Type I collagenase, dexamethasone, 2-phospho-L-ascorbic acid (vitamin C), ITS liquid media supplement and Cytodex 3 microcarrier beads (Sigma-Aldrich, Poole, UK); trypsin, Dulbecco’s modified Eagle’s medium (DMEM)-high glucose (Gibco Life Technologies, Carlsbad, CA, USA); fetal bovine serum (FBS; Beijing Yuan Heng Sheng Ma Biotechnology Institute, Beijing, China); recombinant murine FGF-basic and TGF-β1 (PeproTech EC Ltd., London, UK); type II collagen mouse anti-human monoclonal antibody (Beijing Zhongshan Co., Beijing, China); a rotating bioreactor (Synthecon, Inc., Houston, TX, USA); and an inverted microscope (Olympus IX70; Olympus Corporation, Tokyo, Japan).

The present study and the collection of human fat tissue was under the ethical approval of the Chinese PLA General Hospital Ethics Committee (Beijing, China). The infrapatellar fat pad removed during knee arthroplasty was maintained in strictly aseptic conditions, and stem cells were separated within 30 min of harvest. The superficial fascia and small blood vessels on the surface of the fat tissue were carefully removed with microsurgical scissors, D-Hank’s solution was used to wash the samples three times to remove red cells. The fat was cut into pieces, added to 5X 0.075% type I collagenase, stirred for 30 min at 37°C and centrifuged at 671 × g for 5 min to release the fat and the supernatant. It was then washed again with D-Hank’s solution, filtered through a 100-mesh filter (BD Biosciences, Suzhou, China) and centrifuged at 168 × g for 5 min. Then the precipitation was washed twice in D-Hank’s solution, which was prepared in the Key Laboratory of PLA (Chinese PLA General Hospital, Beijing, China) and contained KCl (0.4 g/l), KH₂PO (0.06 g/l), NaCl (8.0 g/l), NaHCO₃ (0.35 g/l), Na₂HPO₄.7H₂O (0.06 g/l) and phenol red (0.02 g/l). The cells were then resuspended in 10% FBS containing high-glucose DMEM medium, with a change of medium after 48 h. When the cells were at 80% confluency, 0.25% trypsin was used for digestion passage culture. At the second passage, the medium was changed to cartilage-inducing liquid (5 ng/ml FGF-2; 10 ng/ml TGF-β1; 50 μg/ml vitamin C; 10⁻⁷ M dexamethasone; and 1X ITS) for cartilage differentiation.

The dry weight of 50 mg microcarriers was placed into 10-ml clean glass bottles, with 10 ml phosphate-buffered saline (PBS; Sigma-Aldrich) without calcium and magnesium ions, and incubated at room temperature for at least 3 h to expand the microcarriers. The supernatant was discarded with a polyethylene aluminum compound pipe straw, the microcarriers were cleaned twice with PBS without calcium and magnesium ions, which was then discarded prior to the addition of 10 ml fresh PBS without calcium and magnesium ions. The samples then underwent high pressure sterilization for 20 min, the supernatant liquid was removed and the microcarriers were cleaned with culture liquid. They were then stored in a refrigerator at 4°C.

The Cytodex 3 microcarriers (50 mg) and ADSCs (final concentration, 1×10⁵/ml) were added to a 10-ml rotary cell culture system (RCCS; Synthecon, Inc., Houston, TX, USA) culture container, then the cartilage inducing medium was added to fill the container. The container with cell-microcarriers complex was connected to the rotating base. The device was put in a 5% CO₂ incubator and was cultured at 37°C. In order to fully mix cells, the container was rotated at a force of 5 rpm for 5 min and paused for 5 min alternately, which was repeated for several cycles, it was then continuously rotated and gradually adjusted to 10–12 rpm. Once the culture medium exhibited a yellow color and a PH value <7.0, the medium was changed. Static culture was conducted by seeding 1×10⁵/ml ADSCs into five wells of a 6-well plate, 2 ml in each well. During the culture, the medium was changed every 2–3 days to maintain a sufficient quantity of cells.

Human ADSCs were cultured for 1, 3, 5 and 7 days, and under the inverted microscope, the morphology of the ADSCs and the proliferation on the surface of the microcarriers were assessed. The chondrocytes on the surface of the microcarrier were digested, separated and counted with a blood cell counting plate (Yancheng Glass Instrument Company, Yancheng, China), then a cell growth curve was constructed. Cell microcarrier samples were taken on day 3 to be observed by scanning electron microscopy (Hitachi S-4500; Hitachi, Tokyo, Japan), and the cell morphology and matrix secretion was observed. The static monolayer culture of ADSCs in the control group was observed to assess the ADSC morphology and proliferation at days 1, 3, 5 and 7. They were then collected and the survival rate was calculated using a trypan blue dye exclusion test. Finally, a cell growth curve was constructed based on whole cell counts.

The ADSCs growing on the microcarriers and those in monolayer culture were harvested, digested and smeared. They were then subjected to safranin-O and toluidine blue staining to observe the levels of glycosaminoglycan (GAG) synthesis and secretion from the extracellular matrix. The type II collagen monoclonal antibody was incubated with the cells with DAB (Beijing Hepten and Protein Biomedical Institute, Beijing, China) as the substrate, and the immunoperoxidase method was used for development. Brown staining indicated positive staining, and thus the occurrence of type II collagen secretion and synthesis.

Three hours following inoculation, a number of cells had adhered to the surface of microcarriers. After 24 h, the majority of the cells had adhered to microcarriers, and only a small number of dead cells were present in the medium. Cells gradually changed from a spherical and hemispherical to a short fusiform shape, and occasionally there were pseudopodium with irregular shapes (Fig. 1A).

On day 3, the number of cells on the surface of the microcarriers increased significantly, and the adherent cells were apparent on a different focal plane. Cell matrix secretion around the cells, and occasionally connecting cells between the microcarriers, were observed. Cells were spindle-shaped and flat. There were few dead cells in the medium, and cells adhered well to microcarriers (Fig. 1B). On the 5th day, the majority of the microcarrier surface was covered with cells and cells had grown in layers, with abundant extracellular matrix secretion, indistinguishable single cell morphology, and large cell connection bodies between microcarriers, whilst microcarriers displayed cluster growth (Fig. 1C). On the 7th day, microcarrier surfaces were completely covered by cells overlapping as a mass, leading to a rough, uneven surface on the microcarriers. There were widespread cell connections between the microcarriers, and there were almost no dead cells in the medium (Fig. 1D).

Two hours after cells were treated with the induction medium, cells began to adhere to the wall, and after 24 h, all cells were adherent. The cells were spindle-shaped or polygonal, and were slightly larger than non-induced ADSCs. Once fully adherent, cells began to rapidly proliferate, after 3 days they reached a level of 50% fusion, and after 5 days they reached 80% fusion. Along with the increase in cell density, the proliferation slowed down, and after 7 days, almost complete fusion was reached.

On days 1, 3, 5 and 7, ADSCs were harvested from the RCCS container and common culture bottle and were counted, and the growth curves were as presented in Fig. 2. The Trypan blue dye exclusion test indicated that the survival rate of ADSCs harvested from the microcarriers was >90%. According to the blood cell count, it was clear that the growth of ADSCs accelerated from day 3 after RCCS inoculation, and the cell density reached its highest value at day 7, ~19 times the initial inoculation. On day 7, the total number of harvested cells cultured in a static monolayer was ~6 times the original.

It was demonstrated by ADSC smear staining in the two groups, that toluidine blue (Fig. 3) and safranin-O (Fig. 4) staining in the microcarrier culture was strongly positive, and was stronger than that in the static culture group. The positive staining indicated that the extracellular matrix contained a high level of GAGs; and type II collagen staining was also strongly positive (Fig. 5), indicating that the ADSCs cultured on the microcarriers through dynamic induction culture were able to express matrix components characteristic of cartilage.