal stem cell (hMSCs)-loaded injectable gelatin-based hydrogels containing NSC/EC co-spheroids into Sprague-Dawley rats [119]. Improvements in vascular development might be advantageous to NSCs, as the distribution of signals in blood circulation regulates the local NSC niche. However, the benefits of an NSC perspective have not been explored in either of these studies. Microparticle networks that release VEGF and poly(lactic-co-glycolic acid) (PLGA) have been investigated as cellular alternatives to generate a vascularized niche-like implantation site that might be exploited for NSC administration. Although HA-heparin microgels markedly enhanced vessel patency, each trial showed strong revascularization. With many particles lacking VEGF and a subset of particles loaded with a high VEGF content, the density of VEGF inside the microgel network is a crucial design feature. High-density microvascular structures were discovered without meticulously designed VEGF-loading parameterizations. However, they led to increased inflammation and astrogliosis instead of enhanced tissue repair and functional recovery in the VEGF density-controlled HA-heparin biomaterial. Only one of these studies investigated its biomaterial for NSC transplantation; nonetheless, astrocyte differentiation increased because of hypervascularization and inflammation. In each study, a porous biomaterial network was created by loading a network of biomaterial microspheres into a wound. Future research should combine the advantages of angiogenic materials to create an environment conducive for further NSC transplantation. [119]

Biomaterials serving as differentiation mediators of transplants

As previously discussed, NSC differentiation is affected by the composition of the biomaterial and the release of bioactive molecules, such as neurotrophins. As differentiation cannot occur without survival, it may be challenging to separate the effects of these variables on NSC differentiation from proliferation and survival. In addition to using biochemical signals, researchers have attempted to manipulate the resultant NSC progeny by preconditioning NSC populations with support cells known to boost neurogenesis, selecting cells for the preferred progenitor or precursor populations, and providing medications to stabilize microtubules. It is unknown whether the injury site primes the survival of specific NSC phenotypes or if preconditioning or selection of NSCs affects differentiation [120]. Some studies have shown that HA hydrogels with higher BMP-4 levels lead to increased astroglial differentiation and enhanced survival of transplanted cells. Although cell survival decreased in HA with lower BMP-4 concentrations and higher BDNF levels, there was an increase in neuronal differentiation among the surviving population, indicating an inverse link between survival rate and tissue repopulation. This inverse connection has been proposed as a model for selective elimination of some neurons during brain development. Neurons are prone to apoptosis and begin to stretch their axons during development. In this process, caspases that initiate cell death can also generate mitogens that promote the proliferation of NSCs. Co-delivery of neural progenitors and V2 spinal interneurons is a method for addressing the issues of survival and differentiation [121].

Engineered applications for in-vitro and in-vivo models for neuronal repair

Engineered application for in-vitro models in neuronal repair

One of the most significant techniques in neuroscience research is primary neuron culture. Initially, researchers employed conventional Petri dish-based 2D cell culture techniques, which helped to understand neuronal development [122]. For the first time, primary neurons were grown in vitro using a novel technique created by Ross Granville Harrison to produce live tissues from vertebrates [123].

2D models

Two-dimensional (2D) cell culture involves the growth of cells in a flat layer on a surface, such as glass or plastic dishes, using nutrient-rich media. This method, established in the early 1900s, remains stable and successful for cultivation of simple cells. It offers simplicity, efficiency, and cost-effectiveness, and ensures uniform growth by providing consistent access to nutrients and growth factors [124]. These new models include co-cultures (mixing different cell types), sandwich cultures (separating cells with a membrane), and conditioned medium assays (using secreted factors from one cell type to study the effects on another). These models allow researchers to study cell interactions more effectively than traditional monoculture [125, 126] models for ischemic stroke created by researchers employing methods such as glutamate exposure and oxygen deprivation. This enables them to investigate the effects of microglia and astrocytes, two crucial neuronal support cells, after a stroke. A protein named NLPR6 in astrocytes was found to potentially exacerbate neuronal damage following stroke [127]. Co-cultures with microglia and immune cells of the brain also shed light on their roles in various neurological conditions. Studies have shown that microglia can be toxic to neurons during ischemic stroke [128]. Researchers have used new co-culture systems with primary microglia and neurons to study the effects of microglia on neurons. They found that high glutamate levels activated microglia, causing neuronal injury. This study also explored potential treatments to protect neurons and reduce inflammation [129]. Additionally, some models have been developed using neurons from different brain regions to study connections crucial for memory and learning. These models are also valuable for assessing the safety of nanoparticles and drugs by helping identify potential neurotoxic effects. For example, a recent study revealed that some nanoparticles might indirectly harm neurons by activating microglia [130, 131]. Transwell cultures are widely utilized as supplements to 2D co-culture systems because of their ability to study indirect cellular interactions, simplicity of cell separation between upper and lower chambers, and precise control of variables. They play a crucial role in advancing our understanding of neurodegenerative diseases, their molecular mechanisms, and the development of potential therapeutic drugs [132, 133].

Ex vivo model

Ex vivo models offer a valuable approach for studying complex mechanisms in neuroscience research, overcoming the limitations of traditional two-dimensional (2D) cultures. These models involve preserving tissue or organ sections in culture, allowing for a more accurate simulation of the in vivo cellular distribution and interactions [134].

Brain slice cultures grown in a dish from thin sections of brain tissue offer a valuable tool for studying how neurons interact with each other and their surrounding environment. This method bridges the gap between simple 2D cell cultures and complex in vivo studies in living animals. One approach is to investigate the mechanisms of these diseases. Researchers have used hippocampal slices to study the protein leucine-rich glioma-inactivated 1) LGI1, implicated in epilepsy and encephalitis. By manipulating the LGI1 levels in the slices, they observed changes in neuronal activity, suggesting a potential target for treatment [135]. In addition, this culture method is valuable for pharmacological research. Scientists have used these drugs to test their effects on neuronal survival and synaptic function. For example, studies on levetiracetam, an epilepsy drug, have used hippocampal slices to investigate its impact on communication between neurons [136].

Unlike brain slice cultures, which focus on broader brain regions, hippocampal cultures isolate this memory and learning center in a dish. This allowed researchers to examine their development in detail. This technique is particularly useful for studying Fetal Alcohol Spectrum Disorders (FASD). By comparing gene activity in lab-grown hippocampi exposed to alcohol with that in living organisms, scientists can understand how alcohol disrupts memory and learning functions, even showing sex-specific effects. This approach not only helps us understand FASD, but also opens doors for developing treatments [137] (Fig. 5).

Spheroid and patient derived organoid models

Spheroids

Spheroid-based models are a type of 3D cell culture that is gaining traction in neuroscientific research. Unlike traditional flat cultures, these models allow cells to form 3D clusters that better mimic the brain environment. This was achieved by leveraging the natural tendency of cells to clump together [138]. Spheroids play a crucial role in tissue repair through two primary mechanisms: (1) direct involvement in tissue formation by supplying functionally specialized cells and (2) indirect support via the secretion of potent trophic factors that enhance tissue formation and attract essential host cells for regeneration. The spheroid size, determined by cell density, has a significant impact on cell function depending on the cell type and intended application [139]. Several techniques such as hanging drops or low-attachment surfaces can be used to create these spheroids. Researchers have successfully created complex spheroid models that include multiple cell types, mimicking the blood-brain barrier (BBB). This allowed us to study how drugs pass through the brain. Spheroids can be used to model diseases such as Parkinson’s disease by using stem cell-derived neurons [140].

Various cell lines have been used to create spheroids to study neuronal repair. Human Induced Pluripotent Stem Cell (hiPSC)-derived neurons and astrocytes offer a versatile approach that allows the aggregation of cells to form functional neural spheroids that mimic specific regions of the human brain. This capability makes them invaluable for modeling neurological diseases and screening for potential therapeutics [141]. Some studies utilizing human induced pluripotent stem cells (hiPSCs) in animal models have shown promising results in the treatment of neural injuries. These cells can differentiate into various neural lineages, aiding in the repair of damaged neuronal circuitry [142]. Additionally, hiPSCs are valuable for studying human neural development and modeling neurodegenerative diseases, such as Alzheimer’s, as well as neurodevelopmental disorders, such as autism spectrum disorders [143]. Research indicates that hiPSC-derived neural progenitor cells (hiPSC-NPs) can differentiate into tissue-specific neurons, offering the potential for long-term tissue restoration. For example, hiPSC-derived long-term neuroepithelial-like cells (LT-NCLCs) have been found to promote nerve fiber regeneration and enhance host myelination, suggesting a promising avenue for neural repair and regeneration studies [144]. Mesenchymal stem cells (MSCs) have been recognized for their safety and therapeutic potential in cell-based therapies. Acting as “molecular factories,” MSCs secrete trophic factors (NGF, GNDF and NT-3) and anti-apoptotic molecules, (eg, BCL-2, BCL-XL, MCL-1, BFL-1, BCL-W, and BCL2L10) enhancing neural cell survival and neurogenesis, thereby offering superior therapeutic effects for neural repair. Furthermore, MSC infusion exhibits immunomodulatory effects, reducing cerebral inflammatory responses by boosting the release of anti-inflammatory cytokines and interacting with the peripheral inflammatory systems. Researchers have investigated the therapeutic potential of MSC spheroids in neuropathic pain resulting from chronic constriction injury (CCI). They optimized spheroid generation and found that spheroids containing approximately 10,000 cells were the most effective. Transplantation of MSC spheroids near the sciatic nerve in a mouse model resulted in enhanced survival compared to that of traditional monolayer MSCs. [145]. Preclinical studies across various fields, including ischemic kidneys, cartilage regeneration, vascularization, and wound healing, have demonstrated the benefits of cell aggregation. For instance, in a rat model, transplantation of 3D MSC aggregates improved kidney tissue regeneration by augmenting the secretion of angiogenic factors (VEGF and TGF-β), anti-oxidative factors (IGF), and anti-inflammatory proteins (TSG-6). Similarly, MSC spheroid-loaded scaffolds have shown superior cartilage regeneration compared to single cells, likely owing to the formation of a significantly larger amount of cartilage-associated extracellular matrix [146]. This improved survival was correlated with reduced pain and inflammation, suggesting that MSC spheroids could be a promising therapy for neuronal injury. In contrast, when cultured in 3D, primary postnatal rat cortical cells form spheroids that exhibit in vivo-like features, including electrically active neurons that form excitatory and inhibitory synapses. This method is notable for its accessibility, reproducibility, and cost-effectiveness, making it a preferred choice for cortical studies [147]. Neural Crest Stem Cells (NCSCs) cultured in spheroids demonstrate a remarkable potential for neuronal repair and regeneration. This culture method enhances the secretion of soluble factors crucial for neuromuscular regeneration, including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and glial cell line-derived neurotrophic factor (GDNF). These factors create a supportive microenvironment conducive to neuronal survival, axonal outgrowth, and synaptic plasticity, fostering the regeneration of damaged neural circuits. Moreover, NCSC spheroids offer sustained trophic support to the surrounding cells, promoting long-term neuronal integration and function post-transplantation. The close cell-cell interactions within spheroids mimic physiological conditions, facilitating paracrine signalling and promoting the organization of NCSCs into functional neuronal networks upon transplantation. Additionally, encapsulation of NCSCs within spheroids provides protection from immune-mediated rejection, ensuring their survival and engraftment within the host tissue. Overall, NCSC spheroids represent a promising strategy for enhancing neuronal repair, offering potential advancements in the treatment of neurodegenerative diseases and traumatic injuries affecting the nervous system [148]. Peripheral Nerve-Derived Stem Cells (PNSCs), akin to neural-crest stem cells, hold potential for stem cell therapies in spinal cord injury regeneration because of their ability to differentiate into various cell types. Recent advancements in stem cell therapy have shown promising results in the treatment of spinal cord injury (SCI). Specifically, co-administration of peripheral nervous system stem cells (PNSCs) with Resolvin D1 has emerged as a potential therapeutic approach for SCI regeneration. Leveraging their capacity to secrete neurotrophic factors, PNSCs have significant potential for promoting neuronal regeneration and functional recovery in patients with SCI. In an animal model of SCI, PNSC spheroids were found to induce functional recovery and neuronal regeneration, while reducing neuropathic pain associated with SCI following remyelination. These findings underscore the diverse applications of spheroids for understanding and treating neuronal injuries and disorders. The exploration of PNSC spheroids in SCI therapy represents a promising avenue for developing novel therapeutic strategies to improve the outcomes and quality of life of individuals affected by SCI [149].

Spheroid culture conditions typically involve the use of specialized culture plates or scaffolds that facilitate cell aggregation and spheroid formation. The choice of culture medium and supplements depends on the cell type and desired experimental outcomes. Commonly used growth factors and supplements include fetal bovine serum (FBS), growth factors (e.g., EGF and FGF), and extracellular matrix components (e.g., Matrigel and collagen). The culture environment may also incorporate factors that mimic the physiological conditions of specific tissues, such as oxygen tension and pH [150]. Additionally, the duration of spheroid culture can vary depending on the experimental objectives and the cell type used. Spheroids typically require several days to weeks to form and mature, with culture times ranging from a few days to several weeks, or even months. Long-term culture allows for the assessment of complex biological processes such as cell proliferation, differentiation, and drug responses within the 3D microenvironment [151] (Fig. 6).

Fig. 6.

Histological and ultrastructural analysis of peripheral nerve regeneration and biomaterials evaluation i) microscopic results of peripheral nerve regeneration and biomaterials biodegradation: this picture shows how nerve, brain, and spinal cord injuries launch a complex sequence of defensive cellular processes and signalling cascades that biomaterial-based therapy techniques can control. After damage, macrophages (polarising into pro-inflammatory M1 or anti-inflammatory M2 phenotypes), astrocytes, microglia, T cells, and neutrophils activate. Whether secondary harm or repair occurs depends on these cellular responses. The central panel shows key signalling pathways involved in inflammation, apoptosis/survival, regeneration, and stem cell homing (NF-κB, IL-1 R, NLRP3 inflammasome, PI3K, Akt, ERK, MAPK). These pathways are important molecular targets for tissue healing and cell fate in regenerative medicine. Many biomaterials assist and modify these processes. These include natural biomaterials (agarose, alginate, collagen, gelatin, hyaluronic acid, and silk fibroin), semi-synthetic materials (PLGA, PLA, PEG), and synthetic polymers (polypyrrole, polyaniline, PEDOT) with unique chemical, mechanical, and biological properties. These biomaterials can be created into organoids, microporous scaffolds, hydrogels, soft hydrogels, and fibrous scaffolds to mimic the neuronal microenvironment and aid regeneration

Organoids

Organoids are innovative three-dimensional (3D) tissue structures derived from stem cells that offer sophisticated models of organ function and development. Organoids are more advantageous than 2D cell cultures and complex animal models, providing a versatile tool for studying various neurological injuries and complications [152]. Organoids are emerging as powerful tools for neuronal repair and disease modelling. The selection of the cell source plays a pivotal role in significantly influencing the variability, heterogeneity, and functionality of the resultant organoids. Neuronal stem cells or brain tissue from specific regions, such as the memory cortex, and movement and regulation of the substantia nigra. Pluripotent stem cells (PSCs) and adult neural stem cells (NSCs) are used as primary cells in neuronal organoid cultures. Additionally, a high-throughput drug screening platform for Alzheimer’s disease was developed using 1,300 organoids derived from iPS cells sourced from human peripheral blood mononuclear cells, representing the whole cerebrum. This platform incorporates CRISPR-Cas9-edited isogenic lines and Matrigel, facilitating comprehensive drug screening across 11 human donors [153]. Spinal cord organoids derived from iPS and ES cells isolated from early stage embryos were used to study ALS. These organoids, embedded in Matrigel, mimic lower motor neuron-muscular interfaces. ALS-linked cells in these organoids show impairments, shedding light on ALS pathology [154]. Striatal organoids derived from iPS cells of healthy individuals and fibroblasts from patients with Huntington’s disease were used to study the disease. Using this model embedded in Matrigel, researchers identified the overaccumulation of HSF1 in the mitochondria, resulting in Huntington’s disease-like behaviors observed in rodent models [155]. In a novel study utilizing midbrain-like organoids generated from iPS cells of patients with Parkinson’s disease carrying the LRRK2(G2019S) mutation, researchers isolated a neurodevelopmental defect in dopaminergic cells. This defect is indicative of Parkinson’s disease pathology, providing valuable insights into the disease mechanism [156]. A study investigated autism spectrum disorder (ASD) using forebrain organoids derived from induced pluripotent stem (iPS) cells obtained from patients with CNTNAP2-associated ASD. These organoids, cultivated within Matrigel, displayed a unique phenotype of cortical overgrowth, primarily driven by progenitor cells, a hallmark of ASD. This study further demonstrated the potential reversibility of this phenotype through gene editing techniques involving CRISPR-Cas9 technology [157]. To investigate schizophrenia, researchers have developed whole cerebrum organoids using induced pluripotent stem (iPS) cells from human fibroblasts. Cultivated in Matrigel, these organoids served as a model for schizophrenia developmental disruption, driven by abnormal WNT signalling, particularly in cells with DISC1 mutations [158] (Fig. 6).

Another study explored ischemia by generating forebrain organoids, including choroid plexus components, from human donor-derived embryonic stem (ES) cells. These organoids, which were also cultured in Matrigel, were transplanted into a rodent model of middle cerebral artery occlusion. Their transplantation not only promotes repair processes but also stimulates endogenous neurogenesis within the ischemic brain region, suggesting potential therapeutic avenues for stroke recovery [159]. In some instances, organoids are cultured in bioreactors to provide a controlled environment with continuous perfusion of nutrients and removal of waste products. This approach is particularly beneficial for culturing larger organoids and for facilitating long-term culture. Moreover, the culture environment may incorporate factors that replicate physiological conditions, including oxygen levels, pH balance, and mechanical cues, to ensure fidelity of organoid development and function [160]. Moreover, in organoid media, growth factors play a pivotal role in guiding stem cell differentiation to establish and maintain specific types of organoids. Some critical growth factors utilized in organoid culture include R-spondins, Noggin, Wnt-3a, BMP signalling antagonists, Gremlin 1, EGF, FGF10, FGF2, and Activin A. Each factors uniquely contributes to the regulation of cell fate and function, thereby facilitating the successful generation and sustenance of organoids with desired characteristics [161]. Organoid methods typically involve culture times ranging from 2 to 8 weeks. Neuronal organoids serve as invaluable tools for unraveling complex mechanisms and facilitating the healing of neuronal injuries [162].

Notably, seminal work by Mansour et al. showed that human brain organoids transplanted into the mouse cortex could survive long term, vascularize, and establish synaptic connections with host tissue marking a critical step toward functional integration in vivo [163]. Building on this, Revah et al. demonstrated that human cortical organoids implanted into newborn rat brains not only integrated structurally and electrophysiologically but also responded to sensory stimuli and influenced host behavior. These findings underscore the potential of organoids and assembloids to contribute to circuit-level repair and cognitive function restoration after injury or degeneration [164]. Incorporating organoid-based strategies into neural repair frameworks may overcome limitations of traditional stem cell therapies by providing more structurally organized, region-specific grafts that mimic human brain architecture [165]. Studies shown, combining organoid engineering with in vivo transplantation could revolutionize treatment paradigms for conditions such as stroke, traumatic brain injury, and neurodegenerative diseases. Going forward, refining organoid maturation, improving vascular integration, and enhancing functional connectivity will be essential for clinical translation.

Additionally, the study by Sozzi et al. (2022) presents an innovative approach to improve human brain organoid development using silk scaffolding. By integrating a recombinant spider silk matrix biofunctionalized with human laminin, the researchers created a 3D environment that enhances neuroectoderm formation, reduces cellular heterogeneity, and improves oxygenation. This method led to more structurally organized and functionally mature brain organoids, offering a robust platform for modeling human brain development and neurological diseases [72]. Moreover, study demonstrates how human pluripotent stem cell-derived midbrain organoids can model key aspects of dopamine (DA) neuron development. Using single-cell RNA sequencing, the researchers identified three distinct DA neuron subtypes resembling those in the human midbrain. To enhance organoid maturation and reproducibility, they introduced spider silk-based scaffolds functionalized with human laminin. This bioengineered system offers a robust platform for studying human brain development and disorders like Parkinson’s disease [166]. A novel neural assembloid model called Midbrain–Striatum–Cortex Organoids, which integrates region-specific human brain organoids ventral midbrain, striatum, and cortex into a linear arrangement mimicking the brain’s anterior posterior axis. This setup enables the formation of functional long-range dopaminergic projections, allowing the study of neuronal maturation, connectivity, and function. MISCOs effectively address the limitations of traditional organoid models by incorporating inter-regional interactions, providing a physiologically relevant platform to investigate dopaminergic circuit development, drug-induced changes (e.g., cocaine exposure), and potential cell therapies for neurological disorders like Parkinson’s disease [167, 168].

Engineered applications for in-vivo model neuronal repair

The nervous system is a complex network and the repair of neuronal damage can be challenging. However, recent advances in engineering applications show promise for in vivo neuronal repair in rodent and primate models [169]. Various studies have explored the application of genetically modified stem cells, multicomponent polymer scaffolds, and self-assembling peptide hydrogels to enhance neural stem cell differentiation and foster axonal regeneration in rats with spinal cord injury [170–173].

Neural Stem Cells (NSCs), which are improved by neuroprotective factors or enhanced migratory capabilities, exhibit significant potential in rodent stroke and spinal cord injury models. Human NSC transplantation significantly improves motor function in rats [174]. In neural regeneration, neural stem cells (NSCs) play a crucial role in neural regeneration. Neurological system diseases can be treated with cell therapy based on NSC transplantation [175]. Induced Pluripotent Stem Cells (iPSCs) hold immense potential for personalized medicine, especially in neurological disorders, such as Parkinson’s disease. Differentiating iPSCs into specific neuronal subtypes has shown promising results, improving motor recovery in rodent models without the need for immunosuppression or immunological rejection. Thus, iPSC-based interventions offer potential therapeutic benefits against neurological conditions [176, 177].

Hydrogels and scaffolds are biomaterials that function as bridges in rodent spinal cord injury models, promote axonal development, and provide structural support. Rats with 3D-printed biodegradable scaffolds showed enhanced axonal regeneration and functional recovery [178]. Nanoengineered delivery systems: Nanoparticles with therapeutic agents such as growth factors or neuroprotective drugs can target specific cell populations and enhance repair processes [179]. A rodent model of Alzheimer’s disease showed memory improvement with the targeted delivery of anti-amyloid oligomers using nanoparticles [180, 181] (Table 1).

Table 1.

Polymer/Biomaterial, source, applications, advantages and disadvantages

Polymer/Biomaterial	Source	General Applications	Clinical Applications	Advantages	Disadvantages	Ref
Agarose	Natural	Neuronal Neuronal regeneration, 3D neurite growth, neurogenesis	Use in spinal cord injury models to guide axonal regeneration by acting as a neutral, porous scaffold that supports neuron adhesion and alignment	Biocompatible, thermo-reversible, supports neurite growth	Brittle, contracts under stress, requires blending	[182–184]
Alginate	Natural	NSC differentiation in 3D scaffold	Alginate scaffolds support peripheral nerve regeneration by serving as carriers for Schwann cells or exosomes. They maintain structure and allow nutrient exchange while being slowly degradable	Tunable stiffness, biocompatible, supports NSC growth	Lacks cell adhesion sites, requires chemical modification	[184–186]
Collagen	Natural	Nerve conduits, Scaffolds for stem cell transplantation, and drug delivery systems	Collagen is extensively used in nerve conduits for both central and peripheral nervous system repair. It supports axonal growth and mimics the extracellular matrix (ECM) of nerve tissue	FDA-approved, supports nerve growth and cell adhesion	Low mechanical strength, expensive	[184, 187]
Gelatin	Natural	Cell survival, Stem cell therapy, Proliferation	Gelatin-based nerve scaffolds have been shown to enhance axon elongation and support neurotrophic factor delivery, especially when modified with cross-linkers or growth factors	Low cost, supports 3D tissue engineering, printable	Degrades quickly, may lack structural stability alone	[184, 188]
Hyaluronic Acid	Natural	Neural tissue repair, Cognitive recovery post-injury, Modeling neurological disorders	These promotes glial scar reduction and axon regeneration in spinal cord injuries due to its anti-inflammatory and space-filling properties. It’s often used in injectable gels for minimally invasive therapy	ECM component, promotes NSC survival and differentiation	Poor cell adhesion, needs combination with other materials	[184, 189, 190]
Silk Fibroin	Natural	Promotes neuronal differentiation, increase cells attachment	Silk fibroin has been used in nerve guidance conduits for peripheral nerve repair. It combines mechanical strength with cell compatibility and has shown promising results in sciatic nerve regeneration	Biocompatible, good mechanical properties, supports nerve regeneration	Limited availability, batch variation, complex processing	[184, 191, 192]
		Helps stem cell therapy neuronal survival
Polylactic Acid (PLA)	Synthetic	Support differentiation of NSCs and NPCs and neurite out growth	PLA-based scaffolds have been explored for spinal cord repair and nerve guidance, offering biodegradable support with tunable stiffness that can be tailored to match neural tissues	Biodegradable, supports NGF delivery, enhances axon formation	Hydrophobic, structural instability, limited bioactivity	[193, 194]
Poly(lactic-co-glycolic acid) (PLGA)	Synthetic	Neural stem cell scaffolding, Axonal regeneration	PLGA is widely used for controlled delivery of neurotrophic factors and in nerve conduits due to its well-established degradation profile and compatibility with neuron-supporting drugs	Biodegradable, FDA-approved, supports neuron adhesion and migration	Hydrophobic, limited functional groups, low conductivity	[195, 196]
Polyethylene Glycol (PEG)	Synthetic	Stem cell therapy, Cell survival, Axonal regeneration	PEG is used in neural tissue sealing and fusion, especially in acute spinal cord injuries, where it promotes membrane repair and axonal continuity	Hydrophilic, tunable, supports NSC culture, drug delivery potential	Not bioactive alone, poor 3D mimicry of spinal cord	[197, 198]
Polyurethane (PU)	Synthetic	Axonal regeneration, Stem cell therapy	PUs elasticity and biocompatibility make it suitable for nerve regeneration scaffolds, especially in dynamic or load-bearing regions of the nervous system	Biocompatible, thermoresponsive, good for bioprinting	Limited mechanical strength, poor cell adhesion	[199, 200]
Polycaprolactone (PCL)	Synthetic	Differentiation & Neurite outgrowth, Affect NSC/Progenitor proliferation	PCL is used in nerve guidance conduits and 3D-printed neural scaffolds, supporting cell migration and neurite outgrowth due to its long-term stability and porous architecture	Good mechanical strength, supports Schwann cells and neurons	Slow degradation, hydrophobicity affects cell infiltration	[201]
Polyvinyl Alcohol (PVA)	Synthetic	Support neuronal proliferation and differentiation, Stem cell therapy	PVA hydrogels serve as soft, water-rich matrices for nerve regeneration, especially in combination with bioactive agents or as part of hybrid hydrogels	High water retention, biocompatible, potential for neuroprotection	Breaks into acidic products, loses adhesion, limited scale-up	[201, 202]
Hydrogels	Natural/Synthetic	Neuroregeneration scaffold, NSC encapsulation	Various hydrogels (e.g., collagen, HA, PEG-based) are being used in neural regeneration to encapsulate cells, deliver drugs, and mimic ECM for neuron survival and axon guidance	Mimics ECM, tunable, supports NSC encapsulation and delivery	May not mimic all ECM components, complex tuning needed	[202, 203]
Polypyrrole (PPy)	Synthetic	Neural stem cell differentiation, Neurite outgrowth promotion	PPy, a conductive polymer, enhances neural stem cell differentiation and electrical signal transduction, making it valuable for neural interfacing and repair, especially in combination with hydrogels.	Electroconductive, enhances neurite growth, biocompatible	Brittle, limited long-term in vivo data	[204, 205]
Polyaniline (PANI)	Synthetic	Neurite outgrowth enhancement	PANI is being explored for neural regeneration and stimulation, particularly due to its conductivity, but clinical use is still limited due to long-term stability and cytocompatibility concerns.	High conductivity, promotes NSC differentiation	Difficult synthesis, potential toxicity	[206, 207]
PEDOT	Synthetic	Neurite outgrowth, Neural scaffold coating	PEDOT is clinically relevant in cochlear implants and brain-machine interfaces, as it improves electrical conductivity and tissue integration in neural electrodes.	Electroconductive, stable, supports neuroregeneration	Requires dopants, complex polymerization	[208–210]

The many biomaterials investigated in preclinical in vivo studies for cell-based brain repair in the central nervous system are detailed in the following Table 2. These studies used models of neurodegenerative diseases, traumatic brain injuries, strokes, and spinal cord injuries. In neurodegenerative diseases such as Multiple Sclerosis (MS), poly (lactic-co-glycolic acid)-based nanoparticles carrying leukemia inhibitory factors demonstrated increased myelin repair in mice with demyelinating lesions [217]. RADA16-YIGSR nanofibers for Alzheimer’s disease (AD) showed increased memory and learning recovery through improved neuronal survival and development of rat hippocampal-derived neural stem cells (NSCs) [218].

Table 2.

Summary of the features, advantages, and limitations of the primary neuron in vitro model

In Vitro Model	Biomaterials used	Marterial Characteristics	Advantage	Limitation	Reference
2D in Vitro model	Poly-lysine coated plate	Biocompatible, biomimetic surface chemistry	Easy to use, versatile, well-established methods	Lacks 3D complexity and cell-cell interactions Doesn’t replicate the in vivo microenvironment.	[211]
Ex vivo	Porous membrane (1–10 µm)	Biocompatibility, mechanical strength, and permeability	Maintains cell types, 3D networks, and connections	Limited lifespan (1–2 weeks)	[212]
Spheroid	Matrigel or low-attachment plate	Provides cell adhesion site and mimic extracellular matrix	Partially mimics 3D structures, easy to make, high-throughput potential	Uncontrollable cell composition and arrangement, variations between batches	[213]
Scaffold	Natural and synthetic polymers	versatility in scaffold design	Mimics real-life environment, controlled release of substances	Biocompatibility issues, complex to produce	[214]
3D bioprinting	Hydrogels with controlled gelling and printability	Crosslinking capability, biocompatibility	Highly controlled cell arrangement, high-throughput potential	Shear stress can damage neuronal cells	[215]
Microfluidic Chip	Polydimethylsiloxane (PDMS)	Elasticity and flexibility, Gas permeability	Precise control over environment, high reproducibility, high-throughput potential	Expensive equipment needed, difficult to design a realistic environment, Sometime toxic to cells	[216]

In Parkinson’s disease (PD) models, various biomaterials, such as growth factor-reduced Matrigel hydrogel, GDNF-bound poly (l-lactic acid) nanofibers, and HA with RGD and heparin hydrogel exhibited positive outcomes, including increased survival, integration of grafts, and enhanced motor function [219–221]. When GDNF-loaded type 1 collagen hydrogel was applied to rats with 6-OHDA lesions, the immune response was lowered, motor function improved, and striatal reinnervation improved [222].

In traumatic brain injury models, the utilization of type 1 collagen-based scaffolds, type 1 collagen hydrogel scaffolds, and hydrogels featuring a self-assembling poly (RADA) peptide linked with a laminin-derived IKVAV motif resulted in enhanced survival of transplanted cells, improved spatial organization, increased sensorimotor function, and more significant neurite outgrowth [223–225]. Similarly, using a collagen and fibronectin hydrogel scaffold in mice given controlled cortical impact (CCI) increased the migration and survival of neural stem cells (NSCs) [226].

In stroke condition models, the deployment of PLGA microparticles and VEGF-encapsulated PLGA microparticles in rats with middle cerebral artery occlusion (MCAO) facilitated effective integration between the graft and host tissue, accompanied by an augmented angiogenic response in the transplant region [227, 228]. Putting VEGF-producing baby hamster kidney (BHK) cells inside polysulfone hollow fibers leads to increased blood vessel growth, reduced cell death, and improved motor performance [229]. A mixed hydrogel of hyaluronic acid (HA), gelatin, and heparin was also used in mice with focal ischemic injuries. This helps neural progenitor cells (NPCs) live longer and reduce graft-related inflammation [230].

Various biomaterials have demonstrated favorable outcomes in SCI models. Some of these include a methylcellulose hydrogel with chondroitinase ABC, a collagen hydrogel scaffold with an EGFR antibody, hyaluronan, a methylcellulose hydrogel, an LAP hydrogel with neurotrophic factors, a fibrin hydrogel with a mix of growth factors, and a hyaluronan and methylcellulose hydrogel. These biomaterials have shown positive effects on graft maturation, enhancement of forelimb function, promotion of neural plasticity, synapse formation, facilitation of axonal regeneration, and overall functional recovery [231–237].

Additionally, in spinal cord injury models in rats and mice, type 1 collagen, fibrin, alginate, and chitosan microfibers, as well as a fibrin hydrogel enriched with BDNF, have been demonstrated to increase the survival and migration of transplanted cells, further contributing to positive outcomes [238, 239] (Fig. 7).

Fig. 7.

Steps for developing a 3D ex vivo peripheral nerve injury (PNI) model. The figure illustrates the development of a 3D ex vivo peripheral nerve injury (PNI) model. The process begins with threading fibers into a nerve guide conduit (NGC), followed by the extraction of dorsal root ganglia (DRG) from the rat spinal column. The nerve roots are then carefully trimmed from the DRG body, which is placed on top of the fibers within the NGC. This setup is incubated in a suitable media at 37 °C for 21 days to allow for tissue culture and potential nerve regeneration. After the incubation period, the DRG body is removed from the NGC and transferred onto a microscope slide for further analysis. Finally, the nerve repair process is observed under confocal microscopy, focusing on the growth cone, which serves as an indicator of nerve regeneration. This model is designed to study nerve regeneration and repair mechanisms in a controlled laboratory environment

Advantages of in-vivo neuronal repair in rodent and primate models

In vivo animal models have significant advantages in the search for treatment options for neurological disorders. Rodents, such as mice and rats, are cost-effective and readily available for large-scale neuronal repair research. Their characterized neuroanatomical structure, extensive genetic manipulation techniques, rapid data generation, and fast breeding rate help in targeted neuronal repair approaches and practical assessments of functional recovery [240, 241]. The close similarity between the primate neuron and human neuron systems, along with their higher cognitive abilities and complex behavior, makes it essential to translate neuronal repair approaches with promising efficacy in clinical trials [86]. Models such as these contribute to the knowledge of the active cellular and molecular mechanisms driving brain-healing processes by revealing their real-time monitoring [242]. These models significantly evaluate the safety and efficacy of diverse neuroregeneration strategies, such as stem cell therapy, gene therapy, neuromodulation methods, and biomaterials in neuronal repair [243–245]. Behavioral and cognitive evaluations of post-neuronal interventions offer directions for potential clinical benefit. Although each model has limitations, collective efforts to understand neuronal repair mechanisms in rodents and primates have provided valuable insights for developing effective treatments for neurological disorders. [246].

Although each model has limitations, collective efforts to understand neuronal repair mechanisms in rodents and primates have provided valuable insights for developing effective treatments for neurological disorders. Before moving on to clinical translation for human brain repair, it is crucial to build a strong foundation for non-human primate (NHP) models, as many clinical trials for CNS conditions have failed despite positive preclinical research in rodent models. Specific findings in rodent models show promising outcomes, but do not work in models, including primates. [247–249]. Neuroregeneration research has several limitations that impact the translation of findings from animal models, mainly rodents and primates, to human clinical trials. One significant challenge is species differences, as intricate variances in the complexity and function of rodents and human brains can hinder the success of promising strategies identified in animal studies [169, 241]. In addition, because rodents do not have advanced cognitive abilities, it is difficult to show how neuroregeneration affects higher-order cognitive functions essential for neurological diseases in humans, such as Alzheimer’s disease [250]. Furthermore, immune rejection is a significant problem because rodents have a robust immune system, which can render cell transplantation for neuroregeneration less effective and possibly even kill the graft [251, 252]. Additionally, the high cost and limited availability of primate models present challenges for large-scale studies, prolonging the translational process owing to primates’ demanding care and housing needs [253]. Ethical considerations further complicate primate research, necessitating stringent adherence to animal welfare guidelines and a thorough justification for their use in studies [254]. Differential and inconsistent outcomes in how primates respond to neuroregenerative treatments make it difficult to determine the meaning of the data. Genetics, advancing age, and environmental factors are potential causes of these problems [255, 256]. The complexity of the nervous system itself adds another layer of difficulty, as it is highly intricate, with interconnected components that differ between primates and humans [257]. Off-target effects are a notable concern in neuroregeneration because therapeutic agents or procedures may lead to unintended side effects outside the targeted repair area. Biomaterials and stem cell therapies have potential side effects in animals and humans [257]. Finally, ensuring the long-term sustainability of transplanted cells or repaired tissue is challenging because of the dynamic nature of the nervous system, which requires constant adaptation for sustained effectiveness [258]. These limitations collectively underscore the need for a broad understanding of these challenges to enhance the translational success of neuroregenerative strategies (Tables 3 and 4).

Table 3.

Biomaterials in preclinical in vivo studies for cell-based brain repair in the central nervous system

Related Disease	Biomaterials	Animal Model	Cell type	Study output	Reference
1. Neurodegenerative Diseases
Multiple Sclerosis (MS)	Poly(lactic-co-glycolic acid)-based nanoparticles carrying leukaemia inhibitory factor	Mice with demyelinating lesions	Oligodendrocyte Progenitor Cells	Increased myelin repair	[217]
Alzheimer’s Disease (AD)	RADA16-YIGSR (synthetic self-assembling peptide) nanofibers	Rats with Aβ1–40 intra-hippocampal injection	Rat hippocampal-derived NSC	Improved NSC neuronal survival and differentiation as well as the recovery of memory and learning	[97]
Parkinson Disease (PD)	Growth factor-reduced Matrigel hydrogel	Mice	Embryonic stem cell-derived Neural precursor cells (NPCs)	Increased NPC survival and differentiation	[219–221]
Parkinson Disease (PD)	GDNF (glial cell line-derived neurotrophic Factor) bound Poly(l-lactic acid) nanofibers in a xyloglucan hydrogel with	Mice with 6-OHDA lesion	Ventral mDA (Mesencephalic dopamine) progenitors	Enhanced survival and integration of VM (Ventral Mesencephalon) grafts	[222]
	Soluble GDNF
Parkinson Disease (PD)	HA with RGD and heparin hydrogel	Rats	Human pluripotent stem cell (hPSC)-derived mDA progenitors	Enhance graft survival by 5-fold	[220, 222]
Parkinson Disease (PD)	GDNF-loaded type 1 collagen hydrogel	Rats with 6-OHDA lesion (PD)	VM grafts	Reduced immune response from the host, enhanced survival of grafted dopaminergic neurons, heightened striatal re-innervation, and improved motor function	[222]
2.Traumatic brain injury
	Type 1 collagen-based scaffold with the	Mice with CCI (Controlled cortical impact)	Primary fetal-derived NSC	Improved transplant cell survival and better spatial organization were attributed to the laminin scaffold.	[223]
	addition of either laminin or fibronectin
	Type-1 collagen	Rats with CCI	hMSCs (human mesenchymal stem cells)	Enhancement of sensorimotor function and spatial learning, together with a rise in VEGF-positive astrocytes that support brain function	[223, 224]
	hydrogel scaffold
	Type-1 collagen hydrogel scaffold	Rats with CCI	hMSCs	Increased neurite outgrowth, brain metabolism, functional recovery, and cell survival while lowering cell diffusion to non-specific tissues	[225]
	Hydrogel of self-assembling Poly(RADA) peptide linked with laminin-derived IKVAV motif	Rats with biopsy punch of cortex	Rat NSCs	decreased glial astrocyte production and increased NSC survival and differentiation	[259]
	Collagen and fibronectin hydrogel scaffold	Mice with CCI	Mice NSCs	Increased migration and survival of NSCs	[226]
3.Stroke Models
	PLGA microparticles	Rats with MCAO	NSCs	Integration of the graft and host tissue with efficiency	[226]
	VEGF-encapsulated PLGA microparticle	Rats with MCAO	Human NSCs	Angiogenesis in the transplant region increases	[228]
	Capsules made of polysulfone hollow fibers that encapsulate the cells	Rats with MCAO 6 days after cell transplantation	VEGF producing Baby hamster kidney (BHK) cells	Enhanced angiogenesis, decreased apoptotic cell count, and enhanced motor performance	[229]
	Blend of HA, gelatin, and heparin hydrogel	Mice with focal ischemia injury	Mouse cortex-derived NPCs	Improved NPC survival and reduced graft-related inflammation	[230]
	Polyglycolic acid (PGA) fiber scaffold	Mice with hypoxia ischemia injury	NSPCs	Decreased host immunological response, enhanced cortical tissue regeneration and neuronal differentiation
					[260]
4. Spinal cord injury models
	Fibrin hydrogel with BDNF, NT-3, GDNF, EGF, bFGF, aFGF, HGF, IGF-1, VEGF, PDGF-AA, calpain inhibitor	Rhesus monkeys with SCI	Human spinal cord-derived NPCs	Grafts matured and enhanced forelimb function	[231]
	GelMa, PEGDA, LAP hydrogel with BDNF, VEGF, bFGF, and calpain inhibitor	Rats with SCI	Rat spinal cord-derived neural progenitor cells (NPCs)	Host axons synapse onto transplanted NPCs, efficient recovery	[232]
	Methylcellulose hydrogel with chondroitinase ABC (ChABC) to promote neural plasticity and functional recovery	Rats with SCI	Human oligodendrocyte progenitor cells (oNPCs)	Enhanced oNPC survival and differentiation as well as enhanced axon remyelination in sparing areas	[233]
	Collagen hydrogel scaffold with epidermal growth factor receptor (EGFR) antibody	Rats with SCI	Rat brain-derived NSCPCs	Improved synapse formation, neuronal differentiation, axonal regeneration, and functional recovery	[234]
	Hyaluronan and methylcellulose (HAMC) hydrogel	Rats with SCI	Rat brain-derived NSCPCs	Produced increased forelimb function, functioning synapses, and regeneration of corticospinal axons.	[235]
	Hyaluronan and methylcellulose hydrogel modified with RGD peptide and platelet-derived growth factor	Rats with SCI	Human iPSC-derived OPCs	Promoted cell survival and incorporation and attenuated teratoma formation	[236]
	Fibrin hydrogel with BDNF, NT-3, PDGF-AA, IGF-1, EGF, bFDF, aFGF, GDNF, HGF, and calpain inhibitor	Rats with SCI	Rat spinal cord-derived NSPCs	Increased forelimb function, active synapses, and regeneration of corticospinal axons.	[237]
	Type 1 collagen, fibrin, alginate and chitosan microfibers	Mice with SCI	Mice neural stem/progenitor cells (NS/PCs)	Increased cell survival	[238]
	Fibrin hydrogel	Rats with SCI	Bone marrow stromal cell (BMSC)	Increased migration and survival of transplanted cells	[239]

Table 4.

Limitations in-vivo neuronal repair in rodent and primate models

Sr. No.	Limitations	Interpretation	Reference
1	Species differences	Despite extensive characterization, variations in complexity and function between rodent and human brains limit the translation of promising neuroregeneration strategies. For example, successes in rodent models of spinal cord injury may not replicate in clinical trials, suggesting potential disparities in repair mechanisms between rodents and humans.	[169, 241]
2	Limited cognitive abilities	The constrained behavioural range of rodents may not fully represent the impact of neuroregeneration on higher-order cognitive functions relevant to human neurological disorders, as evidenced by differences between rodent models and clinical trials, particularly in Alzheimer’s disease.	[241]
3	Immune rejection	Cell transplantation for neuroregeneration in rodents faces challenges due to their strong immune response, potentially limiting graft survival and therapeutic efficacy, as their immune system may attack and reject transplanted cells.	[241, 250]
4	High cost and limited availability	Primate models pose challenges for research due to their higher costs and limited availability compared to rodents, hampering large-scale studies and prolonging the translational process. These challenges arise from the difficult care and housing needs of primates.	[253]
5	Ethical considerations	Ensuring ethical standards in primate research is crucial, demanding thorough justification and strict adherence to animal welfare guidelines Primates are highly intelligent and social animals, and their use in research must be carefully considered.	[254]
6	Heterogeneity and variable results	Variability in primate responses to neuroregeneration interventions can be significant and create challenges in data interpretation. This complexity arises from factors like genetics, age, and environmental conditions, making outcomes inconsistent.	[255, 256]
7	Complexity of the nervous system	The nervous system is a highly complex system with a variety of interconnected components. This complexity can make it difficult to target specific repair mechanisms without affecting other areas of the brain. These complexities differ between primates and human.	[257]
8	Off-target effects	Off-target effects are a concern in neuroregeneration as therapeutic agents or procedures may cause unintended side effects outside the intended repair area. The use of biomaterial and stem cell therapy in neuroregeneration can introduce potential side effects in animals and humans.	[257]
9	Long-term sustainability	Ensuring the long term survival and functional integration of transplanted cells or repaired tissue is challenging in neuroregeneration. The dynamic nature of the nervous system requires adaptation for long-term effectiveness.	[261]

Clinical translations, challenges and future directions

Disruption of the nervous system by injury or disease can lead to reflective and devastating consequences. Although preclinical research shows promising signs of neuronal regeneration, translating these discoveries into practical clinical applications poses a formidable challenge [52]. Therapies that demonstrate remarkable efficacy in animal models or controlled environments struggle with the unique complexities of the human brain, considering subtle species differences and diverse array of injury patterns [53].

The translational potential of brain organoid and neural assembloid technologies is rapidly gaining momentum, particularly in the context of neurodegenerative diseases such as Parkinson’s disease (PD). While much of the current focus lies in disease modeling and drug screening, significant strides have also been made toward clinical application through cell replacement therapies. Notably, recent transplantation-based studies underscore the promise of stem cell-derived dopaminergic neurons in restoring motor function in PD models and patients. For instance, Kirkeby et al. study demonstrated long-term functional integration of human pluripotent stem cell-derived midbrain dopaminergic progenitors following transplantation into PD patients, revealing safety and sustained clinical benefit [262]. Similarly, Piao et al. study group provided critical insights into immune compatibility and graft maturation, reinforcing the feasibility of such interventions [263]. These advances reflect a maturation of the field from bench to bedside and suggest that organoid-derived cell types may soon play a central role in regenerative strategies. Moving forward, integrating organoid and assembloid systems with transplantation paradigms could help optimize donor cell properties, improve graft-host integration, and personalize therapy. Continued interdisciplinary collaboration and regulatory alignment will be essential to harness the full clinical potential of these technologies.

Notably, batches of these stem cell derived neurons could be reproducibly manufactured with consistent potency, addressing a key scalability challenge in regenerative therapy [262]. These advances have rapidly translated to the clinic: the MSK-DA01/BlueRock product gained FDA clearance to initiate a U.S. trial [264] and the STEM-PD cells entered a first-in-human European trial in 2022 [262]. Early clinical findings are highly encouraging an initial open-label trial of an hESC-derived dopamine neuron graft (bemdaneprocel) in PD patients achieved its primary safety endpoint at 1 year, with no cell-related adverse effects or evidence of tumor formation [265]. At 18 months post-transplant, transplanted cells were shown to survive and function (increased putaminal F-DOPA PET uptake), and treated patients exhibited improved motor scores compared to baseline, especially in a high-dose cohort [265]. In parallel, an allogeneic iPSC-derived dopamine neuron transplant trial in Japan has also reported an absence of serious safety issues and signs of dopaminergic activity in vivo. Together, these milestone studies mark an inflection point in PD therapy, validating the concept that stem cell derived dopaminergic neurons can be delivered safely into the human brain and even impart measurable symptomatic benefits [266].

Comprehensive clinical studies conducted with careful attention to ethical principles play a critical role in striking a delicate balance between safety, effectiveness, and optimal treatment for each patient [53]. Tailoring therapies to the diverse nature of neuronal damage, utilizing biomarkers, and incorporating regenerative strategies, such as stem cell transplantation, biocompatible biomaterials, and neurotrophic factors, aims to maximize treatment effectiveness. This universal approach bridges the gap between research findings and transformative therapies for individuals with neuronal injuries [54].

Innovative methods are required to overcome these obstacles in neural repair treatment. The Blood-Brain Barrier, acting as a shielding barrier between the bloodstream and brain, poses a significant challenge for delivering therapeutic agents to the injury site [267]. Current research efforts are focused on devising novel drug delivery systems, such as biomaterials and nanoparticles, to effectively overcome this barrier [268]. The potential immune response, in which transplanted cells or therapeutic agents may be superficial and trigger rejection and inflammation, requires attention [269]. Implementing approaches involving immunosuppressant drugs or engineering immune tolerance is fundamental for ensuring the success of cell-based therapies [251]. Ethical considerations play an essential role in clinical trials, featuring the necessity for strict adherence to guidelines and obtaining informed consent from participants, thus navigating the delicate balance between probable welfare and safety [254]. Furthermore, the sophisticated task of measuring success in neuronal repair therapies extends beyond enhancements in motor function or cognitive abilities to the ongoing challenge of quantifying restrained changes in neuronal circuitry and connectivity [89, 270]. The development of comprehensive measurement techniques is necessary to accurately assess the effectiveness of these ground-breaking therapies.

Despite these challenges, clinical translation of neuronal repair is rapidly advancing. Innovation in stem cell biology, gene editing, biomaterials, and brain imaging has facilitated the development of targeted therapies. Collaborative efforts among researchers, clinicians, and biotechnological companies are accelerating. Public awareness and engagement support the ongoing research and clinical trials in this field.

Understanding these complexities and ongoing research efforts is essential for keeping hope alive for millions affected by various types of neuronal damage. The day may not be far off when neuronal repair becomes a reality, restoring function or transforming life (Fig. 8).

Fig. 8.

The diagram outlines the use of stem cells and biomaterials in neural regeneration. It showcases various types of stem cells (like NPCs, iPscs, ESCs, NSCs, MSCs), biomaterials (hydrogels, scaffolds, growth factors), and their applications in treating neurological diseases (Parkinson’s, Alzheimer’s, spinal cord injury, stroke, MS). The diagram also illustrates the mechanisms involved, such as stem cell differentiation, biomaterial support, and growth factor signaling

Conclusion

In conclusion, this review highlights the significant contribution of biomaterials, especially when coupled with stem cells, to neural repair and regeneration. Given the current challenges associated with neural tissue damage, these interventions represent a promising path for therapeutic development. Biomaterials can alter therapeutic approaches and provide a foundation for novel therapies. Here, we present an in-depth investigation of various aspects, including the characteristics of efficient biomaterials and the mechanisms that encourage neural healing. Research using in vitro applications and animal models provides valuable insights that strengthen the foundation for further investigation. From preclinical research to prospective clinical use, this study highlights the obstacles in developing more biomaterial-based therapies for human use. This review summarizes the current knowledge on neural repair and regeneration, thereby guiding future research and clinical applications. Instilling hope for transformative progress by integrating biomaterials and stem cells is a significant step towards realizing the potential of these interventions to restore neural function and enhance the lives of individuals affected by neural damage. Moreover, research utilizing in vitro models and animal studies has yielded valuable insights into the mechanisms by which biomaterials and stem cells contribute to neural healing. These preclinical findings are instrumental in shaping the foundation for future research, ultimately guiding the translation of these strategies into clinical applications. However, significant challenges remain in optimizing biomaterials for human use, including issues related to biocompatibility, long-term functionality, and scalability.

Looking ahead, the successful development of biomaterial-based therapies will require multidisciplinary collaboration, integrating advances in materials science, stem cell biology, and clinical research. By addressing these challenges and building on the current knowledge, there is potential to develop therapies that can restore neural function and improve the quality of life for individuals affected by neural damage. This review not only summarizes the current state of the field but also serves as a roadmap for future research, highlighting the need for continued innovation and exploration in the quest to harness the full potential of biomaterials and stem cells in neural repair and regeneration.

Abbreviations

2D

Two-dimensional

3D

Three-dimensional

ADMSCs

Adipose-derived mesenchymal stem cells

Akt

Protein Kinase B

ALS

Amyotrophic lateral sclerosis

AMPA

α-Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic Acid

ASCs

Adipose Tissue-derived Mesenchymal Stem Cells

BBB

Blood-brain barrier

BDNF

Brain-derived neurotrophic factor

BmimCl

1-Butyl-3-methylimidazolium chloride

BMSCs

Bone Marrow-derived Mesenchymal Stem Cells

BMSCs

Bone marrow stromal cells

CaAlg

Calcium alginate

CCI

Controlled cortical impact

CNS

Central Nervous System

CNTNAP2

Contactin associated protein-like 2

CRISPR-Cas9

Clustered regularly interspaced short palindromic repeats, Cas9 nuclease

DISC1

Disrupted in schizophrenia 1

DMF

Dimethylformamide

DMF

N,N-dimethylformamide

DMSO

Dimethyl sulfoxide

DRG

Dorsal root ganglions

EGF

Epidermal growth factor

EGFR

Epidermal growth factor receptor

EO

Ethylene oxide

ESC

Embryonic stem cell

ESCs

Embryonic Stem Cells

ERK

Extracellular signal-regulated Kinase

FA

Formamide

FBS

Fetal bovine serum

FGF

Fibroblast growth factor

GDNF

Glial cell line-derived neurotrophic factor

GFP

Green fluorescent protein

HA

Hyaluronic acid

hiPSC

Human induced pluripotent stem cell

IL

Interleukin

iPS

Induced pluripotent stem cell

iPSCs

Induced Pluripotent Stem Cells

JAK

Janus Kinase

kDa

Kilodalton

LAP

Laminin-derived angiopoietin-1 peptide

LGI1

Leucine-rich glioma-inactivated 1

LT-NCLC

Long-term neuroepithelial-like cells

MCAO

Middle cerebral artery occlusion

MERTK

MER Proto-oncogene Tyrosine Kinase

MHC

Major Histocompatibility Complex

MSC

Mesenchymal stem cell

MSCO

Midbrain Striatum Cortex Organoids

NaAlg

Sodium alginate

NGF

Nerve growth factor

NPC

Neural progenitor cell

NSC

Neural stem cell

NSCs

Neural stem cells

NSCS

Neural stem cells

NHP

Non-human primate

PD

Parkinson’s disease

PEG

Polyethylene glycol

PEGDA

Poly(ethylene glycol) diacrylate

PI3K

Phosphatidylinositol-3-Kinase

PLA

Polylactic acid

PLDL

Polylactic acid

PLGA

Poly (lactic-co-glycolic acid)

PLGA

Poly(lactic-co-glycolic acid)

PLL

Poly (L-lysine)

PLLA-CL

Poly (L-lactide-co-ϵ-caprolactone)

PNSC

Peripheral nerve-derived stem cell

PPy

Polypyrrole

PU

Polyurethane

PU

Polyurethane

RADA16

Self-assembling peptide

RGD

Arginine-glycine-aspartic acid

RSP

Regenerated silk spidroin

SB

Supporting Bibliography

SEM

Scanning electron microscopy

SF

Silk Fibroin

SpC

Spinal cord

SR

Supporting Reference

STAT

Signal Transducer and Activator of Transcription

TAM

Tyro3, Axl, Mertk

TCP

Tissue culture plates

TBI

Traumatic Brain Injury

TGF

Transforming Growth Factor

UC-MSCs

Umbilical Cord-derived Mesenchymal Stem Cells

VGLUT1

Vesicular Glutamate Transporter 1

VGLUT2

Vesicular Glutamate Transporter 2

Author contributions

G. K, S. B, and M. K collected literature and wrote the manuscript. V. R. proofread and finalized the revised manuscript. S.B. and S.R. conceived, prepared, and approved the final version of the manuscript.

Funding

Not available.

Data availability

The datasets used and analyzed during the current study are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

Not required.

Consent for publication

Not Applicable.

Competing interests

The authors declare that they have no competing interests.