Effects Of Hypoxia On Expression Of HIF-1α, VEGF, Aggrecanase-1 And TIMP-3 In Rat Mandibular Condylar Chondrocytes In Vitro

Objectives To isolate, culture and identificate rat condylar chondrocytes and build the chemical hypoxic cells cultivating model induced by Cobalt chloride (CoCl2) in vitro. To investigate the expression of hypoxia inducible factor-lα(HIF-1α), vascular endothelial growth factor (VEGF), aggrecanase-1 and tissue inhibitor of metalloproteinase-3 (TIMP-3) in rat mandibular condylar chondrocytes cultured under hypoxic and normoxia conditions for different time periods, and explore the role of HIF-1α, VEGF, aggrecanase-1 and TIMP-3 in condylar growth.Methods (1)chondrocytes were obtained from the condylar of 3-week-old Sprague-Dawley rats by mechanical dissection and digestion with trypsin andâ…¡collagenase. Primary culture and subculture of chondrocytes were conducted. The morphological changes and growth feature were recorded under the inverted microscope each day. Generational observation and cell growth curve were carried out by cellular MTT method. The chondrocytes were detected by immunohistochemical analysis expression of typeâ…¡collage, and by toluidine blue under light microscope.(2)The chondrocytes were treated with different concentrations of CoCl₂ : 75μmol/L(A group), 125μmol/L(B group), 175μmol/L (C group) and 0μmol/L (the normoxia group) for 48 hours, respectively. Cell viability was determined by lactate dehydrogenase (LDH) assay. CoCl₂ was used as a chemical hypoxia-inducible reagent to mimic hypoxic microenvironment.(3)HIF-1α, VEGF, aggrecanase-1and TIMP-3 mRNA of chondrocytes were detected by real-time PCR at 12, 24 and 48 hours after initiation of hypoxia and normoxia conditions.Results (1)This isolating method of condylar cartilage can obtain 1×10⁴～4×10⁴ cells by each rat. The survival rates of chondrocytes were 95% by trypan blue staining. By toluidine blue staining, chondrocytes nucleus were dyed blue. The cytoplasm and extracellular matrix were dyed purple. Immunohistochemical analysis showed that the positive reaction expression of typeâ…¡collage was located in cytoplasm.(2)The released LDH activities elevated after 48 hours along with the increased concentrations of CoCl2 in the cell culture medium. There was statistically significantly difference of the released LDH activities between C group and the normoxic group (P<0.01). On the other hand, there were no significantly difference of the released LDH activities among B group, A group and the normoxic group (P >0.05). (3)Semiquantitative analysis revealed HIF-1α, VEGF, aggrecanase-1 and TIMP-3 mRNA were expression in condylar chondrocytes under normoxia conditions. Compared with normoxia conditions, HIF-1α(P<0.05)and VEGF(P<0.01)mRNA were increased obviously at 12h, 24h and 48h after hypoxia. There were statistically significantly difference of the expressions of HIF-1α(P<0.05)and VEGF(P<0.05) mRNA between 12h and 24h, 12h and 48h, 24h and 48h after hypoxia. After hypoxia, HIF-1αmRNA expression was gradually increased, while VEGF mRNA reached peak at 12h, and then decreased. However, they were higher than normoxia condition at all time points. On the other hand, there were no significantly difference of the expression of aggrecanase-1 and TIMP-3 mRNA between all time points of the hypoxia and normoxia conditions (P >0.05).Conclusions (1)This isolating method obtains a higher survival rate of cartilage cells.(2)The results of LDH indicate that the cells may suffer serious damage by 175μmol/L of CoCl2 treatment, and many literatures suggest that effect of hypoxia by 125μmol/L of CoCl2 is better than 75μmol/L. Hence, 125μmol/L is selected as the concentration of CoCl2. Using cobalt chloride to build the chemical hypoxic cells cultivating model is stable, and easy to operate.(3)The expressions of HIF-1αand VEGF mRNA are up-regulated under early hypoxia microenvironment in mandibular condylar chondrocytes. However, the chemical hypoxic cells cultivating model can not mimic sophisticated regulation of hypoxic microenvironment in vivo. It may not influence on the expressions of aggrecanase-1 and TIMP-3 mRNA efficiently. HIF-1αis increased with the time lasting, but VEGF is gradually decreased. It may play a significant role in cartilage vascularization and development.