壳聚糖促进牙釉质仿生矿化修复的研究进展

牙釉质的仿生矿化是口腔材料学关注的热点问题.壳聚糖是一种天然的阳离子多糖,它与牙釉质细胞外基质成分具有相似的化学结构和生物学行为.壳聚糖及其衍生物的优异生物相容性和聚电解质作用等使其成为一种非常合适的载体或有机模板.近年来,壳聚糖有机-无机复合材料已应用于牙釉质仿生矿化修复.该文就壳聚糖促进牙釉质仿生矿化原理及其在牙釉...     

牙本质和牙骨质的仿生修复与再生

牙体硬组织缺乏完善的自我修复的能力。在牙本质和牙骨质中,羟基磷灰石(hydroxyapatite,HA)可同时存在于胶原纤维内外。如何在胶原纤维存在的情况下,修复或再生出具有高度有序的定向排列的HA是牙本质和牙骨质修复和再生的难点。目前牙本质和牙骨质的仿生矿化主要从模仿其生物形成过程和其生理结构两个方面来进行,分为源于纤维矿化机制的仿生矿化体系和以HA为主要成分的仿生矿化体系。应用的材料有脱矿牙本质(demineralized dentin matrix,DDM)、煅烧牛骨羟基磷灰石(bovine hydroxyapatite,BHA)等天然材料和聚合物诱导液体前体(polymer-induced liquid precursor,PILP)、合成HA等合成材料。未来应通过将天然材料与合成材料相结合,应用再矿化溶液-HA释放磷酸钙离子仿生矿化的方式来促进牙本质和牙骨质的修复与再生。     

Klinischer Effekt biomimetischer Mineralisation bei Approximalkaries

Background

Caries still has a high prevalence even in western countries with preventive dental care. When treated mechanically carious teeth will be weakened, which in the long-term often results in tooth loss and substantial inherent costs. In recent years various remineralizing products have been introduced which are supposed to supplement fluorides which in turn promote remineralization. Moreover, specific invasive or minimally invasive options are available, such as proximal sealing and caries infiltration.

Methods

This article presents a new method of biomimetic mineralization based on self-assembling peptides (SAPs). Suspended in water the SAPs are applied onto the cleaned and etched enamel surface. From there the SAPs will diffuse into the lesion and trigger the formation of a bioactive scaffold, which in turn encourages natural repair through incorporation of calcium and phosphate ions from the saliva.

Results

According to interim results from a clinical study using biomimetic mineralization, three out of four proximal lesions were stabilized or remineralized. The new method may allow for the first time natural repair of demineralized enamel extending to the enamel-dentin junction.     

牙本质的仿生再矿化

近年来,在修复由龋病等原因引起的牙本质脱矿的研究中,牙本质仿生再矿化技术以其精确控制无定形矿物质前体在脱矿牙本质胶原纤维内有序沉积、形成的磷灰石晶体与天然矿化牙本质相似、自下而上的再矿化方式不依赖于籽晶存在等优点,逐渐成为这一领域的研究热点。本文回顾了脱矿牙本质再矿化的理念和实践的进展,并着重对牙本质仿生再矿化策略的相关研究进行综述,文献复习结果表明,传统的牙本质再矿化方法通常是脱矿牙本质与矿物质晶体的无序混合,这样矿化后的牙本质在形态特征和机械性能上均无法与天然矿化牙本质相媲美;而近年逐渐兴起的牙本质仿生再矿化技术则复现了天然矿化牙本质中矿物质在牙本质胶原纤维内迭序排列的结构特点,其微观结构、理化性能均得到极大提高,有望在树脂⁃牙本质粘结混合层和龋坏牙本质脱矿层的再矿化研究领域实现新的突破。目前牙本质仿生再矿化在临床应用上需要克服的技术障碍在于如何在再矿化过程中持续补充矿化所需的各种有效成份,并在缓慢释放各成份的同时保持母体材料的机械性能不变,研究者们已相继提出了三步法输送仿生再矿化原材料,以及预先制备聚合物稳定的矿化前体、再使用介孔硅纳米材料作为输送矿化成份的系统的构想,为牙本质仿生再矿化策略向临床应用的转化提供了初步体外实验基础。     

In vivo and in vitro study of the effects of chlorpromazine on tooth mineralization in rats and mice

The effects of chlorpromazine (CPZ) on tooth mineralization were examined using incisor dentine in adult rats and cultured tooth germs of mandibular first molars dissected from mouse embryos. CPZ (10, 50 and 250 mg/kg, s.c.) substantially inhibited dentine mineralization as evaluated by contact microradiographs. Plasma calcium and phosphorus concentrations were not decreased by CPZ (10 and 50 mg/kg). Physicochemical effects were not involved in the action of CPZ on the mineralization. In vitro experiments showed that CPZ (1 and 10 μM) inhibited mineralization and alkaline phosphatase (ALP) activity in the tooth germs. As CPZ has the properties of a calmodulin antagonist, the calmodulin antagonists W-7 and W-5 were also examined. Both inhibited mineralization and ALP activity in tooth germs; W-5 had less effect than W-7. These in vivo and in vitro findings suggest that CPZ inhibited cell-mediated mineralization in dentine without affecting the calciumdashregulating system and physicochemical mineral deposition. In addition, calmodulin could be involved in cell-mediated mineralization.     

煅烧牙粉影响人牙周膜干细胞体外矿化的自噬机制研究

目的 探讨自噬在煅烧牙粉调节牙周膜干细胞体外矿化中的作用,为牙周膜干细胞的定向诱导分化和牙周病的治疗提供参考。方法 选用成人完整的牙齿在300 ℃的条件下煅烧后,研磨成粉,与α-MEM混合制备成20 μg/mL牙粉条件培养基,与人牙周膜干细胞共培养。采用流式细胞技术检测其对牙周膜干细胞凋亡的影响,通过Western blot检测、免疫荧光染色、茜素红染色和ALP染色观察检测其对牙周膜干细胞自噬活性及体外矿化的影响。结果 流式细胞技术结果显示牙粉对牙周膜干细胞的凋亡无明显影响（P＞0.05）;Western blot结果显示煅烧牙粉显著上调人牙周膜干细胞的自噬相关蛋白Beclin1,ATG5的表达和LC3Ⅱ/LC3Ⅰ的比值,以及明显下调了P62的蛋白水平（P<0.05）;免疫荧光染色显示牙粉促进人牙周膜干细胞中LC3在胞质内点状聚集;茜素红染色显示牙粉促进牙周膜干细胞的体外矿化;ALP染色显示自噬活性抑制剂氯喹降低牙粉对牙周膜干细胞体外矿化的促进作用。结论 煅烧牙粉通过激活自噬调节牙周膜干细胞的体外矿化作用。     

可注射型热触发仿天然骨材料的基础研究

目的：试图研制可注射型热敏仿天然骨材料,测定其理化特性,并将其初步应用于体外根尖孔破坏的离体牙模型上。方法：采用逆向蒸发法制备载碱性磷酸酶（alkalinephosphatase,ALP）的脂质体,考察其包封率、活性率及相变温度。将载ALP、载钙离子（Ca^24）和磷酸盐（Pi）3种脂质体与β-甘油磷酸盐（β-glycerophosphate,β-GP）混合制备矿化液,观测矿物质的形态、元素分析及红外光谱曲线;将I型胶原与以上矿化液混合制备的复合材料应用于根尖孔破坏的离体牙根尖周围,根管充填并统计分析。结果：载ALP的脂质体平均包封率为25．1％,平均活性率为16．5％,相变温度为37．3～40．1℃,峰值为39．2℃;与未加ALP者相比,所得矿化物形态和Ca／Pi比与天然骨中矿化物更接近,红外光谱曲线表明其为羟基磷灰石;体外模型显示该复合材料可在一定程度上支托根充材料,预防超充。     

Long-term dentin remineralization by poly(amido amine) and rechargeable calcium phosphate nanocomposite after fluid challenges

Objective

Previous studies investigated short-term dentin remineralization; studies on long-term dentin remineralization after fluid challenges mimicking fluids in oral environment are lacking. The objective of this study was to develop a long-term remineralization method to via poly(amido amine) (PAMAM) and rechargeable composite containing nanoparticles of amorphous calcium phosphate (NACP) after fluid challenges for the first time.

Biomimetic transformation of polyphosphate microparticles during restoration of damaged teeth

Objective

In the present study, we investigated the fusion process between amorphous microparticles of the calcium salt of the physiological polymer comprising orthophosphate units, of inorganic polyphosphate (polyP), and enamel.

氯丙嗪对大鼠牙齿矿化抑制作用的实验研究

目的：探讨氯丙嗪(chlorpromazine,CPZ)对大鼠切牙硬组织矿化的影响。方法;采用酶联反应仪及高压液相色谱仪（HPLC）检测CPZ对培养不同时间牙胚ALP及Ca^2 的含量,利用显微放射照相技术观察CPZ对大鼠硬组织矿化的情况。结果：培养0-7d牙胚Ca^2 含量及ALP逐渐上升,培养7-10d的牙胚中,实验组Ca^2 含量及ALP活性的上升幅度显著减弱;CPZ剂量依赖性地抑制大鼠牙本质形成,并且对血浆中Ca、P无明显影响。结论：CPZ对大鼠牙齿矿化过程具有抑制作用。该作用是通过成牙本质细胞的钙调节系统实现的。     

Biological modification of tooth surface by laser-based apatite coating techniques

BackgroundDevelopment of new clinical regenerative procedures is needed for the reconstruction of the connective tissue attachment lost to periodontal disease. Apatite coating on the affected root surfaces could improve root surface biocompatibility and promote the reestablishment of connective tissue attachment.HighlightWe developed two novel techniques that use laser light for coating the tooth surface with apatite. In the laser-assisted biomimetic (LAB) process, a tooth substrate was placed in a supersaturated calcium phosphate solution and irradiated for 30 min with low-energy pulsed laser light. Due to the laser-assisted pseudo-biomineralization, a submicron-thick apatite film was created on the laser-irradiated tooth surface. Furthermore, we created a fluoride-incorporated apatite film on the tooth surface using the LAB process and demonstrated its antibacterial activity against Streptococcus mutans.In the laser-induced forward transfer with optical stamp (LIFTOP) process, a thin apatite film loaded with the cell-adhesion protein, fibronectin, was prepared beforehand as a raw material on the optical stamp (carbon- and polydimethylsiloxane-coated support) by a conventional biomimetic process. After irradiation with a single laser pulse, the film (microchip) was transferred onto a tooth substrate via laser ablation of the carbon sacrificial layer. The LIFTOP process requires only a short processing time and has a minimal heat effect on the film; thus, the film exhibits cell adhesion activity even after the LIFTOP process.ConclusionThe LAB and LIFTOP processes have the potential as novel tools for tooth surface modification in the treatment of periodontal disease.     

Biomimetic remineralization of dentin

Objectives

Remineralization of demineralized dentin is important for improving dentin bonding stability and controlling primary and secondary caries. Nevertheless, conventional dentin remineralization strategy is not suitable for remineralizing completely demineralized dentin within hybrid layers created by etch-and-rinse and moderately aggressive self-etch adhesive systems, or the superficial part of a caries-affected dentin lesion left behind after minimally invasive caries removal. Biomimetic remineralization represents a different approach to this problem by attempting to backfill the demineralized dentin collagen with liquid-like amorphous calcium phosphate nanoprecursor particles that are stabilized by biomimetic analogs of noncollagenous proteins.

Methods

This paper reviewed the changing concepts in calcium phosphate mineralization of fibrillar collagen, including the recently discovered, non-classical particle-based crystallization concept, formation of polymer-induced liquid-precursors (PILP), experimental collagen models for mineralization, and the need for using phosphate-containing biomimetic analogs for biomimetic mineralization of collagen. Published work on the remineralization of resin–dentin bonds and artificial caries-like lesions by various research groups was then reviewed. Finally, the problems and progress associated with the translation of a scientifically sound concept into a clinically applicable approach are discussed.

Results and significance

The particle-based biomimetic remineralization strategy based on the PILP process demonstrates great potential in remineralizing faulty hybrid layers or caries-like dentin. Based on this concept, research in the development of more clinically feasible dentin remineralization strategy, such as incorporating poly(anionic) acid-stabilized amorphous calcium phosphate nanoprecursor-containing mesoporous silica nanofillers in dentin adhesives, may provide a promising strategy for increasing of the durability of resin–dentin bonding and remineralizing caries-affected dentin.     

Matrix Gla protein inhibition of tooth mineralization

Extracellular matrix (ECM) mineralization is regulated by mineral ion availability, proteins, and other molecular determinants. To investigate protein regulation of mineralization in tooth dentin and cementum, and in alveolar bone, we expressed matrix Gla protein (MGP) ectopically in bones and teeth in mice, using an osteoblast/odontoblast-specific 2.3-kb Col1a1 promoter. Mandibles were analyzed by radiography, micro-computed tomography, light microscopy, histomorphometry, and transmission electron microscopy. While bone and tooth ECMs were established in the Col1a1-Mgp mice, extensive hypomineralization was observed, with values of unmineralized ECM from four- to eight-fold higher in dentin and alveolar bone when compared with that in wild-type tissues. Mineralization was virtually absent in tooth root dentin and cellular cementum, while crown dentin showed "breakthrough" areas of mineralization. Acellular cementum was lacking in Col1a1-Mgp teeth, and unmineralized osteodentin formed within the pulp. These results strengthen the view that bone and tooth mineralization is critically regulated by mineralization inhibitors.     

OPG knockout mouse teeth display reduced alveolar bone mass and hypermineralization in enamel and dentin

Recent studies showed that local injection or upregulation of OPG gene would result in early temporal retardation of tooth development. It was assumed that this retardation might cause defective tooth mineralization and pulp formation as the long-term effects. However, since those OPG treatments were transient, any possible long-term effects of OPG addition could not be assessed previously. In the present study, a high-resolution microCT was used to evaluate the long-term effect of OPG gene deprivation on the mineralization and morphology of mouse tooth. Our results showed that the mineralization of alveolar bone in OPG^−/− mouse tooth was decreased while those of enamel and dentin were increased, compared with the wild-type (WT) group. The labial and lingual dentin thicknesses of OPG^−/− group were significantly higher and with larger area in enamel and dentin than those of WT group. The size of pulp chamber was also substantially decreased in OPG^−/− mouse incisor. Different responses in mineralization and morphogenesis to OPG gene deprivation were found between bone and tooth. These effects may be independent of the early odontogenesis, and further studies are warranted to investigate the molecular mechanism of the effect of OPG gene expression on bone formation and later tooth development.     

仿生矿化纳米磷酸钙对牙本质小管封闭性能的评价

目的 制备仿生矿化的纳米磷酸钙,并探讨其对牙本质小管的封闭性能。方法 采用DMEM仿生矿化策略制备无定形磷酸钙材料(DMEM based amorphous calcium phosphate,DACP),运用透射电子显微镜(TEM)、傅里叶变换红外光谱扫描(FTIR)等检测观察其理化表征。选择口腔上皮角质形成细胞(HOK)和牙髓细胞(DPC)分别与材料共培养24 h后,CCK-8法评估材料的生物相容性。收集完整无龋的牙齿制备牙本质片,用DACP材料悬液均匀涂抹牙本质片,设置阳性对照NovaMin组和空白对照组,分别处理1、7 d后扫描电子显微镜(SEM)评估牙本质片的表面和截面封闭效果。结果 TEM显示DACP为均匀球形无定形纳米颗粒,随矿化时间的延长粒径有所增加。CCK-8结果显示HOK和DPC在25、50、100、200 μg/mL DACP下细胞活力均较好,提示材料有较好的生物相容性。牙本质片被处理1 d后DACP组牙本质小管大部分封闭;被处理7 d后DACP组牙本质小管表面呈现完全封闭的状态,且牙本质片截面可见DACP可渗入小管内。结论 运用DMEM仿生矿化策略制备的纳米磷酸钙具有较好的生物相容性和牙本质小管封闭作用,有望成为一种新的脱敏材料。     

Electron microscopy of dentinal tubule sclerosis in the enamel-free region of the rat molar

Ultrastructural changes in dentinal tubule contents were followed for periods up to 6 weeks following tooth eruption into the mouth. Characteristic odontoblast processes were observed in some tubules throughout the enamel-free dentine. Many dentinal tubules contained shrunken odontoblast processes, large collagen fibres and/or accumulations of mineral in various forms. The mineralization patterns suggest mechanisms of tubule occlusion other than simple formation of peritubular dentine. Both the degenerating odontoblast processes and collagen fibres appear to provide an organic framework for deposition of mineral. This region of rat molar dentine is recommended as useful for study of naturally-occurring tubular sclerosis.     

Extracellular matrix mineralization in periodontal tissues: Noncollagenous matrix proteins,enzymes, and relationship to hypophosphatasia and X‐linked hypophosphatemia

As broadly demonstrated for the formation of a functional skeleton, proper mineralization of periodontal alveolar bone and teeth – where calcium phosphate crystals are deposited and grow within an extracellular matrix – is essential for dental function. Mineralization defects in tooth dentin and cementum of the periodontium invariably lead to a weak (soft or brittle) dentition in which teeth become loose and prone to infection and are lost prematurely. Mineralization of the extremities of periodontal ligament fibers (Sharpey's fibers) where they insert into tooth cementum and alveolar bone is also essential for the function of the tooth‐suspensory apparatus in occlusion and mastication. Molecular determinants of mineralization in these tissues include mineral ion concentrations (phosphate and calcium), pyrophosphate, small integrin‐binding ligand N‐linked glycoproteins and matrix vesicles. Amongst the enzymes important in regulating these mineralization determinants, two are discussed at length here, with clinical examples given, namely tissue‐nonspecific alkaline phosphatase and phosphate‐regulating gene with homologies to endopeptidases on the X chromosome. Inactivating mutations in these enzymes in humans and in mouse models lead to the soft bones and teeth characteristic of hypophosphatasia and X‐linked hypophosphatemia, respectively, where the levels of local and systemic circulating mineralization determinants are perturbed. In X‐linked hypophosphatemia, in addition to renal phosphate wasting causing low circulating phosphate levels, phosphorylated mineralization‐regulating small integrin‐binding ligand N‐linked glycoproteins, such as matrix extracellular phosphoglycoprotein and osteopontin, and the phosphorylated peptides proteolytically released from them, such as the acidic serine‐ and aspartate‐rich‐motif peptide, may accumulate locally to impair mineralization in this disease.     

骨涎蛋白、骨桥蛋白在小鼠骨组织和牙胚组织发育中的表达

目的:探讨骨涎蛋(bone sialoprotein,BSP)、骨桥蛋白(osteopontin,OPN)在小鼠骨组织和牙胚组织发育过程中的分布特点及两者在骨组织形成和牙矿化过程中的作用.方法:取E16d胎鼠及出生后第10、30天的BALB/c小鼠共9只,拉颈处死,分离解剖下颌骨及其他骨组织,新鲜配制4%多聚甲醛固定过夜,常规石蜡包埋,近、远中向5μm连续切片,采用免疫组化法检测E16d胎鼠不同骨组织及10d、30d小鼠牙胚组织中BSP、OPN的表达及分布情况.结果:BSP、OPN在牙胚及骨组织发育中的分布模式存在差异.BSP在已经矿化成熟的矿化组织基质中呈阳性表达,而OPN主要在矿化的前沿及矿化活性强的矿化组织中强阳性表达.结论:BSP、OPN参与骨组织的发育和牙胚组织的矿化成熟,并在小鼠牙及骨组织的形成和矿化中具有重要作用.     

Tensile strength of mineralized/demineralized human normal and carious dentin

The bond strengths of resins to caries-affected dentin are low. This could be due to weakened organic matrix. The purpose of this work was to determine if the ultimate tensile strength (UTS) of excavated carious dentin is weaker than that of normal dentin. Soft caries was excavated from extracted human molars, and the tooth was vertically sectioned into slabs. Each slab was trimmed to an hourglass shape, parallel or perpendicular to the tubule direction. Half of the specimens were mineralized, while the other half were completely demineralized in EDTA. ANOVA on ranks showed that the three-factor interactions (mineralization, caries, tubule direction) were all significant (p < 0.0001), indicating that mineralization and tubule direction gave different UTS results in normal and caries-affected dentin. No significant differences were seen between the UTS of normal and and that of caries-affected demineralized dentin in the parallel or perpendicular group. The matrix of demineralized caries-affected dentin was as strong as that of normal demineralized dentin when tested in the same direction.     

Changes in matrix phosphorylation during bovine dentin development

Phosphorylation of the organic matrix proteins of dentin is important for the initiation of mineralization, but its relevance in later mineralization stages is controversial. The objective of this study was to analyze changes in the total matrix phosphate content during dentin development and to identify their origin. Amino acid and total matrix phosphate analyses of microdissected developing mantle and circumpulpal fetal bovine dentin specimens were performed. The amino acid composition showed few changes during mantle and circumpulpal dentin maturation. However, the total matrix phosphate content showed a significant, positive correlation with tissue maturation in both mantle and circumpulpal dentin, with a two- and a three-fold increase, respectively, being observed. The data indicate that changes occur in the pattern of phosphorylation of matrix proteins during dentin maturation, which we suggest may play a functional role in later stages of tooth mineralization.