Bone sialoprotein-induced reparative dentinogenesis in the pulp of rat’s molar

Bone sialoprotein (BSP), an osteogenic protein (OP), mixed with a carrier, was implanted in the pulp of rat first upper molars (OP group). Cavities were prepared with dental burs and pulp perforation was carried out by pressure with the tip of a steel probe. After 8, 14, and 30 days, the rats were killed and the pulps of the OP group were compared with (1) a sham group (S group), (2) a group where the carrier was implanted alone (C group), and (3) capping with calcium hydroxide (Ca group). After 8 days, a few inflammatory cells were seen, mostly located at the pulp surface near the perforation. In the Ca group, a dentin bridge started to form, in contrast to the other groups. After 15 days, globular structures were seen in the pulps of the S and C groups. A reparative osteodentin bridge isolated the pulp from the cavity in the Ca group. Variable reactions were seen in the OP group, with some evidence of cell and matrix alignments or plugs of osteodentin in continuity with an inner layer of reparative dentin. After 30 days, irregular osteodentin formation was observed in the pulps of the S and C groups, with a tendency for globular structures to merge, but with interglobular spaces filled by pulp remnants. In the Ca group, osteodentin was observed in the mesial part of the pulp chamber. In the BSP-implanted group, the osteogenic protein stimulated the formation of a homogeneous dentin-like deposit occupying most of the mesial part of the pulp. Apparently, BSP stimulates the differentiation of cells which secrete an organized extracellular matrix more efficiently than any other capping material used so far. Altogether, the results reported here support that bone sialoprotein displays novel bioactive properties and is capable of stimulating in 1 month’s time the development of a thick reparative dentinal tissue in the pulp, occluding the perforation and filling the mesial third of the pulp chamber. Received: 13 October 1999 / Accepted: 6 December 1999     

Short-term dentin bridging of mechanically-exposed pulps capped with adhesive resin systems

Dentin bridging of 150 mechanically exposed monkey pulps to two adhesive resins [BondWell LC (BW); Clearfil Liner Bond II (LB)] and a calcium hydroxide cement [Dycal (DY)] were histopathologically evaluated at 3, 7, 14, 30, and 60 days after operation (n = 10). The dentin bridge structure was three-dimensionally reconstructed from serial sections using a computer-aided reconstruction system. At three and seven days, in all pulps, no necrotic tissue and slight inflammatory cell infiltration was observed just below the exposure site. At 14 days, spindle-shaped fibroblast cells could be detected at the wound surface. All dentin chips showed reparative dentin deposition along the periphery of the wound surface. From this stage, the formation of secondary dentin from the pulpal wall at the periphery of the exposed area was recognized in all pulps. At 30 days, initial signs of dentin bridging were observed at the wound surface with a well-organized layer of odontoblastoid cells. The exposed area became occluded with a dentin bridge as the observation period increased. Group DY showed significantly higher incidence of dentin bridging than other groups at 30 days (p < 0.05). However, no significant difference of dentin bridge formation was found between Group DY and Groups BW and LB at 60 days. Bacterial penetration along the cavity walls and pulp tissue could not be detected in all groups. Histopathological observations and three-dimensional image analysis suggested that dentin bridge formation may occur following three patterns: (1) formed from the periphery of the residual dentin chip at the wound surface within 14 days, (2) formed within 14 days from the periphery of the cavity floor and with formation of reparative dentin by stimulation during the cavity preparation, and (3) formed from the wound surface within 30 days after exposure.     

Primary mineralization of dentin in rats after pulp capping with calcium-hydroxide

It has been previously reported that short term application of calcium-hydroxide to the dental pulp resulted in the formation of a dentin bridge with matrix vesicle calcification. The present study deals with the induction of reparative dentin in rat molar pulps by direct capping with calcium-hydroxide. The teeth were examined after 21 and 35 days. Transmission electron microscopy (TEM) revealed that the calcification process in the newly formed dentin was characterized by apatite deposition in matrix vesicles and the occurrence of calcifying nodules in the matrix. The persistence of these features of primary mineralization, during the experimental period, was associated with the continuous calcium hydroxide contact with pulp tissue, producing changes in its metabolic state.     

Primary mineralization of dentin in rats after pulp capping with calcium-hydroxide

It has been previously reported that short term application of calcium-hydroxide to the dental pulp resulted in the formation of a dentin bridge with matrix vesicle calcification. The present study deals with the induction of reparative dentin in rat molar pulps by direct capping with calcium-hydroxide. The teeth were examined after 21 and 35 days. Transmission electron microscopy (TEM) Revealed that the calcification process in the newly formed dentin was characterized by apatite deposition in matrix vesicles and the occurrence of calcifying nodules in the matrix. The persistence of these features of primary mineralization, during the experimental period, was associated with the continuous calcium hydroxide contact with pulp tissue, producing changes in its metabolic state.     

Dentonin, a fragment of MEPE, enhanced dental pulp stem cell proliferation

Matrix extracellular phosphoglycoprotein (MEPE) is a SIBLING protein, found in bone and dental tissues. The purpose of this study was to determine whether a 23-amino-acid peptide derived from MEPE (Dentonin or AC-100) could stimulate dental pulp stem cell (DPSC) proliferation and/or differentiation. DPSCs were isolated from erupted human molars, and the mitogenic potential of Dentonin in DPSCs was measured by BrdU immunoassay and cell-cycle gene SuperArray. Differentiation of DPSCs with Dentonin was characterized by Western blot and by osteogenesis gene SuperArray. Dentonin enhanced DPSC proliferation by down-regulating P16, accompanied by up-regulation of ubiquitin protein ligase E3A and human ubiquitin-related protein SUMO-1. Enhanced cell proliferation required intact RGD and SGDG motifs in the peptide. This study shows that Dentonin can promote DPSC proliferation, with a potential role in pulp repair. Further studies are required to determine the usefulness of this material in vivo.     

An in vivo evaluation of hemorrhage control using sodium hypochlorite and direct capping with a one- or two-component adhesive system in exposed nonhuman primate pulps.

OBJECTIVE: This study evaluated the biologic ability of sodium hypochlorite to control hemorrhage via chemical amputation of the coagulum, to remove dentin chips, to assist healing, and to facilitate formation of a dentinal bridge under two adhesive systems. METHOD AND MATERIALS: Ninety Class V cavities with mechanical pulpal exposures were placed in the teeth of five adult monkeys and histologically observed. All exposures were prepared with a No. 330 bur, and hemorrhage was controlled with 3% sodium hypochlorite. Twenty-two exposures were capped with All-Bond 2 and AElitefil, and 26 exposures were capped with One-Step (OS) and Resinomer (RS). Two pulps were excluded from the final data. Forty-two exposures were capped with calcium hydroxide and amalgam as controls. At 7, 27, and 90 days, tissues were obtained by perfusion fixation, demineralized, sectioned, stained, and histologically graded according to published qualitative criteria. RESULTS: For both adhesives, at 7 days, 12 of 16 pulps showed no coagulum remnants or dentin chips at the material interface. No necrotic pulps were observed. At 27 and 97 days, 26 of 30 capped pulps had dentinal bridges at the adhesive interface. Reparative dentin was present in 28 pulps. Four 97-day pulps exhibited necrosis associated with stained bacteria. One 97-day pulp contained dentin chips throughout the pulp and demonstrated no healing, no reparative dentin, and no stained bacterial profiles. CONCLUSION: Normal soft tissue reorganization and dentinal bridge formation were observed in 86% of pulps treated with sodium hypochlorite and either adhesive system.     

The impact of bioactive molecules to stimulate tooth repair and regeneration as part of restorative dentistry

After implantation in the exposed pulp, some molecules of the den-tin extracellular matrix induce the formation of a reparative dentinal bridge in the coronal pulp. In some cases, total occlusion of the root canal also is observed. This is the case for bone sialoprotein, bone morphogenetic protein-7, Dentonin (a fragment from matrix extracellular phosphoglycoprotein), and two small amelogenin gene splice products (A+4 and A-4). Cells implicated in the reparative process are recruited, proliferate, and differentiate into osteoblast-like and odontoblast-like cells. The same results may be obtained by direct implantation of odontoblast progenitor cell into the pulp.     

Pulpal reactions to two experimental bonding systems for pulp capping procedures

This study evaluated the pulpal responses induced by application of two types of bonding system to the exposed dental pulp. One consisted of the following steps: etching with neutralized EDTA, application of an experimental water-based photocuring bonding agent, and restoration with a commercially available photocuring resin composite (EDTA etching system). The other was treatment with an experimental water-based self-etching primer, application of a commercially available bonding agent, and restoration with a commercially available photocuring resin composite (self-etching system). These two systems of treatment were applied to the exposed pulp. Calcium hydroxide was used as a control for the direct pulp-capping material. The pulps in class V cavities in the anterior teeth of beagles were mechanically exposed and then filled using the etching, the self-etching, or the Ca(OH)2 system. The beagles were sacrificed on the 7th, 30th or 90th postoperative day, and pulpal responses were investigated histopathologically using light microscopy. The EDTA etching system induced severe pulp reactions at 7 days after the operation. These reactions did not completely diminish after 90 days. Reparative dentin formation was observed at day 90. The self-etching system showed moderate pulp reactions, which gradually decreased over the experimental period. Reparative dentin bridge formation was observed at day 90. No necrosis of the pulp was observed at any time. Calcium hydroxide induced both moderate and severe initial pulp reactions, with reparative dentin formation evident at day 30. Necrosis was observed in the superficial pulp. It is suggested that the EDTA etching agent caused not only pulpal damage but also re-bleeding because of rinsing and drying. The self-etching system is a promising system for direct pulp capping.     

Octacalcium phosphate-based cement as a pulp-capping agent in rats

OBJECTIVE: The purpose of this study was to evaluate the pulpal response to an octacalcium phosphate (OCP)-based cement used as a pulp-capping material. STUDY DESIGN: The pulps of 60 maxillary first molars of male Sprague-Dawley rats were exposed and then capped directly by using either OCP-based cement or a calcium hydroxide slurry (control). Histologic examinations were performed at 1, 2, and 5 weeks after the surgical procedure, and the results were analyzed statistically by using the Mann-Whitney U test (P<.05). RESULTS: One week after pulp capping, the initial formation of reparative dentin in the exposed areas was more notable in the calcium hydroxide group than in the OCP-based cement group. At 2 weeks, reparative dentin covered by a layer of odontoblast-like cells was observed in both groups. However, at 5 weeks, reparative dentin consisting of regular dentinal tubules was observed more frequently in the OCP-based cement group. CONCLUSION: OCP-based cement allowed favorable healing processes to occur in the dental pulp.     

Expression of amelin and trauma-induced dentin formation

According to recent studies, amelin (ameloblastin, sheathlin) is expressed in young odontoblasts at the initiation of dentin formation during odontogenesis. The purpose of the present investigation was to study whether amelin is also expressed at the onset of trauma-induced reparative dentin formation. The mandibular developing first molars of 5-day-old rats were surgically taken out, and their pulp tissue briefly separated from the inner dentin surface and immediately repositioned. Then the teeth were re-implanted in their alveoli. At 0, 2, 4, 6, 8, 12 or 14 days after surgery, the animals were sacrificed and the experimental teeth evaluated by histology and immunohistochemistry for amelin. At 2, 4, 6 and 8 days after surgery, the detached and traumatized odontoblasts in the experimental teeth exhibited increasing signs of degeneration and loss of intracellular structures. At days 6 and 8 after surgery, immunohistochemistry revealed a strong staining for amelin in the traumatized odontoblastic layer. Twelve and 14 days after replantation, only necrotic cell remnants of the traumatized odontoblasts were discernible. At this stage, no amelin could be detected by immunostaining. A wide zone of an unorganized mineralized tissue surrounded the odontoblastic cell remnants. On the pulpal side of the unorganized tissue, a new, highly organized tubular reparative dentin layer was observed, bordered by columnar odontoblast-like cells abutting on newly formed predentin. The results indicate that the initiation of trauma-induced reparative dentin formation mimics that of primary dentin formation and that amelin seems to be involved in both processes, possibly as a signaling molecule.     

Pulpal response to exposure with Er:YAG laser

OBJECTIVE: The purpose of this study was to evaluate the pulpal response to the Er:YAG laser after accidental exposure of the pulp. STUDY DESIGN: Cavities were prepared, and pulps were exposed by either Er:YAG laser or mechanically by a slow-speed conventional handpiece (control group) in 76 maxillary first molars of male Wistar rats. Rats were killed immediately, at 3 days, 1 week, and 2 weeks. Histopathologic examinations of the pulp at the exposure site were performed and evaluated with the Mann-Whitney U test (P <.05). RESULTS: The Er:YAG laser group showed no bleeding and no dentin chips at the exposure site immediately after pulp exposure. However, they displayed an area of blood extravasation near the exposure site. Subsequently, the Er:YAG laser group formed dentin bridges at the exposure site more frequently than the control group. The Er:YAG laser group demonstrated more reparative dentin formation near the exposure site than the control group, especially at 2 weeks, which was highly significant (P <.01). CONCLUSION: According to the results of this study, Er:YAG laser-exposed pulp tissue demonstrated good healing capacity with the formation of a dentin bridge and reparative dentin. However, further investigations are suggested to study the effect of the blood extravasation, which appeared near the laser exposure sites.     

Differential repair responses in the coronal and radicular areas of the exposed rat molar pulp induced by recombinant human bone morphogenetic protein 7 (osteogenic protein 1)

Bone morphogenetic protein 7 (BMP 7), also termed osteogenic protein 1, a member of the transforming growth-factor superfamily, was examined for its efficacy in inducing reparative dentinogenesis in the exposed pulps of rat molars. To determine if the reaction was dose-dependent, collagen pellets containing 1, 3 or 10 microgram of recombinant BMP 7 were inserted in intentionally perforated pulps (10-12 pulps per group) in the deepest part of half-moon class V-like cavities cut in the mesial aspect of upper first molars. As controls, the collagen carrier (CC group) alone and calcium hydroxide (Ca group) were used as capping agents. All cavities were then restored with a glass-ionomer cement. Half of the animals were killed after 8 days and the other half after 28 days, by intracardiac perfusion of fixative. The molars were processed for histological evaluation by light microscopy. No difference in effect could be detected between the three concentrations of BMP 7 groups at either time interval. After 8 days, all groups showed varying inflammation, from mild of severe, and the Ca group demonstrated early formation of a reparative dentine bridge. At 28 days the CC group displayed irregular osteodentine formation, leaving some unmineralized areas at the exposure site and interglobular unmineralized areas containing pulp remnants. In the Ca-treated pulps, the initial formation of thick reparative osteodentine bridges that sealed more or less completely the pulp perforation was followed, in the deeper part, by irregular tubular dentine. In most BMP 7-treated specimens, the initial inflammation has resolved at 8 days and at 28 days heterogeneous mineralization or osteodentine filled the mesial coronal pulp. They also had complete filling of the radicular pulp by homogenous mineralization in the mesial root; this reaction was found in 11 teeth in the BMP 7 group, one tooth in the CC group an none of the Ca group. These results emphasize the biological differences the coronal and radicular parts of the pulp, and the potential of bioactive molecules such as BMP 7 to provide an a alternative conventional endodontic treatments.     

Immunolocalization of fibronectin during reparative dentinogenesis in rat molor teeth after pulp capping with mineral trioxide aggregate or calcium hydroxide

The aim of this study was to evaluate the immunolocalization of fibronectin during reparative dentinogenesis in rat teeth after pulp capping with mineral trioxide aggregate (MTA) or calcium hydroxide (Ca(OH)2). The pulps of 72 upper and lower first molar teeth from 18 male Wistar rats were experimentally exposed. The pulps were capped with MTA or (Ca(OH)2); final restoration followed with zinc oxide and eugenol cement. The animals were euthanized at, respectively, one, three, seven and fourteen days postoperatively. At day one, all groups showed varying degrees of inflammation, from mild to severe. There was no positive reaction for fibronectin at day one. After three days, a partial acute pulpitis was observed in the Ca(OH)2 group. There was less inflammation in the MTA group (p<0.05), and a layer of fibrin barrier was observed along the pulp walls of the MTA material. The layer of fibrodentin formation showed positive reaction for fibronectin. At seven days, the Ca(OH)2 group showed mild inflammation and demonstrated more immunostaining for fibronectin than the Ca(OH)2 group (p<0.05) at three days. Pulps capped with MTA at seven days showed thicker fibrin barrier formation than the MTA group at three days and more immunostaining for fibronectin in whole groups (p<0.05). At fourteen days, there was no positive reaction for fibronectin in either the MTA or Ca(OH)2 group. It seems MTA showed better biocompability properties with the dental pulp tissue, inducing the expression of reparative molecule fibronectin compared with Ca(OH)2. Therefore, MTA may be a better choice for pulp capping procedures.     

The role of canonical Wnt signaling in dentin bridge formation

ObjectiveWnt signaling has been reported to be involved in dentin bridge formation. However, the detailed mechanism has not yet been clarified. We elucidated the localization of canonical Wnt signaling molecules during dentin bridge formation.MethodsPulp of the maxillary first molar in mice was exposed and directly capped with MTA cement. Maxillae were collected on the 1st, 4th, 7th, 14th, and 28th days after treatment. After μCT analysis, immunohistochemistry for Wnt3a, Wnt10a, β-catenin, F4/80, and osterix was performed in paraffin-embedded sections.ResultsOn the 4th and 7th days after pulp capping, odontoblasts and dental pulp cells expressed Wnt3a, Wnt10a, and β-catenin. On the 14th day, reactionary dentin was formed around the pulp exposure area. Odontoblasts and dental pulp cells express Wnt3a, Wnt10a, and β-catenin. Additionally, F4/80- and Wnt10a-positive macrophages were observed at the center of the dental pulp. When the dentin bridge was formed on the 28th day, reparative odontoblasts expressed Wnt3a, β-catenin and osterix.ConclusionWnt ligands derived from odontoblasts and dental pulp cells are important for the activation of odontoblasts and the differentiation of reparative odontoblasts during dentin bridge formation. Macrophage-derived Wnts are also involved in reparative odontoblast differentiation.     

富血小板血浆用于犬牙直接盖髓的实验研究

目的:观察富血小板血浆(platelet-rich plasma,PRP)直接盖髓后修复性牙本质的形成。方法:成年雄性健康杂种犬2只,分别取静脉血制备PRP后,选取每只犬的所有切牙,尖牙和前磨牙共48个牙作为对象,于颈部制作穿髓模型后,按拆分口设计原则随机分为6组(MTA、Dycal、MTA+PRP、Dycal+PRP、PRP、空白),分别用相应盖髓剂进行直接盖髓治疗,3个月后应用牙科CT观察各组穿髓处硬组织和牙本质桥形成情况。结果:实验3个月后,MTA,MTA+PRP,Dycal+PRP组修复性牙本质形成的效果均优于单纯氢氧化钙组(Dycal)。PRP组、空白组无完整的牙本质桥形成。结论:PRP+Dycal能促进犬富血小板血浆牙髓组织修复,是一种很有希望的复合型生物盖髓剂。     

Histopathologic study on pulp response to single-bottle and self-etching adhesive systems

This study compared the pulp response to seven adhesive resins (three single-bottle and four self-etching primers) and their companion resin composite systems with a commercial calcium hydroxide material when applied to exposed monkey pulps. The control group was capped with Dycal (DY), while the experimental groups were capped with one of the following adhesive resin systems: AQ Bond (AQ), Single Bond (SB), Imperva Fluorobond (IF), One Step (OS), Prime&Bond NT (PBNT), Perme Bond F (PBF) and One-up Bond F (OBF). Histopathologic evaluation of pulp tissue disorganization, inflammatory cell infiltration, reparative dentin formation and bacterial penetration at the 3rd, 30th and 90th post-operative days was done using light microscopy. Data were analyzed using the Kruskal-Wallis test followed by the Least Significant Difference Test to determine differences between the control group and the experimental groups at each observation period. The correlation of inflammatory cell infiltration and bacterial presence was investigated by the Kendall correlation analysis. All tests were performed at a 95% level of confidence. The pulpal responses of groups DY, SB, OS, PBF and OBF were generally characterized by none-to-mild pulp tissue disorganization and inflammatory cell infiltration. Also, initiation of reparative dentin formation was found earlier in Group DY, resulting in more complete dentin bridges at the 30- and 90-day observation periods. Groups AQ, IF and PBNT had significantly more inflammatory cell infiltration and a lower incidence of reparative dentin formation than Group DY. A significant correlation was detected between inflammatory cell infiltration and the presence of bacteria. It is concluded that the pulp response to SB, OS, PBF and OBF is not significantly different from the calcium hydroxide preparation. However, calcium hydroxide capping resulted in a higher incidence and faster rate of reparative dentin formation.     

Expression of mineralization markers in dental pulp cells

There is an increasing interest in the utility of dental pulp stem cells (DPSCs) for dentin regeneration. The mechanisms involved in DPSC differentiation remain poorly understood. The purpose of the study was to investigate the mineralization capacity of human dental pulp cells (DPCs) and identify potential markers for odontoblast differentiation. The isolated DPCs expressed mesenchymal stem-cell markers as shown by flow cytometry and could differentiate in vitro into odontogenic, adipogenic, and chondrogenic lineages. Alkaline phosphatase activity of DPCs elevated over time, with significant upregulation on day 21 in odontogenic induction. Quantitative RT-PCR revealed that osteocalcin, dentin sialophosphoprotein (DSPP), and matrix extracellular phosphoglycoprotein (MEPE) expression also increased time dependently in the induction cultures. In conclusion, we isolated DPCs with stem cell characteristics. MEPE and DSPP showed a similar regulatory pattern of DPCs mineralization. MEPE along with DSPP may be potential odontogenetic differentiation markers.     

牙本质陶瓷粉、非胶原蛋白及其复合材料对修复性牙本质形成作用的研究

目的：观察牙本质陶瓷粉（ceramic demtin powder,CDP)、CDP与非胶原蛋白（noncollagenous proteins,NCPs）复合材料（CDP／NCPs）对修复性牙本质形成的作用。方法：牙本质粉煅烧后与人牙本质NCPs复合，用SD大鼠牙作直接盖髓实验，并用组织学方法评价修复性牙本质形成的效果。结果：术后14d，CDP组：穿髓下方可见少许成牙本质细胞。CDP／NCPs组：穿髓孔周围见成纤维细胞增殖，呈团块状包绕穿髓孔。术后28d，CDP组：穿髓下方可见少量修复性牙本质团块形成。CDP／NCPs组：可见大量修复性牙本质形成。结论：CDP／NCPs能促进大鼠牙体、牙髓组织修复。     

牙本质黏结剂对牙髓直接影响的动物实验

目的：观察两种牙本质黏结剂对活体牙髓组织的影响，并与氢氧化钙直接盖髓结果进行对比。方法：选用54个犬牙，机械穿髓后，无菌蒸馏水冲洗，控制穿髓孔处的出血和渗出。使用优邦(UB)和Adper Prompt Self-etching Adhesive(AP)两种牙本质黏结剂盖髓，Dycal作对照。于7、30、90d拔除实验牙，脱钙切片，HE染色、Mallory三色法染色和Gram快速细菌染色检查?结果：所有处理组在短期都出现了轻到中度的炎症反应，在长期的观察中，UB和AP两组出现持续的慢性炎症反应，均未发现牙本质桥肜成。Dycal组出现更多的修复性牙本质，并有牙本质桥形成(P＜0．05)结论：这两种牙本质黏结剂会引起牙髓的慢性炎症反应，在穿髓孔不能形成完整的牙本质桥，临床上这两种黏结剂不能直接与牙髓接触。     

矿物三氧化物凝聚体用于犬牙直接盖髓的实验研究

目的：观察矿物三氧化物凝聚体(MTA)直接盖髓后牙髓炎症反应和修复性牙本质的形成。方法：人工机械暴露犬牙髓，用MTA或氢氧化钙直接盖髓。结果：实验2周和8周，MTA诱导修复性牙本质形成的效果优于氢氧化钙，炎症反应较轻。结论：MTA是一种效果较好的盖髓剂。