Cementomimetics——constructing a cementum-like biomineralized microlayer via amelogenin-derived peptides

Cementum is the outer-, mineralized-tissue covering the tooth root and an essential part of the system of periodontal tissue that anchors the tooth to the bone. Periodontal disease results from the destructive behavior of the host elicited by an infectious biofilm adhering to the tooth root and left untreated, may lead to tooth loss. We describe a novel protocol for identifying peptide sequences from native proteins with the potential to repair damaged dental tissues by controlling hydroxyapatite biomineralization. Using amelogenin as a case study and a bioinformatics scoring matrix, we identified regions within amelogenin that are shared with a set of hydroxyapatite-binding peptides （HABPs） previously selected by phage display. One 22-amino acid long peptide regions referred to as amelogenin-derived peptide 5 （ADP5） was shown to facilitate cell-free formation of a cementum-like hydroxyapatite mineral layer on demineralized human root dentin that, in turn, supported attachment of periodontal ligament cells in vitro. Our findings have several implications in peptide-assisted mineral formation that mimic biomineralization. By further elaborating the mechanism for protein control over the biomineral formed, we afford new insights into the evolution of protein-mineral interactions. By exploiting small peptide domains of native proteins, our understanding of structure-function relationships of biomineralizing proteins can be extended and these peptides can be utilized to engineer mineral formation. Finally, the cementomimetic layer formed by ADP5 has the potential clinical application to repair diseased root surfaces so as to promote the regeneration of periodontal tissues and thereby reduce the morbiditv associated with tooth loss.

自组装类釉原蛋白两亲性寡肽在大鼠骨缺损中成骨效应的研究

目的:研究自组装类釉原蛋白两亲性寡肽的骨缺损修复作用。方法:构建具有黏附特性的自组装类釉原蛋白寡肽纳米凝胶、骨髓间充质干细胞(BMSCs)细胞膜片和大鼠股骨骨缺损模型,将SD大鼠随机分为空白对照(Control)组、BMSCs种子细胞膜片(Cell sheet)组、类釉原蛋白寡肽生物支架(OPA scaffold)组、BMSCs种子细胞膜片+类釉原蛋白寡肽生物支架(Cell sheet/OPA scaffold)组,将各组材料填充于股骨骨缺损处,在术后8周分别通过大体观察、微型CT扫描重建及组织学来评估骨缺损修复情况。结果:术后大体标本观察发现4组均有不同程度骨修复,Cell sheet组缺损区大致轮廓仍清晰可见且缺损区仍较深,骨修复最差。与Control组相比,其余两组骨修复较好,以Cell sheet/OPA scaffold组骨缺损区域修复最好,缺损轮廓已不明显。微型CT三维重建及定量分析结果显示,Cell sheet组新骨生成率低于Control组(P<0.05);OPA scaffold组和Cell sheet/OPA scaffold组新骨生成率优于Control组(P<0.05),两组缺损区域都有较高的骨小梁数量(Tb.N)、骨小梁厚度(Tb.Th)、相对骨体积(BV/TV)、连接密度(Conn.D)和较低的骨小梁分离度(Tb.Sp),其中以Cell sheet/OPA scaffold组最高。组织学结果显示,OPA scaffold组和Cell sheet/OPA scaffold组有较多趋向成熟的骨小梁生成,Control组和Cell sheet组有少量趋向成熟的骨小梁生成。结论:自组装类釉原蛋白寡肽纳米凝胶具有很好的成骨能力及骨缺损修复能力,具有作为骨组织工程支架材料的潜力加以进一步研究。

Remineralization of enamel caries by an amelogenin-derived peptide and fluoride in vitro

Dental caries is one of the most common oral diseases in the world.This study was tantamount to investigate the combinatory effects of an amelogenin-derived peptide(called QP5)and fluoride on the remineralization of artificial enamel caries.The peptide QP5 was synthesized and characterized,and the binding capability of the peptide on hydroxyapatite(HA)and demineralized tooth enamel surface was analysed.Then,the mineralization function of the peptide and fluoride was studied through the spontaneous mineralization testing and remineralization on enamel caries in vitro.First,the novel peptide QP5 could bind on the hydroxyapatite and demineralized tooth enamel surfaces.Second,QP5 can transitorily stabilize the formation of amorphous calcium phosphate and direct the transformation into hydroxyapatite crystals alone and in combination with fluoride.In addition,compared to blocks treated by peptide QP5 alone or fluoride,the sample blocks showed significantly higher surface microhardness,lower mineral loss and shallower lesion depth after treatment with a combination of QP5 and fluoride at high or low concentrations.The peptide QP5 could control the crystallization of hydroxyapatite,and combinatory application of peptide QP5 and fluoride had a potential synergistic effect on the remineralization of enamel caries.

An amelogenin-based peptide hydrogel promoted the odontogenic differentiation of human dental pulp cells

Amelogenin can induce odontogenic differentiation of human dental pulp cells(HDPCs),which has great potential and advantages in dentine-pulp complex regeneration.However,the unstability of amelogenin limits its further application.This study constructed amelogenin self-assembling peptide hydrogels(L-gel or D-gel)by heating-cooling technique,investigated the effects of these hydrogels on the odontogenic differentiation of HDPCs and explored the underneath mechanism.The critical aggregation concentration,conformation,morphology,mechanical property and biological stability of the hydrogels were characterized,respectively.The effects of the hydrogels on the odontogenic differentiation of HDPCs were evaluated via alkaline phosphatase activity measurement,quantitative reverse transcription polymerase chain reaction,western blot,Alizarin red staining and scanning electron microscope.The mechanism was explored via signaling pathway experiments.Results showed that both the L-gel and D-gel stimulated the odontogenic differentiation of HDPCs on both Day 7 and Day 14,while the D-gel showed the highest enhancement effects.Meanwhile,the D-gel promoted calcium accumulation and mineralized matrix deposition on Day 21.The D-gel activated MAPK-ERK1/2 pathways in HDPCs and induced the odontogenic differentiation via ERK1/2 and transforming growth factor/smad pathways.Overall,our study demonstrated that the amelogenin peptide hydrogel stimulated the odontogenic differentiation and enhanced mineralization,which held big potential in the dentine-pulp complex regeneration.

人釉原蛋白亮氨酸富集片段的冷冻电子显微镜观察及其引导矿化性能研究

目的采用冷冻电子显微镜观察体外重组的人釉原蛋白富亮氨酸片段(LRAP),分析其细微结构和聚集状态,结合矿化实验评估LRAP体外引导羟磷灰石生长的定向成核能力。方法人工合成LRAP基因,与原核表达载体p Cold-SUMO行质粒构建,于宿主菌大肠杆菌BL21plys中诱导表达并纯化,在冷冻电子显微镜下观察LRAP在p H值从3.5到8.0的环境中聚合自组装的过程,并在人工唾液中通过透射电镜观察羟磷灰石晶体的生长规律。结果通过原核表达成功得到纯度90%以上的LRAP蛋白提取物,当p H值为8.0时,LRAP能聚集成多聚体和纳米小球等功能结构,在人工唾液中能诱导羟磷灰石晶体生长成熟。结论作为简化的釉原蛋白功能域,LRAP兼备了自组装为纳米小球和c轴诱导晶体矿化的特性,可作为运用釉基质蛋白多聚物行无细胞修复牙体硬组织缺损的备选材料之一。

釉原蛋白羧基末端肽加速细胞周期促进成釉细胞系ALC细胞增殖

背景:釉原蛋白羧基末端肽(C-terminus of the amelogenin peptide,AMG-CP)作为一种小分子内源性短肽,在物种间的序列高度保守,提示其在牙齿发育过程中参与重要的生理过程。有研究表明AMG-CP对成牙骨质细胞、骨髓间充质干细胞、牙周膜成纤维细胞的增殖分化有调控作用,但是AMG-CP对成釉细胞生物学功能的影响尚未见报道。目的:探究不同质量浓度AMG-CP对成釉细胞系ALC细胞增殖的影响及相关机制。方法:人工合成并通过液相色谱和质谱检测AMG-CP合成情况。使用xCELLigence RTCA实时细胞分析系统观察0,0.5,1,2 mg/L AMG-CP对ALC细胞增殖的影响。流式细胞仪检测0,1,2 mg/L AMG-CP对ALC细胞周期的影响。Real-time PCR检测0,1,2 mg/L AMG-CP对ALC细胞的细胞周期蛋白Cyclin D1、CDK4、MCM2、MCM5 mRNA表达水平的影响。Western blot检测0,1 mg/L AMG-CP对ALC细胞表达细胞增殖相关通路中p-ERK1/2、total-ERK1/2表达水平的影响。利用MAPK-ERK1/2通路抑制实验,在ERK阻断剂U0126作用下阻断ERK1/2磷酸化,检测AMG-CP对ALC细胞增殖能力的影响。结果与结论:①与对照组比较,AMG-CP促进ALC细胞增殖,群体倍增时间降低,并存在浓度梯度依赖性;②与对照组比较,AMG-CP具有加速细胞周期的作用;③与对照组比较,AMG-CP可以上调细胞周期相关基因Cyclin D1、CDK4、MCM2、MCM5的表达;④与对照组比较,AMG-CP可以上调p-ERK1/2表达,激活MAPK-ERK1/2信号通路;⑤U0126抑制MAPK-ERK1/2通路激活后,AMG-CP失去对ALC细胞促增殖作用;⑥以上结果证实AMG-CP可以激活MAPK-ERK1/2通路,加速细胞周期,进而促进ALC细胞增殖,提示AMG-CP具有促进成釉细胞增殖的潜能。

人釉原蛋白全长及其功能片段的体外自组装和矿化行为的研究

目的探讨人釉原蛋白(AM)全长及其N端酪氨酸富集段(TRAP)、C端亮氨酸富集段(LRAP)体外自组装的动态过程及其在羟磷灰石(HA)晶体形成中的作用。方法体外重组、纯化人AM全长及其功能片段TRAP、LRAP,在三氨基甲烷(Tris-HCl)中配制成100μg·mL^(-1)、pH=8的蛋白溶液,室温孵育1~15 min,透射电镜(TEM)下观察比较AM全长、TARP和LRAP的自组装行为;在人工唾液中加入AM全长孵育3 d,扫描电镜(SEM)观察诱导形成的新生晶体形貌,继续加入TARP和LRAP孵育3 d后再次观察。结果pH为8时,人AM全长及TRAP自发启动组装,15 min后均可形成“纳米球”结构,其中TRAP形成的“纳米球”孤立存在,大小均匀,没有明显内部结构;而AM全长分级组装,形成“纳米球”后进一步各向延伸趋势,形成链状结构,随后聚集成网;LRAP的自组装行为不明显,蛋白多以单体形式存在,无“纳米球”生成,仅可见少量低聚物。AM全长诱导3 d后形成棒状HA晶体,加入TRAP和LRAP蛋白继续诱导3 d后晶体在c轴明显伸长,而a、b平面生长欠佳。结论人AM全长、TRAP和LRAP的自组装和矿化行为与体内HA晶体定向生长的机制契合,为它们在HA晶体生长、成熟过程中的作用提供了理论依据。