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Primary hUSCs were obtained from five healthy male adult donors, who were between 23 and 27 years old (mean age: 25 years) without urinary system disease, using the methods that were described previously [8, 10].
To evaluate cell proliferation, hUSCs were seeded into a 96-well plate and incubated in 100 [micro]L of cell culture medium at 37[degrees]C and 5% CO2.
When at passage 4 (P4), hUSCs were harvested using trypsin-EDTA, and 1 x 106 hUSCs were resuspended in Hank's Balance Salt Solution (HBSS) supplemented with 1% (v/v) bovine serum albumin (BSA).
To induce osteogenic differentiation, hUSCs were cultured at a density of 5 x [10.sup.3] cells/well in a 6-well plate for 21 days under the condition of 37[degrees]C and 5% C[O.sub.2] in osteogenic medium (Cyagen Biosciences Inc., USA).
hUSCs (4th passage) were cultured at a density of 5 x [10.sup.3] cells/well in a 6-well plate and induced using adipogenic medium (Cyagen Biosciences Inc., USA).
To induce chondrogenic differentiation, 1 x [10.sup.6] hUSCs were centrifuged for 5 min at 1500 rpm after which the pellet was resuspended in chondrogenic medium (Cyagen Biosciences Inc., USA).
hUSCs and HA were mixed at a ratio of 1 x [10.sup.7]:1 mL.
Cell morphology of hUSCs in HA was compared with that of hUSCs in PBS to assess the cell state.
The following four groups were established: (i) group A (hUSCs plus HA, n = 6), 1 x [10.sup.7] cells and 1mL 1% HA (pH 6.7, 1000 kDa, Furuida, China) were injected into the knee joint cavity; (ii) group B (hUSCs, n = 6), 1 x [10.sup.7] cells and 1 mL of normal saline were injected into knee joints; (iii) group C (HA group, n = 6), only 1 mL 1% HA was injected into knee joints; and (iv) group D is the control group with normal saline injected (n = 6).
Morphology and Characterization of hUSCs. Cell colonies of hUSCs were observed 7-10 days after initial plating, in which the cells had "rice grain"-like appearance (Figure 2(a)).
In Vitro Chondrogenic Differentiation Potential of hUSCs. After 21 days of chondrogenic induction, toluidine blue staining of hUSCs indicated the presence of polysaccharides and proteoglycans (Figure 3(a)).
The morphology of hUSCs in HA was similar to hUSCs in PBS at 0 h, 5 h, and 48 h after the seeding (Figure 4(a)).