目的:诱导牙源性分化和肾小管牙本质形成在牙本质修复和牙齿再生中极为重要。BMP信号在牙本质发育和三级牙本质形成中起关键作用,而BMPR1A介导的信号传导如何影响表达Axin2(Axin2)的牙源性细胞的成牙本质分化和肾小管牙本质形成仍然未知。本研究旨在揭示继发性牙本质形成的细胞和分子机制。
方法:使用在出生后21(P21)收获的Axin2lacZ/+小鼠来定位Axin2+间充质细胞。制作Axin2CreERT2/+;R26RtdTomato/+小鼠和Axin2CreERT2/+;R26RDTA/+;R26RtdTomato/+小鼠,观察Axin2谱系细胞的时空分布规律及Axin2+细胞消融对牙本质发生的影响,分别。用Axin2CreERT2/+;Bmpr1afl/fl;R26RtdTomato/+(cKO)小鼠建立功能丧失模型,以研究BMP信号传导在调节Axin2+细胞中的作用。微型计算机断层扫描,组织学和免疫染色,和其他方法被用来检查生物功能,包括牙本质的形成,cKO小鼠的矿化和细胞分化。
结果:结果显示Axin2在P21时成牙本质细胞中丰富表达。谱系示踪试验证实了在继发性牙本质形成过程中成牙本质层和牙髓中Axin2谱系细胞的广泛分布(P23至P56),提示Axin2+细胞是原代成牙本质细胞的重要细胞来源。Axin2+细胞的消融(DTA小鼠)显著损害了继发性牙本质形成,其特征是牙本质厚度显著降低(对照平均值:54.11μm,DTA平均值:27.79μm,p=0.0101)。此外,在没有Bmpr1a的情况下,畸形的骨牙本质代替了肾小管次生牙本质,具有不规则的细胞形态,异常的细胞过程形成和缺乏细胞-细胞紧密连接。检测到成骨标志物如Runx2和DMP1的表达显著增加,而DSP表达以分散的方式观察到,表明cKO小鼠的牙源性细胞命运受损,无法产生肾小管牙本质。
结论:Axin2+细胞是原发性成牙本质细胞的关键群体,有助于肾小管继发性牙本质形成,BMP信号通路在维持Axin2+细胞牙源性命运中起着至关重要的作用。
OBJECTIVE: Inducing odontogenic differentiation and tubular dentine formation is extremely important in dentine repair and tooth regeneration. Bone morphogenic proteins (BMPs) signalling plays a critical role in dentine development and tertiary dentine formation, whilst how BMPR1A-mediated signalling affects odontoblastic differentiation of Axin2-expressing (Axin2+ ) odontogenic cells and tubular dentine formation remains largely unknown. This study aims to reveal the cellular and molecular mechanisms involved in the formation of secondary dentine.
METHODS: Axin2lacZ/+ mice harvested at post-natal 21 (P21) were used to map Axin2+ mesenchymal cells. Axin2CreERT2/+ ; R26RtdTomato/+ mice and Axin2CreERT2/+ ; R26RDTA/+ ; R26RtdTomato/+ mice were generated to observe the tempo-spatial distribution pattern of Axin2-lineage cells and the effect of ablation of Axin2+ cells on dentinogenesis, respectively. A loss-of-function model was established with Axin2CreERT2/+ ; Bmpr1afl/fl ; R26RtdTomato/+ (cKO) mice to study the role of BMP signalling in regulating Axin2+ cells. Micro-computed tomography, histologic and immunostainings, and other approaches were used to examine biological functions, including dentine formation, mineralization and cell differentiation in cKO mice.
RESULTS: The results showed rich expression of Axin2 in odontoblasts at P21. Lineage tracing assay confirmed the wide distribution of Axin2 lineage cells in odontoblast layer and dental pulp during secondary dentine formation (P23 to P56), suggesting that Axin2+ cells are important cell source of primary odontoblasts. Ablation of Axin2+ cells (DTA mice) significantly impaired secondary dentine formation characterized with notably reduced dentine thickness (Mean of control: 54.11 μm, Mean of DTA: 27.79 μm, p = .0101). Furthermore, malformed osteo-dentine replaced the tubular secondary dentine in the absence of Bmpr1a with irregular cell morphology, abnormal cellular process formation and lack of cell-cell tight conjunction. Remarkably increased expression of osteogenic markers like Runx2 and DMP1 was detected, whilst DSP expression was observed in a dispersed manner, indicating an impaired odontogenic cell fate and failure in producing tubular dentine in cKO mice.
CONCLUSIONS: Axin2+ cells are a critical population of primary odontoblasts which contribute to tubular secondary dentine formation, and BMP signalling pathway plays a vital role in maintaining the odontogenic fate of Axin2+ cells.