A modified glass ionomer cement to mediate dentine repair

ObjectivesGlass ionomer cements (GIC) can be used to protect dentine following caries removal. However, GIC have little biological activity on biological repair processes, which means that neo-dentine formation remains reliant on limited endogenous regenerative processes. Wnt/β-catenin signalling is known to play a central role in stimulating tertiary dentine formation following tooth damage and can be stimulated by a range of glycogen synthase kinase (GSK3) antagonists, including lithium ions.MethodsHere, we created lithium-containing bioactive glass (BG) by substituting lithium for sodium ions in 45S5 BG. We then replaced between 10 and 40% of the powder phase of a commercial GIC with the lithium-substituted BG to create a range of formulations of ‘LithGlassGIC’. In vitro physical properties of the resulting glasses were characterised and their ability to stimulate reactionary dentine formation in mouse molars in vivo was tested.ResultsLithium release from LithGlassGIC increased with increasing lithium content in the cement. In common with unmodified commercial GIC, all formations of LithGlassGIC showed in vitro toxicity when measured using an indirect cell culture assay based on ISO10993:5, precluding direct pulp contact. However, in a murine non-exposed pulp model of tooth damage, LithGlassGIC quickly released lithium ions, which could be transiently detected in the saliva and blood. LithGlassGIC also enhanced the formation of tertiary dentine, resulting in a thickening of the dentine at the damage site that restored lost dentine volume. Dentine regeneration was likely mediated by upregulation of Wnt/β-catenin activity, as LithGlassGIC placed in TCF/Lef:H2B-GFP reporter mice showed enhanced GFP activity.SignificanceWe conclude that LithGlassGIC acts as a biological restorative material that promotes tertiary dentine formation and restores tooth structure.     

Separation of odontoblast Ca2+-ATPase and alkaline phosphatase

Summary Ca²⁺-ATPase activity was solubilized, partly purified, and separated from nonspecific alkaline phosphatase activity (APase) of dentinogenically active rat incisor odontoblasts. Attempts were made to extract the enzymes by various agents, such as Triton X-100, deoxycholate, butanol, EDTA, and buffers of decreasing ionic strength. Solubilization by butanol followed by extraction with low concentrations of EDTA proved to be most effective. Purification and separation were done by molecular sieve chromatography. Ca²⁺-ATPase showed no activity againstp-nitrophenyl phosphate (p-NPP) or inorganic pyrophosphate (PP_i) and was unaffected by R 8231 [(±)-6(m-bromophenyl)-5,6-dihydroimidazo(2,1-b)thiazole oxalate]. It was activated by Ca²⁺ and Mg²⁺ ions in equimolar concentrations with the substrate. The enzyme was rapidly inactivated in the solubilized state. An apparent molecular weight of about 18,000 was obtained from molecular sieve data. APase, showing activity against ATP, PP_i, andp-NPP, was virtually totally inhibited by R 8231. It was activated by Mg²⁺ ions but slightly reduced in activity by Ca²⁺ ions. It had an apparent mol. wt. of 79,000. The results provide direct evidence for earlier suggestions of the existence in hard tissue forming cells of two phosphatases active at alkaline pH.     

Deposition of calcium phosphate into dentin and enamel as shown by radioautography of sections of incisor teeth following injection of45Ca into rats

To find out whether the calcium phosphate of dentin and enamel is elaborated in the cells prior to passing into the organic matrix or is initially deposited into the matrix, rats were injected intravenously with⁴⁵Ca and killed 30 sec or 5 min later by glutaraldehyde perfusion; semithin sections of the undecalcified incisor teeth were then radioautographed for detection of the incorporated⁴⁵Ca. Rat incisor teeth were selected since there is evidence that the calcium they take up is stable; that is, not subject to significant loss by exchange or by other physiocochemical processes. Whendentin is examined after⁴⁵Ca injection, the maximum radioautographic reaction is observed next to the junction with predentin and a gradual decrease up to the dentin enamel border. No radioactivity is detected in odontoblasts. These observations are interpreted as indicating that the initial site of calcium phosphate deposition is in the matrix of dentin. In theenamel, the radioautographic reaction is spread fairly uniformly throughout the matrix, with a weak reaction over ameloblasts attributed to radiation scatter. The interpretation is again that calcium phosphate is deposited into the matrix; and, furthermore, that this deposition begins as soon as the matrix is laid down and continues at about the same rate up to an advanced stage of mineralization.     

Calbindin D-28k distribution in odontoblasts underneath tertiary dentine in human carious teeth

OBJECTIVE: The aim of this study was to examine the calbindin D-28k immunoreactivity in carious teeth to know whether this protein may have a function in tertiary dentine formation. METHODS: Human extracted teeth with or without carious lesions were immersion-fixed with Zamboni fixative, demineralized in 4.13% EDTA solution (pH 7.4), frozen-sectioned, and processed for calbindin immunoreactivity and hematoxylin-eosin stain. The intensity of the immunostaining was evaluated by quantitative densitometry. RESULTS: In intact teeth, numerous odontoblasts were aligned underneath the secondary dentine and their cell bodies showed the immunoreactivity. In carious teeth, tertiary dentine had poor- or rich tubular patterns under the carious lesion. Underneath the tubule-poor tertiary dentine, distinct odontoblasts could not be seen at the central site. However, some cells with a flat appearance were located at this site and were immunonegative for calbindin D-28k. On the other hand, columnar odontoblasts were seen at the peripheral site, and their cell bodies and processes showed strong immunoreactivity. Underneath the tubule-rich tertiary dentine, columnar odontoblasts were abundantly distributed, and the strong immunoreactivity was observed in their cell bodies and processes. The immunoreactivity in odontoblasts underneath the tertiary dentine with poor or rich tubular pattern was more intense than that for the secondary dentine in intact teeth (P<0.05). On the other hand, the intensity of the immunoreactivity in odontoblasts was similar underneath the secondary dentine in intact and carious teeth. CONCLUSIONS: The present study demonstrated that calbidin D-28k was actively synthesised by odontoblasts under the carious lesion. These findings may suggest that this protein plays an important role in the tertiary dentine formation.     

Exploration of the role of the subodontoblastic layer in odontoblast-like cell differentiation after tooth drilling using Nestin-enhanced green fluorescent protein transgenic mice

ObjectivesOriginal odontoblasts and regenerated odontoblast-like cells (OBLCs) may differently regulate Nestin expression. This study aimed to investigate the role of the subodontoblastic layer (SOBL) using green fluorescent protein (GFP) reactivity in the process of OBLC differentiation after tooth drilling in Nestin-enhanced GFP transgenic mice.MethodsA groove-shaped cavity was prepared on the mesial surface of the maxillary first molars of 5- or 6-week-old mice under deep anesthesia. Immunohistochemical staining for Nestin and GFP and Nestin in situ hybridization were conducted on the sections obtained at 1–14 days postoperative.ResultsOdontoblasts showed intense endogenous Nestin protein and mRNA expression, whereas the coronal SOBL cells showed a Nestin-GFP–positive reaction in the control groups. The injured odontoblasts had significantly decreased Nestin immunoreactivity as well as decreased expression of Nestin mRNA 1–2 days after the injury; subsequently, newly differentiated OBLCs were arranged along the pulp–dentin border, with significantly increased Nestin expression as well as increased expression of Nestin mRNA on days 3–5 to form reparative dentin. Nestin-GFP–positive cells at the pulp–dentin border significantly increased in number on days 1 and 2. GFP(+)/Nestin(+) and GFP(?)/Nestin(+) cells were intermingled in the newly differentiated OBLCs.ConclusionsThe commitment of Nestin-GFP–positive cells into Nestin-positive OBLCs suggests that the restriction of endogenous Nestin protein and mRNA expression in the static SOBL cells was removed by exogenous stimuli, resulting in their migration along the pulp–dentin border and their differentiation into OBLCs.     

Removal of the apical one-third of the root improves the fixation process of the dental pulp in teeth

The fixation process in histology is an important process that will eventually determine the quality of the histology slides. Fixation of the dental pulp involves the entry of the fixative agent into the dental pulp through the root canals that are found at the apical part of the root. The objective of this study was to evaluate the effectiveness of four methods of tooth preparation (teeth cut longitudinally or cervically, removal of apical third of root and whole uncut teeth) on the fixation process of the dental pulp for producing high quality histology slides. Among the four methods of tooth preparation, removal of the apical one-third of the root produced high quality histology slides as fixation of the dental pulp was improved and hence, preservation of the pulp’s architecture and contents were achieved. It is concluded that removal of the apical one-third of the root improves the fixation process of the dental pulp in teeth.     

纤维粘连蛋白及骨形成蛋白-2、4在牙胚发育中的表达研究

目的 观察纤维粘连蛋白(fibronectin,FN)和骨形成蛋白-2、4(BMP-2、4)在大鼠牙胚发育中的表达,探讨其在成牙本质细胞分化及成熟过程的时空表达关系,为明确牙齿发育的分子调控网络提供理论和实验基础.方法 获取SD大鼠蕾状期和钟状期第一磨牙牙胚,通过免疫组化LsAB法观察FN和BMP-2、4的表达差异及相互关系.结果 FN在蕾状期和钟状期都有表达,蕾状期主要分布在牙上皮、牙间质,周围非牙间质细胞为阴性,钟状期主要分布在成牙本质细胞分化活跃的区域和分化中的内釉上皮细胞.钟状期BMP-2、4的表达较强,主要分布在成牙本质细胞分化活跃的区域. 结论 BMP-2、4和FN在牙齿发育的蕾状期和钟状期均有表达,但表达的阳性分布区不同.     

Peripherin- and CGRP-immunoreactive nerve fibers in rat molars have different locations and developmental timing

Developing rat molars gain mature sensitivity to electric stimulation at 4-5 weeks after eruption, but the related mechanisms are incompletely understood. Preliminary studies showed weak co-localization of calcitonin gene-related peptide (CGRP) immunoreactivity (IR) with peripherin (PER) or neurofilament protein (NF) in rat molar nerve fibers, while the latter two co-localized extensively. OBJECTIVE: Our goal was to compare timing and location of PER-IR and CGRP-IR innervation in rat first molars during tooth maturation. METHODS: We used single and double immunocytochemistry to study molars of rats aged 10 days to 1 year. Neural patterns were compared with odontoblast maturation stages, dentinogenesis, formation of cell-free and cell-rich zones, and root closure. RESULTS: Spatial and temporal patterns showed that most CGRP-IR and PER-IR have different terminal domains in teeth. PER-IR fibers were well established among immature odontoblasts prior to tooth eruption, but CGRP-IR fibers were absent. Two weeks after eruption of first molars, many CGRP-IR beaded fibers entered dentin, the larger PER-IR fibers began shifting away from odontoblasts towards the pulp, and the symmetrical PER-IR pulpal pattern was being established. The CGRP-IR fibers continued to increase their asymmetric dentinal innervation until root growth was completed, during which time odontoblasts matured, the cell-free and cell-rich zones appeared, and roots closed. CONCLUSIONS: Sensory maturation of rat molars coincides with closed root apices, extensive innervation of dentin by CGRP-IR nerve fibers, and the appearance of the mature avascular odontoblast layer next to cell-free and cell-rich zones in the pulp horns.     

c-jun原癌基因在鼠牙胚中的表达

目的 通过观察ｃ－ｊｕｎ原癌基因在鼠牙胚中的表达,探讨成牙本质细胞分化的转录调节。方法 利用免疫组化方法检测ｃ－ｊｕｎ原癌基因在鼠牙胚中的表达。结果 ｃ－ｊｕｎ原癌基因在小鼠牙胚的蕾状期、帽状期呈阴性表达,在钟状期牙胚的成牙本质细胞中呈阳性表达。结论 ｃ－ｊｕｎ可能参与了成牙本质细胞分化的转录调节,并且可能成为牙本质细胞的标志分子之一。     

Complex cellular responses to tooth wear in rodent molar

The arrangement and roles of the odontoblast and its process in sensing and responding to injuries such as tooth wear are incompletely understood. Evidence is presented that dentine exposure by tooth wear triggers structural and functional changes that aim to maintain tooth integrity.Mandibular first molars from freshly culled 8 week Wistar rats were prepared for light microscopy ground-sections (n = 6), or fixed in 4% paraformaldehyde, decalcified in 17% EDTA, sectioned and stained with antibodies to cyto-skeletal proteins (vimentin (vim), α-tubulin (tub) and α-actin), cellular homeostatic elements (sodium potassium ATPase (NaK-ATPase) and sodium hydrogen exchanger (NHE-1)), and sensory nerve fibres (CGRP) (n = 10) for fluorescence microscopy of worn and unworn regions of the mesial cusp.Immunoreactivity (IR) to vim, actin, NaK-ATPase and CGRP was confined to the pulpal third of odontoblast processes (OPs). IR to tub and nhe-1 was expressed by OPs in full dentine thickness. In areas associated with dentine exposure, the tubules contained no OPs. In regions with intact dentine, odontoblasts were arranged in a single cell layer and easily distinguished from the sub-odontoblast cells. In regions with open tubules, the odontoblasts were in stratified or pseudo-stratified in arrangement.Differences in structural antibody expression suggest a previously unreported heterogeneity of the odontoblast population and variations in different regions of the OP. This combined with differences in OPs extension and pulp cellular arrangement in worn and unworn regions suggests active and dynamic cellular responses to the opening of dentinal tubules by tooth wear.