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Poly(-Caprolactone) Nerve Conduit and Local Delivery of vegf and fgf2 Genes Stimulate NeuroregenerationS. I. Nikolaev*, A. R. Gallyamov*, G. V. Mamin**, and Yu. A. Chelyshev*,**

Translated from Kletochnye Tekhnologii v Biologii i Meditsine, No. 1, pp. 44-48, January, 2014Original article submitted June 4, 2013

We studied regeneration of rat sciatic nerve while overcoming of a 5-mm diastasis with the aid of nanostructured conduit made of biocompatible and biodegradable poly(ε-caprolactone) and fi lled with fi brin hydrogel matrix. Implantation of the conduit into the nerve in combination with local delivery of the expression plasmid carrying genes encoding vascular endothelial growth factor (vegf) and fi broblast growth factor 2 (fgf2) leads to an increase in number of myelinated fi bers and S-100+ cells in the peripheral nerve stump and improved recovery of the nerve function. Under conditions of direct gene therapy, an advantage of electrospun poly(ε-caprolactone) conduit with high-porosity was revealed on the basis of these criteria in comparison with biocompatible silicon conduit.

Key Words: nerve regeneration; vegf; fgf2

*Department of Histology, Cytology, and Embryology, Kazan State

Medical University; **Institute of Physics, Kazan (Volga Region) Fe-

deral University, Russia. Address for correspondence: chelyshev-

kzn@yandex.ru. Yu. A. Chelyshev

Ner ve conduits, i.e. tubular tissue-engineering con-structs that guide and support regeneration of the nerve fi bers, are actively developed for bridging peripher-al nerve defects. Biodegradable polymers ensuring implant resorption after completion of nerve regen-eration are promising materials for these conduits. Poly(ε-caprolactone), one of the best studied synthetic biocompatible polymers for conduit formation, dem-onstrates adequate mechanical properties and biode-gradability. Electrospun nanostructured polymer apart from optimal mechanical properties is characterized by high porosity and hence, is permeable for biomol-ecules maintaining and stimulating neuroregeneration. In some studies, electrospun nanostructured poly(ε-caprolactone) is used as the polymer or copolymer for creation of nerve conduits [5-7,11,13] and for ob-taining of functionalized nanomatrix guiding directed migration of cells and in vitro axonal growth [8].

Hydrogels on the basis of natural or synthetic polymers are optimal fi lling for the conduits [3]. Fibrin hydrogel is an adequate matrix for the growth of nerve

fi bers. Neuroregeneration assisted by nanostructured poly(ε-caprolactone) and fi brin-based hydrogel matrix was never studied.

Higher effi ciency of neuroregeneration in conduit-guided nerve plasty can be explained by the possi-bility of simultaneous using direct gene therapy (in vivo) that implies injection of DNA-vectors into the damaged area in the nerve tissue. For the purposes of neuroregeneration, genes of neurotrophic factors are studied in detail [4]. Local delivery of these genes into the damaged area is a promising approach to stimu-lation of neuroregeneration [9]. Experiments on the model of nerve gap bridging with a biocompatible (but nor biosoluble) silicone conduit fi lled with physiologi-cal saline have demonstrated that single injection of a double-cassette plasmid pBud-VEGF-FGF2 expressing simultaneously cloned vegf and fgf2 genes stimulated the growth of regenerating myelin fi bers and improved functional parameters of regeneration [1]. Neuroregen-eration under conditions of biosoluble conduit with high wall permeability and adequate mechanical properties in combination with local delivery of therapeutic re-generation-stimulating genes was never studied.

Here we evaluated the effi ciency of regeneration of rat sciatic nerve under condition of implantation of

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nanostructured poly(ε-caprolactone) conduit in com-bination with direct local delivery of a plasmid vector carrying cloned genes vegf and fgf2 to the damaged area.

MATERIALS AND METHODS

Experiments were carried out on 25 albino rats weigh-ing 200-250 g. All procedures on animals were per-formed in accordance with the rules recommended by the Physiological Section of the Russian National Committee on Bioethics. The animals were kept under standard conditions with free access to food and water. The rats were intraperitoneally narcotized with chlo-ral hydrate (80 mg/ml, 0.4 ml pr 100 g body weight; Sigma).

Fabrication of tubular nerve conduit by the me-thod of electrospinning Samples of polymeric tubes for nerve conduit were fabricated by electrospinning [5,11,13]. A solution (6%, w/w) of a biocompatible and biosoluble polymer poly(ε-caprolactone) (80 kDa; Sigma) in a chloroform-methanol 3:1 mixture was placed in a syringe with a 1.3-mm needle; the dis-tance between the collector and the end of the needle was 20 cm, polymer feed rate 0.2 ml/min, fi eld form-ing voltage 24 kV, collector rotation speed 3000 rpm. Electrospinning was carried out at room temperature. Structure of the polymer tube wall was analyzed by light microscopy (Axio Imager A1; Сarl Zeiss) and scanning electron microscopy (XL30ESEM; Philips). The tubes were kept in vacuum for 10 min to remove solvent traces and then sterilized [7]. Immediately be-fore the surgery, the polymer tubes were fi lled with fi brin hydrogel under sterile conditions (Tissucol; Bax-ter) prepared according to manufacturer’s instruction.

Reconstruction of the nerve and local delivery of the plasmid. Nerve diastasis (5 mm) was formed in the left sciatic nerve at the mid-thigh level and then a nerve conduit was implanted to bridge the gap. Each end of the implants was fi xed with two epineu-ral stiches (8.0 monothread; atraumatic needle). In animals of groups 1 (n=6) and 2 (n=10), a conduit on the basis of poly(ε-caprolactone) was implanted. In group 3 animals (n=9), a biocompatible silicone tube (A-M Systems) fi lled with fi brin hydrogel (Tissucol; Baxter) was implanted. Immediately after implantation of conduits, a double-cassette plasmid pBud-VEGF-FGF2 expressing vegf and fgf2 [2] was injected to rats of groups 1 and 3 at the sites of proximal and distal sutures of the reconstructed nerve (7.5 μl per injec-tion site; 15 μl total in PBS) with a Hamilton syringe (Sigma). Group 2 animals (n=10) received the same volume of physiological saline.

Functional tests. The effi ciency of nerve rege-neration was evaluated using functional index of the

sciatic nerve and by the recovery of skin sensitivity (pinch test) [1]. The tests were carried out on days 7, 10, 13, 15, 18, 21, 24, 27, and 30 after surgery.

Morphological and immunohistochemical anal-ysis. On day 30 after surgery, two fragments were taken from the peripheral nerve segment adjacent to the implant. The proximal fragment was fi xed in 2.5% glutaraldehyde and 2% OsO4 and embedded in epon araldite. The distal nerve fragment was fi xed in 10% neutral formalin. The number of myelinated fi bers was determined on semithin transverse sections of the nerve stained with methylene blue. Sections of the peripheral fragment (18 μ) were prepared on a Cryo-Star HM 560 cryostat and peroxidase and immunofl uo-rescent reaction with polyclonal antibodies to S-100 protein (1:1200; Dako) was performed. Anti-rabbit Alexa 555 (1:150; Invitrogen) was as the fl uorescent dye. Nuclei were poststained with DAPI (Sigma). The sections were examined under an LSM 510-Meta con-focal microscope (Carl Zeiss). The number of S-100+ cells and myelinated fi bers in the total cross-section of the regenerating nerve was counted on digitized im-ages. The data were processed statistically by ANOVA and using Student’s t test.

RESULTS

Polymer tubes with adequate mechanical character-istics were prepared by electrospinning of poly(ε-caprolactone) solution. Optimal combination of elas-ticity and strength ensures constant gap length as a potential space for the growth of regenerating nerve fi bers during the long-term experiment. Scanning elec-tron microscopy have demonstrated that the walls of the polymer tubes consist of micro- and nanofi bers (Fig. 1) and have highly porous structure determin-ing high permeability for water molecules from the microenvironment.

Under conditions of local injection of the plasmid carrying the therapeutic genes, the mean value of the

Fig. 1. Filamentous structure of the wall of tubular electrospun

poly(-caprolactone) nerve conduit. Scanning electron microscopy,

×1000.

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functional index of sciatic nerve (FISN) calculated for the 30-day postoperation period in group 1 animals with poly(ε-caprolactone) conduit surpassed the correspond-ing parameter in group 3 rats with silicone conduit by 29.5% (p<0.05; Fig 2). By day 30 after surgery, the number of myelinated fi bers in the proximal fragment of the peripheral stump of the regenerating nerve in animals with poly(ε-caprolactone) conduit surpassed the corresponding parameter in rats with silicone con-duit by 40.5% (p<0.05). Thus, nanostructured poly(ε-caprolactone) conduit more effi ciently supports nerve re-generation than silicone conduit probably due to higher permeability of poly(ε-caprolactone) conduit wall. This polymer acts as a matrix supporting cell survival and differentiation [10,12], which in our experiments can be extrapolated to Schwann cells of the regenerating nerve.

Experiments demonstrated more active regenera-tion of the nerve fi bers in animals receiving poly(ε-Fig. 2. Dynamics of FISN under the effect of nerve conduit implan-

tation in combination with delivery of pBud-VEGF-FGF2 plasmid.

Fig. 3. Peripheral segment of the sciatic nerve in 30 days after diastase bridging with the use of tubular electrospun poly(-caprolactone) nerve

conduit, ×400. Myelinated fibers (a and b) on semithin cross-sections (methylene blue) and S-100+ cells (c and d) on longitudinal sections.

Alexa 555 fluorescent dye (yellow), cell nuclei stained with DAPI (blue); confocal microscopy. a) Saline; b-d) pBud-VEGF-FGF2 plasmid.

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caprolactone) conduit in combination with adminis-tration of the therapeutic genes in comparison with animals receiving saline. The mean FISN calculated for the 30-day postoperation period in group 1 animals with local delivery of the plasmid carrying the thera-peutic genes surpassed the corresponding parameter in group 2 by 45% (Fig. 2). In this group, FISN values on days 7, 13, 15, 21, 24, 27, and 30 were signifi cantly higher than in group 2 animals by 32.9, 40.1, 59.0, 65.6, 55.5, 49.7, and 15.0%, respectively. The most pronounced differences were observed on week 3 of the experiment.

According to pinch-test results, the area of plantar surface of the foot with restored sensitivity in group 1 animals exceeded the corresponding parameter in group 2 animals by 26.1% (p<0.05).

The number of myelinated fi bers (Fig. 3, a, b) and S-100+ cells (Fig. 3, c, d) were higher in group 1 ani-mals receiving local administration of the therapeutic genes. By day 30 after surgery, the number of myelin-ated fi bers in the proximal fragment of the regenerat-ing peripheral nerve increased by 46.4% (p<0.05) and the number of S-100+ cells in the distal fragment of the same peripheral segment 2.5-fold surpassed the corre-sponding parameter in group 2. These data suggest that direct local injection of plasmid pBud-VEGF-FGF2 to the site of traumatic injury stimulates regeneration of rat sciatic nerve and recovery of motor and sensory functions. The increase in the number of myelinated fi bers in animals receiving gene therapy can be a result of direct supportive effect of neurotrophic factors on differentiation and survival of Schwann cells and on the process of axon re-myelination. This promoted revascu-larization, stimulates proliferation of Schwann cells and expression of some stimulators of neuroregeneration, such as neurotrophic factors, adhesion molecules, and extracellular matrix molecules by these cells.

The authors are grateful to R. R. Islamov, A. P. Kiyasov, and A. A. Rizvanov for providing plasmid pBud-VEGF-FGF2.

The study was supported by Federal Target Pro-gram “Research and Development on Priority Direc-tions of Russian Scientifi c-Technological Complex Development in 2007-2012” by Ministry of Education and Science of the Russian Federation (State Contract No. 16.512.11.2101).

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