All experimental procedures involving animals conformed with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals and were approved by the Administration Committee of Experimental Animals, Jiangsu, China.

Ten 4-month-old New Zealand white rabbits (2.8–3.5 kg; Jiangsu Academy of Agricultural Sciences, Nanjing, China) were used. Under sterile conditions, adipose tissues (∼10 g) were carefully taken from the rabbits following anesthesia. Anesthesia was induced via an intravenous injection of ketamine hydrochloride (60 mg/kg) and xylazine (6 mg/kg) (Shanghai Tongwei Biological Technology Co., Ltd., Shanghai, China). These samples were minced and then digested for 1 h at 37°C with type I collagenase (2.5 mg/ml; Sigma-Aldrich, St Louis, MO, USA). During the digestion time, these tissues were swiftly shaken every 20 min. The digested samples were passed through a 70-μm cell strainer (Becton Dickinson, Franklin Lakes, NJ, USA) to yield a single-cell suspension. These released cells were washed with phosphate-buffered saline (PBS) and re-suspended in complete culture medium containing low-glucose Dulbecco's modified Eagle's medium (LG-DMEM; Gibco Life Technologies, Carlsbad, CA, USA), 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 μg/ml streptomycin and 2 mM L-glutamine (all from Invitrogen Life Technologies, Carlsbad, CA, USA) at a density of 500 cells/cm². The resuspended cells were cultured in a humidified atmosphere containing 5% CO₂ at 37°C. At day 3 after initial plating, the cells were washed twice with PBS to remove the non-adherent cells. At day seven to ten, these colonies were trypsinized and mixed together as passage 0.

The osteogenic, adipogenic and chondrogenic differentiation potential of adipose-derived stem cells at passage 3 was investigated according to the method of Rui et al (13) with certain modifications, as described below.

ADSCs were plated at 4×10³ cells/cm² in a six-well plate and cultured in complete culture medium until the cells reached confluence. The medium was replaced with complete medium or adipogenic medium, which was complete culture medium and adipogenic reagents. The adipogenic reagents included dexamethasone (500 nM), isobutylmethylxanthine (0.5 mM), indomethacin (50 mM) and insulin (10 mg/ml) (all from Sigma-Aldrich). The culture medium was replaced every 3–4 days. After 21 days of incubation, Oil Red O staining was performed to confirm the formation of oil droplets. For Oil Red O staining, the cells were fixed in 70% ethanol for 10 min and stained with 0.3% fresh Oil Red O solution (Sigma-Aldrich) for 2 h.

ADSCs were plated at the same density as in the previously described adipogenic assays. Then, these cells were cultured with complete medium or osteogenic medium, which were complete culture medium and osteogenic reagents. The osteogenic reagents included dexamethasone (1 nM), ascorbic acid (50 mM) and β-glycerolphosphate (20 mM) (all from Sigma-Aldrich). The culture medium was replaced every 3–4 days. After 28 days of incubation, 0.5% alizarin red (pH 4.1; Sigma-Aldrich) was utilized to stain the cells for 30 min, subsequent to the cells being fixed in 70% ethanol for 10 min.

A pellet culture system was used for chondrogenic differentiation. Approximately 8×10⁵ ADSCs were centrifuged at 450 × g for 10 min in a 15-ml conical polypropylene tube to form a micromass and incubated in complete medium or a chondrogenic medium that comprised complete culture medium and chondrogenic reagents. The chondrogenic reagents comprised LG-DMEM, supplemented with 10 ng/ml TGF-β3 (R&D Systems, Minneapolis, MN, USA), 500 ng/ml bone morphogenetic protein-2 (BMP-2; R&D Systems), 10⁻⁷ M dexamethasone, 50 μg/ml ascorbate-2-phosphate, 40 μg/ml proline, 100 μg/ml pyruvate (all from Sigma-Aldrich) and 1:100 diluted ITS⁺ Premix [6.25 mg/ml insulin, 6.25 mg/ml transferrin, 6.25 mg/ml selenous acid, 1.25 mg/ml bovine serum albumin (BSA) and 5.35 mg/ml linoleic acid; Becton Dickinson]. After 21 days of incubation, the pellet was fixed for histology and the immunohistochemical staining of collagen type II (Col II).

The immunohistochemical staining was performed as follows. Briefly, paraffin-embedded sections were deparaffinized in xylene and dehydrated through a graded series of alcohol. Then, dewaxed slices of cell mass were incubated in 3% hydrogen peroxide at room temperature for 20 min. Antigen retrieval was performed with 2 mg/ml protease (Calbiochem, Bie and Berntsen, Rødovre, Denmark) at 37°C for 30 min for Col II detection. The sections were incubated with mouse monoclonal antibody against rabbit Col II (cat. no. sc-52658, Santa Cruz Biotechnology, Inc., Dallas, TX, USA; 1:100 dilution with 5% goat serum in PBS containing 1% BSA) overnight at 4°C after blocking with 5% goat serum for 20 min at room temperature. The spatial localization of Col II was observed by incubating with goat polyclonal anti-mouse IgG tetramethylrhodamine-conjugated secondary antibody (cat. no T5393, Sigma-Aldrich; 1:200 dilution with 5% goat serum in PBS containing 1% BSA) for 1 h at room temperature, followed by 3,3′-diaminobenzidine tetrahydrochloride (Dako, Glostrup, Denmark) in the presence of H₂O₂. The slides were gently washed with deionized water and rinsed slowly, followed by dehydration through a graded series of ethanol and xylene, and mounted with DPX for light microscopy (Leica DMRXA2; Leica Microsystems, Wetzlar, Germany).

PRP was prepared according to the method described by Nagae et al (14) with certain modifications. The procedures of PRP preparation were as follows (Fig. 1). Under general anesthesia, 10 ml fresh blood was obtained using a syringe containing 1.0 ml acid citrate dextrose solution A as anticoagulant. The whole blood was centrifuged using a centrifugation apparatus (KN70; Kubota, Tokyo, Japan) at 250 × g for 10 min. Subsequently, the single plasma fraction was collected and further centrifuged at 1,000 × g for 10 min. The precipitated platelets at the bottom of the centrifuge tube were collected with 3 ml of the supernatant (platelet-poor plasma) to yield PRP.

ADSCs at passage 3 were utilized, and plated at 1×10⁸ cells/l in 12-well culture plates containing coverslips. These wells were randomly assigned into a PRP group and a control group, each with six wells. The ADSCs in the PRP group were cultured with complete medium containing 10% PRP. In the control group, the ADSCs were cultured with the complete medium. The culture medium was replaced every 3–4 days.

Following two weeks of culture, ADSCs in the PRP group were fixed with methanol for 10 min, then washed with PBS for 5 min. Subsequently, PBS with Tween 20 (PBST) with 5% BSA was utilized to fix the washed cells. Goat polyclonal anti-rabbit Coll II antibody (cat. no. sc-52658; Santa Cruz Biotechnology, Inc.; 1:200 dilution in PBS) was used as a primary antibody for incubation with the induced ADSCs for 1 min; the cells were then incubated with a fluorescein isothiocyanate (FITC) fluorescence-labeled goat anti-mouse secondary antibody (cat. no. F0257; Sigma-Aldrich; 1:200 dilution in PBS) for a further 60 min. Following the incubation, the slides were washed with PBS and observed under a fluorescence microscope (EVOS® FL; Life Technologies, Carlsbad, CA, USA). For toluidine blue staining, the procedures performed to fix the induced ADSCs were the same as those described for immunofluorescence staining. Then, the slides were dyed with 1% toluidine blue for 2 h prior to washing with PBS. Finally, the slides were mounted with neutral gum for observation.

To compare the expression of type II collagen α1 chain (COL2A1) and aggrecan (AGC) mRNA expression between the PRP and control groups, RT-qPCR was performed. Following two weeks' culture, ADSCs from the PRP and control groups were harvested and subjected to RNA extraction with an RNeasy mini kit (Qiagen GmbH, Hilden, Germany). mRNA was reverse-transcribed to complementary DNA (cDNA) using the First Strand cDNA kit (Promega Corporation, Madison, WI, USA). Then, 5 μl of total cDNA from each sample was amplified in a final volume of 25 μl of reaction mixture containing Platinum SYBR Green qPCR SuperMix-UDG ready-to-use reaction cocktail and specific primers for COL2A1, AGC and β-actin (all from Mergene; Table I). Cycling conditions were denaturation at 95°C for 1 min, 45 cycles at 95°C for 20 sec, optimal annealing temperature (as defined in Table I) for 15 sec, 72°C for 45 sec and at 60–95°C with a heating rate of 0.1°C/sec. Target gene expression was normalized to that of β-actin. Relative gene expression was calculated with the 2^−ΔΔCt formula (15).

Comparisons between groups were performed using a paired t-test. All data analysis was conducted using SPSS statistical software (version 17.0; SPSS Inc, Chicago, IL, USA). P≥0.01 was considered to indicate a statistically significant result.

It was shown in the present study that the ADSCs isolated from rabbit adipose tissue were adherent to the plastic culture flask after being cultured for 24 h. Following four days of culture, long spindle cells were observed and the number of cells was significantly increased. Passage 3 ADSCs retained the long spindle morphology and proliferative potential.

Following the various multi-lineage induction processes, ADSCs were successfully induced toward adipogenic, osteogenic and chondrogenic lineages. After 21 days of adipogenic induction, lipid droplets were formed and confirmed by Oil Red O staining (Fig. 2A). This was not observed in the group cultured with complete medium only (Fig. 2B). The osteogenic differentiation potential of the isolated ADSCs was determined in vitro. Alizarin-red staining was positive as calcium nodules were observed after 21 days of induction (Fig. 2C). In the group cultured with complete medium only, alizarin red staining was negative (Fig. 2D). The chondrogenic differentiation potential of the ADSCs was determined in vitro by pellet culture. Following 21 days of chondrogenic induction, the diameter of the pellet was ∼1 mm (Fig. 2E). The induced ADSC pellet was rich in Col II, as indicated by immunohistochemical staining (Fig. 2F).

Following two weeks of induction, ADSCs in the PRP group produced Col II, as indicated by immunofluorescence staining (Fig. 3A). No green fluorescent cells were seen in the control group (Fig. 3B), indicating that there was no significant expression of Col II. Toluidine blue staining indicated that PRP was a potent reagent for inducing ADSCs to produce aggrecan. Toluidine blue staining was positive in the PRP group (Fig. 3C) but negative in the control group (Fig. 3D). RT-qPCR confirmed that CoL2A1 and AGC mRNA expression was significantly upregulated in the PRP group compared with that in the control group (P<0.01; Table II).