Role Of TNF-αin The Excessive Death Of Chondrocytes Within Degraded Cartilage Induced By Abnormal Occlusion

One of the main pathological changes in osteoarthritis (OA) is the enhancedchondrocyte apoptosis or apoptosis-like programmed cell death. Pro-inflammatorycytokines, most notably tumor necrosis factor-α(TNF-α) and interleukin-1β(IL-1β),play a key role. TNF-αhas been established as a pivotal mediator in the progression ofcartilage degeneration. This study was designed to investigate the role of TNF-αin theexcessive death of chondrocyte within degraded cartilage of temporomandibular joint(TMJ) induced by abnormal occlusion.Methods:In vivo study, Fifty-four6-week-old female SD rats were randomly divided intoexperimental and control groups for3time point (2-,4-and8-week). In theexperimental groups, rats were applied with two metal tubes on left incisors in order to create abnormal occlusion. Histomorphological changes of their TMJ cartilages wereassessed by HE staining and the number of death of chondrocytes was tested bytransmission electron microscopy and TUNEL staining at each time point. Protein andmRNA levels of TNF-αand caspase3were investigated by immunohistochemistry andreal-time PCR, while the mRNA levels of caspase8, caspase9, IL-1β, IL-6and LC3were investigated by real-time PCR.In vitro study, chondrocytes from4-week-old female SD rats were exposed inTNF-α(10,50and100ng/ml) for24hours. They were then observed undertransmission electron microscopy and was detected by flow cytometry for cell death.The mRNA levels of caspase8, caspase9and caspase3were investigated by real-timePCR.Results:1. Cartilage degradation in experimental groups----Histological observationThe condylar cartilage in the control group was typically recognized as thefibrous, proliferative cell, hypertrophic chondrocyte and endochondral ossificationlayers. However, in the2-,4-and8-week experimental subgroups, OA-like lesionswere observed in the cartilage, characterized as reduced number and size ofchondrocytes in cartilage, pyknotic nuclei, a disarrangement of cellular disposition,condensed cytoplasm which did not fill the lacune, and even cell free areas.2. Increasing of TNF-αexpression within degraded cartilage in4-week subgroupThe TNF-α-positive cells were located mainly in the hypertrophic layer ofcondylar cartilage from the control groups. But in the experimental group of4-week,TNF-α-positive cells were observed not only in the hypertrophic layer, but also in theproliferative layer. In the experimental subgroups, the mRNA expression level ofTNF-αshowed no significant difference (P=0.244) in2-week, but up-regulated in4-week (P=0.025), and even down-regulated in8-week (P=0.023) compared to theirage-matched controls. The mRNA expression level of IL-1βwas not up-regulated until 8-week (P=0.043), and in the2-week experimental group it was down-regulated(P=0.01) as what IL-6was in the2-week group (P=0.006). The expression level ofIL-1βin4-week experimental group and that of IL-6in4-and8-week experimentalgroup were identical to their age-matched control subgroups (P>0.05). The proteinexpression level of TNF-αsignificantly increased in4-week (P<0.0001) but decreasedin8-week experimental group (P=0.008), while it was identical to their age-matchedcontrol in the2-week (P=0.616).3. Increasing of apoptotic chondrocytes in the experimental groupsIn all control subgroups, only few TUNEL-positive chondrocytes was noticed, butin the2-,4-and8-week experimental subgroups, abundant TUNEL-positive cells werefound (P<0.05) which were located mainly in the proliferative and hypertrophic layersof condylar cartilage. The mRNA expression level of LC3, which participates in theprocess of autophagy, was down-regulated in2-and4-week experimental subgroups(P<0.05), and identical to the control subgroup in8-week (P=0.105). The mRNAexpression of caspase8was up-regulated in4-and8-week experimental subgroups(P<0.05), but not in2-week experimental subgroup (P=0.242), and that of caspase9was up-regulated in all of the2-,4-and8-week experimental groups (P<0.05). ThemRNA expression of caspase3in each of the experimental group was identical to itsage-matched control group (P>0.05). However, the active-caspase3immunohistochemistrically positive cells that were located mainly in the hypertrophiclayers of condylar cartilage were significantly increased (P=0.026) in8-weekexperimental subgroup.4. Exogenous TNF-αincreased chondrocyte death in vitro via TNF-receptorpathwayAfter exposed to TNF-αat10,50and100ng/ml for24h, the FITC+/PI-viableapoptotic cell population increased from0.6%to1.6%,1.5%and1.3%, accordingly.The values of FITC+/PI+non-viable apoptotic cell population were increased which respectively as0.9%,1.6%,6.5%and9.6%when TNF-αdosage increased. Similarresults were found to that of FITC-/PI+necrotic cell population which were1.9%,3.6%,5.4%and9.3%, accordingly.When observed under electron microscope, chondrocytes were found polygonaland with large, uncondensed nucleus in controls groups. In the groups stimulated withTNF-α, typical chondrocytes undergoing apoptosis could be found and seemed moreobvious with dosage. These chondrocytes became rounded. Condensed chromatin wascompacted around the edge of the nucleus. In these chondrocytes, multiple vacuoles,swelling of rough endoplasmic reticulum and clustering of swollen mitochondria werevisible.The mRNA expression of caspase8was significantly up-regulated after TNF-αstimulation for24h at all the three concentration (P<0.05) but that of caspase9was not(P>0.05). The mRNA expression of caspase3was significantly up-regulated at the50and100ng/ml (P<0.05) but not at the10ng/ml (P=0.069) stimulating groups.Conclusion:In the osteoarthritic cartilage there was first an obvious increasing of cell deathwhich is demonstrated to be initiated by the biomechanical dental stimulation viamitochondrial pro-apoptotic signaling, and was secondarily promoted by the elevatedTNF-αthrough extrinsic apoptotic pathways. In this view, anti-TNF-αat verybeginning of OA should be helpful to diminish the inflammation mediated chondrocyteapoptosis, yet this prediction should be confirmed with evidences from in vivocause-effect investigations.