All animal experimental protocols were approved by the Ethics Committee of China Medical University (Shenyang, China) and performed according to China Medical University guidelines. Five Male Sprague-Dawley rats (age, 8–12 weeks; weight, 200–250 g) were obtained from the experimental animal department of China Medical University (Shenyang, China). Rats were pre-medicated with Ketalar (50 mg/ml, 0.2 ml/100 g body weight; Jiangsu Hengrui Medicine Co. Ltd., Lianyungang, China), and sacrificed to harvest inguinal fat pad and dissect adipose tissue. The adipose tissue was digested using 0.1% collagenase type I (V900891; Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) at 37°C for 30 min. Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (both from Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was added for neutralization, and the stromal vascular fraction was separated from the floating adipocytes by centrifugation at 300–500 × g at room temperature for 10 min. The cells in the stromal vascular fraction were resuspended and cultured in DMEM supplemented with 10% FBS. The adherent cells were reserved by changing the culture medium after 24 h, and were passaged 3 times prior to use in further experiments.

Rat adipose-derived stem cells from the 3rd passage were lifted with trypsin and washed with phosphate-buffed saline (PBS) 3 times. The cells were incubated with fluorescein isothiocyanate (FITC)-conjugated primary antibodies: Anti-CD29 (SAB4700397, 1:1,000), anti-CD44 (SAB4700189, 1:500), anti-CD45 (SAB4700480, 1:500) and anti-CD11b (SAB4700388, 1:500) (all from Sigma-Aldrich; Merck KGaA), in the dark at room temperature for 20 min. Following this, the cells were washed with PBS and flow cytometry (FACSAria; BD Biosciences, Franklin Lakes, NJ, USA) was performed, the results were analyzed with WinMDI2.9 (developed by Joe Trotter; Purdue University Cytometry Laboratories, West Lafayette, IN, USA).

Rat adipose-derived stem cells from the 3rd passage were seeded into 96-well plates at a density of 2×10⁴ cells/ml with 100 µl hyaluronic acid based gel (Shandong Freda Biotechnology Co. Ltd., Linyi, China), and cultured in DMEM supplemented with 10% FBS. Following culture for 1, 2, 3, 4, 5, 6 or 7 days, cells were incubated with 10 µl Cell Counting kit-8 (CCK-8) solution (Beyotime Institute of Biotechnology, Haimen, China) at 37°C for 3 h. The samples were measured at a wavelength of 450 nm with an Infinite M200PRO Microelisa reader (Tecan Group Ltd., Männedorf, Switzerland). Adipose-derived stem cells were cultured in 100 µl DMEM supplemented with 10% FBS alone as a control group.

For the cytotoxicity study, 100 µl hyaluronic acid-based gel and 0.5×10⁴ adipose-derived stem cells were cultured in 100 µl DMEM supplemented with 10% FBS, and subsequently seeded into 96-well plates. CCK-8 solution (10 µl) was added following 3 days of culture. Following incubation for 3 h, absorbance values were detected by a spectrophotometer at a wavelength of 450 nm. Adipose-derived stem cells were cultured in 100 µl DMEM supplemented with 10% FBS alone as a control group.

For the proliferation study, 100 µl hyaluronic acid based gels and 0.2×10⁴ adipose-derived stem cells were cultured in 100 µl DMEM supplemented with 10% FBS, and then seeded into 96-well plates. CCK-8 solution (10 µl) was added 1, 3 or 6 days following this into the co-cultured system. Following incubation for 3 h, absorbance values were detected by a spectrophotometer at a wavelength of 450 nm.

An Annexin V-fluorecein isothiocyanate Apoptosis Detection kit (Sigma-Aldrich; Merck KGaA) was used to evaluate the effect of hyaluronic acid based gels on apoptosis in adipose-derived stem cells, and 100 µl hyaluronic acid based gel and 1×10⁶ adipose-derived stem cells were cultured in 100 µl DMEM supplemented with 10% FBS, and subsequently seeded into 24-well plates. The cells were lifted with trypsin 1, 3 or 6 days later, and resuspended in binding buffer. Annexin-V FITC and propidium iodide were added and incubated in the dark for 15 min. The FACScan flow cytometer (BD Biosciences) was used to detect the effect on apoptosis and the results were analyzed using WinMDI2.9. Adipose-derived stem cells cultured in 100 µl DMEM supplemented with 10% FBS alone served as a control group.

To detect the multipotential differentiation capacity of the adipose-derived stem cells within the hyaluronic acid based gel, rat adipose-derived stem cells from the 3rd passage were used. Once the cells reached 80% confluence, they were cultured in adipogenic induction medium [DMEM supplemented with 10% FBS, isobutyl-methylxanthine (0.5 mM), dexamethasone (1 µM), insulin (10 µM) and indomethacin (200 µM)] for 14 days, orosteogenic induction medium [DMEM supplemented with 10% FBS, dexamethasone (0.1 µM), ascorbate-2-phosphate (50 µM) and β-glycerophosphate (10 mM)] for 21 days. Oil-Red O and Alizarin Red S staining were subsequently used to confirm adipogenic and osteogenic differentiation of the rat adipose-derived stem cells, respectively.

To evaluate the cell attachment capacity of hyaluronic acid based gels, scanning electron microscopy was applied. Briefly, 3% glutaraldehyde solution in cacodylate buffer (0.1 M) was used to fix the adipose-derived stem cells with hyaluronic acid for 3 h. The fixed complex was subsequently washed with PBS three times. Graded ethanol solutions (30, 50, 70, 85, 90, 95 and 100%) and hexamethyl disilazane were used for dehydration. The dried complex was coated with Pt/Pd, and scanning electron microscopy was applied.

All data were expressed as the mean ± standard deviation. One-way analysis of variance followed by Tukey's post hoc test was used to analyze the differences between groups. Statistical analyses were conducted with SPSS13.0 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a significant difference.

Rat adipose-derived stem cells isolated from adipose tissue were adhered following 3–4 h culture, and the suspended cells were removed following 24-h culture. The spindle cells demonstrated proliferation from 3 days and colony formation from 5 days, and were passaged after 7 days when they reached 80–90% confluence. Rat adipose-derived stem cells were successfully obtained at the 3rd passage and were used in subsequent experiments (Fig. 1). Flow cytometry demonstrated that rat adipose-derived stem cells from the 3rd passage were CD29 and CD44 positive, but CD45 and CD11 negative (Fig. 2).

The results of the cell viability assay indicated that rat adipose-derived stem cells within hyaluronic acid proliferated slowly for the first 48 h, but proliferation accelerated from 3 days onwards and appeared to peak at 6 days, which was similar to the results observed for adipose-derived stem cells alone (Fig. 3).

Following 3 days of culture, the mean absorbance value of adipose-derived stem cells within hyaluronic acid was 0.7471±0.0129, and the control group was 0.2989±0.0190 (Fig. 4). This indicated that the cell activity of the group with hyaluronic acid was significantly higher. The results demonstrated that hyaluronic acid was not cytotoxic to adipose-derived stem cells.

The mean absorbance value of adipose-derived stem cells within hyaluronic acid at 6 days was significantly higher than that at 3 days (0.9829±0.0185 and 0.7471±0.0129, respectively; Fig. 5). This indicated that cell activity of adipose-derived stem cells at 6 days was significantly higher than at 3 days. The results suggested that hyaluronic acid may promote adipose-derived stem cell proliferation.

Cell apoptosis rates at 1, 3 and 6 days were 20.34±1.53, 28.59±0.48 and 37.95±0.93%, respectively. The apoptosis activity of adipose-derived stem cells within hyaluronic acid increased mildly with time, and was higher than the control group, which indicated that hyaluronic acid may induce a fair level of apoptosis in adipose-derived stem cells (Fig. 6).

To verify the maintenance of multipotent differentiation in the adipose-derived stem cells within the hyaluroinc acid gel, adipogenic differentiation was verified by Oil-Red O staining, which demonstrated the presence of intracellular lipid droplets, while osteogenic differentiation was confirmed by Alizarin Red S staining, which demonstrated calcium deposits (Fig. 7).

Scanning electron microscopy indicated that adipose-derived stem cells maintained an intact structure on the surface of hyaluronic acid gel as well as inside it, and demonstrated abundant cell attachments (Fig. 8).