Procurement of adipose tissues
Adipose tissue samples were obtained from patients during routine abdominoplasty following informed patient consent and according to the guidelines set by our institution’s Committee on Human Research. Rat adipose tissue was obtained from the epididymal fat pad of 2-month-old male Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA). All animal care, treatments and procedures were approved by our Institutional Animal Care and Use Committee.
Decellularization of adipose tissue
The adipose tissue in a 50-ml conical tube was dipped into liquid nitrogen for 5 min and then immediately placed in a 37°C water bath for 10 min. After repeating this freeze-and-thaw step two more times, the tissue was centrifuged at 1500 rpm for 10 min at room temperature (RT). After removing the liquid fatty portion, the tissue was washed with 70% ethanol and PBS three times each. It was then incubated in 0.05% trypsin, 0.05% ethylenediaminetetraacetic acid, 20 ng/ml DNAse I, and 20 ng/ml RNAse for 4 h with slow rotation at RT. After washing with PBS twice, the tissue was incubated in 0.1% sodium dodecyl sulphate (SDS) for 12 h at RT. After washing with PBS three times, the tissue was incubated in 1% penicillin and streptomycin for 12 h at 4°C.
Preparation of acellular matrix
The decellularized tissue from above was further processed into various forms of acellular matrices as follows. In one example, the decellularized tissue was cut to ~0.5mm × 1mm × 8mm threads and dried for 12 h for the preparation of adipose tissue-derived acellular matrix thread (ADMT). In another example, the decellularized tissue was homogenized in PBS, placed into a 10-cm plastic dish, and dried for the preparation of adipose-derived acellular matrix sheet (ADMS). The resulting ADMT or ADMS was further UV-sterilized inside a tissue culture hood.
Seeding of acellular matrix thread with adipose-derived stem cells
ADMT was washed with PBS three times, dried at RT for 2 h, and placed in a 6-well cell culture dish. ADSCs were isolated as described previously 20
. Approximately 1×104
ADSCs in 200 µl of DMEM was then evenly added to the ADMT. The culture dish was then placed in a humidified 37°C incubator with 5% CO2
. Four h later, 3 ml of DMEM supplemented with 10% FBS was added to the ADMT, and the dish was returned to the incubator. At 24 h and 1 week, the seeded ADMT was stained with 1 µg/ml Calcein AM (Invitrogen, Carlsbad, CA) for 10 min at 37°C and examined with Nikon Eclipse E600 fluorescence microscope.
Subcutaneous transplantation of seeded matrix
ADSCs were grown to 60% confluence and then labeled with 10 µM of EdU for 24 h as previously described 21
. The cells were then washed three times with PBS and then seeded onto an allogenic ADMT by the above-described procedure. Forty-eight h later, the seeded ADMT was transplanted into an autologous host (from which the ADSC was isolated). The transplantation procedure was as follows. Under inhalant anesthesia, an incision was made in the lower abdomen to expose the subcutaneous space, into which the seeded ADMT was transplanted. Ten days later, the rat was sacrificed and the transplanted tissue examined by HE staining and microscopy. The transplanted ADMT was also retrieved and stained with an EdU detection cocktail that contained Alexa-594 (Invitrogen) and with 4',6-diamidino-2-phenylindole (DAPI, for nuclear staining, 1 µg/ml, Sigma-Aldrich). The stained matrix was examined with Nikon Eclipse E600 fluorescence microscope and photographed with Retiga 1300 Q-imaging camera.
Grafting of unseeded and seeded matrix in nerve injury rats
Thirty 2-month-old male Sprague-Dawley rats were randomized into three equal groups: Control, ADMT, and ADMT+ADSC. Under inhalant anesthesia, a midline incision was made in the lower abdomen and the periprostatic space containing the major pelvic ganglion (MPG) and the cavernous nerves (CN) were exposed. A 5-mm-long nerve segment, starting 5 mm distal from the origin of the CN at the MPG was isolated and resected. In the control group, the abdomen was then closed in layers without further treatment. In the treatment groups, either acellular ADMT, or ADMT/ADSC construct was microsurgically interposed and fixed against the prostatic capsule using 10-0 nylon sutures to bridge the nerve gap.
Determination of erectile function
Three months after CN injury with and without grafting, all rats were examined for erectile function by standard protocol 10
. Under Ketamine-Midazolam anesthesia, the MPG and CN were exposed bilaterally via a midline laparotomy. A 23G butterfly needle was inserted into the proximal left corpus cavernosum, filled with 250 U/mL heparin solution and connected to a pressure transducer (Utah Medical Products, Midvale, UT, USA) for intracavernous pressure (ICP) measurement. The ICP was recorded at a rate of 10 samples/second using a computer with LabView 6.0 software (National Instruments, Austin, TX). A bipolar stainless-steel hook electrode was used to stimulate the origin of the CN at the MPG proximally from the nerve gap (in controls) or the interposed construct (in treatment groups). The electrode was connected to a signal generator (National Instruments) and custom-built constant-current amplifier generating monophasic rectangular pulses with stimulus parameters of 1.5 mA, 20 Hz, pulse width 0.2 ms, and duration 50 seconds. Three stimulations were conducted per side and the erection with the maximum increase in ICP was included for statistical analysis in each animal. For the calculation of ICP increase/mean arterial pressure (MAP) ratio, systemic blood pressure was recorded using a 23G butterfly needle inserted into the aorta at the level of the iliac bifurcation.
Immunohistochemical and immunofluorescence staining
Tissue samples were fixed in cold 2% formaldehyde and 0.002% saturated picric acid in 0.1 M phosphate buffer, pH 8.0, for 4 hours followed by overnight immersion in buffer containing 30% sucrose. The specimens were then embedded in OCT Compound (Sakura Finetec USA, Torrance, CA) and stored at −70 °C until use. Fixed frozen tissue specimens were cut at 7 microns, mounted onto SuperFrost-Plus charged slides (Fisher Scientific, Pittsburgh, PA) and air dried for 5 min. For immunostaining, the slides were placed in 0.3% H2O2/methanol for 10 min, washed twice in PBS for 5 min and incubated with 3% horse serum in PBS/0.3% Triton X-100 for 30 min at RT. After draining this solution, the slides were incubated at RT with anti-nNOS antibody (Santa Cruz Biotechnology, Santa Cruz, CA) or anti-S100 antibody (Leica Microsystems Inc., Bannockburn, IL) for 1.5 h. After rinses with PBS, the sections were incubated with FITC-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA). After rinses with PBS, the slides were incubated with freshly made EdU detection cocktail (Invitrogen) for 30 min at RT followed by staining with DAPI. The stained tissue was examined with Nikon Eclipse E600 fluorescence microscope and photographed with Retiga 1300 Q-imaging camera.
Data was analyzed with Prism 4 (GraphPad Software, Inc., San Diego, CA) using one-way ANOVA followed by Tukey-Kramer test for post-hoc comparisons. All data are reported as mean ± standard deviation. Significance was set at p<0.05.