Cell isolation and characterization
MVSCs were isolated from the tunica media layers of the aorta of transgenic Sprague-Dawley (SD) rats expressing enhanced green fluorescence protein (GFP) (University of Missouri, Columbia). The cell isolation methods were described previously, using a tissue explant culture method [7]. Briefly, the tissue segments were washed three times with phosphate-buffed saline (PBS) supplemented with 1% penicillin/streptomycin (P/S). The surrounding connective tissues and adventitia were removed under a dissecting microscope. The endothelium was removed by scraping off the cell layer on the luminal surface with sterile scalpel blades. Using a tissue explant culture method, the tunica media was cut into millimeter size and placed onto the surface coated with 1% CellStart (Invitrogen) in 6-well plates. The cells were initially cultured in DMEM with 2% chick embryo extract (MP Biomedical), 1% fetal bovine serum, 1% N2 (Invitrogen), 2% B27 (Invitrogen), 100 nM retinoic acid (Sigma-Aldrich), 50 nM 2-mercaptoethanol (Sigma-Aldrich), 1% P/S, and 20 ng/ml bFGF (R&D Systems) (as phenotype maintenance medium). Cells were then seeded onto CellStart-coated dishes and maintained at 37 °C in an incubator with 5% CO2. Among all isolated cells, small population of cells (less than 10%) were small, round, and negative for smooth muscle myosin heavy chain (SM-MHC) expression, a mature marker of vascular SMCs [8]. These SM-MHC− cells, mostly MVSCs [6], were expandable with enhanced telomerase activity as compared to SM-MHC+ cells.
For immunostaining, cells were fixed with 4% paraformaldehyde (PFA), permeabilized with 0.5% Triton-100 (Sigma-Aldrich), and blocked with 1% bovine serum albumin (Sigma-Aldrich). For the staining of cell markers, samples were incubated with specific primary antibodies: Sox10 (ab155279, Abcam), Sox17 (ab84990, Abcam), Ki67 (ab197234, Abcam), smooth muscle α-actin (⍺-SMA) (ab5694, Abcam), calponin-1 (CNN1) (ab233854, Abcam), CD31 (ab28364, Abcam), peripherin (ab4666, Abcam) or S100β (ab4066, Abcam) for 2 h at room temperature, washed with PBS for 3 times, and then incubated with appropriate Alexa 488- and/or Alexa 546-labeled secondary antibodies (Molecular Probes). The nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI) (Invitrogen). Fluorescence images were collected using a Zeiss LSM710 confocal microscope.
Nerve conduit fabrication
Electrospinning technique was used to produce nanofibrous nerve conduits as previously described [4]. Non-woven aligned nanofibrous nerve conduits composed of poly(l-lactide-co-caprolactone) (PLCL) (70:30, Purac Biomaterials), poly(propylene glycol) (Acros Organics), and sodium acetate (Sigma) were fabricated by using a customized electrospinning process. To make tubular scaffolds with aligned nanofibers in the longitudinal direction on the luminal surface, a rotating mandrel assembly with two electrically conductive ends and a central non-conductive section was used. The jet stream of polymer solution from the spinneret whipped between the two conductive ends, resulting in longitudinally aligned nanofibers, forming a tubular scaffold on the non-conductive portion of the mandrel. To enhance the mechanical strength of the scaffolds, the outer layers of random nanofibers were deposited onto this inner layer of longitudinally aligned fibers [9].
Matrigel matrix formulation and preparation of tissue engineered conduits
The Matrigel we used in this study was Growth Factor Reduced (GFR) BD Matrigel Matrix (Cat. No.356230). The PLCL conduits were cut to 1.1cm-long and sterilized with ethylene oxide gas before use. MVSCs were detached and re-suspended in serum-free MVSC maintenance medium to the density of 40 million cells/ml, modified from previous protocol [10]. The cell suspension was then mixed with cold GFR matrigel solution at 1:1 ratio (volume to volume). Cells in 25 μL of matrigel was used to fill up one 1.1-cm PLCL conduit and thus the transplanted cell number was 1 million per conduit. The filled conduits were incubated in 37°C for 1 h to allow matrigel solidification. Serum-free MVSC maintenance medium was then added to cover the tissue-engineered nerve conduits for overnight culture in 37°C incubator before surgery.
In vivo transplantation of stem cells and nerve conduits
To investigate the in vivo therapeutic effect, 18 adult SD rats were randomly divided into three treatment groups (acellular, MVSC, and autograft, N = 6 in each group). MVSC transplantation was performed using allogenic cells which were isolated from GFP rats. All animal experimental procedures were approved by the Animal Care and Use Committee at UC Berkeley and were carried out according to the institutional guidelines. Adult female SD rats (Charles River) weighing 200–250 g were used in all treatment groups. For nerve conduit implantation, an incision was made over the skin above the hip joint with a sterile scalpel. Under a surgical microscope, the sciatic nerve was isolated and severed with a microscissor at two spots to make a 10-mm gap. Then, the tissue-engineered nerve conduits (11 mm in length, 1.5 mm in inner diameter), with or without MVSCs, were inserted between the two nerve stumps and then sutured with 9-0 nylon monofilament sutures (Ethilon, Ethicon). The overlying muscle layers and skin were sutured with 4-0 absorbable sutures (polydioxanone, Ethicon) to close the surgery site. For the autograft group, 10-mm segment of sciatic nerve was transected and microscopically repaired in a reverse fashion. After 1-month transplantation, nerve regeneration was assessed by electrophysiology. To confirm the histologic evidence, all animals were euthanized and the conduits were harvested, fixed in 4% PFA for immunohistochemistry (IHC) analysis.
Electrophysiology
Electrophysiology testing was performed to evaluate the functional recovery of regenerated nerve by following the previous method [4]. In brief, the rat sciatic nerve was exposed, and electrical stimuli (single-pulse shocks, 1 mA, 0.1 ms) were applied to the native sciatic nerve trunk at the point 5 mm proximal to the graft suturing point. Amplitude of the depolarization was recorded as compound muscle action potential (CMAP) on the gastrocnemius belly from 1 to 12 V or until a supramaximal CMAP was reached. Normal CMAP from the unoperated contralateral side of the sciatic nerve was also recorded for comparison, at the same level of the sciatic nerve. Grass Tech S88X Stimulator (Astro-Med Inc.) was used for the test, and PolyVIWE16 data acquisition software (Astro-Med, Inc.) was used for recording. Recovery rate is the ratio of injured hindlimb’s CMAP to contralateral normal hindlimb’s CMAP of a rat [4]. After electrophysiological evaluation, the rats were sacrificed to harvest the regenerated nerve for histological examination.
IHC analysis
The nerve conduits were harvested and fixed in 4% PFA at 4 °C for 2 h. After being washed with PBS, the tissues were cryoprotected with 30% sucrose in PBS at 4 °C overnight, and were then embedded in optimum cutting temperature compound, followed by being frozen at − 80 °C. The frozen samples were cryosectioned longitudinally and transversely at − 20 °C for the thickness of 10 μm. The slices were placed onto Superfrost plus slides and stored at − 20 °C. IHC staining was performed for histological analysis. The slices were permeabilized with 0.5% Triton X-100 in PBS for 30 min, blocked with 4% normal goat serum in PBS for 1 h, and then incubated overnight at 4 °C with primary antibodies. The slides were then washed with PBS and incubated with secondary antibodies for 1 h at room temperature. After further PBS washing, the coverslips were mounted and viewed with a fluorescent microscope (Zeiss). The primary antibodies used for IHC analysis in this study were as follows: neural filament-medium polypeptide (NFM) (ab7794, Abcam), S100β (ab4066, Abcam), Claudin-1 (ab15098, Abcam), CD31 (ab28364, Abcam), ⍺-SMA (ab5694, Abcam), and CNN1 (ab233854, Abcam). Fluorescence-tagged anti-mouse and anti-rabbit secondary antibodies (Abcam) were used.
Statistical analysis
The data was reported as mean ± standard error of mean, unless otherwise described. Comparisons among values for groups greater than two were performed by one-way analysis of variance, and differences between the groups were then determined using a Tukey’s post hoc test. For all experiments, a value of p < 0.05 was considered statistically significant. GraphPad Prism software (version 8.0) was used for all statistical analyses.