Injuries and diseases of the peripheral nervous system (PNS) often result in irreversible functional deficits. Current therapeutic approaches demonstrate limited efficacy, which has driven the development of regenerative medicine strategies. This review systematizes contemporary gene and cell therapy approaches aimed at PNS repair and regeneration. Key neurotrophic factors (NGF, BDNF, GDNF, VEGF, etc.) and the molecular mechanisms underlying their regenerative effects are discussed. Gene delivery strategies employing viral and plasmid vectors are analyzed, along with the therapeutic application of various cell populations, including Schwann cells, mesenchymal stromal cells, and derivatives of induced pluripotent stem cells. Particular attention is given to combined gene-cell-based approaches, which enable localized and sustained expression of therapeutic molecules. The integration of advances in genetic engineering, cell biology, and tissue engineering is shaping a new treatment paradigm focused on pathogenetic restoration of nerve tissue. These promising strategies pave the way toward achieving complete functional regeneration following PNS injuries.

The intrinsic ability of peripheral nerves to regenerate after injury is extremely limited, especially in case of severe injury. This often leads to poor motor function and permanent disability. Existing approaches for the treatment of injured nerves do not provide appropriate conditions to support survival and growth of nerve cells. This drawback can be compensated by the use of gene therapy and cell therapy-based drugs that locally provide an increase in the key regulators of nerve growth, including neurotrophic factors and extracellular matrix proteins. Each growth factor plays its own specific angiotrophic or neurotrophic role. Currently, growth factors are widely studied as accelerators of nerve regeneration. Particularly noteworthy is synergy between various growth factors, that is essential for both angiogenesis and neurogenesis. Fibroblast growth factor 2 and vascular endothelial growth factor are widely known for their proangiogenic effects. At the same time, fibroblast growth factor 2 and vascular endothelial growth factor stimulate neural cell growth and play an important role in neurodegenerative diseases of the peripheral nervous system. Taken together, their neurotrophic and angiogenic properties have positive effect on the regeneration process. In this review we provide an in-depth overview of the role of fibroblast growth factor 2 and vascular endothelial growth factor in the regeneration of peripheral nerves, thus demonstrating their neurotherapeutic efficacy in improving neuron survival in the peripheral nervous system.

Several methods for the stimulation of skin wound repair have been proposed over the last few decades. The most promising among them are gene and stem cell therapy. Our present experiments combined several approaches via the application of human umbilical cord blood mononuclear cells (hUCB-MC) that were transfected with pBud-VEGF165-FGF2 plasmid (gene-cell therapy) and direct gene therapy using pBud-VEGF165-FGF2 plasmid to enhance healing of full thickness skin wounds in rats. The dual expression cassette plasmid pBud-VEGF165-FGF2 encodes both VEGF and FGF2 therapeutic genes, expressing pro-angiogenic growth factors. Our results showed that, with two weeks post-transplantation, some transplanted cells still retained expression of the stem cell and hematopoietic markers C-kit and CD34. Other transplanted cells were found among keratinocytes, hair follicle cells, endothelial cells, and in the derma. PCNA expression studies revealed that transplantation of transfected cells terminated proliferative processes in regenerating wounds earlier than transplantation of untransfected cells. In the direct gene therapy group, four days post-operatively, the processes of flap revascularization, while using Easy LDI Microcirculation Camera, was higher than in control wounded skin. We concluded that hUCB-MC can be used for the treatment of skin wounds and transfection these cells with VEGF and FGF2 genes enhances their regenerative abilities. We also concluded that the application of pBud-VEGF165-FGF2 plasmids is efficient for the direct gene therapy of skin wounds by stimulation of wound revascularization.

Vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2) are well-known growth factors involved in the regeneration of various tissues and organs, including peripheral nerve system. In the present study, we elucidated the local and systemic effects of plasmid construct рBud-coVEGF165-coFGF2 injected into the epineurium of intact rat sciatic nerve. Results of histological examination of sciatic nerve and multiplex immunoassays of serum showed the absence of immunogenicity and biosafety of plasmid рBud-coVEGF165-coFGF2. Moreover, local administration of plasmid DNA construct resulted in significantly decreased levels of pro-inflammatory cytokines in the peripheral blood, including tumor necrosis factor α (TNFα) and interleukin-12, and significantly increased levels of cytokines and chemokines including Regulated upon Activation, Normal T Cell Expressed and Presumably Secrete (RANTES), epidermal growth factor, interleukin-2, and monocyte chemoattractant protein 1. These changes in the peripheral blood on day 7 after injection of plasmid construct рBud-coVEGF165-coFGF2 show that the plasmid construct has systemic effects and may modulate immune response. At the same time, reverse transcription-polymerase chain reaction revealed transient expression of coFGF2, coVEGF165, ratFGF2 and ratVEGFA with direct transport of transcripts from distal part to proximal part of the sciatic nerve. Immunohistochemical staining revealed prolonged presence of VEGFA in sciatic nerve till 14 days post-injection. These findings suggest that local administration of plasmid construct рBud-coVEGF165-coFGF2 at a concentration of 30 ng/µL results in the formation of pro-angiogenic stimuli and, and the plasmid construct, used as a drug for gene therapy, might potentially facilitate regeneration of the sciatic nerve. The study was approved by the Animal Ethics Committee of Kazan Federal University, procedures were approved by the Local Ethics Committee (approval No. 5) on May 27, 2014.

Objective − to evaluate the therapeutic properties of cells of the stromal vascular fraction isolated from adipose tissue and the gene-therapeutic plasmid pBUD-VEGF165-FGF2 expressing vascular endothelial growth factor and fibroblast growth factor in traumatic injury of the rat sciatic nerve. Materials and methods. The study was carried out on 45 male outbred white rats. Five mm diastasis in sciatic nerve was formed and the defect was replaced with an autologous insert. Stimulation of regeneration was carried out by injection of cells of the stromal vascular fraction or plasmid DNA pBUD-VEGF165-FGF2. The efficiency of regeneration was assessed using electromyography, morphological methods and the number of neurons in the L5 spinal ganglia. Results and discussion. On the 56th day after injury to the sciatic nerve in the groups with stimulation of regeneration, the threshold for the occurrence of the M-response practically corresponded to that of intact animals, the area of scar tissue in the area of the autonomic nerve insertion of the sciatic nerve decreased, and the number of neurons of the spinal ganglion L5 significantly exceeded that in the control group. Conclusion. Cells of the stromal vascular fraction from adipose tissue and the pBUD-VEGF165-FGF2 gene-therapeutic plasmid DNA appear to be promising stimulators for the regeneration of nervous tissue.

Traumatic injuries of peripheral nerves lead to profound disability in patients with partial or total loss of limb function. There remains the question about the use of technologies for detecting defects of the peripheral nerve with concurrent of its regeneration. In the study it has been investigated the effect of the gene-therapeutic plasmid construct pBud-VEGF165-FGF2 with various methods of overcoming 5 mm diastasis of the sciatic nerve: nerve autograft and tubulation with the NeuraGen® tube. In the study groups, assessment of sciatic nerve regeneration was based on functional and morphometric parameters. Direct injection of plasmid pBud-VEGF165-FGF2 stimulates regeneration and restoration of motor function in both groups, but with different efficacy. Comparative analysis of nerve defect replacement in combination with direct gene therapy showed the most effective approach with autologous insertion replacement by comparison to the NeuraGen. Thus, on the basis of the obtained data, we can assert that nerve autograft of the peripheral nerve remains the "gold standard” and provides the best hope of research in combination with the use of various regeneration stimulants.

Delayed union and non-union are widespread complications of bone fracture healing which are worse in cases of concomitant chronic diseases (diabetes mellitus, alcoholism, etc.). Osteonecrosis of different etiology is another cause of persistent bone defects. In the current research, we tested gene-activated grafts (GAGs) carrying dual expression cassette plasmid constructs encoding human vascular endothelial growth factor A (VEGF-A) and bone morphogenetic protein 2 (BMP2) genes. We modeled a thighbone diaphyseal defect on male Wistar rats and tested either GAG based on collagen and hydroxyapatite granules (Collapol CP-3) or GAG octacalcium phosphate (OCP) grafts. Histological examination revealed significant increases in vascularization and bone tissue formation in the GAG Collapol CP-3 graft experimental group, while neither the OCP nor the GAG OCP group showed a positive effect on callus formation. We conclude that GAG Collapol CP-3 bone grafts are a potential therapeutic solution for bone tissue regeneration and further clinical studies are required.

Here, we provide a first comparative study of the therapeutic potential of allogeneic mesenchymal stem cells derived from bone marrow (BM-MSCs), adipose tissue (AD-MSCs), and dental pulp (DP-MSCs) embedded in fibrin matrix, in small (rat) and large (pig) spinal cord injury (SCI) models during subacute period of spinal contusion. Results of behavioral, electrophysiological, and histological assessment as well as immunohistochemistry and real-time polymerase chain reaction analysis suggest that application of AD-MSCs combined with a fibrin matrix within the subacute period in rats (2 weeks after injury), provides significantly higher post-traumatic regeneration compared to a similar application of BM-MSCs or DP-MSCs. Within the rat model, use of AD-MSCs resulted in a marked change in: (1) restoration of locomotor activity and conduction along spinal axons; (2) reduction of post-traumatic cavitation and enhancing tissue retention; and (3) modulation of microglial and astroglial activation. The effect of an autologous application of AD-MSCs during the subacute period after spinal contusion was also confirmed in pigs (6 weeks after injury). Effects included: (1) partial restoration of the somatosensory spinal pathways; (2) reduction of post-traumatic cavitation and enhancing tissue retention; and (3) modulation of astroglial activation in dorsal root entry zone. However, pigs only partially replicated the findings observed in rats. Together, these results indicate application of AD-MSCs embedded in fibrin matrix at the site of SCI during the subacute period can facilitate regeneration of nervous tissue in rats and pigs. These results, for the first time, provide robust support for the use of AD-MSC to treat subacute SCI.

Mesenchymal stem cells (MSCs) hold a great promise for cell therapy. To date, they represent one of the best choices for the treatment of post-traumatic injuries of the peripheral nervous system. Although autologous can be easily transplanted in the injured area, clinical advances in this filed have been impaired by lack of preservation of graft cells into the injury area after transplantation. Indeed, cell viability is not retained after injection into the blood stream, and cells injected directly into the area of injury either are washed off or inhibit regeneration through scar formation and neuroma development. This study proposes a new way of MSCs delivery to the area of traumatic injury by using fibrin glue, which not only fixes cells at the site of application but also provides extracellular matrix support. Using a sciatic nerve injury model, MSC derived from adipose tissue embedded in fibrin glue were able to enter the nerve and migrate mainly retrogradely after transplantation. They also demonstrated a neuroprotective effect on DRG L5 sensory neurons and stimulated axon growth and myelination. Post-traumatic changes of the sensory neuron phenotype were also improved. Importantly, MSCs stimulated nerve angiogenesis and motor function recovery. Therefore, our data suggest that MSC therapy using fibrin glue is a safe and efficient method of cell transplantation in cases of sciatic nerve injury, and that this method of delivery of regeneration stimulants could be beneficial for the successful treatment of other central and peripheral nervous system conditions.

In this study we applied autologous fat tissue stromal vascular fraction (SVF) cells in combination with microfracturing technique in a 36-year-old man with an osteochondral lesion of the medial femoral condyle 8 months after the injury. Cell material was generated by fat tissue liposuction from the anterior abdominal wall with subsequent extraction of the SVF and injected through a mini-arthrotomy portal with subsequent fibrin sealant fixation. The follow-up period was 2 years. Clinical score improved from 23 to 96 according to IKDC and from 10 to 90 according to EQ-VAS at 24 months follow-up. Magnetic resonance imaging (MRI) before the surgery revealed an osteochondral lesion with development of significant trabecular edema that remained unchanged for 6 months despite conservative treatment. MRI 1 and 2 years after the surgery showed the recovery of the damaged cartilage thickness with somewhat uneven structure and a decrease in the trabecular edema of the femoral condyle. The use of SVF cells with fibrin sealant fixation might be a promising approach in the treatment of osteochondral joint lesions. Further studies are required.

We examined the effect of transplantation of allogenic adipose-derived stem cells (ADSCs) with properties of mesenchymal stem cells (MSCs) on posttraumatic sciatic nerve regeneration in rats. We suggested an approach to rat sciatic nerve reconstruction using the nerve from the other leg as a graft. The comparison was that of a critical 10 mm nerve defect repaired by means of autologous nerve grafting versus an identical lesion on the contralateral side. In this experimental model, the same animal acts simultaneously as a test model, and control. Regeneration of the left nerve was enhanced by the use of ADSCs, whereas the right nerve healed under natural conditions. Thus the effects of individual differences were excluded and a result closer to clinical practice obtained. We observed significant destructive changes in the sciatic nerve tissue after surgery which resulted in the formation of combined contractures in knee and ankle joints of both limbs and neurotrophic ulcers only on the right limb. The stimulation of regeneration by ADSCs increased the survival of spinal L5 ganglia neurons by 26.4%, improved sciatic nerve vascularization by 35.68% and increased the number of myelin fibers in the distal nerve by 41.87%. Moreover, we have demonstrated that S100, PMP2, and PMP22 gene expression levels are suppressed in response to trauma as compared to intact animals. We have shown that ADSC-based therapy contributes to significant improvement in the regeneration.