| Our previous studies show that unilateral anterior crossbite?UAC? can lead to temporomandibular joint osteoarthritis?TMJOA? and some degenerative changes had occurred in mandibular condyle cartilage of rat, such as the reduction of cartilage thickness and the degradation of cartilage matrix, etc. Therefore, there may have a abnormal differentiation of chondrocyte in UAC cartilage. The literatures reported that Ca MK? was expressed in chondrocyte of growth plate cartilage. Ca MK?was one kind of multifunctional protein kinase and which was allosteric activated by Ca2+ /Ca M complex. After the holoenzyme was allosteric activated, the active center and the sites of substrate binding of the enzyme would be exposed, thus it own the ability to phosphorylate substrates. Indian hedgehog?Ihh? is one most important downstream signal molecules of Ca MK?. The chondrocyte will be promoted from proliferation to differentiation through activating Ihh by activated Ca MK?. In growth plate cartilage, there has a negative feedback adjustment between Ihh and PTHr P. The enhancement of activity of Ihh can promote PTHr P synthesis and secretion by perichondrium and the chondrocyte which at the early stage of differentiation. PTHr P reduces the number of chondrocyte which can secrete Ihh through controlling the differentiation of the chondrocyte at early stage of differentiation. This is how the negative feedback adjustment Ihh function work by PTHr P. This study is about characteristics of Ca MK? expression changes influence by UAC stimulation and the relationship between Ca MK?and Ihh-PTHr P signal axis, by using TMJ local injected Ca MK? inhibitor KN93 method to observe the influence of condylar cartilage by Ca MK?and discuss the role of Ca MK?in degenerative condylar cartilage of rat caused by UAC stimulation.Objective: To investigate the changes of Ca MK?expression in degenerative condylar cartilage of rat and the influences of condylar cartilage after Ca MK?was inhibited in local TMJ.Methods: Ninety-six female rats which about 6-week-old were randomly and equally divided into Control group?Con?, Experimental group?UAC?, Experimental injection group?UAC+KN93? and the Control injection group?Con+KN93?. The left incisors of the UAC group rats was cemented the metal prosthesis to create the unilateral anterior crossbite relation, the injection group rats was injected KN93 at 4th or 8th week, administrating injection every other day for 4 weeks, the control rats without any treatment. At the end of 8th or 12 th week after UAC stimulation, the animals were sacrificed.Hematoxylin-eosin staining and the safranin O/fast green?SO? staining and the von kossa staining were carried out for studying the morphological changes of the condylar cartilage, super microstructural changes of chondrocyte was observed by transmission electron microscopy?TEM?, immunohistochemical?IHC? staining used to observe the expression and distribution of Ihh and PTHP, and Real-time q PCR analysis used to detect the expression of Ca MK?, Ihh/Patched, PTHr P/PPR and differertiation, proliferation and calcification related genes in condylar cartilages, the protein expression changes of phosphorylation Ca MK? and Ihh and PTHP were detected by Western Blot. Results 1. Compared with the control groups, at the two time point, the thickness of the full cartilage and the hypertrophic layer of rat condylar cartilage was decreased, the density of chondrocyte was decelined, and the positive areas of SO staining was also decreased.The number of hypertrophic chondrocyte which surrounded by calcium salt deposited was widely increased in the shallow layer of hypertrophic layer of cartilage, the number of aging and apoptosis hypertrophic chondrocyte were increased in rat cartilage of UAC groups. The degeneration was going to get worse with the time of UAC continuous stimulation. The m RNA and protein expression levels of Ihh and that of Ca MK?in UAC condylar cartilage was up-regulation at 8th or 12 th week, the m RNA and protein expression levels of patched the receptor of Ihh had no changes at 8th week, but at 12 th week it was down-regulation, the m RNA and protein expression levels of PTHr P was increased at 8th week and was decreased at 12 th week, the m RNA and protein expression levels of PPR the receptor of PTHr P was down-regulation at the two time point. In UAC condylar cartilage, the m RNA expression levels of the proliferation related molecule Pcna and the cartilage matrix synthesis related molecules Col2a1, Aggrecan were down-regulation at at 8th or 12 th week, the m RNA expression levels of the Cartilage matrix degradation related enzymes as Admnts5, MMP-13 and the differentiation related molecules as Ocn, Alp were up-regulation at 8th week and were down-regulation at 12 th week.2. Compared with the same period control groups, in control injection group, the thickness of full cartilage and the hypertrophic layer and the positive areas of SO staining of rat condylar cartilage were decreased at the two time point. The genes and protein expression levels of Ca MK? and Ihh-PTHr P signaling pathway related molecules were down-regulation, the differentiation related genes as Alp, Ocn and the cartilage matrix related genes as Aggrecan, Adamts5, MMP-13 were statistically significant decreased. The m RNA expression levels of Col2?1 and Pcna had no statistically significant changes in condylar cartilage of the control injection groups at the 8th or 12 th, though the m RNA expression levels of Col2?1 showed a trend of descending and that of Pcna showed a trend of rising.3. Compared to the same period UAC groups, in UAC injection groups, the thickness of the full cartilage and the hypertrophic layer of rat condylar cartilage were increased, the positive areas of SO staining in cartilage were also increased at the two time point. The m RNA and protein expression levels of Ca MK? and Ihh/Patched were down-regulation at the two time point. The expression levels of PTHr P m RNA was decreased at 8th week and that was up-regulation at 12 th week. The m RNA expression levels of the cartilage matrix related genes as Col2?1, Aggrecan and the proliferation related gene Pcna were up-regulation at 8th and 12 th week; The Cartilage matrix degradation related genes as Adamts5, MMP-13 and the differentiation related genes as Alp, Ocn m RNA expression levels were down-regulation at 8th week and that had no changes at 12 th week.4. Though there had no influence about PPR expression levels, it had reduced the number of hypertrophic chondrocyte which arrounding with calcium salt deposit and the number of aging or apoptosis hypertrophic chondrocyte in KN93 injection groups at the two time point.Conclusion 1. UAC stimulation can lead to condylar cartilage degeneration, it shows that the chondrocyte differentiation was increased, and it proliferation was declined, the number of apoptosis chondrocyte was increased, and the density of chondrocyte was decreased, and the cartilage matrix was lost, the thickness of cartilage was decreased, and the expression levels of differentiation related molecules such as Ca MK?and Ihh were up-regulation. But with the degeneration of condylar cartilage was get worse that the capacity of chondrocyte expressing such related molecules were falling down. 2. The gene transcription and the protein phosphorylation of Ca MK?were be inhibited by KN93 injection in rat cartilage, the expression levels of molecules which related to differentiation, calcification and the matrix catabolism were down-regulation, and upregulated the expression levels of molecules which related proliferation and matrix synthesis metabolism, so that increased the thickness of the condylar cartilage to improve the degeneration induced by UAC stimulation.The treatment effect depended on the numbers and the conditions of the survival chondrocyte in the degenerative condylar cartilage, so it mainly acted on the early phase of OA. 3. The thickness of full cartilage and hypertrophic layer and the calcified layer and the positive areas of cartilage matrix were reduced in normal condylar cartilage when KN93 was injection, it reflects that there was a toxic and side effect about KN93 which can intervene chondrocyte normal differentiation, prompting that Ca MK?is necessary for the maintenance of normal cartilage. |
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