Unilateral anterior crossbite induces aberrant mineral deposition in degenerative temporomandibular cartilage in rats

Abstract

Purpose: Rrecently, we have developed a unilateral anterior crossbite (UAC) procedure that could induce osteoarthritic (OA)-like changes in the temporomandibular joints (TMJs) cartilage of rats and mice. Herein, we employed imaging, histological, and molecular biology techniques, and also in vitra assay, to investigate whether there is mineral deposition in the UAC induced OA cartilage, what the composition the mineral deposition has, and what a role of the minerals may take in OA cartilage. Methods: Spraguee-Dawley rats were subjected to a unilateral anterior crossbite (UAC) procedure. Histology, electron microscopy, and energy dispersive spectroscopy (EDS) were used to examine cartilage matrix structures and compositions of mineral deposit in TMJs. Protein and/or RNA expression of modulators of phenotypic markers, mineralization and matrix degradation was analyzed by immunohistochemistry and/or real-time PCR. Synthetic BCP and CPPD crystals were used to stimulate chondrogenic ATDC5 cells for their impact on activities of cellular differentiation and secretion. Results: The proteoglycan content in UAC cartilage began to decrease as early as 2 weeks post-operation and nearly absent at 20 weeks post-UAC without significant inflammatory response. The collagen arrangement was obviously interrupted and the cartialge thickness reduced from 8 weeks but the thickness of the calcified zone was increased. The expression of Col-II, Col-X, and aggrecan was decreased. The control group chondrocytesunder transmission electron microscopy (TEM) were found in well-delimited lacunae that were surrounded by the tightly-organized collagen fibers. In UAC cartilage, however, collagen fibers were disorganized and fragmented with increasing inter-fibers spaces and were worsened with time. Mineral deposition was observed around the hypertrophic chondrocytes, beginning 2 weeks after the UAC procedure. Those peri-cellular minerals were contained in abundant matrix vesicles (with diameters ranging from 50-200 nm) that were located in close proximity to the cell member of hypertrophic chondrocytes, supporting the cellular origin of the vesicles. Immunohistochemistry and/or real-time PCR analyses demonstrated biphasic changes in the expression of MGP mRNA in TMJ joints of UAC rat, which increased at 2 and 4 weeks post-operation and decreased after 8 weeks post-operation when compared to age-matched controls. The expression of CD73 and Ank was significantly reduced in TMJ cartilage of UAC group at all time points. The expression of Npp1 was significantly reduced in UAC cartilage at 2 and 4 weeks post-operation. In contrast to the expression of anti-mineralization molecules, the expression of Tnap, which hydrolyzes PPi into Pi to promote the formation of BCP crystals, was up-regulated in the UAC groups at 4, 8, and 12 weeks post-operation, and this effect waned at 20 weeks post-operation. The expression of matrix metalloproteinase, Mmp13, which cleaves type II collagen, was also up-regulated in the UAC groups in the similar time-dependent manner as seen in the expression of Tnap. Using EDS technology we confirmed an increase in calcium (Ca) and phosphorus (P) content in UAC cartilage vs their age-matched controls, beginning at 2 weeks post-operation. Only one of four joints tested in 12- and 20-week UAC group was noticed an isolated mineral which was located closing to the deep zonal mineral deposition. They were rich in calcium and phosphorus, but had a low carbon content. Their averaged Ca/P ratio was 1.42 (ranging from 1.38 to 1.48) and 1.44 (range from 1.39 to 1.49) at 12- and 20-weeks UAC group, respectively, closely resemble to those of octacalcium phosphate (Ca/P=1.33) and tricalcium phosphate (Ca/P=1.50), two subtypes of BCP crystals. We then exposed ATDC5 cells to the synthetic BCP crystals. We found that BCP crystals (50μg/ml) reduced Col-II, Col-X and aggrecan expression, but upregulated the expression of Adamts5, Mmp3 and Mmp9, however, only transiently stimulated Tnf-α and Il-1β expression. Conclusions: The present data indicated that mechanical stimuli by UAC enhance mineral accumulation of BCP-like minerals in the cartilage matrix of TMJ condyle at deep layer. The UAC induced aberrant mineralization deposition of BCP-like minerals can stimulate matrix degradation via promoting cartilage-degrading enzymes, and may contribute, at least in part, to the thinning of articular cartilage by expansion of the calcified cartilage, facilitate osteoarthritis progression.