Dentin demineralization

Abstract Preliminary work has shown that the rate of dentin demineralization increases with increasing concentrations of citric acid. This rate subsequently diminishes at much higher concentrations. The purpose of this study was to more precisely identify the citric acid concentration which produces peak dentin demineralization and to determine if this demineralization process is time dependent. Flat dentin surfaces were prepared on the buccal and lingual sides of 15 bovine molars. 8 depressions were made in each dentin surface using a #8 round bur in a high-speed handpiece with air-water coolant. Various concentrations of citric acid solutions (weight per cent) were prepared, e.g., 0%, 50%, 20%, 25%, 30%, 35%, 40%, 65% and their respective pH's recorded. 3 microliters of each citric acid solution were placed in individual depressions on the dentin surfaces and left undisturbed for 1, 2 or 3 min. Cotton pellets were used to soak up the citric acid solution, along with any dissolved calcium, and were subsequently placed in 10 ml of 18 MeΩ water. The parts per million calcium found in each water sample were determined using atomic absorption spectrophotometry. Peak dentin demineralization for 1-, 2- and 3-min application times occurred at 30% (pH = 1.55), 25% (pH=1.62) and 25% (pH=1.62) citric acid concentrations/(pH), respectively. Dentin demineralization was found to be time-dependent for all citric acid solution concentrations. The clinical significance of these findings is discussed.     

Reparative Dentin Formation by Dentin Matrix Proteins and Small Extracellular Vesicles

IntroductionVital pulp therapy aims at preserving pulp vitality and regenerating dentin. Therefore, the purpose of this study was to explore the effects of a combination of treated dentin matrix (TDM) proteins and dental pulp cell (DPC)-derived small extracellular vesicles (sEVs) on pulp-dentin complex repair.MethodsWe prepared TDM by chemical demineralization and mechanical disruption of teeth to a powder preparation. The sEVs were isolated from culture supernatants of DPCs and identified by nanoparticle tracking analysis, Western blotting, and transmission electron microscopy. The effect of a combination of TDM proteins and DPC-derived sEVs on DPC proliferation, migration, and odontogenic differentiation was evaluated in vitro. A minipig model of pulp injury was used to compare the clinical outcomes and tissue responses attributed to 4 materials including TDM, sEV-TDM, sEVs, and mineral trioxide aggregate.ResultsThe sEV isolated from the cell supernatant promoted DPC proliferation and migration. The combination of TDM extracts and sEV synergistically promoted the migration of DPCs but suppressed their proliferation. Real-time polymerase chain reaction and Western blot revealed that sEV-TDM enhanced the odontoblast-related protein expressions in DPCs. In in vivo studies, TDM and sEV-TDM promoted the formation of continuous reparative dentin. Furthermore, odontoblastlike high columnar cells were observed on the pulp side of the dentin bridge.ConclusionsThe sEV-TDM complex exhibits intrinsic biological activities, which has potential applications as a bioactive pulp-capping material.     

Microtissue Engineering Root Dentin with Photodynamically Cross-linked Nanoparticles Improves Fatigue Resistance of Endodontically Treated Teeth

IntroductionMicrotissue engineering root canal dentin with biopolymeric nanoparticles has the potential to improve mechanical properties of iatrogenically compromised root dentin. This study aims to characterize the surface mechanical property, bulk biomechanical response, and fatigue resistance of microtissue-engineered root dentin using photodynamically (photodynamic-activated [PDA]) cross-linked chitosan nanoparticles (CSnps).MethodsExperiments were conducted in 3 parts: part 1, root canal dentin sections were subjected to nanoindentations before/after treatment with CSnps and chemically (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide [EDC] cross-linked CSnps) and photodynamically cross-linked CSnps to determine the properties of treated surfaces (n = 84 points/group); part 2, root canal dentin specimens treated with PDA cross-linked CSnps were subjected to strain analysis using customized moiré interferometry (n = 5/group); and part 3, root canal dentin specimens treated with EDC cross-linked CSnps, PDA cross-linked CSnps, and instrumented controls were tested using an accelerated fatigue loading protocol to evaluate the sustained loads and cycles at failure (n = 15/group). Data were analyzed using the paired sample t test, trend analysis, and Kaplan-Meier with log-rank tests at a significance of .05 in each experiment.ResultsRoot dentin microtissue engineered with PDA cross-linked CSnps showed a 16.8% increase in elastic modulus and a conspicuous decrease in strain distribution in cervical root dentin (P < .01). There was a significant reduction in the tensile strain formed at the apical region of the instrumented root dentin after treatment (P < .05). Survival analysis showed a statistically significant difference (P < .05) among evaluated conditions in fatigue resistance (ie, PDA cross-linked CSnps > EDC cross-linked CSnps > control).ConclusionsThis study highlighted the potential of root canal dentin microtissue engineering with PDA cross-linked CSnps to diminish radicular strain distribution and improve resistance to fatigue loads in endodontically treated teeth.     

Cell Homing for Pulp Tissue Engineering with Endogenous Dentin Matrix Proteins

Introduction

Compelling evidence pinpoints that pulp tissue engineering after the transplantation of stem cells is possible. Although intriguing, severe problems regarding clinical feasibility remain. Cell homing has been proposed as a viable alternative in which dentin-derived growth factors in a conducive scaffold may attract resident cells to form pulplike tissue. In this study, an ectopic animal model for in situ dental pulp tissue engineering was developed to evaluate whether pulplike tissue formation in empty root canals after the attraction of stem cells was possible and whether this could be enhanced by dentin-derived growth factors.

Demineralized Dentin as a Semi‐Rigid Barrier for Guiding Periodontal Tissue Regeneration

Background: Guided tissue regeneration (GTR) is an accepted approach in the correction of periodontal bone loss. Nonetheless, the deficiencies of commonly applied absorbable membrane, such as flexibility and limited osteoconductive and osteoinductive capability, still leave much room for improvement. Thus, the feasibility of applying demineralized dentin tissue to improve the therapeutic effect of GTR in periodontal regeneration was explored. Methods: Demineralized dentin was harvested after acid treatment, and its physiochemical properties were assessed in terms of mineralization density, contact angle, three‐point test, and cell attachment. Because of its similar characteristics with bone tissue, dentin that had been acid‐treated for 6 hours was chosen to repair a periodontal defect using an induced‐periodontitis canine model. Histologic measurements were taken to compare its therapeutic effects to an absorbable membrane group and an untreated group. Results: The demineralized dentin displayed continually decreased hardness and density as the acid etching time was prolonged. Enhanced attachment and spreading of bone marrow mesenchymal stem cells were observed on the 6‐hour processed dentin. Furthermore, in the demineralized dentin group, more periodontal tissues were newly formed compared with the biomembrane and untreated groups. Conclusion: Acid etching represents an easy and promising approach to obtain demineralized dentin with desirable properties, similar to bone, for clinical application to promote periodontal tissue regeneration.     

Human Freeze-dried Dentin Matrix as a Biologically Active Scaffold for Tooth Tissue Engineering

IntroductionDentin is a suitable scaffold for tooth tissue engineering and can provide the appropriate combinations and concentrations of bioactive proteins to accommodate odontogenic cells. Freeze drying is a possible strategy to treat dentin because it ensures long-term stability of biopharmaceutical products and can increase the stability of dentin for improved distribution and storage. We hypothesized that freeze-dried dentin matrix (FDDM) constitutes a novel bioinstructive scaffold for tooth tissue engineering.MethodsThis study used a modified technique to prepare an FDDM that preserved the mechanical and biological properties of dentin. The compression resistance and microhardness of FDDM were determined. Similarly, various biological characteristics, including cell morphology, cell proliferation, collagen secretion, alkaline phosphatase activity, and gene and protein expression, were investigated. To assess the inductive capacity of FDDM in vivo, FDDM and human dental pulp stem cell (DPSC) sheets were subcutaneously implanted in the dorsal pockets of nude mice. At 8 weeks postimplantation, the transplants were removed and histologically studied.ResultsThe results show that FDDM has mechanical and biological characteristics similar to those of dentin (P > .05). DPSCs cultured on FDDM and dentin showed superior attachment, growth, viability, and collagen secretion capacity but decreased mineral capability compared with DPSCs cultured with alpha minimum essential medium or hydroxyapatite (P < .05). The histologic results show that FDDM, which is similar to dentin, supported dentin/pulplike tissue regeneration in vivo as shown by the related expression levels of dentin markers, such as dentin sialoprotein and alkaline phosphatase.ConclusionsThese results suggest that FDDM constitutes a novel superior scaffold for tooth tissue engineering.     

牙本质基质在组织再生中的应用

组织器官病变严重影响到人们的生活质量,组织器官缺损可能威胁人们的生命,组织、器官缺损的修复和功能重建是现代医学面临的挑战。组织工程为再生组织器官带来希望。组织工程支架为细胞的生长、增殖和分化提供了微环境,而且影响着形成组织的大小和形态。牙本质基质作为一种天然的生物活性支架,具有良好的生物相容性,在组织工程中得到广泛的应用,成为研究热点。学者们以牙本质基质为支架,开展了包括牙根、牙周、牙髓、骨等软硬组织缺损修复的研究,取得了一系列重要进展。本文对牙本质生物学特性、牙本质基质在组织再生中的应用等进行综述,为临床应用牙本质基质进行组织再生提供参考。     

永生化成牙本质细胞表达牙本质基质蛋白的实验研究

目的　探讨永生化人成牙本质细胞样细胞系hTERT-hOd-l表达牙本质基质蛋白的情况。方法　矿化液培养hTERT-hOd-l细胞5周,检测骨钙素(OC)分泌量和碱性磷酸酶(ALP)活性。采用免疫组织化学、RT-PCR和原位杂交方法检测Ⅰ型胶原、骨涎蛋白(BSP)、牙本质基质蛋白1 (DMP1)以及成牙本质细胞标志物牙本质涎磷蛋白(DSPP) 和牙本质涎蛋白(DSP)在细胞中的表达。结果　在矿化液诱导下,hTERT-hOd-l细胞ALP活性和OC分泌量升高。 hTERT-hOd-l细胞在mRNA水平上表达BSP、DMP1和DSPP,在蛋白质水平上表达DSP和Ⅰ型胶原。结论　hTERT- hOd-l细胞在体外表达牙本质基质蛋白,具有矿化的潜能。     

Biomimetic Approach to Perforation Repair Using Dental Pulp Stem Cells and Dentin Matrix Protein 1

Introduction

Dentin regeneration could be an ideal treatment option to restore tissue function. This study was conducted to evaluate the ability of dental pulp stem cells (DPSCs) and dentin matrix protein 1 (DMP1) impregnated within a collagen scaffold to regenerate dentin.

Methods

Simulated perforations were created in 18 dentin wafers made from freshly extracted human molars. Six groups were established. They were (1) empty wafers, (2) mineral trioxide aggregate, (3) collagen scaffold, (4) scaffold with DMP1, (5) scaffold with DPSCs, and (6) scaffold with DPSCs and DMP1. One sample was placed subcutaneously in each mouse with three mice in each group. After 12 weeks, the samples were subjected to radiographic, histological, and immunohistochemical evaluations.

Results

DPSCs impregnated within a collagen scaffold differentiated into odontoblast-like cells forming a highly cellular, vascular, and mineralized matrix in the presence of DMP1.

Conclusions

A triad consisting of DPSCs, DMP1, and a collagen scaffold promotes dentin regeneration in a simulated perforation repair model.     

脱矿牙本质基质用于根尖诱导成形术的临床研究

目的　观察同种脱矿牙本质基质(DDM)用于根尖诱导成形术的临床效果。方法　将人牙本质去脂、脱矿粉碎、冻干等处理制备成DDM,作为根尖诱导剂应用于临床,并以氢氧化钙糊剂作为对照,共治疗患牙57颗。分别于术后1年、2年拍X线片及临床检查。结果　根尖诱导成形术后1年,患牙X线片有不同程度的根尖屏障形成,或虽无明显的X线改变但根管内探测有明显阻力,此时更换永久性根管充填材料。术后2年的临床观察发现, DDM组的治愈率为92·86%,略高于氢氧化钙组的91·30%,但无显著性差异。结论　脱矿牙本质基质可作为一种新的根尖诱导剂应用于临床。     

基质金属蛋白酶-1对根面牙本质有机质降解作用的超微结构观察

目的　观察基质金属蛋白酶-1(MMP-1)对根面牙本质有机质的降解作用。方法　收集临床拔除的健康无 龋阻生牙,切取根颈1/3处约5 mm厚的牙体,制成牙本质组织块,随机分为4组。第1组用酸溶液脱矿处理21 d 后,放入MMP-1溶液中孵育7 d;第2组仅用酸溶液脱矿处理21 d;第3组仅用MMP-1溶液酶解7 d;第4组为正常牙 本质标本对照组,不作任何处理。将各标本切割制作样本,脱水干燥,喷金,扫描电镜观察。结果　酸和酶溶液共 同处理的标本牙本质硬组织脱矿明显,牙本质小管管腔失去原有形态,边界不清,周围暴露的胶原纤维断裂不连 续,排列杂乱不规则。酸或酶溶液单独处理组未发生明显的基质纤维降解现象。结论　内源性蛋白酶参与了根面 龋的发生发展过程,MMP-1能够明显降解脱矿后的牙本质有机质。     

Tetracycline demineralization of dentin: the effects of concentration and application time

Abstract The current investigation was initiated to study the effect concentration and application time has on the rate of tetracycline demineralization of dentin. Buccal and lingual surfaces of extracted bovine molars were ground to a smooth flat dentin surface using wetted silicon carbide discs. Standardized depressions were made in the dentin surface with a #909-055 diamond round wheel. Fresh tetracycline HCl (TTC-HCl) (Flavine Int. Inc.) solutions, i.e., 0, 25, 50, 75, 100, 125 and 150 mg/ml were prepared. A 30% citric acid solution was used as a positive control. The pH of each solution was recorded. 7 μl of each solution were pipetted into a depression and remained undisturbed for 1, 3, or 5 min. At the end of each application time period a fresh #3 cotton pellet was placed in the depression, once every 20 s for 1 min, to soak up the solution. The 3 pellets were placed in a 2.00 ml of 18 Mω H₂O sample. As a measure of the rate of demineralization the parts per million calcium (ppm Ca⁺⁺) found in each sample were determined using atomic absorption spectrophotometry. Two-way analysis of variance was used to determine effects of TTC-HC1 concentration and time on the rate of demineralization. No significant differences were found in the mean ppm Ca⁺⁺ released at 1-, 3- and 5-min application times for 0, 25, or 50 mg/ml TTC. No significant differences were found in the mean ppm Ca⁺⁺ released (i) between 3- and 5-rmn application times for 75, 100, 125 and 150 mg/ml TTC-HCl solutions and (ii) between 75, 100, 125 and 150 mg/ml TTC-HCl solutions within either the 3- or 5-min application times. The mean ppm Ca⁺⁺ released at 3- and 5-min application times for 75, 100, 125 and 150 mg/ml TTC-HCl solutions were all significantly greater than the respective readings at the 1-min application time. The mean ppm Ca⁺⁺ recorded for the 30% citric acid solution for all 3 application times were 3 to 5.5 × greater than the highest mean ppm Ca⁺⁺ recording for TTC-HCl. The results of this study show that a 3-min application time of 75 mg/ml TTC-HCl solution is equally as effective at demineralizing dentin as is higher concentrations and/or longer application times, but was far less effective than a 30% citric acid solution.     

Dentin induction by implants of autolyzed antigen-extracted allogeneic dentin on amputated pulps of dogs

Abstract Implantation of autolyzed antigen-extracted allogeneic (AAA) dentin matrix gelatin powder caused homeoinduction on amputated dental pulps. This event began with migration of spindle-shaped mesenchymal cells into the cavity on the amputated pulp. This was followed by proliferation of undifferentiated large cells concomitantly with vascular invasion and attachment of spindle-shaped cells and large cells to the AAA dentin. The adhering cells differentiated into osteodentinoblasts and/or preodontoblasts to form osteodentin matrix and predentin respectively. Osteodentinocytes and odontoblasts, then, formed osteodentin and tubular dentin. The healing of pulp dressed with inactivated AAA dentin using guanidine HCl was delayed. These results suggested that AAA dentin matrix powder may have chemotactic and mitogenic activity for undifferentiated mesenchymal cells and may provide a suitable scaffolding for fixation of these cells. Compartments of microenvironment formed by AAA dentin and the enveloping hard substances may play some role in differentiation into odontoblasts.     

Evaluation of the Apical Complex and the Coronal Pulp as a Stem Cell Source for Dentin-pulp Regeneration

IntroductionThis study compared the stemness and differentiation potential of stem cells derived from the apical complex (apical complex cells [ACCs]) and coronal pulp (dental pulp stem cells [DPSCs]) of human immature permanent teeth with the aim of determining a more suitable source of stem cells for regeneration of the dentin-pulp complex.MethodsACC and DPSC cultures were established from 13 human immature permanent teeth using the outgrowth method. The proliferation capacity and colony-forming ability of ACCs and DPSCs were evaluated. ACCs and DPSCs were analyzed for mesenchymal stem cell markers using flow cytometry. The adipogenic and osteogenic differentiation potential of ACCs and DPSCs were evaluated using the quantitative real-time polymerase chain reaction and histochemical staining. ACCs and DPSCs were transplanted subcutaneously in immunocompromised mice using macroporous biphasic calcium phosphate as a carrier. The histomorphologic characteristics of the newly formed tissues were verified using hematoxylin-eosin staining and immunohistochemical staining. Quantitative alkaline phosphatase analysis and quantitative real-time polymerase chain reaction using BSP, DSPP, POSTN, and Col XII were performed.ResultsACCs and DPSCs showed similar cell proliferation potential and colony-forming ability. The percentage of mesenchymal stem cell markers was similar between ACCs and DPSCs. In the in vitro study, ACCs and DPSCs showed adipogenic and osteogenic differentiation potential. In the in vivo study, ACCs and DPSCs formed amorphous hard tissue using macroporous biphasic calcium phosphate particles. The quantity and histomorphologic characteristics of the amorphous hard tissue were similar in the ACC and DPSC groups. Formation of periodontal ligament–like tissue, positive to Col XII, was observed in ACC transplants, which was absent in DPSC transplants.ConclusionsACCs and DPSCs showed similar stemness, proliferation rate, and hard tissue–forming capacity. The notable difference was the periodontal ligament–like fiber-forming capacity of ACCs, which indicates the presence of various lineages of stem cells in the apical complex compared with the coronal pulp. Regarding regeneration of the dentin-pulp complex, the coronal pulp can be a suitable source of stem cells considering its homogenous lineages of cells and favorable osteo/odontogenic differentiation potential.     

脱矿牙本质基质材料对大鼠颅骨缺损修复的研究

目的：探讨生物相容性较好的脱矿牙本质基质材料对大鼠颅骨缺损的修复效果。方法：①拔除健康大鼠上下前牙,脱矿,脱脂,粉碎后提取脱矿牙本质基质粉末,后与熟石膏以重量2：1比例混合。②大鼠头盖骨正中建立直径约8mm的圆形骨缺损,实验组：在缺损区植入脱矿牙本质基质粉末与熟石膏复合物,用生理盐水调和。对照组：无修复治疗。术后4周和8周分组处死取标本,进行大体标本, X线和组织学HE染色光镜下观察。结果：脱矿牙本质基质粉末与熟石膏具有良好的组织相容性和骨引导性,并与新骨紧密结合,熟石膏可以较好的抑制脱矿牙本质基质牙粉末颗粒离散。结论：脱矿牙本质基质粉末与熟石膏复合骨材料对大鼠颅骨缺损有良好的修复作用,可促进新骨形成,缩短骨缺损修复时间。     

异种脱细胞真皮基质膜在引导牙周组织再生中应用的临床效果评价

目的：评价异种脱细胞真皮基质膜在引导牙周组织再生中应用的临床效果。方法：30例牙周缺损区患牙随机分两组：实验组20例,异种脱细胞真皮基质膜和羟基磷灰石修复;对照组10例仅羟基磷灰石修复。统计学比较6个月时两组各项临床指标和牙槽骨水平改变。结果：实验组牙周袋深度、临床附着丧失的减少量和牙槽骨水平的增加量均较对照组高（P〈0.05）,牙龈退缩量低于对照组（P〈0.05）。结论：异种脱细胞真皮基质膜应用于引导牙周组织再生有较好的临床效果,值得推广。     

白藜芦醇对脱矿牙本质基质影响的相关性研究

目的:探讨白藜芦醇对基质金属蛋白酶-2活性及耐脱矿力的影响,以期为白藜芦醇的临床应用提供实验依据。方法:设置实验组白藜芦醇的浓度梯度:12.5 μmol/L、25 μmol/L、50 μmol/L、100 μmol/L、200 μmol/L、400 μmol/L,阳性对照组0.2%氯己定,阴性对照组去离子水。每组取10 μL与提取出来的牙源性mmp-2 30 μL混合孵育30 min,用体液基质金属蛋白酶-2活性比色法定量检测各组混合20 min后的酶活性,每组重复5次。 牙本质片经pH循环后,用激光共聚焦电子显微镜3D图像观察牙本质脱矿深度。结果:实验组白藜芦醇浓度在50 μmol/L以上时有明显抑制作用,且在100 μmol/L、200 μmol/L、400 μmol/L时的酶活性抑制率与阳性对照组间无显著性差异。激光共聚焦电子显微镜3D图像显示100 μmol/L Res的染色深度明显低于阴性对照组,统计学分析结果显示100 μmol/L Res的平均脱矿深度显著低于阴性对照组(P<0.05),与阳性对照组间并无显著性差异。结论:白藜芦醇可抑制基质金属蛋白酶-2活性及牙本质脱矿。     

Citric acid demineralization of cementum and dentin: the effect of the storage medium

Abstract The purpose of this study was to see if the root surface topography of teeth, stored in saline and subsequently treated with citric acid, differred from the root surface topography of teeth that were treated immediately upon extraction. 12 freshly extracted adult human permanent teeth, with proximal surfaces free of caries and periodontal disease, were treated in succession. The crowns were removed at the level of periodontal attachment, the teeth sectioned buccallingually and a treatment area deligniated on each proximal section. The treatment area of 6 teeth was root planed to expose dentin (D) and scaled to remove adherent tissue and leave a cementum surfaces (C) on the other 6 teeth. A coronalapical groove down the middle of the treatment area divided it into approximately equal parts or experimental regions. One proximal section of each tooth was placed in physiologic saline (S) and treated after 6 weeks of storage while the other proximal section was freshly treated (F). Treatment consisted of applying a 30% citric acid (CA) solution (pH= 1.60) for 5 min. Cotton pellets soaked in the citric acid solution were placed (P) on one half of the experimental area and heavily burnished (B) on the other half. Treatment areas were subsequently prepared for scanning electron microscopy analysis. Assessment was made of (i) the % of surface area tufted, (ii) fibril tufting depth (0–3) and (iii) fibril tufting density (1–3). Similarities were found in the data for both storage methods (F and S) across each application technique (P or B) and each tooth surface (D or C) with respect to the (i) % area tufted and (ii) frequency distribution of tufting depth scores. As for the application techniques, the data for burnishing was greater than placed across each storage method (F or S) and each tooth surface (D or C) for the same two parameters. The results of the study indicated that 6-week physiologic saline storage does not affect root surface demineralization by citric acid, as assessed by SEM.