Materials
Poly (L-lactic acid)-co-poly(ϵ-caprolactone) [P(LLA-CL)] with a ratio of 70:30 was purchased from Evonik (Germany). Atelocollagen (COL) was purchased from KOKEN (Japan). 1,1,1,3,3,3-hexafluoro-2-propanol (HFP) was obtained from Fluorochem (UK). Polyaniline (PANI) and (1S)-(+)-10-Camphorsulfonic acid (CSA) were purchased from Sigma-Aldrich (USA).
Fabrication of electrospun tubular fibrous scaffolds
A 10 % (w/v) solution of P(LLA-CL) and Col [ratio of 85 to 15] in HFP was mixed with 10 % PANI/CSA [50:50] in HFP solution in the ratio of 7 to 3. The composite solution was then electrospun in the NANON-01 apparatus (MECC, Japan) at a voltage of 15 kV using a needle to tip a working distance of 80 mm and feed rate of 1.0 ml/h. A stainless-steel mandrel with a 2 mm diameter rotating with a speed of 100 rpm was used as a collector. The 21 G needle – spinneret was moving with a speed of 100 mm/sec and the spinneret width was set to 160 mm. The electrospinning process was conducted for 70 minutes. Electrospun tubes after 48 h drying in vacuum drier (25°C, 50 mbar) were extracted from the mandrel.
Characterization of the electrospun scaffolds
The morphology of the obtained fibrous tubes was observed, after sputter coating with gold, using a scanning electron microscope (SEM, PhenomX, The Netherlands) at an accelerating voltage of 10 kV. The diameters of obtained nanofibers were analyzed from the SEM images using Image Analysis Software (ImageJ, National Institute of Health, USA). The average diameter of the fibers was determined by measuring the diameters of 50 randomly selected fibers. The thickness of the walls and the outer diameter of the tubes were measured using a micrometer screw in 5 different places of the tube.
The tensile properties of the electrospun nanofibrous tubes were evaluated using a tensile testing machine Instron 5943 (Instron, USA) at a crosshead speed of 5 mm /min under ambient conditions. The samples for mechanical tests were 20 mm long, and the end of each side of 5mm was attached to the hydraulic clamps.
Hydrophilicty of the tubes was evaluated by the static contact angle measurement method using a contact angle goniometer (OCA 20, Dataphysics, Germany). A volume of 2 µl of DI water was placed on the surface of the samples and 3 seconds after the droplet touched the surface the image was taken. Measurements were taken at nine different positions and contact angles were calculated using SCA20 software.20.
Harvesting, isolation, differentiation, and seeding of ASCs into the scaffold
10 male Lewis rats were sacrificed and euthanized for fat pad harvest. Fat was harvested in a sterile manner from 4 regions – inguinal, gonadal, pararenal, and nuchal (parascapular). Fat pads were collected in 1 % PBS and immediately transferred on wet ice to the Department of Regenerative Medicine for further ASC isolation. Each fat pad underwent isolation protocol with 0,075 % Type I collagenase consistent with the original protocol proposed by Zhu et al. and previous studies [16, 17]. Isolated stromal vascular fraction (SVF) pellet was dissolved in 2 ml DMEM. Cells underwent immediate and delayed (after 3-5 passage) cytometric analysis (CD29, CD11b, CD90, CD45 and CD34). SVF was also subjected to a differentiation test with alizarin red, Masson's Trichrome, and oil red staining. Finally, MTS and doubling time tests were applied to compare the survival rate of fat harvested from different regions. Ultimately, there were no statistically significant differences in the survival rate of cells and contents in ASCs in any region. Thus, cell culture after 3-5th passage from all regions was used to prepare ASCs stocks for conduits enrichment in further steps.
Animal study design
Animal care and handling were carried out in accordance with the UK’s Animals (Scientific Procedures) Act 1986 and associated guidelines, the EU Directive 2010/63/EU for animal experiments, and comply with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines. The experiments were approved by the Second Local Ethics Committee in Warsaw (Protocol no 72/2015). All surgical procedures were performed using aseptic techniques. Inbred male Lewis rats aged 4-6 months (n = 28), were acclimatized to 12 h light/dark cycle at 19°C, with unlimited water and standard food. The rats weighed 366 to 486 g at the time of surgery.
Twenty eight animals were randomly assigned into 4 even groups, each receiving a unilateral sciatic nerve transection and resection of 10 mm of its trunk and further: A) control, 10 mm gap remained, B) reconstructed with autograft, C) reconstructed with P(LLA-CL)-COL-PANI conduit, D) reconstructed with P(LLA-CL)-COL-PANI conduit and enriched with ASCs (See Figure 1). Animals were anesthetized with Isoflurane. The randomly chosen limb was shaved, sterilized and a 3 cm cut was performed along the dorsal side of a rat’s thigh. Gluteal muscles (GM) were divided to expose the sciatic nerve (SN). SN was cleared from the surrounding tissues and measured to obtain a continuous 10 mm trunk to be resected with at least a 5 mm margin from its divisions or branches. The previously marked fragment of SN was resected with a sharp blade. In rats assigned to group A SN was left without coaptation, with both stumps unsecured. Group B was reconstructed with the resected trunk of the sciatic nerve, which was rotated 180° and used as an autograft, finally coaptated with 2 epineural sutures on each side (10/0 Prolene). In group C P(LLA-CL)-COL-PANI conduit was placed between proximal and distal nerve stump, which were further advanced about 2 mm into the lumen of the conduit and secured with 2 epineural sutures on each side (10/0 Prolene). In group D 3x106 ASCs were seeded into a P(LLA-CL)-COL-PANI conduit and incubated for 3 days in 37°C, 5% CO2 and 95% humidity in separate Petri dishes in DMEM with 10% FBS. Implantation was managed similarly to group C. After conduit securing an additional portion of 0,3ml of cultured ASCs stock containing 1x106 cells was injected with a 32G needle in the lumen of the conduit (in the space between both nerve stumps) (see Figure 2). Afterward, gluteal muscles were secured with two 4/0 Vicryl sutures, and skin was closed with interrupted 4/0 Vicryl sutures. Rats were administered postoperatively with analgesics and remained under strict observation for 6 months. Operated limbs were secured for the prevention of autotomy or self-cannibalism. Scaffolds were sterilized with 25 kGy dose radiation prior to the implantation.
Samples harvest and histological analyses
At 6 months of observation, rats were sacrificed by inhalation using isoflurane overdose (>4% v/v). Samples of conduits (or nerve autografts), ipsilateral and contralateral gastrocnemius muscle were harvested. In group A, discontinuation of SN was confirmed during the harvest. Sciatic nerves were cut out with a 5 mm margin on both proximal and distal sides of conduits, in case of Group B harvest was performed likewise. Additionally, SN and its surroundings were inspected for the presence of inflammation, scarring, fibrosis, or conduit dissolution. Freshly harvested muscles were weighted on a RADWAG AS220 electronic balance (Puszczykowo, Poland) and results were expressed as a ratio of ipsilateral to the contralateral muscle (denervated to innervated). Nerve samples were divided into 3 portions – (A) proximal to the conduit, (B) body of conduit, and (C) distal to the conduit. Further, samples were immediately immersed in a 10% Buffered formalin solution and transferred to the Department of Methodology (Medical University of Warsaw) for further processing. Samples underwent a standard protocol for sample embedding in paraffin. Subsequently, nerves were cut into 4 µm slices and muscles into 10 µm slices. The samples of the muscles were stained with hematoxylin and eosin (HE) with standard protocol and covered with a coverslip. Images of muscle sections were captured under 400x magnification.
Nerve sections in the center of conduit/autograft were stained immunohistochemically with NF-200 primary antibody (N4142, Sigma-Aldrich, Munich, Germany,). Staining was performed according to manufacturer protocol. Samples were counterstained with hematoxylin. Images of stained nerve sections were captured under 400x magnification. Quantitative assessment was performed for sections B. Nerve fiber were identified with the aid of semi-automatic analysis tool – CellProfiler and adjusted protocol reported by Paskal et al. [18]. Nerve fiber density was described as the number of fibers in an mm2 (axons/mm2).
Statistical analysis
Data from each study were collected in Excel (Microsoft, Redmond, WA). Kolmogorov-Smirnov test and Shapiro-Wilk test were used to determine the distribution of data. T-test, univariate ANOVA, post-hoc Turkey’s tests were used for the analyses accordingly. GraphPad Prism 6 (GraphPad Prism, San Diego, CA) was used for statistical analyses and plots preparation. The threshold of statistical significance is set at p ≤ 0.05.