Objective    To culture subchondral bone osteoblasts and to study the biological characteristic differences between the osteoblasts in sclerotic area and non-sclerotic area in osteoarthritis (OA) patients.     Methods    We collected the bone tissue of tibial plateaus resected from 7 OA patients (5 men and 2 women)

with an average age of 68 years (55-79)

who received total knee replacement between October 2009 and March 2010 in our hospital. Subchondral bone osteoblasts were obtained from the sclerotic area and non-sclerotic area of the tibial plateaus through type-Ⅰ collagenase digestion and adherent culture. The appearances of the osteoblasts were observed under a light microscope. Immunohistochemistry

NBT/BCIP staining

alizarin red staining

and oil red O staining were performed on the osteoblasts to detect type-I collagen (COL1)

alkaline phosphate (ALP)

mineralized nodules

and fat

respectively. Real-time RT-PCR was performed to evaluate the mRNA expression levels of ALP

osteocalcin (OCN)

transforming growth factor-β1 (TGF-β1)

insulin-like growth factor-1 (IGF-1)

COL1A1

COL1A2

matrix metalloproteinase-13 (MMP-13)

osteopontin (OPN)

tumor necrosis factor-α (TNF-α)

IL-6

and IL-8 of the osteoblasts in the sclerotic area and non-sclerotic area. Alizarin red staining was performed to compare the mineralization degrees of the osteoblasts in the sclerotic area and non-sclerotic area.     Results    The growth of subchondral bone osteoblasts could be observed under the light microscope. COL1

ALP

and mineralized nodules were detected in the osteoblasts

while no fat granules were found. The alizarin red staining showed that the subchondral bone osteoblasts in the

sclerotic area had a lower mineralization degree than those in the non-sclerotic area[（0.92±0.06）

（1.32±0.15）

P0.01

]

. Compared with those in the non-sclerotic area

the osteoblasts in the sclerotic area had significantly higher mRNA expression levels of OPN[（2.62±1.47）×10

-2

（9.63±3.37）×10

-4

（P0.05）

]

OCN[（6.04±6.27）×10

-6

（4.84±2.18）×10

-6

P0.05

]

ALP[（73.62±15.75）×10

-4

（3.10±0.33）×10

-4

P0.01

]

IGF-1[（14.84±1.23）×10

-4

（6.33±2.80）×10

-4

P0.01

]

IL-6[（8.24±1.05）×10

-4

（2.64±0.37）×10

-4

P0.01

]

IL-8[（14.68±6.97）×10

-6

（2.60±0.89)×10

-6

P0.01

]

MMP-13[（1.50±0.11）×10

-3

（1.23±0.10）×10-3

P0.05

]

TGFβ1[（4.41±0.42）×10

-3

（2.96±0.36）×10

-3

P0.01

]

and COL1A1[（55.44±21.61）×10

-2

（9.17±3.30）×10

-2

P0.01

]

while no significant differences(P0.05) were found in terms of the mRNA expression levels of TNF-α[（5.62±2.03）×10

-6

（5.62±3.146）×10

-6

]

and COL1A2[0.64±0.07

0.46±0.12

]

.     Conclusion    Type-I collagenase digestion in conjugation with adherent culture is an effective means of culturing subchondral bone osteoblasts. The biological characteristic changes of the subchondral bone osteoblasts in sclerotic area are important pathological characteristics of OA patients. A further research on these changes and their causes will provide a great help for the research and therapy of OA.