Mineral composition of hypothermally induced ankylosis in rat molars.

Abstract

This study used Backscattered electron imaging (BSE) and X-ray Microanalysis to qualitatively and quantitatively investigate morphology and elemental composition of ankylotic adaptation in the periodontium following hypothermic insult to their maxillary first molars. This method has been shown in previous studies to induce aseptic root resorption along with ankylotic changes within the periodontal ligament (PDL). A secondary objective was to assess the pulpal changes that occurred concurrent with the changes in the periodontium. Twenty-eight eight week old Sprague Dawley rats were divided into four groups of seven animals corresponding to one of four observation periods i.e.: t₁= 7 days, t₂= 14 days, t₃= 21 days, t₄ = 28 days. At t=0 days, six animals in each group received a thermal insult as a continuous 20 minute application of dry ice (CO₂ at -81⁰C) to the crowns of their upper right maxillary molar. The untreated left molars were used as controls. The remaining rat within each group did not receive the dry ice. All rats were given two sequential bone labels, calcein 5mg/kg and alizarin red 30mg/kg, administered intraperitoneally 8 days apart. The timing of the labels was such that all rats were euthanased 2 days after the last label. Following sacrifice, the maxillae were dissected out, fixed in ethanol and embedded in methylmethacrylate. Ten microns thick, undecalcified maxillary first molar coronal sections through the furcation were obtained. For every 3 out of 10 sections: the first was left unstained and undecalcified; the second stained with Von Kossa/haematoxylin & eosin; and the third decalcified and stained with haematoxylin & eosin. Unstained sections were viewed under fluorescence, while transmitted light microscopy was used for the other sections. Following initial analysis, the unstained, undecalcified sections were de-coverslipped and carbon coated. These sections were investigated with scanning electron microscopy and Energy Dispersive X-ray Spectrometry (EDS). Quantitative spot analysis and element mapping was performed on alveolar bone, ankylotic areas, cementum and dentine. A linear mixed effects model was employed to investigate any interaction between the four tissues of interest. A focal pattern of ankylosis was observed at days 14 and 21 in three rats. No ankylosis was observed in the control teeth. SEM revealed a focal type of ankylosis with central nodules of mineralized tissue forming within the PDL. Bridging between bone and dentine occurred with fine trabeculae which extended from the central mineralized nodule. Bridging was progressive and was more extensive at day 21 compared to day 14. At day 28, no ankylosis was observed. EDS analysis revealed that the ankylotic tissue was composed of major constituents Calcium and Phosphorous along with trace elements of Mg and Na. This was comparable to the surrounding alveolar bone, cementum and dentine. There was no statistically significant difference in the Ca/P ratios, Mg when ankylotic material was compared to bone. There was a trend towards elevated Na levels in ankylosis but this was not statistically significant relative to bone. Mg in dentine was lower than for all the other tissues and Na was higher in dentine when compared to bone and cementum. In the pulp, hypothermic injury demonstrated alteration of the odontoblast layer, reduction in cellularity, vascular alterations and tertiary dentin formation. At the 28 day observation period, the cellular and vascular changes had returned to levels comparable to the control teeth, indicating successful pulpal healing and regeneration. Marked tertiary dentine deposition was also observed at days 14, 21 and 28. Pulp chambers were visibly smaller due to tertiary dentine formation, however no pulp necrosis was observed. Thus the aseptic root resorption model, using a continuous 20 minute application of dry ice, suggested the occurrence of reversible pulpal tissue alterations compatible with an inflammatory repair process. The observation of ankylosis initiating as centralised nodules within the PDL suggest that the origins may be a consequence of osteogenic potential from PDL stem cells. The null hypothesis that a single, prolonged thermal insult on a rat molar does not have an effect on mineralized tissue formation and that ankylotic tissue is similar to bone was rejected. ABSTRACT There is a presumption that the ankylotic region formed after periodontal ligament (PDL) injury represents an unremarkable bony repair process. Essentially, the injury triggers an osteogenic repair response and the ankylotic tissue merely represents alveolar bone. There is uncertainty whether the oestogenic event is predominantly osteoblast driven, cementoblast directed, or a consequence of osteogenic potential from PDL stem cells. In this study, twenty-eight Sprague Dawley rats were divided into four groups of six animals, corresponding to one of four observation periods, and received a thermal insult as a continuous 20 minute application of dry ice to the crowns of their upper right maxillary molar. The appearance of ankylotic tissues was examined using backscattered electron images using a scanning electron microscope (SEM) equipped with an Energy Dispersive X-ray Analyser (EDS). The Ca, P, and trace elements contents were determined by EDS from four different hard tissue regions: ankylosis; bone; dentine and cementum, and the Ca/P ratios were calculated. Ankylosis was observed at days 14 and 21 in 3 rats and was not seen at day 28. No ankylosis was observed in the control teeth. BSE imaging revealed a focal type of ankylosis with central nodules of mineralized tissue forming within the PDL. The morphological features of ankylotic tissue differed to that of alveolar bone and dentine. Bridging between bone and dentine occurred with fine trabeculae which extended from the central mineralized nodule. EDS analysis showed that the ankylotic tissue was composed of major constituents Ca and P along with trace elements of Mg and Na. This was comparable to the surrounding alveolar bone, cementum and dentine. There was no statistically significant difference in the Ca/P ratios, Mg, and Na between ankylotic material and bone. Statistically significant differences was evident in Mg content between ankylotic material compared to dentine and cementum. Na content was higher in dentine than in ankylotic material. The results of this study indicate that, histochemically, ankylotic material is similar to bone. However, the appearance of ankylotic material as centralised foci with a morphology different from bone suggest that ankylosis may originate from an osteoblastic phenotype originating within PDL.