Absorbance or organization into ankylosis: a microarray analysis of haemarthrosis in a sheep model of temporomandibular joint trauma

Abstract

Background Traumatic haemarthrosis was hypothesized to be the etiology of temporomandibular (TMJ) ankylosis. Here, taking haematoma absorbance as a control, we aimed to reveal the molecular mechanisms involved in haematoma organizing into ankylosis using transcriptome microarray profiles. Material/methods Disk removal was performed to building haematoma absorbance (HA) in one side of TMJ, while removal of disk and articular fibrous layers was performed to induced TMJ ankylosis through haematoma organization (HO) in the contralateral side in a sheep model. Haematoma tissues harvested at days 1, 4 and 7 postoperatively were examined by histology, and analyzed by Affymetrix OviGene-1_0-ST microarrays. The DAVID were recruited to perform the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis for the different expression genes (DEGs). The DEGs were also typed into protein–protein interaction (PPI) networks to get the interaction data. Six significant genes screened from PPI analysis, were confirmed by real-time PCR. Results We found 268, 223 and 17 DEGs at least twofold at days 1, 4 and 7, respectively. At day 1, genes promoting collagen ossification (POSTN, BGN, LUM, SPARC), cell proliferation (TGF-β), and osteogenic differentiation of mesenchymal stem cells (BMP-2) were up-regulated in the HO side. At day 4, several genes involved in angiogenesis (KDR, FIT1, TEK) shower higher expression in the HO side. While HA was characterized by a continuous immune and inflammatory reaction. Conclusions Our results provide a comprehensive understanding of the role of haematoma in the onset and progress of TMJ ankylosis. The study will contribute to explaining why few injured TMJs ankylose and most do not from the molecular level.

Temporal gene expression profiling during early-stage traumatic temporomandibular joint bony ankylosis in a sheep model

“…Some genes that promote collagen matrix mineralisation, such as FMOD and LUM, reached peak expression during the cartilage formation phase , which is consistent with the findings of our previous study [ 17 ]. Furthermore, we found that the Wnt signaling pathway was significantly expressed at all time points in the TMJ ankylosis formation process (Table 1 and 2 ).…”

“…In this study, we identified significant BP and pathways via genome-wide transcriptional analysis of the entire fibrous–chondral phase of traumatic TMJ ankylosis in a sheep model. Days 1, 4, 7, 9, 11, 14 and 28 were selected as time points based on the findings of previous studies [ 8 , 9 , 17 ] on the early stages of TMJ ankylosis include inflammation, hematoma organisation and TMJ fibrous-chondral ankylosis formation. The timeline was confirmed using serial histological sections and gene expression profiles.…”

“…The animals were euthanized in the same manner as in our previous study [ 6 ], that is, euthanized with a lethal dose of pentobarbital sodium (120 mg/kg) through the external jugular vein, and animal death was confirmed by observing respiratory and heartbeat arrest and loss of pupillary light and nerve reflexes. The haematoma or newly generated ankylosed callus in the joint spaces was harvested as described in a previous study [ 17 ].

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“…The RNA preparation and microarray assay were performed by the CNKINGBIO Corporation (Beijing, China), as described in previous studies [ 6 , 17 ]: TRIzol reagent (Invitrogen Life Technologies, Carlsbad, USA) was used to extract total RNA, and a RNEasy small kit (Qiagen, Valencia, USA) was used to purify the extracted total RNA. Gene expression was analysed using the Affymetrix Ovine Gene 1.0 ST Array (Affymetrix, Santa Clara, CA, United States), which comprised over 22,047 known transcripts and expressed sequence tags.…”

“…Previous studies have demonstrated that several important genes that regulate bone formation and angiogenesis (such as vascular endothelial growth factor (VEGF), VEGF receptor 2 (VEGFR2), Ang1, CYR61, Wnt and BMP signaling) are involved in the pathogenesis of the disease at several specific time points [ 8 , 9 , 16 ]. Recently, we put forward a comprehensive elucidation of the role of haematoma in the onset of traumatic TMJ ankylosis through a comparison of differential gene transcription profiles between the groups of haematoma absorbance and haematoma organisation at Days 1 and 4 postoperatively in a sheep model [ 17 ]. However, to the best of our knowledge, there are no published reports on genome-wide temporal transcriptional analysis of the entire fibrous–chondral phase of traumatic TMJ ankylosis.…”