Insights into the structure and composition of the peritubular dentin organic matrix and the lamina limitans

Dentin is a mineralized dental tissue underlying the outer enamel that has a peculiar micro morphology. It is composed of micrometer sized tubules that are surrounded by a highly mineralized structure, called peritubular dentin (PTD), and embedded in a collagen-rich matrix, named intertubular dentin. The PTD has been thought to be composed of a highly mineralized collagen-free organic matrix with unknown composition. Here we tested the hypothesis that proteoglycans and glycosaminoglycans, two important organic structural features found in dentin, are key participants in the microstructure and composition of the PTD. To test this hypothesis dentin blocks were demineralized with 10 vol% citric acid for 2 min and either digested with 1mg/ml TPCK-treated trypsin with 0.2 ammonium bicarbonate at pH 7.9 (TRY) or 0.1 U/mL C-ABC with 50mM Tris, 60mM sodium acetate and 0.02% bovine serum albumin at pH 8.0 (C-ABC). TRY is known to cleave the protein core of dentin proteoglycans, whereas C-ABC is expected to selectively remove glycosaminoglycans. All specimens were digested for 48 h in 37°C, dehydrated in ascending grades of acetone, immersed in HMDS, platinum coated and imaged using an FE-SEM. Images of demineralized dentin revealed a meshwork of noncollagenous fibrils protruding towards the tubule lumen following removal of the peritubular mineral and confirmed the lack of collagen in the peritubular matrix. Further, images revealed that the peritubular organic network originates from a sheet-like membrane covering the entire visible length of tubule, called lamina limitans. Confirming our initial hypothesis, after the digestion with C-ABC the organic network appeared to vanish, while the lamina limitans was preserved. This suggests that glycosaminoglycans are the main component of the PTD organic network. Following digestion with TRY, both the organic network and the lamina limitans disappeared, thus suggesting that the lamina limitans may be primarily composed of proteoglycan protein cores. In summary, our results provide novel evidence that (1) PTD lacks collagen fibrils, (2) PTD contains an organic scaffold embedded with mineral and (3) the PTD organic matrix is manly composed of glycosaminoglycans, whereas the lamina limitans is primarily made of proteoglycans protein cores.