Enhancing Peripheral Nerve Regeneration with Rehabilitation and Biomaterial-Driven Drug Delivery Strategies

1.1. Classification of PNI

Nerve traction or stretching injury can occur during severe movements. Though the nerve fibers have inherent elasticity due to the abundance of elastin and collagen, over-stretching of peripheral nerves usually disrupts the nerve-blood barrier and results in axonal disconnection, and may even cause complete loss of continuity in severity, such as in a brachial plexus avulsion [6, 7]. Nerve compression is another common trauma-related PNI. Depending on the duration of the injuries, the compressive neuropathies can be either acute or chronic [8]. Acute nerve compression can be caused by traumatic crush injuries and requires from weeks to years for complete recovery [9]. In comparison, chronic compression, such as carpal tunnel syndrome, has more complex etiologies and typically involves the entrapment of nerves, leading to gradually deteriorating symptoms in the absence of appropriate intervention [10]. Other than traction and compression, nerve lacerations or transections are more serious cases [11]. Laceration injuries can be classified as either sharp, which are generally caused by cuts from knives or glass, or blunt, which can involve nerve avulsion, severe contamination, and soft tissue injury, resulting in partial or complete nerve discontinuity.

Depending on the damage severity of nerve injuries, Seddon and Sunderland described 3 broad categories and 5 grades of nerve injury [12, 13]. Neuropraxia and Sunderland grade I injuries are the mildest forms of traumatic PNIs, in which injuries cause a transient conduction block and mild myelin disruption while the axonal structure is intact. Axonotmesis indicates disconnected axons but with intact surrounding connective tissue. The extent of the damage to axons, endoneurium, and perineurium is denoted as Sunderland grade II, III, and IV injuries, respectively. Based on the degree of nerve tissue discontinuity, recovery from the injuries can be complete or incomplete and may require proper surgical intervention. Neurotmesis corresponds to Sunderland grade V injuries, which are the most severe form of nerve injuries, indicating complete disruption of the nerve. Nerve regeneration after these injuries is almost impossible, and clinical outcomes are poor. Sunderland III/IV injuries also generally have poor recovery without surgical intervention.

Overall, the Seddon–Sunderland scale shows that each extra layer of damaged tissue including axon, endoneurium, perineurium and epineurium makes conduction block last longer, raises the risk of neuroma, and shortens the therapeutic window. This means that early, grade-specific triage to conservative care or microsurgical reconstruction is important for the treatment outcome.

1.2. Wallerian degeneration and nerve regeneration

Neural responses following axon damage, including Wallerian degeneration and axon regeneration, have been well and extensively summarized in reviews [1, 14–18]. Briefly, a rapid breakdown of disconnected axons and myelin occurs distal to the injury site within 24–48 hours following the PNI [16]. Meanwhile, mature Schwann cells (SCs) transdifferentiate into active repair SCs, which coordinate with recruited immune cells to phagocytose axonal and myelin debris [15]. Repair SCs can further proliferate and line up in the remaining endoneurial tubes to form bands of Büngner, which contribute to both structural and trophic support for axon regeneration. When axons elongate along the bands of Büngner, the interaction between SCs and regrown axons induces redifferentiation into myelinating SCs, eventually leading to the formation of the myelin structure, known as the process of remyelination [15].

1.3. PNI-induced neuropathic pain

A lesion or disease of the somatosensory nervous system can result in neuropathic pain. More than half of patients who went through surgery or with peripheral nerve trauma suffer from chronic neuropathic pain. Neuropathic pain can be evoked pain and spontaneous pain, often manifesting as ongoing burning, shooting, stabbing, or freezing pain. Upon mechanical or thermal stimulation, patients with PNIs may have allodynia, pain due to a stimulus that does not normally provoke pain, or hyperalgesia, which is increased pain from a stimulus that normally provokes pain. The PNI-induced neuropathic pain stems from a series of hypersensitive changes in the afferent pathway of nociception, including ectopic discharge from the injured nerve, increased excitability of SNs, loss of inhibitory regulation in the dorsal horn, and maladaptive changes in descending inhibitory inputs to spinal nociceptive circuitry [65, 66]. Neuroimmune interactions and injury-induced oxidative environments are essential for pain generation, as well as affecting pain perception, intensity, and persistence. The roles of general inflammation and ROS in pain mechanisms involving the peripheral nerves, spinal cord, and brain are summarized in Fig. 1. Besides neuropathic pain, a complete transection of nerves in events like amputation also produces phantom limb pain, which is a different type of pain and can occur in up to 80% of cases.

2.1. Physical therapy

Physical activity is a conservative and affordable method that has been widely used in clinic to improve functional recovery for diseases of the nervous system [124]. In respect to PNIs, for example, patients with Carpel Tunnel Syndrome reported reduced pain and improved functions by doing nerve gliding exercises that may help them avoid surgical intervention [125]. Rehabilitation, including range of motion stretching exercises, hydrotherapy, or resistance training exercises, showed beneficial effects in patients with traumatic brachial plexus injuries to improve overall recovery [126, 127]. Consistent with the clinical outcomes, considerable research also reported that varied types of exercise, either passive or active, improved nerve regeneration, functional recovery, and relieved pain in animals following traumatic PNIs [128]. The influence of exercise can be affected by the time before beginning training after the injuries, the pattern of exercise (i.e., intensity and duration), and even sex differences [129–132]. For example, Kong et al. demonstrated that running on a treadmill at a low intensity and a late initiation effectively facilitated nerve regeneration in a rat model with a long-gap sciatic nerve injury and autograft implantation [133]. Apart from the assessment of the sciatic nerve and muscles, in vivo magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) were performed in this study to investigate structural and metabolite changes in the brain at 16 weeks post-injury, as described in the overall experimental design and timeline schematic in Fig. 2A. Rats was transected with a10 mm-long sciatic nerve segment which was inverted and orthotopically transplanted by an end-to-end coaptation at week 0 and underwent behavior tests (e.g. von Frey and Hargreaves tests) every week until week 16 for evaluating their sensory function after injury (Fig. 2A). They started receiving low-intensity treadmill treatment and gait-based assays for testing locomotor function at week 10 after the injury, when meaningful paw placement results typically re-emerges due to early re-innervation and initial axonal bridging around that time (Fig. 2A). The results confirmed that the PNI induced the volumetric, microstructural, metabolite, and neuronal activity changes in supraspinal regions of interest (i.e., somatosensory cortex, motor cortex [MC], hippocampus, etc.) after the injury and autograft implantation [133]. For example, manganese-enhanced MRI (MEMRI), which utilizes Mn²⁺ transport into excitable nervous system cells via voltage-gated Ca²⁺ channels to detect neural activity, could reveal the T1 signal in various brain regions, with stronger signals (darker red color) reflecting higher neural activity (Fig. 2B). Further semi-quantitative analysis of the ROIs results in Fig. 2B showed that the T1 signal intensities were significantly increased in the primary somatosensory cortex of the hind limbs (S1HL), MC, and CC, while a decrease was observed in the periaqueductal gray (PAG) region following nerve injury (Fig. 2C). This indicates that neural activity was altered in these regions following PNI. The treadmill exercise could effectively reverse some of these changes.

In clinical applications, it has been shown that physical therapies significantly enhanced sensory recovery and alleviated discomfort in patients with mild to median nerve injury [134]. Dr. Ballestero-Pérez has reported that patients with carpal tunnel syndrome showed significantly enhanced sensory test results and reduced pain intensity after nerve gliding and stretching exercises [135]. Similarly, patients with traumatic brachial plexus injury improved in shoulder abduction and elbow flexion after motion exercises and resistance training protocols [136]. For chronic compressive neuropathies, conservative rehabilitation protocols (gym- based exercise and muscle strength) have been proved to postpone or prevent the necessity for surgical decompression [137]. Recent studies have also demonstrated that patients with peripheral nerve damage were markedly reduced in injury-induced neuronal hyperexcitability by treadmill exercise [138].

Electrical stimulation (EStim), as another option that was frequently integrated into PNI surgical repair clinical protocols, showed evidence for its ability for enhancing axonal outgrowth and neuromuscular junction reformation [139]. Patients with digital nerve transection were improved in sensory recovery after receiving low-frequency Estim [140]. It has also been shown that non-invasive transcutaneous electrical nerve stimulation is capable in mitigating allodynia in patients with post-surgical PNI [141]. Ahn et al. recently used a fully bioresorbable dual-site stimulator which delivers therapeutic electrical bursts to both proximal and distal sciatic-nerve stumps for a week that markedly accelerated axonal bridging and functional recovery in rats [142]. A piezoelectric spiral conduit has also recently demonstrated to provide self-powered bursts that speeded axonal bridging in 15-mm rat gaps [143].

Underlying mechanisms in the neuro-protective and regenerative effects of physical activities converge on the modulation of neurotrophin expression [144]. Expression of BDNF and GDNF have been reported to be increased after exercise in both muscles and nerves following nerve transection and repair, which may be critical for the maintenance of SC activity, axonal elongation, and reinnervation [145]. However, the same NTFs are also pro-nociceptive, and their expressions are induced by PNIs, which can increase the excitability of SNs and maladaptive plasticity, thus exacerbating symptoms of pain [146]. In accordance with the analgesic effects of physical activity, treadmill running can reduce the expression of pro-nociceptive NTFs, including NGF and BDNF in the DRG [147, 148]. Moreover, physical exercise can further modulate central inflammation and plasticity in the CNS. Short-term treadmill running reduced microglia and astrocyte activation and the expression of BDNF in lumbar spinal cord [149, 150]. Increasing-intensity treadmill exercise can also regulate the PAG-RVM descending pathway by enhancing the activity of serotonergic and noradrenergic inhibition to reduce nerve transection-induced pain [151].

Patients with severe PNIs commonly suffer from impaired mobility and are incapable of performing many rehabilitation exercises. Besides, the recovery efficacy of exercise at a late stage, when patients may regain some motor functions, will be poor. In comparison, EStim -based therapy can be applied to the majority of patients. There are several clinical reports about the application of 1 hour of 20 Hz EStim following surgery in patients with cubital tunnel syndrome [152], carpal tunnel syndrome [153], or complete digital nerve transection [154]. Patients who received the EStim showed reduced pain, better muscle reinnervation, and improved sensorimotor functions. Recently, EStim therapy for peripheral neuropathy has gained much attention in preclinical studies, and has been reported to accelerate nerve regeneration, facilitate reinnervation, prevent muscle atrophy, improve functional recovery, and alleviate pain [155]. EStim is commonly applied at the injury site and terminal target organs, including muscle and skin, and can also be induced in DRG and the spinal cord. EStim applied at injured sites can induce the expression of NTFs, such as NGF, BDNF, and GDNF, in DRGs and nerves [148, 156, 157]. EStim can also increase SC activity by changing intracellular Ca²⁺ concentrations to promote the secretion of SC exosomes that contain pro-regenerative molecules [157]. EStim applied to the muscle increased intramuscular expression of BDNF and GDNF, which are critical in maintaining the activity of muscle stem cells and satellite cells as well as facilitating the formation of neuromuscular junctions [158, 159]. Transcutaneous EStim is commonly used to treat neuropathic pain, which can be achieved by modulating the RVM-mediated descending pain inhibition pathway to increase serotonin and gamma-aminobutyric acid (GABA) in the spinal cord [160–163]. Spinal cord stimulation has been clinically used in patients with chronic pain or a spinal cord injury [164]. In animal models with PNIs, spinal cord stimulation can support nerve regeneration by protecting SNs in DRGs as well as motor neurons in the spinal cord [165] and can also attenuate neuropathic pain through reducing neuroinflammation in both DRGs and the spinal cord [166, 167].

Overall, physical therapy has shown promising efficacy in the treatment with PNIs in both preclinical studies and clinical usage. However, their mechanisms for observed improvements in PNI recovery are still largely unknown, especially with respect to maladaptive plasticity CNS. Moreover, local EStim on injured nerves or muscles requires implantation of electrodes, which is expensive and can induce many complications, such as inflammation and wound dehiscence [155]. On the other hand, the effects of rehabilitation exercise are still under debate. It has also been reported that overly intense exercise is excitotoxic, which can lead to neuronal hyperexcitability and induction of pain [168, 169]. Therefore, more studies are needed to accurately evaluate the effects of different exercise regimens (e.g., starting time, interval, duration, intensity) on PNI recovery.

Neuromodulatory techniques like transcutaneous auricular vagus nerve stimulation (taVNS) have also shown increasing promise in managing inflammation [170]. As a non-invasive method, taVNS acts through stimulates the auricular branch of the vagus nerve through the skin of the outer ear and modulates the activity of the cholinergic anti-inflammatory pathway. Studies have demonstrated that taVNS could reduce systemic inflammatory marker expression in clinical models of subarachnoid hemorrhage, rheumatoid arthritis and Crohn’s disease [171–173]. Although taVNS has not been widely studied in PNI models particularly, its success in these neuroinflammatory diseases in anti-inflammation may suggest its translational potential for PNI treatment.

For clinical studies, low-load nerve-gliding and range-of-motion programs are used for relieving entrapment neuropathies and mild traction injuries. The rehabilitation programs, including hydrotherapy, resistance training and treadmill walking, are recommended after microsurgical repair to accelerate sensorimotor return and curb central sensitization. For the device-based modalities deliver targeted currents or fields, low-frequency intra-operative or early post-operative ES primes axonal outgrowth and re-innervation. Transcutaneous ES or spinal-cord stimulation mitigates chronic allodynia. And emerging non-invasive techniques like taVNS extend the anti-inflammatory reach beyond the lesioned limb. For preclinical studies, low-intensity treadmill running in long-gap autograft models amplifies neurotrophin waves and also normalizes supraspinal activity maps, whereas precisely dosed ES or magnetic stimulation could boost Schwann cell exosome release, which preserves neuromuscular junctions and inhibit DRG as well as spinal neuro-inflammation.

In summary, although exercise or stimulation-based rehabilitation consistently enhances neurotrophic signaling and functional recovery, its clinical impact ultimately depends on tailoring timing, intensity and patient adherence of the therapies, which highlights the need for clarifying the mechanistic pathways behind the physical therapies.

2.2. Pharmacological-based treatments

Although currently there are no clinically available pharmacological treatments for nerve regeneration, extensive studies have reported promising potentials of various neurotrophic and growth factors as well as functional small molecules, such as anti-inflammatory and antioxidant compounds, for PNI recovery. Exogenous neurotrophic and growth factors, such as NGF, BDNF, ciliary neurotrophic factor, and basic fibroblast growth factor (bFGF), have be locally delivered to injured nerves through osmotic pumps [174–178] or embedding them in nerve grafts [19, 179]. As each NTF has a unique mechanism for affecting nerve regeneration, administration of a single NTF may not be sufficient to achieve overall improvement. Hence, combined delivery of multiple NTFs or with other therapeutic agents would be more efficacious [180–183]. However, NTFs undergo proteolytic degradation and rapid clearance in the body, and therefore, continuous administration of neurotrophic factors is required to achieve adequate concentration. This limitation severely hinders their application in clinical medicine. In addition, too high of a concentration of NTFs and an inappropriate administration time may entrap regenerating axons and inhibit their further growth [184, 185].

Many anti-inflammatory compounds and antioxidant agents have gained increased attention for treating peripheral neuropathy, particularly induced by chemotherapy or diabetes [186, 187]. It has also been reported that these neuroprotective agents reduced PNI-induced pain and improved functional recovery [188]. For instance, a potent anti-inflammatory compound, minocycline, can decrease perineural inflammation and reduce scar formation around injured sciatic nerves, thus preventing nerve adhesion and attenuating the pain [189, 190]. An antioxidant agent, alpha lipoic acid, has been exhibited to protect SNs from nerve axotomy and accelerate nerve recovery [191–194]. Recently, alpha lipoic acid has been reported to improve clinical and neurophysiologic outcomes in patients with carpal tunnel syndrome after decompression surgery [195]. Phenolic compounds are secondary metabolites of plants and have strong anti-inflammatory and antioxidant properties. Naturally derived phenolic compounds, such as curcumin (Cur), quercetin, and epigallocatechin-3-gallate, have been reported to improve sensory and motor outcomes in different PNI models [196–199]. Moreover, the uptake of various vitamins and minerals, including magnesium and selenium, can also regulate inflammation and redox status in the nervous system and show beneficial effects in PNI recovery [200]. During clinical application for PNI, the administration of drugs like Alpha-lipoic acid, a mitochondrial antioxidant, had improved nerve conduction velocity and alleviated paresthesia for patients with carpal tunnel syndrome in a placebo-controlled clinical trial [201]. Patients with mild traumatic PNI or diabetic neuropathy showed reduced pain perception and improved motor strength after receiving plant-derived antioxidants like curcumin and quercetin in early-phase clinical studies [202, 203]. However, most antioxidant and anti-inflammatory macromolecules are unstable and can be rapidly removed through metabolic activities. Additionally, poor absorbance of some compounds also limits their bioavailability for target tissues, thereby reducing their effects. Agents with these limitations often need frequent administration or more invasive delivery methods.

2.3. Overall strategies for PNI

The PNI recovery strategies are diverse and are usually based on multiple underlying biological mechanisms. Overall, the main strategies contain: surgical approaches (like neurorrhaphy, autografting, allografting and synthetic nerve conduits), physical therapy-based interventions (exercise, EStim, ultrasound stimulation and magnetic stimulation), pharmacological therapies (anti-inflammatory drugs, antioxidant agents and exogenous neurotrophic factors), gene therapy (viral vector-based delivery), exosome-based therapy, and biomaterial-based delivery systems (Fig. 3).

Physical therapy interventions act through different neurobiological pathways. For example, ultrasound stimulation achieves its therapeutic effect by inducing mechanical perturbations in cellular membranes and cytoskeletal structures, which activates mechanosensitive ion channels and intracellular signaling to supports axonal outgrowth and remyelination [204]. Ultrasound has been suggested to promote local angiogenesis, enhancing oxygen and nutrient delivery to regenerate nerves after PNI. While the mechanism of magnetic stimulation (particularly repetitive transcranial and peripheral magnetic field exposure) involves altering the neuronal excitability through electromagnetic induction. It may alleviate chronic pain by regulating synaptic activity and modifying the neurochemical milieu, including levels of GABA and glutamate in pain-related tissue [205].

Pharmacological therapies primarily achieve their effects by interfering with oxidative and inflammatory cascades triggered by nerve injury. Anti-inflammatory agents like minocycline could inhibit microglial activation and prevent leukocyte infiltration by reducing the production of cytokines like TNF and IL-1β which induce neuropathy and demyelination [206]. Antioxidants like alpha-lipoic acid help scavenger ROS accumulation that damages mitochondrial membranes and ion channels in neurons and Schwann cells [207]. Application of neurotrophic factors like NGF act by binding to TrkA receptor to initiate MAPK/ERK and PI3K/Akt pathways to support neuronal survival and neurite extension [208].

Although each of these strategies often target different aspects of the injury and repair process, they may converge mechanistically. For example, both EStim and exercise cloud increase neurotrophic factor levels, while both biomaterials and exosomes may act by modulating immune cell phenotypes and reducing oxidative stress. However, the extent to which these shared mechanisms contribute to functional recovery remains unclear and warrants further investigation.

Despite their promise, surgical repair needs additional interventions to modulate the inflammatory or oxidative stress for neurons under long-term recovery [209]. Physical therapies usually have variable efficacy due to heavy dependence on patient condition and individual differences [210]. Pharmacological intervention is the main area for achieving high efficacy in PNI treatment but needs to prevent rapid degradation and poor bioavailability at the injury site.

Unlike biomaterial-driven drug delivery strategies, physical therapies are only suitable for early and mild injuries. Exercise requires active patient participation. For exercise and EStim, risk of excitotoxicity or worsening symptoms still exist, not to mention that the mechanisms behind them are not fully understood. The usage of biomaterial delivery techniques, like injectable hydrogels or nanoparticle systems, provides a reliable scaffold for localized, controlled and prolonged release of various type of drugs that could be antioxidants, neurotrophic factors, or anti-inflammatory agents. These delivery strategies are necessary especially when it is required to preserve therapeutic concentrations at the site of injury or to precisely target different stages of inflammation, cell debris clearance and nerve regeneration [211].

Biomaterial-based platforms and biotechnologies can achieve localized and sustained release of therapeutic agents to address these limitations with greater precision and flexibility. Thus, in this review, we will focus on the biomaterial-assisted drug and gene delivery systems in the following sections.

Collectively, PNI care depends on strategies including surgery, physical modulation (exercise, E-Stim, ultrasound, magnetic fields), pharmacology (anti-inflammatory, antioxidant, neurotrophic agents), gene transfer, exosome signaling, and biomaterial delivery (hydrogels, fibers, nanoparticles). They face different limitations like structural gap, neurotrophic deficit, cytokine/ROS surge, or prolonged protein expression. Yet they share mechanisms such as cAMP/PI3K-Akt activation and M2 macrophage polarization. The following sections focus on the biomaterial, which offers a precise method to program when and where drugs are delivered.

3.1. Nano-/micro- particles for long-lasting drug release for PNI recovery

Pharmaceutical nano-/micro- particle technologies have gained increased interest for drug delivery in the past few decades. The advantages of nano-/micro- particles are attributed to their small sizes that can facilitate deep-tissue penetration and cellular uptake as well as their large surface area per unit volume, which is applicable for modification for multifunctional drug delivery [244]. Depending on different physical and chemical properties of drugs and biomaterials, drugs can be released from the particles in various kinetics, which generally can be affected by the degree of drug diffusion, solvent transportation, particle degradation, or stimuli-induced destruction [245].

Biodegradable polyesters can be used to encapsulate diverse ingredients for controllable delivery and release. Particularly, PLGA synthesized by the polymerization of cyclic lactide and glycolide monomers is one of the few biodegradable polymers with FDA approval for clinical applications due to its acceptable biocompatibility and tunable biodegradability [246]. Many studies [219–222] have reported the encapsulation of a single or a combination of NTFs in PLGA nano-/micro- particles, which achieve a long-lasting release of payloads for more than one month. Implantation of particles either within nerve guidance conduits or into injured nerves improved axon and myelin sheath regeneration and promoted functional recovery. For example, Ding et al.,[223] encapsulated Rapamycin, an immunosuppressant, within PLGA microparticles that were further incorporated into nerve conduits. The application of the Rapamycin-PLGA-scaffolds reduced PNI-induced pain and neuron sensitization. Fadia et al.,[224] developed GDNF-loaded double-walled PLGA/PLA microspheres. Embedding GDNF microspheres into nerve conduits sustained the release of GDNF for more than 50 days. The application of the GDNF microsphere-embedded nerve conduits improved never conductive functions and promoted SC activity and axonal remyelination in a rhesus macaque model with a 5 cm nerve defect at one-year post-surgery. Encapsulation of water-soluble factors in particles is challenging because they cannot be dissolved in the hydrophobic solvent and thus can easily leak out of the core of the particles. To enhance the encapsulation efficiency, Hanwright et al.,[225] made insulin-like growth factor-1 (IGF-1) and dextran sulfate polyelectrolyte complexes and further encapsulated the complexes into polyethylene glycol (PEG)-b-PLGA nanoparticles. Local application of the IGF-1 nanoparticles through intramuscular injection showed improved neuromuscular reinnervation, reduced muscle atrophy, and greater functional recovery.

Natural biodegradable polymers have also been studied to function as drug carriers due to their extraordinary biocompatibility. Chang et al.,[226] used BDNF-loaded gelatin nanoparticles to establish a neurotrophic gradient in a nerve conduit. By functioning together with NGF released from the conduit scaffold, NTFs showed guiding effects during axon growth, thus leading to better nerve regeneration and ameliorated muscle atrophy that is comparable to the autograft repair in a long-gap nerve defect. Chitosan is a positively charged polysaccharide that is naturally derived from chitin, which is abundant in crustacean exoskeletons, and it is one of the major natural polymers with a broad usage in diagnosis and drug delivery systems [247]. Wei et al.,[227] encapsulated NGF within chitosan nanoparticles and then embedded them into the matrix of nerve conduits. The sustained release of NGF from the chitosan nanoparticles in the nerve conduit was comparable to the autograft repair in terms of nerve regeneration and functional recovery after nerve transection. In addition, as chitosan has the capacity to neutralize the acidic environment during the degradation of polyesters that may deactivate bioactive proteins, Zeng et al.,[228] modified PLGA microspheres with tripolyphosphate (TPP) crosslinked chitosan. The extended the release of NGF to more than 49 days. Implantation of nerve conduits filled with the TPP-chitosan-PLGA-NGF microspheres enhanced sciatic nerve regeneration and prevented gastrocnemius muscle atrophy at 12 weeks post-surgery.

Some newly developed nano- and micro-technologies have also been exploited in the area of peripheral nerve trauma [248, 249]. It has been demonstrated that nanostructured mineral coatings can effectively stabilize protein structures and thus prevent them from denaturation [250]. Hellenbrand et al.,[229] processed β-tricalcium phosphate microparticles in modified simulated body fluid, and they found that the derived mineral coating microparticles stabilized and released NGF and GDNF for at least 22 days in vitro. They incorporated the NGF and GDNF microspheres into the distal end of sciatic nerve isografts, which showed beneficial effects in functional recovery, axon regrowth, and remyelination. Magnetic nanoparticles have also been studied with increasing interest for time- and spatially-controlled drug release since they are guidable through the application of a remote magnetic field [251]. Giannaccini et al.,[230] produced polyethylenimine (PEI)-coated magnetic iron oxide nanoparticles for the delivery of NGF and VEGF within nerve guidance conduits. They placed a magnetic tape around the conduits to localize neurotrophic factors at the center of the conduits, thus promoting axon sprouting and motor functions. And according to Li et al., non-invasive photostimulation by embedding near-infrared up-conversion nanoparticles has significantly increased myelinated-fiber density after sciatic crush [252].

3.2. Hydrogels for regional long-lasting drug release for PNI recovery

A hydrogel, formed by crosslinking between hydrophilic molecule chains, is a 3D network with a highly porous structure and contains large amounts of water [253, 254]. Hydrogels are composed of versatile matrices with varying components and interaction arrangements, enabling them to exhibit a wide range of structural, chemical, and physical properties. These characteristics directly influence the release kinetics of hydrogel-based drug delivery systems [253]. Particularly, hydrogels with injectability and responsiveness to external stimuli greatly extend the operative possibility for in situ drug delivery and controlled release. A PLGA-PEG-PLGA copolymer is thermosensitive and can convert from a liquid phase to hydrogel at a physiologically relevant temperature [255]. Manto et al.,[234, 235] reported that payload-PLGA-PEG injected into injured sciatic nerves formed a hydrogel that gradually degraded for around 20 days in vivo. The PLGA-PEG-PLGA thermosensitive hydrogel has been used to control the long-term release of therapeutic cargos for 14 days [232, 233], and implantation of the hydrogel supported neuron survival, promoted nerve regeneration, and preserved the integrity of neuromuscular junctions in a brachial plexus avulsion model. And an injectable thermosensitive pluronic F127/nano-hydroxyapatite-polydopamine hydrogel further accelerated crush-injury repair and reduced oxidative stress after crush injury [256]. Fibrin is FDA approved for hemostasis and wound healing, and the mechanical properties of the fibrin hydrogel can be modulated based on the constituent ratio of fibrinogen and thrombin. Liu et al.,[236] filled nerve conduits with a fibrin hydrogel with a payload of Wnt5a, a crucial factor in regulating nerve growth and SC activity. The incorporation of Wnt5a and fibrin hydrogel resulted in superior outcomes in terms of nerve fiber growth and myelination, conductive functions, vascularization, muscle atrophy, and motor functions compared to groups with either Wnt5a or fibrin hydrogel alone. In addition, Tajdaran et al.,[237, 238] first encapsulated neurotrophic factors and an FDA-approved immunosuppressant, FK506, within microparticles and then further incorporated these microparticles into fibrin hydrogels to enhance the controllability of drug release. Direct application of fibrin hydrogels to transected nerve not only promoted nerve growth across the coaptation sites but also effectively restricted drug accumulation in the repaired nerve, muscle, and spinal cord, thereby avoiding the systemic distribution and preventing the toxic effects of FK506. Naturally occurring polysaccharides have also been exploited to develop hydrogel technologies in biomedical applications due to their biocompatibility, nontoxicity, and non-immunogenicity. Zhang et al.,[239] fabricated chitosan hydrogels and incorporated sericin, a component derived from silkworm silk, to enhance mechanical endurance. The chitosan/sericin hydrogels released most of the loaded NGF within 8 days in vitro, while they underwent gradual degradation after implantation in vivo for 8 weeks. Application of the chitosan/sericin hydrogel induced pro-regenerative reactions in the nerve, prevented inflammation, protected nerve microstructure and conductive functions, and provided pain relief in a chronic nerve compression model. Yao et al.,[240] grafted hyaluronic acid (HA) with pamidronate, a bisphosphonate that can bind with various metal ions, to deliver Mg²⁺ for PNI repair. Due to the dynamic property of the coordination bonds between Mg²⁺ and pamidronate [257], the hydrogel is shear thinning, injectable, self-healing, and progressively disassembling along with the dissipation of Mg²⁺. The HA-pamidronate-Mg²⁺ hydrogel was injected to fill nerve conduits during the surgery, followed by gradual degradation within 2 weeks, but the effects accelerating nerve regeneration and functional restoration lasted for 12 weeks. Complementary work also showed that Mg-sputtered collagen conduits enhanced bio-electric coupling and functional recovery across 20-mm gaps [258]. Alginate is a natural polysaccharide extracted from brown sea algae, and ionic Ca²⁺ can induce chain-chain crosslinking that leads to its gel formation. An alginate-based copolymer hydrogel has been reported to have enhanced cell compatibility[259]. Chen et al.,[241] incorporated alginate into a Pluronic F127 thermosensitive hydrogel with the addition of positively charged lysine-dextran to control the release of adipose-derived stem cell-derived extracellular vesicles. Recently, a chirality-engineered D-matrix hydrogel has demonstrated the ability to influence Schwann cell redifferentiation and enhance the sciatic functional index by around 30% in a 10 mm rat gap model [260]. The pluronic-alginate hydrogel exhibited an improved stability, and a longer release profile compared to a single Pluronic F127 hydrogel. The application of the hydrogel around coaptated nerves facilitated nerve regeneration and prevented muscle atrophy. Gelatin is denatured collagen and contains abundant Arg-Gly-Asp sequences and integrin-binding motifs, which can support cell attachment and proliferation. The introduction of methacryloyl substituent groups to gelatin makes it have photo-triggered crosslinking to form a GelMA hydrogel [261]. Xu et al.,[242, 243] encapsulated VEGF or VEGF-mimetic peptides-loaded nanoliposomes in a GelMA hydrogel, which was applied to injured nerves through injection with irradiation-induced in situ gelation to enhance regional vascularization and consequently benefit nerve regeneration.

3.3. Virus-based gene delivery for PNI recovery

Gene therapy performed by delivering therapeutic genetic components with viral vectors to lesion tissue has been reported for PNI treatment, aiming to sustainably support nerve regeneration and neuron protection. Genes encoding various neurotrophic factors have been delivered to the lesion area. In situ injection of AAV8 that carried a gene for CNTF transduced SCs around the lesion and enhanced nerve regeneration and myelination [262]. However, a huge hurdle exists with neurotrophic factor-dependent gene therapy due to their potential side effects. Persistent expression of high levels of GDNF has entrapped axons and prevented them from further growth and innervation, due to the so-called “candy store” effect. Meanwhile, the long-term effects of NGF may induce nociception [263]. Besides neurotrophic factors, the intraspinal delivery of AAV that carried a gene for G-CSF showed protection effects on motor neurons in SOD-1 (G93A) transgenic mice, a disease model of Amyotrophic Lateral Sclerosis with progressive degeneration of motor neurons, and it also supported motor axon regeneration and reinnervation after a crush injury [264]. In addition to nerve regeneration, diverse genes have been studied for pain management after PNI. Small hairpin RNA for TRPV1 [265], a nociceptive receptor, and sodium channels [266] have been designed to downregulate their expression, and AAV carrying genes coding for peptides that coupled with NaV_1.7 blocked the Na⁺ current [267, 268], decreasing the excitability of sensory neurons and thus relieving PNI-induced pain. Moreover, the regulation of neuroinflammation after PNI is also feasible by gene delivery. Gushchina et al.,[269] reported that gene delivery of non-functional soluble colony stimulating factor-1 receptor (sCSF1R) decreased microglia and macrophage accumulation in both the spinal cord and DRGs after a PNI. The neuroplastic changes in the spinal cord after a PNI could also be further modulated based on AAV gene therapy. Takahiro et al.,[270] found that subpial delivery of a GABA synthesizing-releasing system, including genes encoding for GAD65 and vesicular GABA transporter, switched spinal dorsal excitatory neurons to an inhibitory phenotype, thereby achieving a long-lasting reversal of PNI-induced pain.

3.4. Exosome-based therapy

Exosomes (Exos) are one of the extracellular vehicles generated and released from cells through the endocytic pathway. Exos encapsulate various bioactive molecules, predominantly nucleic acids and proteins, and thus, they are thought to play a role in modulating intercellular communication. Meanwhile, as Exos have demonstrated favorable features for delivering therapeutic cargos, including stability, biocompatibility, low immunogenicity, and prolonged blood circulation, their therapeutic potential has been explored in neurodegenerative diseases in recent years [271–273]. Mesenchymal stem cells (MSCs) are multipotent cells that secrete many bioactive molecules, such as immunomodulatory factors and growth factors. They have been transplanted to injury and pathological areas, mediating cell response through paracrine factor secretion [274]. MSC-derived Exos may have similar effects as MSC-based therapy and have less ethical and safety concerns. Exos from bone marrow-derived MSCs (BMSC-Exos) have been suggested to regulate the inflammatory activity of macrophages [275]. In addition, BMSC-Exos exhibited functions in neuritogenesis, with miRNA-dependent mechanisms [276]. Exos derived from induced pluripotent stem cells [277] and adipose-derived MSCs (hADMSCs) [278] also benefited the recovery after a PNI. After injury, SCs differentiated, proliferated, and secreted factors to support axonal regeneration. An injection of SC-derived Exos (SC-Exos) to a crushed sciatic nerve resulted in internalization of the Exos into nerve fibers and thus facilitated the growth of axons, which was significantly faster 4 days after injury [279]. Recently, Jun et al.,[280] reported that hypoxia induced an upregulation of miR-21–5p in SC-Exos, which further regulated endothelial metabolism, including glycolysis, oxidative phosphorylation, and intraneural revascularization. Our group and others [278, 281, 282] collected Exos from hADMSC-derived SC-like cells (hADMSC-SCLCs). Our group decellularized porcine sciatic nerves, digested the matrix into a neutralisable pre-gel and blended it with 25 kDa polyethyleneimine (PEI) to yield a modified decellularized nerve hydrogel (pDNH) [278]. Differentiated hADMSCs (denoted as dExo) isolated by ultracentrifugation were electrostatically entrapped in the pDNH, which cross-linked within 1 h at 37 °C to form an injectable in-situ–gelling matrix. The dExo-loaded pDNH was locally injected around a mouse sciatic-nerve crush to envelope the lesion and provid slow exosome release. Then behavioural tests, histology and TEM over 21 days was tested to confirm faster axon regrowth and inhibited macrophage activation. The process of how Exos derived from the differentiated dExo which was further encapsulated within a pDNH is shown in Fig. 4A–C [278]. Then the dExo-loaded pDNH was applied around injured nerves in a mouse sciatic nerve crush injury model (Fig. 4A). The incorporation of the positively charged PEI enhanced the in vivo retention time of the dExo (Fig. 4 D). The group treated with the dExo-loaded pDNH, but not those treated with dExo or pDNH alone, demonstrated a significant improvement in motor function recovery (Fig. 4E, F). Macrophages play a pivotal role in the regeneration process following traumatic nerve injury. Nerves from the Sham group showed few CD68 and colony-stimulating factor-1 receptor (CSF-1R) positive cells, whereas, the Crush, pDNH, and dExo groups exhibited a significant increase in macrophage numbers, particularly in the Crush group (Fig. 4G–J). Notably, the dExo-loaded pDNH treatment reduced CD68 and CSF-1R intensities and the positive areas, indicating that the dExo-loaded pDNH may enhance axon continuity and preserve neural integrity by alleviating long-term inflammation. Vascularization after PNI is an indispensable process for oxygen and nutrient supplementation and debris clearance. Exos from endothelial cells (EC-Exos) were taken up by SCs and maintained the repair phenotype of SCs by upregulating miR199–5p and activating the proliferative pathway[283]. To further enhance the pro-regenerative effects of EC-Exos, Huang et al.,[284] developed ECs with an overexpression of Netrin-1 (NTN1), which is upregulated after PNI and functions in axon guidance. Treatment with NTN1-EC-Exos could enhance the neuronal regenerative ability by down-regulating let7a-5p and activating pro-regenerative pathways. Meanwhile, NTN1-EC-Exos also facilitated the formation of a pro-regenerative environment with increased proliferative vascular cells and SCs with repair-related phenotypes. Exos from fibroblasts (f-Exos) have been studied for PNI recovery [285]. After pre-conditioning with chitosan oligosaccharides (COS), COS-f-Exos were enriched with TFAP2C that targets miR-132–5p, thus disinhibiting the capability of neurite outgrowth.

3.5. Overall biotechnologies strategies for PNI

Various biotechnological delivery toolkits have been developed during the recent decade. At the materials level, polymeric micro-/nanoparticles (e.g., PLGA, PEG-PLGA, gelatin, chitosan) and injectable or in situ–gelling hydrogels (thermosensitive PLGA-PEG-PLGA, fibrin, chitosan/sericin, HA-pamidronate, Pluronic-alginate, photo-cross-linked GelMA) are the most frequently used methods that is capable of releasing trophic factors, immunomodulators or ions from days to months while preventing cargos from premature degradation and systemic toxicity. They are designed based on degradable backbones, enzyme-responsive linkages or mineral coatings that translate polymer erosion, protease activity or ion exchange into controllable burst-and-sustain kinetics. While magnetic or mineral coatings could add remote localization and protein-stabilizing functions. As for genetic-engineering vector technologies, AAV or lentiviral constructs could drive expression of neurotrophic genes or pain-modulating shRNAs during weeks to months. The exosomes derived from MSC, Schwann or endothelial cells could deliver miRNAs without immunogenicity. When these platforms are delivered locally such as inside guidance conduits, within muscle or intrathecally, they consistently improve pain relief, neuromuscular junction integrity, axon extension, myelination, and vascularization. They usually share requirements for modulating cargo specificity, regulating release half-life, and placement consideration.